Cancer treatment

Abstract

The present invention relates to structure-specific endonuclease subunit 4 (SLX4) inhibitors for use in a method of treating an individual with cancer, particularly a cancer with high levels of replication stress and / or defective DDR mechanisms, as well as methods for selecting individuals suitable for such treatments.

Description

[0001] CANCER TREATMENT

[0002] FIELD

[0003] The present invention relates to structure-specific endonuclease subunit 4 (SLX4) inhibitors for use in a method of treating an individual with cancer, particularly a cancer with high levels of replication stress and / or defective DDR mechanisms, as well as methods for selecting individuals suitable for such treatments.

[0004] BACKGROUND DNA replication is a fundamental cellular process. In eukaryotic cells, the entire genome is duplicated once and only once during the S phase of the cell cycle. Accurate DNA replication is essential for the faithful transmission of genetic information through cell divisions.

[0005] Under normal physiological conditions, DNA replication is a high-fidelity process. However, exogenous and endogenous stresses can interfere with this process. A variety of cellular stresses interfering with DNA replication, which are collectively termed replication stress (Saxena and Zou (2022) Mol Cell 82(12):2298-2314. doi: 10.1016 / j.molcel.2022.05.004; Zeman and Cimprich (2014) Nat Cell Biol 16(l):2-9. doi: 10.1038 / ncb2897, each of which are herein incorporated by reference), can impact genomic stability in normal and cancer cells. Cells have evolved a complex network of cellular responses to alleviate and tolerate replication stress, in order to maintain genomic stability.

[0006] Replication stress can arise from a variety of sources, including direct barriers to fork progression such as DNA lesions and secondary DNA structures, R-loops, nucleotide imbalance, etc., as well as the products of DNA damage repair pathways.

[0007] DNA double-strand breaks (DSBs) in mitotic cells may persist from interphase due to replication stress or may occur directly in mitosis. If left unrepaired, these DSBs lead to gross chromosome breakage, genomic rearrangements, and polyploidy, all hallmarks of cancer. Established anti-cancer therapeutic approaches exploit this by induction of replication associated DNA damage, driving cells with unrepaired DNA into mitosis, promoting mitotic catastrophe and cell death.

[0008] Cells deficient in homologous recombination (HR), experience heightened replication stress, and are particularly prone to the accumulation of mitotic DNA double-strand break (DSBs). Recent studies have identified the role of DNA polymerase theta (Pol0) mediated microhomology mediated end joining (MMEJ) and mitotic DNA synthesis (MiDAS) as critical repair pathways in mitosis, functioning redundantly to HR. These pathways present a targetable vulnerability in tumours that exhibit heightened DNA replication stress, including those that are HR deficient. Despite the potential to target the mechanisms of DSB repair in mitosis, to-date these mechanisms remain inadequately defined, limiting the ability to fully exploit these mitotic mechanisms for anti-cancer therapy. Further, clinical resistance to conventional and emerging therapeutic modalities around HR deficiencies is a growing issue and underscores the gap in our mechanistic understanding of DNA repair outside of interphase.

[0009] TOPBP1, is an essential adaptor protein, with key roles in the maintenance of genome stability, largely attributed to its nine BRCT domains, which facilitate phosphorylation-dependent protein interaction throughout the cell cycle, including in mitosis. Notably, TOPBP1 interacts with DNA polymerase theta (Pol0), facilitating MMEJ-mediated double-strand break (DSB) repair during mitosis. TOPBP1 also plays a role in regulating the BTR (BLM-TOP3a-RMIl / 2) dissolvase complex, supporting chromosomal segregation through phosphorylation events driven by CDK1 and PLK130. However, trials to-date suggest that inhibitors of Pol0 are not as effective as might be hoped. Such diminished effectiveness is clinically undesirable not only because of the reduced therapeutic benefit, but also because of the potential increased risk of resistance to such agents. Drug resistance is a common phenomenon that impairs the overall effectiveness of cancer treatments.

[0010] Furthermore, recently a mitotic specific DNA repair complex consisting of MDC1, TOPBP1 and CIP2A has been identified which functions to tether broken chromosomes to prevent fragmentation and mitotic catastrophe. This mechanism is independent of Pol0, POLD3, or the MRN complex components, although their loss increases mitotic DNA damage. CIP2A and TOPBP1, interact directly and exhibit mutual dependence for chromatin recruitment during mitosis. Recent studies have underscored CIP2A and its interaction with TOPBP1 as a highly penetrant mitotic specific synthetic lethal (SL) target in BRCA1 / 2 deficient cells. The mechanistic underpinning of this synthetic lethal interaction is unknown; although, it has been suggested that CIP2A's role in the mitotic DNA tethering complex may be a contributing factor. However, this explanation is challenged by the observation that MDC1 loss, another key DNA tethering factor does not induce the same synthetic lethality in BRCA1 / 2 deficiency. An alternative, hypothesis is that CIP2A is a key regulator of yet undefined mitotic DNA repair. However, although the interaction between TOPBP1 and Pol0 is essential for MMEJ-dependent DSB repair in mitosis, CIP2A appears to be dispensable for MMEJ-driven telomere fusions, even though it is critical in regulating TOPBP1 recruitment to chromatin.

[0011] Therefore, there remains an unmet need in the art to identify targetable factors that drive synthetic lethality between CIP2A-TOPBP1 and mutations common in cancers with high levels of replication stress, such as in homologous recombination defective (HRD) cancers, including HRD caused by / associated with a mutation and / or deficiency in BRCA1 / 2. Further, identification of new draggable targets that could be combined with agents targeting the DNA damage response (DDR) pathway, such as Pol0 inhibitors would also offer potentially significant clinical benefits.

[0012] It is an object of the present invention to address one or more of the above challenges.

[0013] SUMMARY

[0014] The present inventors have conducted comprehensive unbiased co-precipitation / mass spectrometry (Co-IP / MS) analysis to map the TOPBP1 interactome across different cell cycle phases. In particular, the present inventors have identified for the first time a number of key interactions in mitosis with SMX complex components (SLX4, ERCC1, XPF, EMEI, MUS81), CIP2A, MDC1, and PLK1.

[0015] As well as determining that TOPBP1 is required for the recruitment of MUS81 in BRCA2 deficiency, the inventors have shown for the first time previously uncharacterised vulnerabilities in replication stress-dependent tumour cells, emphasising a therapeutic potential not addressed by current DNA repair targeting strategies. In particular, they have shown that CDK1 dependent phosphorylation of SLX4 at Thr1260 is critical for its interaction with BRCT1 and 2 of TOPBP1. This interaction functions to recruit SLX4, MUS81 and ERCC1 to mitotic chromatin marked by the CIP2A-TOPBP1 complex in response to replication stress. Cells with a Thr1260Ala mutation show defective MiDAS, leading to increased genome instability, indicated by elevated micronuclei levels.

[0016] Critically, the inventors have identified a genetic interaction network that defines the CIP2A-TOPBP1 complex as a master regulator that facilitates mitotic DNA repair pathway choice between MiDAS and MMEJ. The CIP2A-TOPBP1 complex achieves this by recruiting not only components of the SMX complex (SLX4, MUS81 and ERCC1) but also Pol0 to mitotic chromatin. Concurrently, they have demonstrated that CIP2A loss impairs both break induced replication (BIR)-like / MiDAS and MMEJ, establishing a synthetic lethality that could be therapeutically exploited in a broad range of cancer with high replicative stress and / or a defective DNA damage response (DDR) mechanism. In particular, the inventors have shown that inhibiting the TOPBP1-SLX4 interaction, exemplified herein using a minimal SLX4 fragment containing Thr1260 and SLX4 siRNA is sufficient to impair proliferation in BRCA1 / 2 deficient cells. This indicates that targeting the TOPBP1-SLX4 interaction is a promising avenue for the development of anti-cancer therapies for a broader range of cancers having high levels of replication stress, and in particular HRD cancers.

[0017] Further, the inventors have demonstrated that pharmacological inhibition of Pol0, combined with loss of the TOPBP1-SLX4 interaction, exacerbates genome instability (particularly chromosomal instability (CIN)) and reduces cellular proliferation under replication stress. Notably, SLX4, Pol0, and CIP2A are essential for cellular proliferation in BRCAl / 2-deficient cells. Therefore, the data provided herein supports the clinical potential of combination therapies comprising agents targeting SLX4, and particularly the T0PBP1-SLX4 interaction, and agents targeting the DDR pathway, such as Pol0 inhibitors.

[0018] Accordingly, the present disclosure provides a structure-specific endonuclease subunit 4 (SLX4) inhibitor for use in a method of treating an individual with a cancer with high levels of replication stress. The present disclosure provides a Topoisomerase beta binding protein 1 (TOPBP1) inhibitor for use in a method of treating an individual with a cancer with high levels of replication stress.

[0019] The replication stress may be associated with a mutation and / or deficiency in one or more gene associated with replication stress, optionally wherein said one or more gene associated with replication stress is selected from BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, and Liglll. The high levels of replication stress may associated with a mutation and / or deficiency in BRCA2 and / or BRCA1.

[0020] The SLX4 inhibitor may be a peptide, a small molecule, a proteolysis-targeting chimeric molecule (PROTAC), a macrocyclic molecule, a molecular glue, a nucleic acid inhibitor, an antibody, or an antibody-drug conjugate.

[0021] The SLX4 inhibitor may target the TO PBP1 binding site of SLX4, which site comprises an amino acid corresponding to Thr1260 of SEQ ID NO: 1. The TOPBP1 inhibitor may target the SLX4 binding site of TOPBP1. The SLX4 binding site of TOPBP1 typically comprises the BRCT1 and / or BRCT2 of TOPBP1.

[0022] The SLX4 inhibitor may: (a) inhibit the phosphorylation of the amino acid corresponding to Thr1260 of SEQ ID NO: 1; (b) inhibit the interaction of the amino acid corresponding to Th rl260 of SEQ ID NO: 1 with BRCT1 and / or BRCT2 of TOPBP1; and / or (c) inhibit the expression of SLX4.

[0023] The SLX4 inhibitor may be a peptide between about 6 to about 100 amino acids in length, which comprises or consists of an SLX4 fragment comprising a threonine at a position corresponding to Thr1260 of SEQ ID NO: 1. Optionally said peptide may comprise or consist of: (a) the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence with at least 90% identity to SEQ ID NO: 7, or a fragment thereof, provided that said peptide retains a threonine at a position corresponding to Thr1260 of SEQ ID NO: 1; or (b) the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence with at least 90% identity to SEQ ID NO: 8, or a fragment thereof, provided that said peptide retains a threonine at a position corresponding to Thr1260 of SEQ ID NO: 1.

[0024] The SLX4 inhibitor may be oligonucleotide, such as an antisense oligonucleotide or an siRNA. Optionally said siRNA may have an antisense strand that is at least 80% reverse complementary to an equal length portion of at least 12 contiguous nucleobases within the nucleobase sequence of SEQ ID NO: 2. The SLX4 inhibitor may be an siRNA which has an antisense strand which comprises or consists of a nucleobase sequence that is at least 80% identical to SEQ ID NO: 9 or 10, and optionally a sense strand which comprises or consists of a nucleobase sequence that is at least 80% identical to SEQ ID NO: 11 or 12.

[0025] The SLX4 inhibitor may be an antibody or antigen-binding fragment thereof that specifically binds to SLX4. Optionally said antibody or antigen-binding fragment thereof: (a) specifically binds to an epitope comprising or consisting of the TOPBP1 binding site of SLX4, which site comprises an amino acid corresponding toThr1260 of SEQ ID NO: 1; (b) specifically binds to SLX4 that is phosphorylated at a position corresponding to Thr1260 of SEQ ID NO: 1; and / or (c) binds to an epitope comprising or consisting of SEQ ID NO: 13, or which competes for binding to SLX4 with an antibody that binds to such an epitope.

[0026] Treatment with an SLX4 inhibitor according to the disclosure may specifically inhibit mitotic DNA synthesis (MiDAS) and / or break induced replication (BIR) in a cancer cell.

[0027] Treatment with an SLX4 inhibitor according to the disclosure may be combined with one or more further anti-cancer therapies. An SLX4 inhibitor may be used in conjunction with: (a) one or more further chemotherapeutic agent(s); and / or (b) one or more immunotherapeutic agent, which is optionally selected from immune checkpoint inhibitors, monoclonal antibodies, cytokines, cell therapy, cancer vaccines, oncolytic viruses, antisense oligodeoxynucleotides, antibody-drug conjugates and / or any modulator of the cytosolic nucleic acid sensing pathways. The one or more further anti-cancer therapy may be: (a) an inhibitor of a component of the DNA damage repair (DDR) pathway; and / or (b) an inhibitor of microhomology mediated end-joining (MMEJ). The one or more further anti-cancer therapy may be an inhibitor of any one of Pol0, CHK1, ATR, WEE1, PARP, RAD51, or ATM. The one or more further anti-cancer therapy may be an inhibitor of a component of the DDR pathway, and wherein the treatment has increased clinical efficacy compared with treatment with the inhibitor of a component of the DDR pathway alone. Alternatively or in addition, the one or more further anti-cancer therapy may be an inhibitor of a component of the DDR pathway, and wherein the treatment reduces resistance to the inhibitor of a component of the DDR pathway compared with treatment with the inhibitor of component of the DDR pathway alone.

[0028] Treatment with an SLX4 inhibitor according to the disclosure may be used in conjunction with radiotherapy.

[0029] The invention also provides a method of reducing resistance to an anti-cancer therapy with an inhibitor of a component of the DDR pathway, said method comprising administering an SLX4 inhibitor in conjunction with the inhibitor of a component of the DDR pathway. The SLX4 inhibitor and the one or more further anti-cancer therapy may be administered separately, sequentially or simultaneously.

[0030] The invention also provides a method of treatinga cancerwith high levels of replication stress, said method comprising administering a therapeutically effective amount of an SLX4 inhibitor to an individual in need thereof.

[0031] The invention further provides use of an SLX4 inhibitor in the manufacture of a medicament for treating a cancer with high levels of replication stress.

[0032] The invention also provides a method of selecting an individual having cancer for treatment with a structure-specific endonuclease subunit 4 (SLX4) inhibitor, the method comprising: (a) determining in a sample obtained from the individual whether the cancer is a cancer with high levels of replication stress; (b) selecting the individual for treatment with the SLX4 inhibitor where the cancer is a cancer with high levels of replication stress; and (c) providing an SLX4 inhibitor suitable for administration to the individual; wherein said method optionally further comprises administering a therapeutically effective amount of the SLX4 inhibitor to the individual. Treatment with an SLX4 inhibitor may be combined with one or more further anti-cancer therapies.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Fig.l: Enrichment of TOPBP1 interactions with SMX complex components during mitosis

[0035] (A). Schematic representation of endogenous TOPBP1 Co-immunoprecipitation experiments across different cell cycle phases from independent HEK293TN cells (created with BioRender.com). (B). Dot plot illustrating mean Iog2 fold change (FC) of proteins detected in T0PBP1 Co-IP samples from M phase versus asynchronous [A] cells, by-MS label-free mass-spectrometry. (C). similar analysis as in B, comparing S phase with M phase. Samples analysed in A, B and C were from three independent experiments. (D). Dot plot representing mean of TMT quantitative mass spectrometry analysis showing statistically significant enrichment of T0PBP1 interactions in asynchronous versus M phase cells, samples were from three independent experiments. Statistical significance was determined by two tailed t-test. (E). Gene Ontology (GO)-term enrichment analysis of the mitotic T0PBP1 interactome-loglO enrichment false discovery rate (FDR). (F). Western blot analysis of BLM, ERCC1, MUS81 and SLX4 interactions with eGFP-TOPBPl in GFP-TRAP Co-IPs from HEK293TN cells transiently transfected with a eGFP-TOPBPl WT expression construct followed with or without mitotic synchronisation by 100 ng / ml nocodazole for 16 hours. 1% of input was used for analysis by western blot of input lysate. Fig.2: Elevation of mitotic TOPBP1 dependent recruitment of SMX complex components in BRCA2 deficiency

[0036] (A). Representative images and a dot plot showing the number of MUS81 and TOPBP1 colocalising foci in DLD1 and DLD1 BRCA2‘Z‘ prometaphase cells (WT: n=74and BRCA2‘Z‘: n=73, from three independent experiments). (B). Representative images and a dot plot showing the number of ERCC1 and TOPBP1 colocalising foci in DLD1 and DLD1 BRCA2‘Z‘ cells (WT: n=73, BRCA2‘Z‘: n=74, from three independent experiments). (C). Representative images and a dot plot showing the number of MUS81 localisation in DLD1 and DLD1 BRCA2‘Z‘ prometaphase cells, treated with siCTRL, siTOPBPl or siSLX4 (DLD1 (siCTRL: n=75, siTOPBPl: n=82, siSLX4: n=86) and DLD1 BRCA2‘Z‘ (siCTRL: n=65, siTOPBPl: n=69, siSLX4: n=64) from three independent experiments). (D). Representative images and a dot plot showingTOPBPl foci in DLD1 BRCA2‘Z‘ prometaphase cells (siCTRL: n=72 and siSLX4: n=73, from three independent experiments). (E). Representative images and a dot plot showing number of MUS81 foci in prometaphase HCT116-TOPBPl-mAID-Clover cells with and without 2 hour IAA (500pM) dependent degradation of TOPBP1 ("-": n=81 and "+": n=84, from three independent experiments. Statistical significance in A, B, D and E was determined by two-tailed unpaired t-test. Statistical significance in C was determined by the two-way ANOVA with Tukey's post-hoc test. In A-E individual measurements are represented by grey dots whereas black dots indicate medians of each experiment, and the mean is presented as bars with error bars showing S. E. M. Scale bars equivalent to 10 pm. (F). Representative images and dot plot of TOPBP1 foci in DLD1 and DLD1 BRCA2‘Z‘ prometaphase cells (WT: n=74, BRCA2‘ n=73 from three independent experiments, statistical significance was determined by two tailed unpaired t-test). Grey dots represent individual measurements, black dots indicate medians from individual experiments and bars represent the mean with S. E. M displayed. (G). Western blot analysis of DLD1 and DLD1 BRCA2‘Z‘ cells treated with siCTRL or siTOPBPl. (H). Western blot analysis of DLD1 and DLD1 BRCA2‘Z‘ cells treated with siCTRL or siSLX4. (I). Representative images and violin plot ofSLX4 and TOPBP1 colocalising foci in DLD1 WT and DLD1 BRCA2- / - prometaphase cells synchronised with 60 ng / ml nocodazole for 2 hours (WT: n=74, BRCA2- / --. n=75 from three independent experiments). (J). Representative images and violin plot of SLX4 and TOPBP1 colocalising foci in RPE1 p53- / - PAC- / -BRCA1- / - prometaphase cells synchronised with 60 ng / ml nocodazole for 2 hours (WT: n=52, BRCA1- / -: n=52 from two independent experiments). (K). Representative images and violin plot of MUS81 and TOPBP1 colocalising foci in RPE1 p53- / - PAC- / - BRCA1- / - prometaphase cells synchronised with 60 ng / ml nocodazole for 2 hours (WT: n=50, BRCA1- / -: n=54 from two independent experiments). (L).

[0037] Representative images and violin plot of SLX4 and TOPBP1 colocalising foci in RPE1 p53- / - FRT / TR prometaphase cells treated with or without aphidicolin then synchronised with 60 ng / ml nocodazole for 2 hours (UT: n=73, Aph: n=76 from three independent experiments). (M). Representative images and dot plot of the number of TOPBP1 foci per prometaphase cell nucleus of RPE1 p53- / - PAC- / -BRCA1- / - cells treated with siCTRL or siSLX4 by synchronisation with 60 ng / ml nocodazole for two hours (siCTRL: n=50, siTOPBPl: n=51 from two individual experiments). (N). Western blot analysis of SLX4 in RPE1 p53- / - PAC- / - BRCA1- / - cells treated with siCTRL or siSLX4. (O) Representative images and dot plot of the number of MUS81 foci per prometaphase cell nucleus of RPE1 p53- / - PAC- / - BRCA1- / - cells treated with siCTRL or siTOPBPl followed by synchronisation with 60 ng / ml nocodazole for two hours (siCTRL: n=72, siTOPBPl: n=50 from three individual experiments). (P) Western blot analysis of TOPBP1 in RPE1 p53- / - PAC- / - BRCA1- / - cells treated with siCTRL or siTOPBPl.

[0038] Fig.3: SLX4 interacts with BRCT1 / 2 of TOPBP1 via pT1260 in mitotic cells

[0039] (A). eGFP-TOPBPl domain architecture schematic (B). Western blot analysis of eGFPTOPBPl interacting SMX complex proteins in eGFP-TOPBPl truncation mutant Co-IP from HEK293TN cells transiently transfected with WT or C-terminally truncated eGFP-TOPBPl expression constructs followed by 16 hours 100 ng / ml nocodazole synchronisation. Mock Co-IP from non-transfected HEK293TN cells were used as negative control (CTRL). (C). Western blot analysis of SMX component interactions with TOPBP1 in eGFPTOPBPl Co-IPs from HEK293TN cells transiently transfected with WT or N-terminally truncated A314 (3-8) eGFP-TOPBPl expression constructs followed by 16 hours 100 ng / ml nocodazole synchronisation. Mock Co-IP from non-transfected HEK293TN cells were used as negative control (CTRL). (D). Domain architecture of SLX4 and relative position of the identified T1260, TOPBP1 BRCT 1 recognition motif. (E). Fluorescence polarisation analysis of recombinant TOPBP1 BRCTO / 1 / 2, BRCT 4 / 5 and BRCT 7 / 8 domain containing fragments in the presence of a fluorescein tagged SLX4 pT1260-containing peptide. For all IP experiments 1% of input was used for analysis by western blot of input lysate. (F). Fluorescence polarisation analysis of recombinant TOPBP1 BRCTO / 1 / 2 fragment in the presence of fluorescein tagged SLX4 pT1260-containing peptide with or without lambda (A) phosphatase treatment. (G). Fluorescence polarisation analysis of recombinant TOPBP1 WT or conserved lysine to glutamic acid mutations in BRCT1 (K155E) or 2 (K250E) or in BRCT 1 +2 (K155E +K250E) fragment in the presence of fluorescein tagged SLX4 pT1260 containing peptide. (H). Table showing the dissociation constant of TOPBP1 BRCT 0-1-2 WT or K250E with SLX4 pT1260. (I). TOPBP1 BRCT 0-1-2 and SLX4 pT260 interaction modelled in AlphaFold 3. (J). TOPBP1 BRCT 0-1-2 and RAD9 pS387 crystal structure (PDB: 6HM5).

[0040] Fig.4: Mitotic interaction of the SMX complex components and TOPBP1 is driven by CDK1 activity (A). Western blot analysis of MDC1 and SMX complex component interaction with eGFPTOPBPl in CoIPs from HEK293TN cells treated with 100 ng / ml of nocodazole for 16h after transient transfection of eGFP-TOPBPl WT, BRCT 1 (K155E), 2 (K250E) or 1+2 (K155E +K250E) expression constructs. Mock Co- IP from non-transfected HEK293TN cells were used as negative control (CTRL). (B). Western blot analysis of TOPBP1 and SMX component interaction with eGFP-SLX4 in Co-IPs from HEK293TN cells treated with 100 ng / ml of nocodazole for 16h after transient transfection of eGFP-SLX4 WT and T1260A expression constructs. Mock Co-IP from non-transfected HEK293TN cells were used as negative control (CTRL). (C). Western blot analysis of CDK1 dependent phosphorylation and interaction between SMX components and TOPBP1 in eGFP-SLX4 Co-IPs. HEK293TN cells were transfected with an eGFP-SLX4 expression construct and treated with or without 9 pM RO-3306 (CDKli) 30 minutes after 16h 100 ng / ml nocodazole treatment. Mock Co-IP from non-transfected HEK293TN cells act as negative control (CTRL). (D). As in C, but reciprocal eGFP-TOPBPl Co-IP analysis. For all IP experiments 1% of input was used for analysis by western blot of input lysate. (E). SDS-PAGE of in vitro reconstitution of CDKl-Cyclin B-CKS1 driven phosphorylation of a SLX4 T1260 containing biotinylated peptide and its phosphorylation-dependent interaction with a recombinant TOPBP1 BRCT 0-1-2 containing fragment. (F). Western blot analysis of fractionated HEK293TN cells treated with 100 ng / ml of nocodazole for 18 hours, 24 hours after transient transfection with eGFP-TOPBPl WT, or 1+2 (K155E+K250E) expression constructs and western blot analysis of SLX4, MUS81 and ERCC1 in Co-IPs from the chromatin fraction of these cells. Mock Co-IP from non-transfected HEK293TN cells was conducted as negative control (CTRL). G-H Representative images and violin plot of number of TOPBP1 (G). and MUS81 (H). foci per prometaphase nucleus of RPE1 p53- / - FRT / TR cells treated with 400 nM aphidicolin for 18 hours followed by 60 ng / ml nocodazole for 4 hours, then DMSO or 5 pM RO-3306 (CDKli) for 30 min ((G) DMSO: n=54, CDKli: n=52; (H) DMSO: n=54, CDKli: n=60 from two individual experiments, statistical significance was determined by Mann-Whitney test). Grey dots represent individual values, red line indicates median, and scale bars represent 10 pm. (I) Western blot analysis of fractionated HEK293TN cells transfected with eGFP-SLX4, followed by synchronisation with 100 ng / ml nocodazole for 18 hours, then with DMSO or 7 pM RO-3306 (CDKli) for 30 min. d-e Representative images and violin plot of number of TOPBP1.

[0041] Fig.5: Mitotic localisation of the SMX complex components are CIP2A-TOPBP1 dependent and facilitates unscheduled DNA synthesis to safeguard genome stability

[0042] (A). Representative images and (top right panel) a bar plot showing the mean percentage of eGFP-SLX4 WT foci colocalising with CIP2A per RPE1 p53z- SLX4‘ / ‘ FRT / TR +eGFP-SLX4 WT / T1260A prometaphase cell after induction with 10 ng / ml doxycycline for 24 hours followed by treatment with or without 400 nM / 18 h aphidicolin followed by synchronisation with 60 ng / ml nocodazole for 2 hours (UT: n=85, Aph: n=76). Black dots represent means from each experiment and bars represent mean of three-experiments; (bottom right panel) a dot plot showing the number of eGFP-SLX4 WT or eGFP- SLX4 T1260A foci colocalising with CIP2A foci per prometaphase cell after treatment as above (eGFP-SLX4 WT (UT: n=85, Aph: n=76);) and eGFP-SLX4 T1260A (UT: n=70, Aph: n=76) from three independent experiments, statistical significance was determined by two-way ANOVA). Grey dots represent individual measurements, black dots represent the medians of each experiment and bars show the mean with error bars showing S. E. M. (B). Representative images and a dot plot showing CIP2A and MUS81 colocalising foci in RPE1 p53‘z‘ FRT / TR WT and SLX4 T1260A prometaphase cells treated with 400 nM aphidicolin followed by synchronisation with 60 ng / ml nocodazole for 2 hours (Parental WT: n=80, T1260A Cl.l: n= 76, T1260A Cl.2 n=85, from three independent experiments, statistical significance was determined by one-way ANOVA test with Dunnet's post-hoc test). Grey dots represent individual measurements, black dots represent the medians of each experiment and bars show the mean with error bars showing S. E. M. (C). Representative images and violin plot of the number of EdU foci in RPE1 p53 / _FRT / TR WT and T1260A prometaphase cells (Parental WT: n=90, T1260A Cl.l: n= 87, T1260A Cl.2 n=82, from three independent experiments, statistical significance was determined by Mann-Whitney U test). (D). Representative images and bar plot of the mean of the percentage of micronuclei-positive RPE1 p53- / - FRT / TR WT, SLX4- / -and SLX4 T1260A cells (Parental WT n=336, SLX4- / - n=346, T1260A Cl.l: n= 384, T1260A CI.2 n=377 from three independent experiments, statistical significance was determined by one-way ANOVA with Dunnet's post-hoc test), black dots represent the mean of each experiment and bars show the mean with error bars showing S. E. M. Scale bars in all A- D are equivalent to 10 pm.

[0043] Fig.6: CIP2Ais required for the regulation of redundant mitotic repair pathways

[0044] (A). Representative images and (B). a bar plot showing the percentage of eGFP-SLX4 foci colocalising with TOPBP1 in RPE1 p53‘z‘FRT / TR SLX4 / _prometaphase cells and (C). number of eGFP-SLX4 and TOPBP1 colocalising foci in RPE1 p53‘z‘FRT / TR prometaphase cells induction with 10 ng / ml doxycycline for 24 hours, 48 hours after siRNA treatment. (B and C). Cells were treated with siCTRL or siCIP2A (siCTRL: n=73, siCIP2A: n=73, statistical significance was determined by two tailed unpaired t-test).

[0045] (D). Representative images and dot plot of EdU foci in prometaphase cells DLD1 BRCA2‘ / ‘cells (siCTRL: n=75, siCI P2A: n= 74 from three independent experiments, statistical significance was determined by two tailed unpaired t test). (E). Reporter system schematic (created with BioRender.com) and bar plot of BIR dependent repair efficiency following sgRNP-CAS9 facilitated cleavage of an l-Scel site in the pBIR-GFP BIR reporter U2OS cell line, following treatment with siCTRL, siP0LD3, siCIP2A or siSLX4. Measurements are from four independent experiments. (F). Representative images and dot plot of eGFP-Pol0 foci in prometaphase DLD1 BRCA2‘ / ‘eGFP-Pol0 cells after induction with 100 ng / ml doxycycline for 24 hours, 48 hours after siRNA treatment. (siCTRL: n=86, siCIP2 A: n= 83 from three independent experiments, statistical significance was determined by two tailed unpaired t-test). In C, D and F grey dots represent individual measurements; black dots represent medians of each experiment and bars the mean with S. E. M displayed. Scale bars equivalent to 10 pm. (G). Reporter system schematic (created with BioRender.com) and bar plot of MMEJ repair efficiency following sgRNP-CAS9 generated cleavage of an l-Scel site in the EJ2 reporter U20S cell lines following treatment with siCTRL, siPolO or siCIP2A. Measurements are from three independent experiments. Statistical significance in E and G was determined by one-way ANOVA with Dunnet's post-hoc test and the mean is presented as bars with S. E. M. (H). Representative images and dot plot showing the number of T0PBP1-MUS81 colocalising foci in RPE1 p53- / - FRT / TR WT or CIP2A- / - prometaphase cells following 400 nM aphidicolin for 18 h and synchronisation with 60 ng / ml nocodazole for 2 h (WT: n=78; CIP2A- / - Cl.l: n=79; Cl.2: n=80; three independent experiments). (I). Representative images and dot plot of EdU foci in prometaphase RPE1 p53- / - WT or CIP2A- / - cells after following 400 nM aphidicolin followed by 30 min synchronisation with 60 ng / ml nocodazole (WT: n=77; Cl.l: n=69; Cl.2: n=74; three experiments). (J). Representative images and dot plot of CIP2A-yH2AX colocalising foci in RPE1 p53- / -FRT / TR WT cells untreated or treated with 400 nM aphidicolin for 18 h followed by synchronisation with 60 ng / ml nocodazole for 2 h (UT: n=82; Aph: n=69; three experiments; two-tailed unpaired t-test). (K). Representative images and dot plot of FANCD2-CIP2A colocalising foci in RPE1 p53- / - WT cells under the same conditions as (J) (UT: n=75; Aph: n=70; three experiments; two-tailed unpaired t-test).

[0046] Fig.7 CIR2A dependent orchestration of DNA repair is required for genome stability and cellular proliferation in the absence of BRCA1 or 2.

[0047] (A). Representative images and bar plot illustrating the number of micronuclei per RPE1 p53‘z‘FRT / TR WT or SLX4T1260A cells. Cells were treated with siCTRLor siCI P2A, 400 nM aphidicolin and with either DMSO or 5 pM ART558 (si PolOi) for 18 hours (Parental WT: (siCTRL + DMSO: n =343, siCI P2 A + DMSO: n=394, siCTRL + si PolOi: n=372) and SLX4 T1260A CI2: (siCTRL +DMSO: n= 399 and siCTRL + si PolOi: n =378) from three independent experiments). Statistical significance was determined using by the Mann-Whitney test, and the mean of three experiments is presented as bars with S. E. M. (B). Proliferation analysis of RPE1 p53‘z‘FRT / TR WT and T1260A cells, performed using the Incucyte live cell analysis system. Cells were treated with or without 5 pM ART558 (si Pol ©i ) in the presence of 400 nM aphidicolin. The mean confluence divided by confluence at day 0, from three independent experiments, is displayed with S. E. M. Scale bars equivalent to 10 pm. (C). Proliferation analysis using the Incucyte SX5 live cell imaging system, of RPE1 p53- / - FRT / TR BRCA1- / - cells treated with siCTRL, siSLX4 or siCI P2A followed by treatment with DMSO or 5 pM ART558 (PolOi). The mean confluence divided by confluence at day 0, from three independent experiments, is displayed with error bars for S. E. M. (D). Proliferation analysis of DLD1 BRCA2‘Z‘ cells using the Incucyte live cell analysis system. Cells were treated with siCTRL, siSLX4 or siCIP2A in the presence of DMSO or 5 pM ART558 (Pol0i). The mean confluence divided by confluence at day 0, from three independent experiments, is displayed with S. E. M. (E). Model: Cells enter mitosis with DNA damage, under replicated DNA or recombination intermediates where the CIP2A-TOPBP1 complex is recruited. CDK1 phosphorylation of SLX4 atT1260 regulates interaction with TOPBP1 BRCT 1 / 2 facilitating SMX component recruitment and MiDAS / BIR to safeguard genome stability. PLK1 phosphorylation of POLQ. in mitosis facilitates TOPBP1 interaction and MMEJ. Loss of CIP2A impairs both pathways and leads to simultaneous deficiency in mitotic MiDAS / BIR and MMEJ promoting genome instability (created with BioRender.com). (F). Proliferation analysis using the Incucyte S3 live cell analysis system of DLD1 BRCA2‘ / ‘cells with inducible expression of empty vector (EV) or a SLX4 T1260A fragment, incubated in the presence of 1 pg / ml doxycycline. Data points represent mean of two independent experiments with error bars representing S. E. M. (G). Proliferation analysis using the Incucyte S3 live cell analysis system of SUM149PT BRCAl ^ cells with inducible expression of empty vector (EV) or a SLX4 T1260A fragment, incubated in the presence of 1 pg / ml doxycycline. Data points represent mean of two independent experiments with error bars representing S. E. M. (H). Representative images and violin plots of the number of yH2AX foci per interphase nucleus of RPE1 p53- / - FRT / TR WT or T1260A cells treated with DMSO or 5 pM ART558 (Pol0i) and 400 nM aphidicolin for 18 hours (WT DMSO: n= 201, WT Pol0i: n=201, T1260A DMSO: n= 201, T1260A Pol0i: n= 201 from two independent experiments, statistical significance was determined by Mann-Whitney test). (I). Representative images and violin plots of the number of 53BP1 foci per interphase nucleus of RPE1 p53- / - FRT / TR WT or T1260A cells treated with DMSO or 5 pM ART558 (Pol0i) and 400 nM aphidicolin for 18 hours (WT DMSO: n= 200, WT Pol0i: n=200, T1260A DMSO: n= 200, T1260A Pol0i: n= 200 from two independent experiments, statistical significance was determined by Mann-Whitney test). In Figures 7H and 71, red line indicates medians, grey dots indicate individual values, representative image scale bars are equivalent to 10 pm.

[0048] Fig.8 Elevated formation of TORBR1, SLX4, and MUS81 foci during interphase in BRCA2-deficient DLD1 cells compared to wild-type controls, as well as in non-cancerous RPE1 cells treated with aphidicolin.

[0049] (A). Representative images and dot plot of the number of TOPBP1 foci per interphase cell nucleus of DLD1 WT and BRCA2‘Z‘ cells (WT: n=200, BRCA2‘Z‘: n=200 from two individual experiments). (B).

[0050] Representative images and dot plot of the number of SLX4 foci per interphase cell nucleus of DLD1 WT and BRCA2‘Z‘ cells (WT: n=300, BRCA2‘Z‘: n=300 from three individual experiments). (C). Representative images and dot plot of the number of MUS81 foci per interphase cell nucleus of DLD1 WT and BRCA2‘Z‘ cells (WT: n=299, BRCA2‘Z‘: n=306 from three individual experiments). (D). Representative images and dot plot of the number of TOPBP1 foci per interphase cell nucleus of RPE1 p53‘z‘FRT / TR cells treated without or with 400 nM aphidicolin for 18 hours (UT: n=592, Aph: n=459 from three individual experiments). (E). Representative images and dot plot of the number of SLX4 foci per interphase cell nucleus of RPE1 p53‘z‘FRT / TR cells treated without or with 400 nM aphidicolin for 18 hours (UT: n=338, Aph: n=302 from three individual experiments). (F). Representative images and dot plot of the number of MUS81 foci per interphase cell nucleus of RPE1 p53‘z‘FRT / TR cells treated without or with 400 nM aphidicolin for 18 hours (UT: n=443, Aph: n=360 from three individual experiments). In Figures 8A-F, scale bars represent 10 pm, grey dots represent individual measurements, black dots indicate medians from individual experiments and bars represent the mean with SEM displayed, statistical significance was determined by two-tailed unpaired t-test.

[0051] Fig.9 Increased colocalisation events between SLX4 / MUS81 and TORBR1 in mitosis compared to interphase, both in BRCA2-deficient DLD1 cells and RPE1 cells treated with or without aphidicolin.

[0052] (A). Bar graph of the mean percentage of SLX4 foci colocalising with TOPBP1 in interphase compared to mitotic cell nuclei in DLD1 WT and BRCA2‘Z‘ cells (Interphase: WT: n=864, BRCA2‘Z‘: n=1046; Mitosis: WT: n=74, BRCA2‘Z‘: n=75 from three individual experiments). (B). Bar graph of the mean percentage of MUS81 foci colocalising with TOPBP1 in interphase compared to mitotic cell nuclei in DLD1 WT and BRCA2‘ / ‘ cells (Interphase: WT: n=1057, BRCA27’: n=1071; Mitosis: WT: n=74, BRCA2‘ / ‘: n=73 from three individual experiments). (C). Bar graph of the mean percentage of SLX4 foci colocalising with TOPBP1 in interphase compared to mitotic cell nuclei in RPE1 p53‘z‘FRT / TR cells treated without or with 400 nM aphidicolin for 18 hours (Interphase: UT: n=553, Aph: n=367; Mitosis: UT: n=73, Aph: n=76 from three individual experiment). (D). Bar graph of the mean percentage of MUS81 foci colocalising with TOPBP1 in interphase compared to mitotic cell nuclei in RPE1 p53‘z‘ FRT / TR cells treated without or with 400 nM aphidicolin for 18 hours (Interphase: UT: n=608, Aph: n=376; Mitosis: UT: n=96, Aph: n=133 from two individual experiments). In Figures 9A-D, Black dots represent median percentage of colocalising foci per cell nucleus in each individual experiment; bars represent mean of these individual experiments with error bars depicting SEM. Statistical significance was determined by two-tailed unpaired t-test. (E). Representative images and dot plot of the number of SLX4 foci per Cyclin A2 positive interphase cell nucleus of DLD1 BRCA2‘Z‘ cells treated with siCTRL or siTOPBPl (WT: n=384, BRCA2‘Z‘: n=357 from three individual experiments). (F). Representative images and dot plot of the number of MUS81 foci per Cyclin A2 positive interphase cell nucleus of DLD1 BRCA2‘ / ‘cells treated with siCTRL or siTOPBPl (WT: n=239, BRCA2‘Z‘: n=161 from three individual experiments). (G). Representative images and dot plot of the number of SLX4 foci per Cyclin A2 positive interphase cell nucleus of RPE1 p53‘z‘ FRT / TR cells treated with siCTRL or siTOPBPl followed by 400 nM aphidicolin for 18 hours (UT: n=188, Aph: n=231 from two individual experiments). (H). Representative images and dot plot of the number of MUS81 foci per Cyclin A2 positive interphase cell nucleus of RPE1 p53‘ FRT / TR cells treated with siCTRL or siTOPBPl followed by 400 nM aphidicolin for 18 hours (UT: n=513, Aph: n=368 from three individual experiments). In Figures 9E-H, Grey dots represent individual measurements, black dots indicate medians from individual experiments and bars represent the mean with SEM displayed, statistical significance was determined by two-tailed unpaired t-test, representative image scale bars represent 10 pm.

[0053] Fig.10 Representative images of CIP2A localisation in prometaphase mitotic cells, Cyclin A2 negative and positive interphase RPE1 p53- / - FRT / TR WT cells and CIP2A- / - cells treated with 400 nM aphidicolin for 18 hours.

[0054] Fig.ll Line graph showing the survival of RPE1 p53- / - FRT / TR WT or SLX4 T1260A endogenous knock in mutant cells in response to cisplatin treatment for 6 days, determined by the CellTiter-Glo® Luminescent Cell Viability Assay (Promega).

[0055] Values normalised to measurements from cells incubated in the absence of cisplatin. Data points are the mean of three independent experiments and error bars display SEM.

[0056] DETAILED DESCRIPTION

[0057] Definitions

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary.

[0059] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognise. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0060] The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.

[0061] As used herein, the term "capable of' when used with a verb, encompasses or means the action of the corresponding verb. For example, "capable of interacting" also means interacting, "capable of cleaving" also means cleaves, "capable of binding" also means binds and "capable of specifically targeting" also means specifically targets.

[0062] Numeric ranges are inclusive of the numbers defining the range. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0063] As used herein, the articles "a" and "an" may refer to one or to more than one (e.g. to at least one) of the grammatical object of the article. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In this application, the use of "or" means "and / or" unless stated otherwise. Furthermore, the use of the term "including", as well as other forms, such as "includes" and "included", is not limiting.

[0064] "About" may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. Preferably, the term "about" shall be understood herein as plus or minus (±) 5%, preferably ± 4%, ± 3%, ± 2%, ± 1%, ± 0.5%, ± 0.1%, of the numerical value of the number with which it is being used.

[0065] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the invention.

[0066] As used herein the term "consisting essentially of" refers to those elements required for a given invention. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that invention (i.e. inactive or non-immunogenic ingredients).

[0067] Embodiments described herein as "comprising" one or more features may also be considered as disclosure of the corresponding embodiments "consisting of" and / or "consisting essentially of" such features.

[0068] Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0069] Amino acids are referred to herein using the name of the amino acid, the three-letter abbreviation or the single letter abbreviation.

[0070] Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0071] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogues, regardless of its size or function. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogues of the foregoing. As used herein, the terms "polynucleotides", "nucleic acid" and "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analogue thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured doublestranded DNA Alternatively, it can be a single-stranded nucleic acid not derived from any doublestranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including siRNA, shRNA, and antisense oligonucleotides. The terms "transgene" and "gene" are also used interchangeably and both terms encompass fragments or variants thereof encoding the target protein.

[0072] Minor variations in the amino acid sequences of the proteins encoded by genes associated with homologous recombination deficiency are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence(s) maintain at least 60%, at least 70%, more preferably at least 80%, at least 85%, at least 90%, at least 95%, and most preferably at least 97% or at least 99% sequence identity to the amino acid sequence of the invention or a fragment thereof as defined anywhere herein. The term homology is used herein to mean identity. As such, the sequence of a variant or analogue sequence of an amino acid sequence of the invention may differ on the basis of substitution (typically conservative substitution) deletion or insertion. Proteins comprising such variations are referred to herein as variants.

[0073] Proteins encoded by genes associated with homologous recombination deficiency according to the invention may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or non-conserved positions. Variants of protein molecules disclosed herein may be produced and used in the present invention. Following the lead of computational chemistry in applying multivariate data analysis techniques to the structure / property-activity relationships [see for example, Wold, et al. Multivariate data analysis in chemistry. Chemometrics-Mathematics and Statistics in Chemistry (Ed.: B. Kowalski); D. Reidel Publishing Company, Dordrecht, Holland, 1984 (ISBN 90-277-1846-6] quantitative activity-property relationships of proteins can be derived using well-known mathematical techniques, such as statistical regression, pattern recognition and classification [see for example Norman et al. Applied Regression Analysis. Wiley-lnterscience; 3rd edition (April 1998) ISBN: 0471170828; Kandel, Abraham et al. Computer-Assisted Reasoning in Cluster Analysis. Prentice Hall PTR, (May 11, 1995), ISBN: 0133418847; Krzanowski, Wojtek. Principles of Multivariate Analysis: A User's Perspective (Oxford Statistical Science Series, No 22 (Paper)). Oxford University Press; (December 2000), ISBN: 0198507089; Witten, Ian H. et al Data Mining: Practical Machine Learning Tools and Techniques with Java Implementations. Morgan Kaufmann; (October 11, 1999), ISBN:1558605525; Denison David G. T. (Editor) et al Bayesian Methods for Nonlinear Classification and Regression (Wiley Series in Probability and Statistics). John Wiley & Sons; (July 2002), ISBN: 0471490369; Ghose, Arup K. et al. Combinatorial Library Design and Evaluation Principles, Software, Tools, and Applications in Drug Discovery. ISBN: 0-8247-0487-8]. The properties of proteins can be derived from empirical and theoretical models (for example, analysis of likely contact residues or calculated physicochemical property) of proteins sequence, functional and three-dimensional structures and these properties can be considered individually and in combination.

[0074] Amino acids are referred to herein using the name of the amino acid, the three-letter abbreviation or the single letter abbreviation. The term "protein", as used herein, includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3-letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0075] Amino acid residues at non-conserved positions may be substituted with conservative or nonconservative residues. In particular, conservative amino acid replacements are contemplated.

[0076] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the amino acid substitution is considered to be conservative. The inclusion of conservatively modified variants in a protein of the invention does not exclude other forms of variant, for example polymorphic variants, interspecies homologs, and alleles.

[0077] "Non-conservative amino acid substitutions" include those in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, lie, Phe or Vai), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Vai, His, lie or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).

[0078] "Insertions" or "deletions" are typically in the range of about 1, 2, or 3 amino acids. The variation allowed may be experimentally determined by systematically introducing insertions or deletions of amino acids in a protein using recombinant DNA techniques and assaying the resulting recombinant variants for activity. This does not require more than routine experiments for a skilled person.

[0079] A "fragment" of a polypeptide comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or more of the original polypeptide. For example, a fragment may comprise at least 5, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 75, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250 or more amino acids of the protein from which it is derived. A fragment may be continuous or discontinuous, preferably continuous. By way of non-limiting example, SLX4 fragments (also referred to interchangeably herein as SLX4 peptides) may be between about 6 to about 250 amino acids in length, such as between about 6 to about 100 amino acids in length.

[0080] The nucleic acid molecules of the present invention may be prepared by any means known in the art. For example, large amounts of the nucleic acid molecules may be produced by replication in a suitable host cell. The natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually, the DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured insect, mammalian, plant or other eukaryotic cell lines.

[0081] The nucleic acid molecules of the present invention may also be produced by chemical synthesis, e.g. by the phosphoramidite method or the tri-ester method, and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

[0082] When applied to a nucleic acid sequence, the term "isolated" in the context of the present invention denotes that the polynucleotide sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences (but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators), and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment.

[0083] In view of the degeneracy of the genetic code, considerable sequence variation is possible among the polynucleotides of the present invention. Degenerate codons encompassing all possible codons for a given amino acid are set forth below:

[0084] Amino Acid Codons Degenerate Codon

[0085] Cys TGC TGT TGY

[0086] Ser AGC AGT TCA TCC TCG TCT WSN

[0087] Thr ACA ACC ACG ACT ACN

[0088] Pro CCA CCC CCG CCT CCN

[0089] Ala GCA GCC GCG GCT GCN

[0090] Gly GGA GGC GGG GGT GGN

[0091] Asn AAC AAT AAY

[0092] Asp GAC GAT GAY

[0093] Glu GAA GAG GAR

[0094] Gin CAA CAG CAR

[0095] His CAC CAT CAY

[0096] Arg AGA AGG CGA CGC CGG CGT MGN

[0097] Lys AAA AAG AAR

[0098] Met ATG ATG

[0099] lie ATA ATC ATT ATH

[0100] Leu CTA CTC CTG CTT TTA TTG YTN

[0101] Vai GTA GTC GTG GTT GTN

[0102] Phe TTC TTT TTY

[0103] Tyr TAC TAT TAY

[0104] Trp TGG TGG

[0105] Ter TAA TAG TGA TRR

[0106] Asn / Asp RAY

[0107] Glu / Gin SAR

[0108] Any NNN One of ordinary skill in the art will appreciate that flexibility exists when determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of the present invention.

[0109] A "variant" nucleic acid sequence has substantial homology or substantial similarity to a reference nucleic acid sequence (or a fragment thereof). A nucleic acid sequence or fragment thereof is "substantially homologous" (or "substantially identical") to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 70%, 75%, 80%, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more% of the nucleotide bases. Methods for homology determination of nucleic acid sequences are known in the art.

[0110] Alternatively, a "variant" nucleic acid sequence is substantially homologous with (or substantially identical to) a reference sequence (or a fragment thereof) if the "variant" and the reference sequence they are capable of hybridizing under stringent (e.g. highly stringent) hybridization conditions. Nucleic acid sequence hybridization will be affected by such conditions as salt concentration (e.g. NaCI), temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions are preferably employed, and generally include temperatures in excess of 30°C, typically in excess of 37°C and preferably in excess of 45°C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. The pH is typically between 7.0 and 8.3. The combination of parameters is much more important than any single parameter.

[0111] Methods of determining nucleic acid percentage sequence identity are known in the art. By way of example, when assessing nucleic acid sequence identity, a sequence having a defined number of contiguous nucleotides may be aligned with a nucleic acid sequence (having the same number of contiguous nucleotides) from the corresponding portion of a nucleic acid sequence of the present invention. Tools known in the art for determining nucleic acid percentage sequence identity include Nucleotide BLAST (as described below).

[0112] A "fragment" of a nucleic acid molecule comprises a series of consecutive nucleotides from the sequence of said full-length nucleic acid molecule. By way of example, a "fragment" of a nucleic acid molecule may comprise (or consist of) at least 600 consecutive nucleotides from the sequence of said nucleic acid molecule (e.g. at least 50, 60, 70, 80, 85, 90, 95, 100 or more consecutive nucleic acid residues of said nucleic acid molecule). Typically, a fragment as defined herein retains the same function as the full-length nucleic acid molecule.

[0113] The terms "decrease", "reduced", "reduction", or "inhibit" are all used herein to mean a decrease by a statistically significant amount. The terms "reduce," "reduction" or "decrease" or "inhibit" typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, "reduction" or "inhibition" encompasses a complete inhibition or reduction as compared to a reference level. "Complete inhibition" is a 100% inhibition (i.e. abrogation) as compared to a reference level.

[0114] The terms "increased", "increase", "enhance", or "activate" are all used herein to mean an increase by a statically significant amount. The terms "increased", "increase", "enhance", or "activate" can mean an increase of at least 25%, at least 50% as compared to a reference level, for example an increase of at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, at least about 95%, or at least about 98%, or at least about 99%, or at least about 100%, or at least about 250% or more compared with a reference level, or at least about a 1.5-fold, or at least about a 2-fold, or at least about a 2.5-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 1.5-fold and 10-fold or greater as compared to a reference level. In the context of a yield or titre, an "increase" is an observable or statistically significant increase in such level.

[0115] References herein to the level of a particular molecule (e.g. SLX4 or any of the SLX4 inhibitors described herein) encompass the actual amount of the molecule, such as the mass, molar amount, concentration or molarity of the molecule. Preferably in the context of the invention, references to the level of a particular molecule refer to the concentration of the molecule.

[0116] The level of a molecule may be determined in any appropriate physiological compartment. Preferred physiological compartments include a tissue sample, e.g. tumour biopsy, plasma, whole blood and / or serum. The level of a molecule may be determined from any appropriate sample from an individual, e.g. a plasma sample, a blood sample and / or a serum sample. Other non-limiting examples of samples which may be tested are tissue or fluid samples urine and biopsy samples. Formalin-Fixed Paraffin-Embedded (FFPE) or formalin-fixed (FF) tissue samples are particularly preferred. Thus, by way of non-limiting example, the invention may reference the level (e.g. concentration) of a molecule (e.g. SLX4 or a phosphorylated form of SLX4) in a tissue sample an individual. The level of a molecule pre-treatment with an agent of the invention may be interchangeably referred to as the "baseline".

[0117] The level of a molecule may be compared with any appropriate control. For example, a control may be obtained from a healthy individual. Alternatively, the control may be obtained from the same individual prior to treatment, or from a different individual with a tumour in the same tissue type as to be treated, but wherein the different individual has not been treated with the SLX4 inhibitor.

[0118] The level of a molecule after treatment with an SLX4 inhibitor of the invention may be compared with the level of the molecule in the individual pre-treatment with the SLX4 inhibitor. Thus, the invention may be concerned with the relative level of the molecule pre- and post-treatment. The level of a molecule pre-treatment may be used to identify an individual as suitable for treatment according to the invention. Other parameters may also be used, either alone or in combination with the level of a molecule as described above, to identify an individual as suitable for treatment according to the invention. Suitable parameters to identify an individual as suitable for treatment according to the invention are known to the skilled person.

[0119] The level of a molecule may be measured directly or indirectly, and may be determined using any appropriate technique. Suitable standard techniques are known in the art, for example Western blotting and enzyme-linked immunosorbent assays (ELISAs).

[0120] As used herein, the term "R-loop" refers to a three-stranded nucleic acid structure, consisting of two antiparallel DNA strands plus one RNA strand. The RNA strand is base-paired to the template DNA strand to form a DNA: RNA hybrid, which is associated with the corresponding non-template single-stranded DNA.

[0121] As used herein, the term "cell-free DNA (cfDNA) refers to fragments of DNA that are present outside of cells that can be detected within bodily fluids, such as the blood. In blood plasma, cfDNA typically consists of double-stranded DNA fragments of around 140-170 base pairs (bp) in length that mostly originate from leukocytes.

[0122] As used herein, the term "circulating tumour DNA" (ctDNA), refers to the portion of cfDNA derived from cancer cells, which typically comprises strands of <145 bp in length and is responsible for the substantially higher plasma cfDNA concentrations often seen in patients with cancer.

[0123] The terms "individual", "subject", and "patient", are used interchangeably herein to refer to a mammalian subject for whom diagnosis, prognosis, disease monitoring, treatment, therapy, and / or therapy optimisation is desired. The mammal can be (without limitation) a human, non-human primate, mouse, rat, dog, cat, horse, or cow. In preferred embodiments, the individual, subject, or patient is a human. An "individual" may be an adult, juvenile or infant. An "individual" may be male or female. A "subject in need" of treatment for a particular condition can be an individual having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0124] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications or symptoms related to such a condition, and optionally, have already undergone treatment for a condition as defined herein or the one or more complications or symptoms related to said condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition as defined herein or one or more or symptoms or complications related to said condition. For example, a subject can be one who exhibits one or more risk factors for a condition, or one or more or symptoms or complications related to said condition or a subject who does not exhibit risk factors.

[0125] As used herein, the term "healthy individual" refers to an individual or group of individuals who are in a healthy state, e.g. individuals who have not shown any symptoms of the disease, have not been diagnosed with the disease and / or are not likely to develop the disease e.g. cystic fibrosis (CF) or any other disease described herein). Preferably said healthy individual(s) is not on medication affecting CF and has not been diagnosed with any other disease. The one or more healthy individuals may have a similar sex, age, and / or body mass index (BMI) as compared with the test individual. Application of standard statistical methods used in medicine permits determination of normal levels of expression in healthy individuals, and significant deviations from such normal levels.

[0126] Herein the terms "control" and "reference population" are used interchangeably.

[0127] The term "treat" or "treating" as used herein encompasses prophylactic treatment (e.g. to prevent onset or recurrence of cancer) as well as corrective treatment (treatment of an individual already / currently suffering from cancer). Preferably, the term "treat" or "treating" as used herein means corrective treatment. The term "treat" or "treating" encompasses treating both cancer, symptoms thereof and diseases / disorder associated therewith.

[0128] A "therapeutically effective amount" is any amount of an SLX4 inhibitor of the invention which, when administered alone or in combination to a patient for treating a cancer with high levels of replication stress or a symptom thereof or a disease associated therewith is sufficient to provide such treatment of the HRD cancer, or symptom thereof, or associated disease. A "prophylactically effective amount" is any amount of an SLX4 inhibitor of the invention that, when administered alone or in combination to an individual inhibits or delays the onset or reoccurrence of a cancer with high levels of replication stress, or a symptom thereof or disease associated therewith. In some embodiments, the prophylactically effective amount prevents the onset or reoccurrence of such a cancer entirely. "Inhibiting" the onset means either lessening the likelihood of onset of a cancer with high levels of replication stress (or symptom thereof or disease associated therewith) or preventing the onset entirely. In some embodiments, the cancer with high levels of replication stress is a homologous recombination defective ( H RD) cancer.

[0129] The term "pharmaceutically acceptable" as used herein means approved by a regulatory agency of the Federal or a state government, or listed in the U. S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia.

[0130] An "analogue" of a chemical structure, as the term is used herein, refers to a chemical structure that preserves substantial similarity with the parent structure, although it may not be readily derived synthetically from the parent structure. A related chemical structure that is readily derived synthetically from a parent chemical structure is referred to as a "derivative."

[0131] A "hydrate" is a compound that exists in a composition with water molecules. The composition can include water in stoichiometric quantities, such as a monohydrate or a dihydrate, or can include water in random amounts. As the term is used herein a "hydrate" refers to a solid form, i.e., a compound in water solution, while it may be hydrated, is not a hydrate as the term is used herein.

[0132] A "solvate" is a similar composition except that a solvent other that water replaces the water. For example, methanol or ethanol can form an "alcoholate", which can again be stoichiometric or non-stoichiometric. As the term is used herein a "solvate" refers to a solid form, i.e., a compound in solution in a solvent, while it may be solvated, is not a solvate as the term is used herein.

[0133] A "prodrug" as is well known in the art is a substance that can be administered to an individual where the substance is converted in vivo by the action of biochemicals within the patient's body, such as enzymes, to the active pharmaceutical ingredient. Examples of prodrugs include esters of carboxylic acid groups, which can be hydrolysed by endogenous esterases as are found in the bloodstream of humans and other mammals. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs", ed. H. Bundgaard, Elsevier, 1985.

[0134] A "salt" as is well known in the art includes an organic compound such as a carboxylic acid, a sulfonic acid, or an amine, in ionic form, in combination with a counterion. For example, acids in their anionic form can form salts with cations such as metal cations, for example sodium, potassium, and the like; with ammonium salts such as NH4+ or the cations of various amines, including tetraalkyl ammonium salts such as tetramethylammonium, or other cations such as trimethylsulfonium, and the like. A "pharmaceutically acceptable" or "pharmacologically acceptable" salt is a salt formed from an ion that has been approved for human consumption and is generally non-toxic, such as a chloride salt or a sodium salt. A "zwitterion" is an internal salt such as can be formed in a molecule that has at least two ionisable groups, one forming an anion and the other a cation, which serve to balance each other. For example, amino acids such as glycine can exist in a zwitterionic form. A "zwitterion" is a salt within the meaning herein. The SLX4 inhibitors of the present invention may take the form of salts. The term "salts" embraces addition salts of free acids or free bases which are SLX4 inhibitors of the invention. Salts can be "pharmaceutically-acceptable salts. " The term "pharmaceutically-acceptable salt" refers to salts which possess toxicity profiles within a range that affords utility in pharmaceutical applications.

[0135] Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which have utility in the practice of the present invention, such as for example utility in process of synthesis, purification or formulation of compounds of the invention.

[0136] Suitable pharmaceutically-acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, p-hydroxybutyric, salicylic, galactaric and galacturonic acid. Examples of pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafiuoroborates.

[0137] Suitable pharmaceutically acceptable base addition salts of SLX4 inhibitors of the invention include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N, N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts. Although pharmaceutically unacceptable salts are not generally useful as medicaments, such salts may be useful, for example as intermediates in the synthesis of Formula (I) compounds, for example in their purification by recrystallization. All of these salts may be prepared by conventional means from the corresponding compound according to Formula (I) by reacting, for example, the appropriate acid or base with the compound according to Formula (I). The term "pharmaceutically acceptable salts" refers to nontoxic inorganic or organic acid and / or base addition salts, see, for example, Lit et al., Salt Selection for Basic Drugs (1986), Int J. Pharm., 33, 201-217, incorporated by reference herein. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0138] Disclosure related to the various methods of the invention are intended to be applied equally to other methods, therapeutic uses or methods, the data storage medium or device, the computer program product, and vice versa.

[0139] Structure-specific endonuclease subunit 4 (SLX4)

[0140] Structure-specific endonuclease subunit 4 (SLX4) is a regulatory subunit that interacts with and increases the activity of different structure-specific endonucleases. SLX4 has several distinct roles in protecting genome stability by resolving diverse forms of deleterious DNA structures originating from replication and recombination intermediates and from DNA damage. SLX4 has been shown to be a critical regulator of the BIR-like MiDAS pathway. It is required for the assembly of the SMX trinuclease complex, which drives resolution of recombination intermediates to facilitate sisterchromatid exchange / rearrangement. Mutations in SLX4 lead to Fanconi anaemia, characterised by bone marrow failure, physical abnormalities and pre-disposition to cancer development. SLX4 also coordinates the repair of inter-strand DNA crosslinks via coordination of the XPF-ERCC1 complex. Furthermore, SLX4 has been shown to form phase-separated condensates which facilitate sumoylation, ubiquitylation and chromatin extraction of Topoisomerase 1 DNA protein cross-links. The condensation of SLX4 also promotes nucleolytic processing of nascent DNA.

[0141] An exemplary SLX4 protein is human SLX4, a reference sequence for which is found as UniProt Accession No. Q8IY92 (version 3, last updated 02 September 2008, accessed 24 October 2024). The sequence of this SLX4 protein is given in SEQ ID NO: 1.

[0142] An exemplary SLX4 is human SLX4, a reference sequence for which is found as NCBI Accession No: NM_032444.4 (RefSeqGene, Gene ID: 84464, last updated 09 October 2024, accessed 24 October 2024). The sequence of this SLX4 mRNA is given in SEQ ID NO: 2.

[0143] Any and all references to SLX4 herein refer to any SLX4 gene, SLX4 mRNA and / or SLX4 protein sequence, particularly human SLX4, including the exemplary sequences described herein and functional variants and fragments thereof.

[0144] As described herein, the present inventors have demonstrated that inhibiting the TOPBP1-SLX4 interaction is sufficient to impair proliferation in BRCA1 / 2 deficient cells or cells exposed to replication stress induced by aphidicolin treatment. This indicates that targeting the TOPBP1-SLX4 interaction is a promising avenue for the development of anti-cancer therapies for a broader range of cancers exhibiting high levels of replication stress, including HRD cancers. Accordingly, the invention provides an inhibitor of the SLX4 - TOPBP1 interaction for use in a method of treating an individual with a cancer with high levels of replication stress. The SLX4 - TOPBP1 interaction typically refers to binding of SLX4 with TOPBP1 (which could interchangeably be described as the binding of TOPBP1 with SLX4). Binding of SLX4 and TOPBP1 may be quantified / determined as described herein. Any inhibitor of the SLX4 - TOPBP1 interaction may be used according to the invention. The inhibitor of the SLX4 - TOPBP1 interaction may be an SLX4 inhibitor and / or a TOPBP1 inhibitor, as described herein. All disclosure herein in relation to SLX4 inhibitors applies equally and without reservation to the broader category of inhibitors of the SLX4 - TOPBP1 interaction, and to TOPBP1 inhibitors unless expressly stated to the contrary. In some preferred embodiments the inhibitor is an SLX4 inhibitor. Thus, preferably the invention relates to an SLX4 inhibitor for use in a method of treating an individual with a cancer with high levels of replication stress.

[0145] SLX4 inhibitors

[0146] The present inventors have now demonstrated that SLX4 inhibitors elicit synthetic lethality in cancer cells with high levels of replication stress. In particular, the inventors have shown that inhibiting the TOPBP1-SLX4 interaction, exemplified herein using a minimal SLX4 fragment containing Thr1260 and SLX4 siRNA is sufficient to impair proliferation in BRCA1 / 2 deficient cells. This indicates that targeting the TOPBP1-SLX4 interaction is a promising avenue for the development of anti-cancer therapies for a broader range of cancers having high levels of replication stress, and in particular HRD cancers. Further, the inventors have demonstrated that pharmacological inhibition of Pol0, combined with loss of the TOPBP1-SLX4 interaction, exacerbates genome instability and reduces cellular proliferation under replication stress.

[0147] Without being bound by theory, it is believed that cells with high levels of replication stress, and HRD cells in particular, such as cells with a BRCA1 / 2 deficiency or loss of function have difficulty repairing DNA.

[0148] One function of SLX4 is to facilitate recruitment of the SMX complex components to mitotic chromatin marked by CIP2A, through a CDKl-dependent interaction between TOPBP1 BRCT 1 / 2 and SLX4 phospho-threoninel260, that drives MiDAS.

[0149] Therefore, the inventors' current hypothesis is that SLX4 inhibition results in inhibition of MiDAS. Whilst normal cells can tolerate such MiDAS inhibition due to the function of redundancy with other DNA repair pathways such as MMEJ, cells with high levels of replication stress (such as HRD cancer cells or cells in which DDR pathways are disrupted) cannot, with the result that cells with high levels of replication stress die in the face of such agents. This is supported by the data herein, which demonstrate that SLX4 inhibition reduces proliferation of BRCA1 / 2 deficient cells. Further, the data herein suggests the use of SLX4 inhibitors with inhibitors of DDR pathway components, such as Pol0, can have a synergistic effect, further reducing proliferation of BRCA1 / 2 deficient cells.

[0150] Thus, the present invention provides for the first time inhibition of the interaction between TOPBP1 and SLX4 as a means for treating cancers with high levels of replicative stress. In particular, this interaction is a mitotic interaction, meaning that the effect is pronounced in replicating cells, such as cancer cells, and particularly cells of cancers that that exhibit elevated replicative stress.

[0151] In the present disclosure, the term "SLX4 inhibitor" refers to any agent, compound or substance that inhibits the expression levels and / or a biological activity of SLX4. Some inhibitors are of different modalities are described and / or exemplified herein. Further examples may be found by the application of screening technologies to these targets. Examples of such screening methods are described herein.

[0152] An SLX4 inhibitor according to the disclosure may directly or indirectly inhibit SLX4 as described herein. Unless explicitly stated, references herein to inhibition of SLX4 encompass both direct and indirect inhibition of SLX4. Preferably an SLX4 inhibitor of the disclosure directly inhibits SLX4.

[0153] "Direct inhibition of SLX4" as used herein means inhibition of the expression and / or activity of SLX4 directly, i.e. without any intermediary step. By way of non-limiting example, direct inhibition of SLX4 may elicited by competitive or non-competitive inhibitors of the SLX4 enzyme or by inhibition of a gene encoding SLX4.

[0154] "Indirect inhibition of SLX4" as used herein means inhibition of the expression and / or activity of SLX4 indirectly, i.e. through the modulation or delivery of genes / enzymes upstream of SLX4 and / or through the generation or delivery of intermediaries which directly inhibit SLX4. Indirect inhibition may, for example, be elicited by upregulating the expression of an enzyme which generates an endogenous direct inhibitor of SLX4.

[0155] An SLX4 inhibitor according to the disclosure may selectively inhibit SLX4. This is typically the case for agents which directly inhibit SLX4. For such agents which directly inhibit SLX4, selectivity may mean that the agent binds selectively (also referred to interchangeably herein as specifically) with SLX4. By "binds selectively", it will be understood that said agent binds to SLX4 (e.g. SLX4 DNA, RNA or protein), with no significant cross-reactivity to any other molecule. Cross-reactivity may be assessed by any suitable method. By way of non-limiting example, cross-reactivity of an SLX4 inhibitor with a molecule other than SLX4 may be considered significant if the agent binds to the other molecule at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% as strongly as it binds to SLX4. An SLX4 inhibitor that directly inhibits SLX4 and that binds selectively to SLX4 may bind to another molecule at less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% the strength that it binds to SLX4. Preferably, the agent binds to the other molecule at less than 20%, less than 15%, less than 10% or less than 5%, less than 2% or less than 1% the strength that it binds to SLX4.

[0156] An SLX4 inhibitor of the disclosure may have off-target effects. An off-target effect is activity against a target other than SLX4. Typically compounds with off-target effects are encompassed by the present disclosure if the activity against the non-SLX4 target is not significant compared with the activity against SLX4. Whether an off-target effect is significant may depend on the intended use of the compound. As a non-limiting example, a compound which may exert an off-target effect on the central nervous system would not be significant for a compound used in an ex vivo method as disclosed herein, but may be significant (depending on the magnitude of the off-target effect) for an in vivo therapeutic indication as disclosed herein. The presence and magnitude of any potential off-target effects can be readily assessed using standard methods known in the art.

[0157] As exemplified herein, mutation of the TOPBP1 BRCT1 / 2 interface (e.g. K155E in BRCT1; K155E+K250E) disrupts binding to SLX4 pT1260 and reduces SMX association on mitotic chromatin. Cellular MiDAS defects are demonstrated with the SLX4-T1260A knock-in and upon CIP2A perturbation. These results support the conclusion that blocking the SLX4-TOPBP1 interface impairs MiDAS. Without being bound by theory, it is believed that this data provides proof of concept that blocking this interaction (by any means) will result in defective MiDAS. Accordingly, any SLX4 inhibitor that is capable of inhibiting this interaction (e.g. by blocking the binding of SLX4 and TOPBP1) may be used in accordance with the present invention.

[0158] An SLX4 inhibitor of the disclosure typically targets the TOPBP1 binding site of SLX4. The TOPBP1 binding site of SLX4 is the region of SLX4 which interacts with a region of the TOPBP1 protein comprising the BRCT1 domain of TOPBP1. In particular, (e.g. wherein an SLX4 inhibitor of the disclosure is a direct inhibitor of SLX4), an SLX4 inhibitor of the disclosure may bind to the TOPBP1 binding site of SLX4.

[0159] An exemplary TOPBP1 protein is human TOPBP1, a reference sequence for which is found as UniProt Accession No. Q92547 (version 3, last updated 15 May 2010, accessed 25 October 2024). The sequence of this TOPBP1 protein is given in SEQ ID NO: 15. The BRCT1 domain of TOPBP1 corresponds to amino acid residues 101-189 of SEQ ID NO: 15, and is SEQ ID NO: 16 herein. The BRCT2 domain of TOPBP1 corresponds to amino acid residues 195-284 of SEQ ID NO: 15, and is SEQ ID NO: 17 herein.

[0160] The TOPBP1 BRCT1 binding site of SLX4 may comprise or consist of the amino acid motif SWLVPATPLA (SEQ ID NO: 3), which corresponds to amnio acid residues 1254-1263 of SEQ ID NO: 1.

[0161] Therefore, a SLX4 inhibitor of the disclosure may targets the TOPBP1 binding site of SLX4, which site comprises or consists of SEQ ID NO: 3. In particular, (e.g. wherein an SLX4 inhibitor of the disclosure is a direct inhibitor of SLX4), an SLX4 inhibitor of the disclosure may bind to the TOPBP1 binding site of SLX4, which site comprises or consists of SEQ ID NO: 3.

[0162] In particular, the present inventors have shown that phosphorylation of the amino acid corresponding to Thr1260 of SEQ ID NO: 1 is important for SLX4 activity. Therefore, a TOPBP1 binding site of SLX4 according to the disclosure typically comprises an amino acid corresponding to Thr1260 of SEQ ID NO: 1.

[0163] Therefore, a SLX4 inhibitor of the disclosure may targets the TOPBP1 binding site of SLX4, which site comprises an amino acid corresponding to Thr1260 of SEQ ID NO: 1. In particular, (e.g. wherein an SLX4 inhibitor of the disclosure is a direct inhibitor of SLX4), an SLX4 inhibitor of the disclosure may bind to the TOPBP1 binding site of SLX4, which site comprises an amino acid corresponding to Thr1260 of SEQ ID NO: 1.

[0164] The region corresponding to amino acid residues 1175-1375 of SEQ ID NO: 1 is highly conserved and / or hyperphosphorylated, indicating it is involved in the interaction of SLX4 with TOPBP1. Accordingly, the TOPBP1 binding site of SLX4 may comprise or consist of SEQ ID NO: 4, which corresponds to amnio acid residues 1175-1375 of SEQ ID NO: 1. The TOPBP1 binding site of SLX4 may comprise or consist of a fragment of SEQ ID NO: 4, which fragment may be from about 6 amino acids to about 100 amino acids in length, such as from about 10 amino acids to about 100 amino acids; from about 10 amino acids to about 80 amino acids; from about 10 amino acids to about 70 amino acids; from about 10 amino acids to about 60 amino acids; from about 10 amino acids to about 50 amino acids; from about 10 amino acids to about 40 amino acids; from about 10 amino acids to about 30 amino acids; from about 15 amino acids to about 100 amino acids; from about 15 amino acids to about 80 amino acids; from about 15 amino acids to about 70 amino acids; from about 15 amino acids to about 60 amino acids; from about 15 amino acids to about 50 amino acids; from about 15 amino acids to about 40 amino acids; from about 15 amino acids to about 30 amino acids; from about 20 amino acids to about 100 amino acids; from about 20 amino acids to about 80 amino acids; from about 20 amino acids to about 70 amino acids; from about 20 amino acids to about 60 amino acids; from about 20 amino acids to about 50 amino acids; from about 20 amino acids to about 40 amino acids; from about 20 amino acids to about 30 amino acids; from about 25 amino acids to about 100 amino acids; from about 25 amino acids to about 80 amino acids; from about 25 amino acids to about 70 amino acids; from about 25 amino acids to about 60 amino acids; from about 25 amino acids to about 50 amino acids; or from about 25 amino acids to about 40 amino acids of SEQ ID NO: 4. Said fragment will comprise an amino acid corresponding to Thr1260 of SEQ ID NO: 1.

[0165] The region corresponding to amino acid residues 1235-1296 of SEQ ID NO: 1 is a particularly conserved region. Accordingly, the TOPBP1 binding site of SLX4 may comprise or consist of SEQ ID NO: 5, which corresponds to amnio acid residues 1235-1296 of SEQ ID NO: 1. The TOPBP1 binding site of SLX4 may comprise or consist of a fragment of SEQ ID NO: 5, which fragment may be from about 6 amino acids to about 60 amino acids in length, such as from about 10 amino acids to about 50 amino acids; from about 10 amino acids to about 40 amino acids; from about 10 amino acids to about 30 amino acids; from about 10 amino acids to about 20 amino acids; from about 15 amino acids to about 50 amino acids; from about 15 amino acids to about 40 amino acids; from about 15 amino acids to about 30 amino acids; from about 15 amino acids to about 20 amino acids; from about 20 amino acids to about 50 amino acids; from about 20 amino acids to about 40 amino acids; from about 20 amino acids to about 30 amino acids; from about 25 amino acids to about 50 amino acids; or from about 25 amino acids to about 40 amino acids of SEQ ID NO: 5. Said fragment will comprise an amino acid corresponding to Thr1260 of SEQ ID NO: 1.

[0166] The inventors have shown that the region corresponding to amino acid residues 1253-1270 of SEQ ID NO: 1 is a particularly conserved region. Accordingly, the TOPBP1 binding site of SLX4 may comprise or consist of SEQ ID NO: 6, which corresponds to amnio acid residues 1253-1270 of SEQ ID NO: 1. The TOPBP1 binding site of SLX4 may comprise or consist of a fragment of SEQ ID NO: 6, which fragment may be from about 6 amino acids to about 16 amino acids in length, such as from about 6 amino acids to about 10 amino acids; from about 10 amino acids to about 16 amino acids; about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 amino acids of SEQ ID NO: 6. Said fragment will comprise an amino acid corresponding to Thr1260 of SEQ ID NO: 1.

[0167] An SLX4 inhibitor according to the disclosure may act by inhibiting the phosphorylation of the amino acid corresponding to Thr1260 of SEQ ID NO: 1. Inhibition of the phosphorylation of the amino acid corresponding to Thr1260 of SEQ ID NO: 1 may be a decrease of phosphorylation by an amount as described herein, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, up to complete (100%) inhibition of the phosphorylation of the amino acid corresponding to Thr1260 of SEQ ID NO: 1. The level of phosphorylation of the amino acid corresponding to Thr1260 of SEQ ID NO: 1 may be determined and / or quantified using any appropriate technique or method. Standard phosphorylation assays are known in the art and exemplified herein and include the use of a phospho-specific antibody raised against SLX4 pThr1260 used in western blotting (such as exemplified herein), flow cytometry, immunohistochemistry, immunofluorescence microscopy. In addition, other suitable techniques for the detection of phosphorylated peptides include mass-spectrometry analysis, in gel kinase assays, and in vitro reconstitution. Alternatively or in addition, an SLX4 inhibitor according to the disclosure may act by inhibiting the interaction of the amino acid corresponding toThr1260 of SEQ ID NO: 1 with BRCT1 and / or BRCT2 of TOPBP1. Inhibition of the interaction of the amino acid corresponding to Thr1260 of SEQ ID NO: 1 with BRCT1 and / or BRCT2 of TOPBP1 may be a decrease of the interaction by an amount as described herein, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, up to complete (100%) inhibition of the interaction of the amino acid corresponding to Thr1260 of SEQ ID NO: 1 with BRCT1 and / or BRCT2 of TOPBP1. The level of interaction of the interaction of the amino acid corresponding to Thr1260 of SEQ ID NO: 1 with BRCT1 and / or BRCT2 of TOPBP1 may be determined and / or quantified using any appropriate technique or method. Standard assays are known in the art and exemplified herein and include fluorescence polarisation, co-immunoprecipitation-western blotting / mass spectrometry, localisation of SMX via immunofluorescence microscopy of mitotic cells, western blot analysis of chromatin bound SMX by fractionation-western blotting / mass spectrometry, proximity ligation assays (PLA), Fluorescence Resonance Energy Transfer (FRET), Far western, and label transfer protein interaction analysis.

[0168] Further alternatively or in addition, an SLX4 inhibitor according to the disclosure may act by inhibiting the expression of SLX4. Inhibition of the expression of SLX4 may be a reduction of expression by an amount as described herein, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, up to complete (100%) decrease of the expression of SLX4. The level of expression of SLX4 may be determined and / or quantified using any appropriate technique or method. Standard assays are known in the art and exemplified herein and include qPCR, western blotting, mass-spectrometry, and RNA-sequencing. SLX4 inhibitors which inhibit the expression of SLX4 are still considered to inhibit the interaction of SLX4 and TOPBP1 according to the present invention. This is because a reduction in the expression of SLX4 will reduce the amount of SLX4 protein that is available to bind to TOPBP1, and hence the interaction between SLX4 and TOPBP1 will be reduced.

[0169] Further alternatively or in addition, SLX4 inhibitor SLX4 inhibitor may specifically inhibit mitotic DNA synthesis (MiDAS) and / or break induced replication (BIR) in a cancer cell. Inhibition of MiDAS may be a reduction of MiDAS by an amount as described herein, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, up to complete (100%) in MiDAS. The level of MiDAS may be determined and / or quantified using any appropriate technique or method. Standard assays are known in the art and exemplified herein and include the analysis of EdU incorporation in mitosis. Inhibition of BIR may be a reduction of BIR by an amount as described herein, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, up to complete (100%) in BIR. The level of BIR may be determined and / or quantified using any appropriate technique or method. Standard assays are known in the art and exemplified herein and include the use of BIR repair reporter constructs or cell lines, and break junction sequencing of defined breakpoint loci. As MiDAS is a sub-pathway falling within the broader BIR pathway, quantifying BIR inhibition will by definition quantify MiDAS inhibition (in addition to other, BIR sub-pathways).

[0170] An SLX4 inhibitor typically decreases the expression and / or activity of SLX4. In the context of the present disclosure, when referring to decreasing the expression and / or activity of SLX4, the degree of decrease may be as defined above.

[0171] Expression may be quantified in terms of gene and / or protein expression, and may be compared with the expression of a control (e.g. housekeeping gene or protein). As a non-limiting example, in the context of SLX4 expression, the actual amount of an SLX4 gene, mRNA transcript and / or protein, such as the mass, molar amount, concentration or molarity of an SLX4 gene, mRNA transcript and / or protein, or the number of mRNA molecules per cell in a sample obtained from an individual treated according to the disclosure and the control may be assessed, and compared with the corresponding value from the control. Alternatively, the expression of an SLX4 gene and / or protein in a sample obtained from an individual treated according to the disclosure may be compared with that of the control without quantifying the mass, molar amount, concentration or molarity of the one or more gene and / or protein.

[0172] Typically, the control is an equivalent sample in which no inhibition of SLX4 expression has been effected. As a non-limiting example, in the case where an individual is treated with an SLX4 inhibitor that inhibits SLX4 expression, a suitable control would be a different individual to which the SLX4 inhibitor has not been administered or the same individual prior to administration of the compound. Conventional methods for the assessment of gene and / or protein expression are well known in the art and include RT-qPCR, ELISA, DNA microarray, RNA-Seq, serial analysis of gene expression (SAGE) and western blotting.

[0173] As SLX4 acts as a scaffold for the SMX, the recruitment of downstream SMX components (SLX4, SLX1, MUS81, EMEI, ERCC4 / XPF, ERCC1) to mitotic chromatin can be used an indicator of SLX4 activity, and may be compared with the activity of a control (i.e. recombinant enzyme of known concentration). SLX4 activity defined in terms of SMX component recruitment may be quantified using any appropriate technique, examples of which are known in the art and exemplified herein, such as: immunofluorescence microscopy including PLA, western blot using antibodies against the SMX components and mass spectrometry. Alternatively or in addition, nuclease activity of interacting structure specific endonuclease EME1-MUS81 and / or XPF-ERCC1 may be used as an indicator of SLX4 activity. Again, this may be compared with the activity of a control (i.e. recombinant enzyme of known concentration). SLX4 activity defined in terms of nuclease activity of EME1-MUS81 and / or XPF-ERCC1. In particular, SLX4 is known to enhance the activity of XPF-ERCC1 and MUS81-EME1 in processing multiple DNA structures including: Holliday junctions, D loops, replication fork structures, three-stranded ICL structures, cruciform, 5' flap structures and 3' flap structures. This activity may be quantified using any appropriate technique, examples of which are known in the art and exemplified herein, such as: biochemical reconstitution, structure specific endonuclease substrate processing in the presence of human cell extracts; microscopy, protein analysis, DNA processing analysis and mass spectrometry. Sister chromatid exchange (sister chromatid rearrangement) assays can also be used to quantify rates of sister chromatid exchange which is mediated through SLX4's regulation of the wider SMX complex.

[0174] Inhibition of SLX4 leads to increased genome instability (including hallmarks of chromosomal instability (CIN)), and consequently SLX4 function and expression is tightly regulated. Even mild changes in SLX4 expression will have consequences to genome stability. Further, overexpression of SLX4 is extremely toxic. Therefore, typically, decreasing the expression and / or activity of SLX4 refers to a decrease in SLX4 expression and / or activity of at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, preferably at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more, up to complete inhibition of SLX4 expression and / or activity.

[0175] An SLX4 inhibitor of the disclosure may result in a decrease in the level of SLX4 within the tissue to be treated. In this context, the level of SLX4 encompasses, the actual amount of SLX4, such as the mass, molar amount, concentration or molarity of SLX4 (for a set sample size or in individual cells of said sample). Typically, the level of SLX4 is determined in a sample obtained from an individual treated according to the disclosure and the control may be assessed quantitatively, and compared with the corresponding value from the control. Alternatively, the level of SLX4 in a sample obtained from an individual treated according to the disclosure may be compared qualitatively with that of the control i.e. without quantifying the mass, molar amount, concentration or molarity of SLX4.

[0176] A combination of SLX4 inhibitors may be used to inhibit SLX4. By way of non-limiting examples, a combination of SLX4 inhibitors may comprise: a direct inhibitor of SLX4 and an indirect inhibitor of SLX4; at least two direct inhibitors of SLX4; or at least two indirect inhibitors of SLX4. Any suitable SLX4 inhibitor may be used according to the present disclosure. Non-limiting examples of suitable agents include peptides, small molecules, proteolysis-targeting chimeric molecules (PROTAC), macrocyclic molecules, molecular glues, nucleic acid molecules (nucleic acid inhibitors), antibodies and antigen-binding fragments thereof, antibody-drug conjugates, and peptidomimetics, and aptamers, as described herein.

[0177] An SLX4 inhibitor may be selected from a peptide, a small molecule, a PROTAC, a macrocyclic molecule, a molecular glue, a nucleic acid molecule (nucleic acid inhibitor), an antibody or antigenbinding fragment thereof, an antibody-drug conjugate, a peptide or peptidomimetic, and an aptamer. Preferably an SLX4 inhibitor is a peptide or a small molecule.

[0178] Peptides and Peptidomimetics

[0179] The disclosure encompasses the use of peptide and peptidomimetic inhibitors of SLX4. For example, the use of peptides, stapled peptides, peptoids and peptidomimetics that would directly or indirectly inhibit SLX4 is embraced by the present disclosure.

[0180] A peptide SLX4 inhibitor of the disclosure may be a fragment of SLX4. Said fragment typically comprises or consists of the TOPBP1 binding site of SLX4 as defined herein.

[0181] A peptide SLX4 inhibitor of the disclosure may be a variant of SLX4 or a fragment of such a variant. Said variant typically comprises or consists of the TOPBP1 binding site of SLX4 as defined herein, or a variant thereof.

[0182] Peptide inhibitors of SLX4 that are fragments and / or variants of SLX4 are not particularly limited provided that they retain the ability to inhibit SLX4 expression and / or activity as described herein.

[0183] Accordingly, a peptide SLX4 inhibitor of the disclosure typically comprises or consists of an SLX4 fragment comprising a threonine at a position corresponding toThr1260 of SEQ ID NO: 1.

[0184] A peptide SLX4 inhibitor may comprise or consist of the amino acid sequence of SEQ ID NO: 3. A peptide SLX4 inhibitor may comprise or consist of an amino acid sequence with at least 80% (e.g. at least 90%, or 100%) identity to SEQ ID NO: 3 (i.e. be a variant of SEQ ID NO: 3). A peptide SLX4 inhibitor may comprise or consist of a fragment of about 6, about 7, about 8 or about 9 amino acids of SEQ ID NO: 3 or said variant. Said fragment or variant will comprise an amino acid corresponding to Thr1260 of SEQ ID NO: 1.

[0185] A peptide SLX4 inhibitor may comprise or consist of the amino acid sequence of SEQ ID NO: 4. A peptide SLX4 inhibitor may comprise or consist of an amino acid sequence with at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%) identity to SEQ ID NO: 4 (i.e. be a variant of SEQ ID NO: 4). A peptide SLX4 inhibitor may comprise or consist of a fragment of from about 6 amino acids to about 100 amino acids in length, such as from about 10 amino acids to about 100 amino acids; from about 10 amino acids to about 80 amino acids; from about 10 amino acids to about 70 amino acids; from about 10 amino acids to about 60 amino acids; from about 10 amino acids to about 50 amino acids; from about 10 amino acids to about 40 amino acids; from about 10 amino acids to about 30 amino acids; from about 15 amino acids to about 100 amino acids; from about 15 amino acids to about 80 amino acids; from about 15 amino acids to about 70 amino acids; from about 15 amino acids to about 60 amino acids; from about 15 amino acids to about 50 amino acids; from about 15 amino acids to about 40 amino acids; from about 15 amino acids to about 30 amino acids; from about 20 amino acids to about 100 amino acids; from about 20 amino acids to about 80 amino acids; from about 20 amino acids to about 70 amino acids; from about 20 amino acids to about 60 amino acids; from about 20 amino acids to about 50 amino acids; from about 20 amino acids to about 40 amino acids; from about 20 amino acids to about 30 amino acids; from about 25 amino acids to about 100 amino acids; from about 25 amino acids to about 80 amino acids; from about 25 amino acids to about 70 amino acids; from about 25 amino acids to about 60 amino acids; from about 25 amino acids to about 50 amino acids; or from about 25 amino acids to about 40 amino acids of SEQ ID NO: 4 or said variant. Said fragment or variant will comprise an amino acid corresponding toThr1260 of SEQ ID NO: 1.

[0186] A peptide SLX4 inhibitor may comprise or consist of the amino acid sequence of SEQ ID NO: 5. A peptide SLX4 inhibitor may comprise or consist of an amino acid sequence with at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%) identity to SEQ ID NO: 5 (i.e. be a variant of SEQ ID NO: 5). A peptide SLX4 inhibitor may comprise or consist of a fragment of from about 6 amino acids to about 60 amino acids in length, such as from about 10 amino acids to about 50 amino acids; from about 10 amino acids to about 40 amino acids; from about 10 amino acids to about 30 amino acids; from about 10 amino acids to about 20 amino acids; from about 15 amino acids to about 50 amino acids; from about 15 amino acids to about 40 amino acids; from about 15 amino acids to about 30 amino acids; from about 15 amino acids to about 20 amino acids; from about 20 amino acids to about 50 amino acids; from about 20 amino acids to about 40 amino acids; from about 20 amino acids to about 30 amino acids; from about 25 amino acids to about 50 amino acids; or from about 25 amino acids to about 40 amino acids of SEQ ID NO: 5 or said variant. Said fragment or variant will comprise an amino acid corresponding to Thr1260 of SEQ ID NO: 1.

[0187] A peptide SLX4 inhibitor may comprise or consist of the amino acid sequence of SEQ ID NO: 6. A peptide SLX4 inhibitor may comprise or consist of an amino acid sequence with at least 80% (e.g. at least 82%, at least 88%, at least 94%, or 100%) identity to SEQ ID NO: 6 (i.e. be a variant of SEQ ID NO: 6). A peptide SLX4 inhibitor may comprise or consist of a fragment of from about 6 amino acids to about 16 amino acids in length, such as from about 6 amino acids to about 10 amino acids; from about 10 amino acids to about 16 amino acids; about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 amino acids of SEQ ID NO: 6 or said variant. Said fragment or variant will comprise an amino acid corresponding to Thr1260 of SEQ ID NO: 1.

[0188] A peptide SLX4 inhibitor may comprise or consist of the amino acid sequence of ENRGSLGRRGAPWLFCDRESSPSEASTTDTSWLVPATPLASRSRDCSSQTQISSLRSGLAVQAVTQHTPRASVGNR EG (SEQ ID NO: 7). A peptide SLX4 inhibitor may comprise or consist of an amino acid sequence with at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%) identity to SEQ ID NO: 7 (i.e. be a variant of SEQ ID NO: 7). Said variant may have at least 90% identity to SEQ ID NO: 7. A peptide SLX4 inhibitor may comprise or consist of a fragment of from about 6 amino acids to about 70 amino acids in length, such as from about 10 amino acids to about 60 amino acids; from about 10 amino acids to about 50 amino acids; from about 10 amino acids to about 40 amino acids; from about 10 amino acids to about 30 amino acids; from about 10 amino acids to about 20 amino acids; from about 15 amino acids to about 60 amino acids; from about 15 amino acids to about 50 amino acids; from about 15 amino acids to about 40 amino acids; from about 15 amino acids to about 30 amino acids; from about 15 amino acids to about 20 amino acids; from about 20 amino acids to about 60 amino acids; from about 20 amino acids to about 50 amino acids; from about 20 amino acids to about 40 amino acids; from about 20 amino acids to about 30 amino acids; from about 25 amino acids to about 60 amino acids; from about 25 amino acids to about 50 amino acids; or from about 25 amino acids to about 40 amino acids of SEQ ID NO: 7 or said variant. Said fragment or variant will comprise an amino acid corresponding to Thr1260 of SEQ ID NO: 1.

[0189] A peptide SLX4 inhibitor may comprise or consist of the amino acid sequence of TSWLVPATPLASRSRDC (SEQ ID NO: 8). A peptide SLX4 inhibitor may comprise or consist of an amino acid sequence with at 80% (e.g. at least 82%, at least 88%, at least 94%, or 100%) identity to SEQ ID NO: 8 (i.e. be a variant of SEQ ID NO: 8). Said variant may have at least 88% identity to SEQ ID NO: 8. A peptide SLX4 inhibitor may comprise or consist of a fragment of from about 6 amino acids to about 16 amino acids in length, such as from about 6 amino acids to about 10 amino acids; from about 10 amino acids to about 16 amino acids; about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 amino acids of SEQ ID NO: 8 or said variant. Said fragment or variant will comprise an amino acid corresponding to Thr1260 of SEQ ID NO: 1. A peptide SLX4 inhibitor may be of any appropriate length. By way of non-limiting example, a peptide SLX4 inhibitor may be from about 6 to about 200 amino acids in length, such as from about 6 amino acids to about 150 amino acids in length; from about 6 amino acids to about 100 amino acids in length; from about 10 amino acids to about 100 amino acids; from about 10 amino acids to about 80 amino acids; from about 10 amino acids to about 70 amino acids; from about 10 amino acids to about 60 amino acids; from about 10 amino acids to about 50 amino acids; from about 10 amino acids to about 40 amino acids; from about 10 amino acids to about 30 amino acids; from about 15 amino acids to about 100 amino acids; from about 15 amino acids to about 80 amino acids; from about 15 amino acids to about 70 amino acids; from about 15 amino acids to about 60 amino acids; from about 15 amino acids to about 50 amino acids; from about 15 amino acids to about 40 amino acids; from about 15 amino acids to about 30 amino acids; from about 20 amino acids to about 100 amino acids; from about 20 amino acids to about 80 amino acids; from about 20 amino acids to about 70 amino acids; from about 20 amino acids to about 60 amino acids; from about 20 amino acids to about 50 amino acids; from about 20 amino acids to about 40 amino acids; from about 20 amino acids to about 30 amino acids; from about 25 amino acids to about 100 amino acids; from about 25 amino acids to about 80 amino acids; from about 25 amino acids to about 70 amino acids; from about 25 amino acids to about 60 amino acids; from about 25 amino acids to about 50 amino acids; or from about 25 amino acids to about 40 amino acids in length.

[0190] Modifications to peptide SLX4 inhibitors are also encompassed by the present disclosure. Many modifications intended to facilitate the clinical utility (e.g. modify pharmacokinetic and / or pharmacodynamic properties) of peptide therapeutics are known in the art, and it would be within the routine practice of one of ordinary skill in the art to make such modifications. Non-limiting examples of such modifications are described, for example at 0 00000.1ebj.ac.uk), and include cyclic peptides (including head to tail; side chain to side chain; terminus to side chain); D-amino acids; peptoids; N-methylation; side-chain halogenation; peptide bonds; amide bioisosteres; terminus protection; lipidation; PEGylation; polymer-peptide conjugations; peptide stapling (including hydrocarbon staples; polyfluorinated staples; Click triazole staples; CLIPS staples); and beta amino acids.

[0191] A peptide SLX4 inhibitor may be a linear peptide or a cyclic peptide.

[0192] Peptidomimetics are compounds which mimic a natural peptide or protein with the ability to interact with the biological target and produce the same biological effect. Peptidomimetics may have advantages over peptides in terms of stability and bioavailability associated with a natural peptide. Peptidomimetics can have main- or side-chain modifications of the parent peptide designed for biological function. Examples of classes of peptidomimetics include, but are not limited to, peptoids and -peptides, as well as peptides incorporating D-amino acids.

[0193] Stapled peptides comprise an external brace that forces the peptide to adopt an a-helical conformation. Starting from the native peptide sequence, two amino acids on the same face of the helix are substituted for non-native amino acids which have side chains that can be crosslinked. This crosslinking forms the external brace which holds the peptide in the desired a-helical conformation. A peptide may comprise multiple "staples", e.g. crosslinking may occur between two, three or more pairs of modified amino acids, with each crosslinked pair forming a staple. Peptides with multiple staples are sometimes referred to as stitched peptides. Stapling peptides can increase target affinity, increase cell penetration, and protect against proteolytic degradation.

[0194] Methods for producing synthetic peptides and peptidomimetics (such as peptoids) are known in the art, as are the sequences of SLX4 and its ligands. Techniques for stapling peptides are also described in the art, and include both covalent and non-covalent stapling strategies, with techniques that generate all-hydrocarbon covalent links being preferred. Non-limiting examples include ringclosing metathesis and olefin metathesis. Thus, it would be routine for one of skill in the art to produce suitable synthetic peptides, including stapled peptides, and peptidomimetics which directly or indirectly inhibit SLX4 using known techniques and based on the known sequence and structures of SLX4 and SLX4 targets.

[0195] siRNA inhibitors

[0196] Another class of SLX4 inhibitors useful for treatment of cancers with high levels of replication stress includes nucleic acid inhibitors which inhibit activity or function by down-regulating production of active SLX4 polypeptide. This can be monitored using conventional methods well known in the art, for example by screening using real time PCR.

[0197] Expression of SLX4 may be inhibited using anti-sense or RNAi technology. The use of these approaches to down-regulate gene expression is now well-established in the art.

[0198] Thus, an SLX4 inhibitor according to the disclosure may be a nucleic acid as defined herein. A nucleic acid inhibitor (nucleic acid molecule) of the disclosure may inhibit SLX4 expression. Such nucleic acid inhibitors include "antisense nucleic acids", by which is meant an RNA or DNA molecule that binds to another RNA or DNA (target RNA, DNA), whether an SLX4 RNA or DNA as defined herein (e.g. in the case of direct SLX4 inhibition), or a non-SLX4 RNA or DNA (e.g. in the case of indirect inhibition). Non-limiting examples of antisense nucleic acids include, for example, antisense RNA or DNA molecules, interference RNA (RNAi), micro RNA, decoy RNA molecules, siRNA, enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid (such as the SLX4 gene). As such, these nucleic acids may be introduced in the form of single-stranded, doublestranded, partially single-stranded, or circular oligomeric compounds.

[0199] Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of the base excision repair pathway component so that its expression is reduced or completely or substantially completely prevented. In addition to targeting coding sequence, anti-sense techniques may be used to target control sequences of a gene, e.g. in the 5' flanking sequence, whereby the anti-sense oligonucleotides can interfere with expression control sequences. The construction of anti-sense sequences and their use is described for example in Peyman & Ulman, Chemical Reviews, 90:543-584, 1990 and Crooke, Ann. Rev. Pharmacol. Toxicol., 32:329-376, 1992.

[0200] Oligonucleotides may be generated in vitro or ex vivo for administration or anti-sense RNA may be generated in vivo within cells in which down-regulation is desired. Thus, double-stranded DNA may be placed under the control of a promoter in a "reverse orientation" such that transcription of the anti-sense strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the sense strand of the target gene. The complementary anti-sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein. Whether or not this is the actual mode of action is still uncertain. However, it is established fact that the technique works.

[0201] The complete sequence corresponding to the coding sequence in reverse orientation need not be used. For example, fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding or flanking sequences of a gene to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon. A suitable fragment may have about 14-23 nucleotides, e.g., about 15, 16 or 17 nucleotides.

[0202] An alternative to anti-sense is to use a copy of all or part of the target gene inserted in sense, that is the same orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression (Angell & Baulcombe, The EMBO Journal 16(12):3675-3684, 1997 and Voinnet & Baulcombe, Nature, 389: 553, 1997). Double stranded RNA (dsRNA) has been found to be even more effective in gene silencing than both sense or antisense strands alone (Fire et al, Nature 391, 806-811, 1998) dsRNA mediated silencing is gene specific and is often termed RNA interference (RNAi). Methods relating to the use of RNAi to silence genes in C. elegans, Drosophila, plants, and mammals are known in the art (Fire, Trends Genet., 15: 358-363, 19999; Sharp, RNA interference, Genes Dev.

[0203] 15: 485-4902001; Hammond et al., Nature Rev. Genet. 2: 110-1119, 2001; Tuschl, Chem. Biochem. 2: 239-245, 2001; Hamilton et al., Science 286: 950-952, 1999; Hammond, et al., Nature 404: 293-296, 2000; Zamore et al., Cell, 101: 25-33, 2000; Bernstein, Nature, 409: 363-366, 2001; Elbashir et al, Genes Dev., 15: 188-200, 2001; WO01 / 29058; WO99 / 32619, and Elbashir et al, Nature, 411: 494-498, 2001). RNA interference is a two-step process. First, dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs (~2nt). The siRNAs target the corresponding mRNA sequence specifically for destruction (Zamore, Nature Structural Biology, 8, 9, 746-750, 2001.

[0204] RNAi may also be efficiently induced using chemically synthesized siRNA duplexes of the same structure with 3'-overhang ends (Zamore et al, Cell, 101: 25-33, 2000). Synthetic siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologous genes in a wide range of mammalian cell lines (Elbashir et al, Nature, 411: 494-498, 2001).

[0205] Another possibility is that nucleic acid is used which on transcription produces a ribozyme, able to cut nucleic acid at a specific site and therefore also useful in influencing gene expression, e.g., see Kashani-Sabet & Scanlon, Cancer Gene Therapy, 2(3) 213-223, 1995 and Mercola & Cohen, Cancer Gene Therapy, 2 (1) 47-59, 1995.

[0206] Small RNA molecules may be employed to regulate gene expression. These include targeted degradation of mRNAs by small interfering RNAs (siRNAs), post transcriptional gene silencing (PTGs), developmentally regulated sequence-specific translational repression of mRNA by micro-RNAs (miRNAs), and targeted transcriptional gene silencing.

[0207] A role for the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci has also been demonstrated. Double- stranded RNA (dsRNA)-dependent post transcriptional silencing, also known as RNA interference (RNAi), is a phenomenon in which dsRNA complexes can target specific genes of homology for silencing in a short period of time. It acts as a signal to promote degradation of mRNA with sequence identity. A 20-nt siRNA is generally long enough to induce gene-specific silencing, but short enough to evade host response. The decrease in expression of targeted gene products can be extensive with 90% silencing induced by a few molecules of siRNA.

[0208] In the art, these RNA sequences are termed "short or small interfering RNAs" (siRNAs) or "microRNAs" (miRNAs) depending on their origin. Both types of sequence may be used to downregulate gene expression by binding to complimentary RNAs and either triggering mRNA elimination (RNAi) or arresting mRNA translation into protein. siRNA are derived by processing of long double stranded RNAs and when found in nature are typically of exogenous origin. Micro-interfering RNAs (miRNA) are endogenously encoded small non-coding RNAs, derived by processing of short hairpins. Both siRNA and miRNA can inhibit the translation of mRNAs bearing partially complimentary target sequences without RNA cleavage and degrade mRNAs bearing fully complementary sequences.

[0209] The siRNA ligands are typically double stranded and, in order to optimise the effectiveness of RNA mediated down-regulation of the function of a target gene, it is preferred that the length of the siRNA molecule is chosen to ensure correct recognition of the siRNA by the RISC complex that mediates the recognition by the siRNA of the mRNA target and so that the siRNA is short enough to reduce a host response.

[0210] miRNA ligands are typically single stranded and have regions that are partially complementary enabling the ligands to form a hairpin. miRNAs are RNA genes which are transcribed from DNA, but are not translated into protein. A DNA sequence that codes for a miRNA gene is longer than the miRNA. This DNA sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reverse-complement base pair to form a partially double stranded RNA segment. The design of microRNA sequences is discussed in John et al, PloS Biology, 11 (2), 1862-1879, 2004. Typically, the RNA ligands intended to mimic the effects of siRNA or miRNA have between 10 and 40 ribonucleotides (or synthetic analogues thereof), more preferably between 17 and 30 ribonucleotides, more preferably between 19 and 25 ribonucleotides and most preferably between 21 and 23 ribonucleotides. In some embodiments of the disclosure employing double-stranded siRNA, the molecule may have symmetric 3' overhangs, e.g. of one or two (ribo)nucleotides, typically a UU of dTdT 3' overhang. Based on the disclosure provided herein, the skilled person can readily design suitable siRNA and miRNA sequences, for example using resources such as Ambion's siRNA finder, see http: / / www.ambion.com / techlib / misc / siRNA finder.html. siRNA and miRNA sequences can be synthetically produced and added exogenously to cause gene downregulation or produced using expression systems (e.g. vectors). Preferably, the siRNA is synthesized synthetically.

[0211] Longer double stranded RNAs may be processed in the cell to produce siRNAs (e.g. see Myers, Nature Biotechnology, 21: 324- 328, 2003). The longer dsRNA molecule may have symmetric 3' or 5' overhangs, e.g. of one or two (ribo)nucleotides, or may have blunt ends. The longer dsRNA molecules may be 25 nucleotides or longer. Preferably, the longer dsRNA molecules are between 25 and 30 nucleotides long. More preferably, the longer dsRNA molecules are between 25 and 27 nucleotides long. Most preferably, the longer dsRNA molecules are 27 nucleotides in length. dsRNAs 30 nucleotides or more in length may be expressed using the vector pDECAP (Shinagawa et al., Genes and Dev., 17:1340-5, 2003). Another alternative is the expression of a short hairpin RNA molecule (shRNA) in the cell. shRNAs are more stable than synthetic siRNAs. A shRNA consists of short inverted repeats separated by a small loop sequence. One inverted repeat is complimentary to the gene target. In the cell the shRNA is processed by DICER into a siRNA which degrades the target gene mRNA and suppresses expression. Preferably, the shRNA is produced endogenously (within a cell) by transcription from a vector. shRNAs may be produced within a cell by transfecting the cell with a vector encoding the shRNA sequence under control of an RNA polymerase III promoter such as the human HI or 7SK promoter or an RNA polymerase II promoter.

[0212] Alternatively, the shRNA may be synthesised exogenously (in vitro) by transcription from a vector. The shRNA may then be introduced directly into the cell. Preferably, the shRNA sequence is between 40 and 100 bases in length, more preferably between 40 and 70 bases in length. The stem of the hairpin is preferably between 19 and 30 base pairs in length. The stem may contain G-U pairings to stabilise the hairpin structure.

[0213] The siRNA, longer dsRNA or miRNA may be produced endogenously (within a cell) by transcription from a vector. The vector may be introduced into the cell in any of the ways known in the art. Optionally, expression of the RNA sequence can be regulated using a tissue specific promoter. Alternatively, the siRNA, longer dsRNA or miRNA may be produced exogenously (in vitro) by transcription from a vector.

[0214] siRNA molecules may be synthesized using standard solid or solution phase synthesis techniques, which are known in the art. Linkages between nucleotides may be phosphodiester bonds or alternatives, e.g., linking groups of the formula P(O)S, (thioate); P(S)S, (dithioate); P(O) N R'2; P(O)R'; P(O)OR6; CO; or CONR'2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotides through-O-or-S-.

[0215] A nucleic acid SLX4 inhibitor of the disclosure may be an antisense oligonucleotide, optionally wherein said antisense oligonucleotide comprises or consists of a nucleobase sequence that is at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%) reverse complementary to an equal length portion of at least 12 contiguous nucleobases, preferably at least 14 (e.g. 14, 15, 16, 17 or 18 contiguous nucleobases) within the nucleobase sequence of SEQ. ID NO: 2. In some embodiments, the antisense oligonucleotide comprises or consists of a nucleobase sequence that is at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%) reverse complementary to an equal length portion of at least 12 contiguous nucleobases, preferably at least 14 (e.g. 14, 15, 16, 17 or 18 contiguous nucleobases) within a nucleobase sequence corresponding to the 3'UTR of SEQ ID NO: 2, which may be defined as corresponding to residues 6155 to 7315 of SEQ ID NO: 2.

[0216] A nucleic acid SLX4 inhibitor of the disclosure may be an siRNA, optionally wherein said siRNA has an antisense strand that comprises or consists of a nucleobase sequence that is at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%) reverse complementary to an equal length portion of at least 12 contiguous nucleobases, preferably at least 14 (e.g. 14, 15, 16, 17 or 18 contiguous nucleobases) within the nucleobase sequence of SEQ ID NO: 2. In some embodiments, the siRNA has an antisense strand that comprises or consists of a nucleobase sequence that is at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%) reverse complementary to an equal length portion of at least 12 contiguous nucleobases, preferably at least 14 (e.g. 14, 15, 16, 17 or 18 contiguous nucleobases) within a nucleobase sequence corresponding to the 3'UTR of SEQ ID NO: 2, which may be defined as corresponding to residues 6155 to 7315 of SEQ ID NO: 2. Said siRNA may additionally comprise a sense strand that comprises or consists of a nucleobase sequence that is at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%) identical to an equal length portion of at least 12 contiguous nucleobases, preferably at least 14 (e.g. 14, 15, 16, 17 or 18 contiguous nucleobases) within the nucleobase sequence corresponding of SEQ ID NO: 2. In some embodiments, the siRNA has a sense strand that comprises or consists of a nucleobase sequence that is at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%) identical to an equal length portion of at least 12 contiguous nucleobases, preferably at least 14 (e.g.

[0217] 14, 15, 16, 17 or 18 contiguous nucleobases) within a nucleobase sequence corresponding to the 3'UTR of SEQ ID NO: 2, which may be defined as corresponding to residues 6155 to 7315 of SEQ ID NO: 2. The antisense strand and the sense strand may respectively be reverse complementary and identical to the same nucleobase sequence within SEQ ID NO: 2.

[0218] An SLX4 siRNA may have an antisense strand which comprises or consists of a nucleobase sequence that is at least 80% identical (e.g. at least 83%, at least 88%, at least 94%, or more, up to 100%) to TTGTTCTGGGCCCTTGTGC (SEQ ID NO: 9) or TACATATGAACCTGGTGC (SEQ ID NO: 10). An SLX4 siRNA may have an antisense strand which comprises or consists of a nucleobase sequence that is identical to TTGTTCTGGGCCCTTGTGC (SEQ ID NO: 9) or TACATATGAACCTGGTGC (SEQ ID NO: 10). An SLX4 siRNA may have an antisense strand which comprises a nucleobase sequence that is identical to TTGTTCTGGGCCCTTGTGC (SEQ ID NO: 9) or TACATATGAACCTGGTGC (SEQ ID NO: 10). An SLX4 siRNA may have an antisense strand which consists of a nucleobase sequence that is identical to TTGTTCTGGGCCCTTGTGC (SEQ ID NO: 9) or TACATATGAACCTGGTGC (SEQ ID NO: 10).

[0219] In addition, an SLX4 siRNA may have a sense strand which comprises or consists of a nucleobase sequence that is at least 80% identical (e.g. at least 84%, at least 89%, at least 94%, or more, up to 100%) to GCACAAGGGCCCAGAACAA (SEQ ID NO: 11) or GCACCAGGUUCAUAUGUA (SEQ ID NO: 12). An SLX4 siRNA may have a sense strand which comprises or consists of a nucleobase sequence that is identical to GCACAAGGGCCCAGAACAA (SEQ ID NO: 11) or GCACCAGGUUCAUAUGUA (SEQ ID NO: 12). An SLX4 siRNA may have a sense strand which comprises a nucleobase sequence that is identical to GCACAAGGGCCCAGAACAA (SEQ ID NO: 11) or GCACCAGGUUCAUAUGUA (SEQ ID NO: 12). An SLX4 siRNA may have a sense strand which consists of a nucleobase sequence that is identical to GCACAAGGGCCCAGAACAA (SEQ ID NO: 11) or GCACCAGGUUCAUAUGUA (SEQ ID NO: 12).

[0220] An SLX4 siRNA may have an antisense strand which comprises or consists of a nucleobase sequence that is at least 80% identical (e.g. at least 83%, at least 88%, at least 94%, or more, up to 100%) to TTGTTCTGGGCCCTTGTGC (SEQ ID NO: 9) and a sense strand which comprises or consists of a nucleobase sequence that is at least 80% identical (e.g. at least 84%, at least 89%, at least 94%, or more, up to 100%) to GCACAAGGGCCCAGAACAA (SEQ ID NO: 11). An SLX4 siRNA may have an antisense strand which comprises or consists of a nucleobase sequence that is to TTGTTCTGGGCCCTTGTGC (SEQ ID NO: 9) and a sense strand which comprises or consists of a nucleobase sequence that is identical to GCACAAGGGCCCAGAACAA (SEQ ID NO: 11).

[0221] An SLX4 siRNA may have an antisense strand which comprises or consists of a nucleobase sequence that is at least 80% identical (e.g. at least 83%, at least 88%, at least 94%, or more, up to 100%) to TACATATGAACCTGGTGC (SEQ ID NO: 10) and a sense strand which comprises or consists of a nucleobase sequence that is at least 80% identical (e.g. at least 84%, at least 89%, at least 94%, or more, up to 100%) to GCACCAGGUUCAUAUGUA (SEQ ID NO: 12). An SLX4 siRNA may have an antisense strand which comprises or consists of a nucleobase sequence that is identical to TACATATGAACCTGGTGC (SEQ ID NO: 10) and a sense strand which comprises or consists of a nucleobase sequence that is identical to GCACCAGGUUCAUAUGUA (SEQ ID NO: 12).

[0222] A nucleic acid SLX4 inhibitor according to the disclosure many also be a guide RNA (gRNA) for using in directing CRISPR-mediated knock-out of SLX4. For example, CRISPR interference (CRISPRi) using deactivated Cas9 nuclease (dCas) may be used to specifically knock down SLX4. Thus, a gRNA may comprise an oligonucleotide which has a nucleobase sequence that is at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%) reverse complementary to an equal length portion of at least 12 contiguous nucleobases, preferably at least 14 (e.g. 14, 15, 16, 17 or 18 contiguous nucleobases) within the nucleobase sequence of SEQ ID NO: 2. Byway of example, suitable guide RNA sequences are given in SEQ ID NOs: 18, 19, 20, 21, 25, 26, 27 and 28; with SEQ ID NOs: 18+19, 20+21, 25+26, and 27+28 being used in pairs.

[0223] Modified nucleotide bases can be used in addition to the naturally occurring bases in a nucleic acid SLX4 inhibitor, and may confer advantageous properties on siRNA molecules containing them.

[0224] For example, modified bases may increase the stability of the siRNA molecule, thereby reducing the amount required for silencing. The provision of modified bases may also provide siRNA molecules, which are more, or less, stable than unmodified siRNA.

[0225] The term 'modified nucleotide base' encompasses nucleotides with a covalently modified base and / or sugar. For example, modified nucleotides include nucleotides having sugars, which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3'position and other than a phosphate group at the 5'position. Thus, modified nucleotides may also include 2'substituted sugars such as 2'-O-methyl-; 2-Oalkyl; 2-O-allyl; 2'-S-alkyl; 2'-S-allyl; 2'-fluoro-; 2'-halo or 2; azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars and sedoheptulose.

[0226] Modified nucleotides are known in the art and include alkylated purines and pyrimidines, acylated purines and pyrimidines, and other heterocycles. These classes of pyrimidines and purines are known in the art and include pseudoisocytosine, N4, N4-ethanocytosine, 8-hydroxy-N6-methyladenine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil, 5 fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, dihydrouracil, inosine, N6-isopentyl-adenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 2,2-dimethylguanine, 2methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5-methoxy amino methyl-2-thiouracil, -D-mannosylqueosine, 5-methoxycarbonylmethyluracil, 5methoxyuracil, 2 methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, psueouracil, 2-thiocytosine, 5-methyl-2 thiouracil, 2-thiouracil, 4-thiouracil, 5methyluracil, N-uracil-5-oxyacetic acid methylester, uracil 5-oxyacetic acid, queosine, 2-thiocytosine, 5-propyluracil, 5-propylcytosine, 5-ethylu racil, 5ethylcytosine, 5-butyluracil, 5-pentyluracil, 5-pentylcytosine, and 2, 6, diaminopurine, methylpsuedouracil, 1-methylguanine, 1-methylcytosine.

[0227] Antibodies

[0228] Antibodies may be employed in the present disclosure as an example of a class of inhibitor useful for treating cancers with high levels of replication stress, and more particularly as inhibitors of SLX4. An antibody which is an inhibitor of SLX4 may be described as an anti-SLX4 antibody. Such antibody inhibitors of SLX4 may be an antibody or antigen-binding fragment thereof that specifically binds to SLX4. Specific binding may be defined as elsewhere herein.

[0229] Typically, an antibody inhibitor of SLX4, or an antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of the TOPBP1 binding site of SLX4. The TOPBP1 binding site of SLX4 is described above. Thus, an antibody inhibitor of SLX4, or an antigen-binding fragment thereof typically specifically binds to an epitope within SLX4 which comprises an amino acid corresponding to Thr1260 of SEQ ID NO: 1.

[0230] An antibody inhibitor of SLX4, or an antigen-binding fragment thereof may specifically bind to an epitope within SLX4 which comprises an amino acid corresponding to Thr1260 of SEQ ID NO: 1, wherein the amino acid at a position corresponding to Thr1260 of SEQ ID NO: 1 is phosphorylated.

[0231] An antibody inhibitor of SLX4, or an antigen-binding fragment thereof may specifically bind to an epitope which comprises or consists of CTTDTSWLVPA{pT}PLASR (SEQ ID NO: 13), or which competes for binding to SLX4 with an antibody that binds to such an epitope.

[0232] An antibody inhibitor of SLX4, or an antigen-binding fragment thereof may specifically bind to an epitope which comprises or consists of CTTDTSWLVPATPLASR (SEQ ID NO: 14), or which competes for binding to SLX4 with an antibody that binds to such an epitope.

[0233] The ability of an antibody or antigen-binding fragment thereof to bind to the same epitope or compete for binding to SLX4 with an antibody or antigen-binding fragment thereof of the disclosure can be readily determined by one of ordinary skill in the art without undue burden. Examples of suitable competition assays are known in the art, including HTRF and AlphaScreen. By way of further example, to determine if a test antibody competes with a reference anti-SLX4 antibody of the disclosure, the reference antibody is allowed to bind to a SLX4 protein or peptide under saturating conditions. Next, the ability of a test antibody to bind to the SLX4 protein molecule is assessed. If the test antibody is able to bind to SLX4 following saturation binding with the reference anti-SLX4 antibody, it can be concluded that the test antibody binds to a different epitope than the reference anti-SLX4 antibody. On the other hand, if the test antibody is not able to bind to the SLX4 protein following saturation binding with the reference anti-SLX4 antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference anti-SLX4 antibody of the disclosure. To determine if an antibody competes for binding with a reference anti-SLX4 antibody, the abovedescribed binding methodology is performed in two orientations: In a first orientation, the reference antibody is allowed to bind to a SLX4 protein under saturating conditions followed by assessment of binding of the test antibody to the SLX4 molecule. In a second orientation, the test antibody is allowed to bind to a SLX4 molecule under saturating conditions followed by assessment of binding of the reference antibody to the SLX4 molecule. If, in both orientations, only the first (saturating) antibody is capable of binding to the SLX4 molecule, then it is concluded that the test antibody and the reference antibody compete for binding to SLX4. As will be appreciated by a person of ordinary skill in the art, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

[0234] Competition between binding members may be assayed easily in vitro, for example by tagging a specific reporter molecule to one binding member which can be detected in the presence of other untagged binding member(s), to enable identification of binding members which bind the same epitope or an overlapping epitope. Competition may be determined for example using ELISA in which SLX4 is immobilized to a plate and a first tagged or labelled binding member along with one or more other untagged or unlabelled binding members is added to the plate. Presence of an untagged binding member that competes with the tagged binding member is observed by a decrease in the signal emitted by the tagged binding member.

[0235] Competition assays can also be used in epitope mapping. In one instance epitope mapping may be used to identify the epitope bound by a binding member which optionally may have optimized neutralizing and / or modulating characteristics. Such an epitope can be linear or conformational. A conformational epitope can comprise at least two different fragments of SLX4, wherein said fragments are positioned in proximity to each other when the SLX4 peptide is folded in its tertiary or quaternary structure to form a conformational epitope which is recognized by an inhibitor of SLX4, such as an SLX4 binding member. In testing for competition a peptide fragment of the antigen may be employed, especially a peptide including or consisting essentially of an epitope of interest. A peptide having the epitope sequence plus one or more amino acids at either end may be used. Binding members according to the present disclosure may be such that their binding for antigen is inhibited by a peptide with or including the sequence given.

[0236] The present disclosure also provides for isolated antibody molecules and antigen-binding fragments thereof that bind to the same epitope as an antibody or antigen-binding fragment thereof of the present disclosure.

[0237] Two antibodies may be considered to bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50% but preferably 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 199050: 1495-1502). Alternatively, two antibodies may be considered to have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies may be considered to have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art. Other suitable techniques to determine whether two antibodies bind to the same epitope include hydrogen / deuterium exchange (HDX) and X-ray crystallography.

[0238] Antibody inhibitors of SLX4 may also be used in the methods disclosed herein for assessing an individual having cancer or predicting the response of an individual having cancer, in particular for determining whether the individual has a cancer with high levels of replication stress that might be treatable according to the present disclosure.

[0239] As used herein, the term "antibody" includes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein comprising an antibody binding domain. Antibody fragments which comprise an antigen binding domain are such as Fab, scFv, Fv, dAb, Fd; and diabodies. It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP 0184187 A, GB 2,188,638 A or EP 0239400 A.

[0240] Antibodies can be modified in a number of ways and the term "antibody molecule" should be construed as covering any specific binding member or substance having an antibody antigen-binding domain with the required specificity. Thus, this term covers antibody fragments and derivatives, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP 0120694 A and EP 0 125023 A.

[0241] It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E. S. et al., Nature 341, 544- 546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F (a b') 2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242; 423-426, 1988; Huston et al, PNAS USA, 85: 5879-5883, 1988); (viii) bispecific single chain Fv dimers (WO 93 / 11161) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO 94 / 13804; Holliger et al, P. N. A. S. USA, 90: 6444-6448, 1993); (x) immunoadhesins (WO 98 / 50431). Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains (Reiter et al, Nature Biotech, 14: 1239-1245, 1996). Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al, Cancer Res., 56: 3055-3061, 1996).

[0242] Preferred antibodies used in accordance with the present disclosure are isolated, in the sense of being free from contaminants such as antibodies able to bind other polypeptides and / or free of serum components. Monoclonal antibodies are preferred for some purposes, though polyclonal antibodies are within the scope of the present disclosure.

[0243] The reactivities of antibodies on a sample may be determined by any appropriate means. Tagging with individual reporter molecules is one possibility. The reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals. The linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or noncovalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule. One favoured mode is by covalent linkage of each antibody with an individual fluorochrome, phosphor or laser exciting dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine.

[0244] Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes which catalyse reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin / avidin or biotin / streptavidin and alkaline phosphatase detection systems may be employed.

[0245] An antibody of the disclosure and antigen-binding fragments thereof may be derived from any species by recombinant means. For example, the antibodies or antigen-binding fragments may be mouse, rat, goat, horse, swine, bovine, chicken, rabbit, camelid, donkey, human, or chimeric versions thereof. For use in administration to humans, non-human derived antibodies or antigen-binding fragments may be genetically or structurally altered to be less antigenic upon administration to the human patient.

[0246] Especially preferred are human or humanized antibodies, especially as recombinant human or humanized antibodies as defined herein.

[0247] An antibody of the disclosure and antigen-binding fragments thereof disclosed herein can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and / or recombination(s) and / or any other modification(s) known in the art, either alone or in combination. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art.

[0248] The antibodies of the disclosure or antigen-binding fragments thereof may have any antibody format. For example, the antibody may have a "conventional" format described above. Alternatively, the antibody may comprise or consist of a Fab fragment. The antibody according to the disclosure can also be a Fab', an Fv, an scFv, an Fd, a V NAR domain, an IgNAR, an intrabody, an IgG CH2, a minibody, a single-domain antibody, an Fcab, an scFv-Fc, F(ab')2, a di-scFv, a bi-specific T-cell engager (BiTE®), a F(ab')3, a tetrabody, a triabody, a diabody, a DVD-lg, an (scFv)2, or a mAb2.

[0249] Antibodies according to the present disclosure may be used in screening for the presence of a polypeptide, for example in a test sample containing cells or cell lysate as discussed, and may be used in purifying and / or isolating a polypeptide according to the present disclosure, for instance following production of the polypeptide by expression from encoding nucleic acid. Antibodies may modulate the activity of the polypeptide to which they bind and so, if that polypeptide has a deleterious effect in an individual, may be useful in a therapeutic context (which may include prophylaxis).

[0250] Antibody-Drug Conjugates

[0251] An ADC comprises or consists of three components— a monoclonal antibody, a linker group and a cytotoxic drug.

[0252] An antibody of the disclosure may be present in an antibody-drug conjugate (ADC). In such ADCs the drug may be any which is suitable for use in the treatment of an HRD cancer, such as those described herein.

[0253] Alternatively, a non-antibody SLX4 inhibitor of the disclosure may be conjugated to an antibody which can specifically bind to a target cancer cell with high levels of replication stress of the disclosure. In such ADCs, the SLX4 inhibitor is the "drug" part of the ADC. An ADC of the disclosure may comprise a cleavable or non-cleavable linker. Non-limiting examples of cleavable linkers include hydrazone, disulphide and peptide linkers. Non-limiting examples of non-cleavable linkers include those based on a maleimide-type structure such as maleimidocaproyl and 4-maleimidomethyl cyclohexane- 1-carboxylate linkers.

[0254] Small Molecule Inhibitors

[0255] Small molecules may be used to inhibit SLX4 as described herein. As defined herein, small molecules are low molecular weight compounds, typically organic compounds. Typically, a small molecule has a maximum molecule weight of 900 Da, allowing for rapid diffusion across cell membranes. The maximum molecular weight of a small molecule may be 500 Da. Typically, a small molecule has a size in the order of 1 nm.

[0256] Standard techniques are known in the art for the production of small molecules, which can then readily be tested for the ability to inhibit SLX4 as described herein.

[0257] Any small molecule which exerts an inhibitory effect on SLX4 expression and / or activity may be used as an SLX4 inhibitor according to the present disclosure. Such small molecule inhibitors may also bind to SLX4.

[0258] It will be understood that when small molecule agents of the present disclosure contain one or more chiral centres, the compounds may exist in, and may be isolated as pure enantiomeric or diastereomeric forms or as racemic mixtures. The present disclosure therefore includes any possible enantiomers, diastereomers, racemates or mixtures thereof of small molecule agents of the disclosure.

[0259] Small molecule agents of the present disclosure may have rotameric forms, or may not have rotational activity. Rotameric forms include slow rotating forms and fast rotating forms. In some preferred embodiments, fast rotating forms of the small molecule agents of the present disclosure are preferred.

[0260] A small molecule agent or a salt thereof may exhibit the phenomenon of tautomerism whereby two chemical compounds that are capable of facile interconversion by exchanging a hydrogen atom between two atoms, to either of which it forms a covalent bond. Since the tautomeric compounds exist in mobile equilibrium with each other they may be regarded as different isomeric forms of the same compound. The disclosure encompasses any tautomeric form of a small molecule agent and is not to be limited merely to any one tautomeric form. Thus, small molecule agents according to the disclosure encompass tautomers (including keto-enol and amide-imidic acid forms). Small molecule agents may be used in the form of pro-drugs which convert into active small molecule agents in the body, analogues or derivates, as well as in salt, hydrate and solvate forms, as defined in the Definitions section herein.

[0261] PROTACs

[0262] Proteolysis targeting chimeric molecules (also referred to as PROTACs or PROTAC reagents) may be used to inhibit SLX4 activity as described herein. PROTACs are heterobifunctional small molecules that simultaneously bind a target protein and ubiquitin ligase, enabling ubiquitination and degradation of the target. In more detail, a PROTAC reagent typically comprises a ligand for the target protein (in the case of the present disclosure, SLX4) and a ligand for an E3 ligase recognition domain. Through the use of such a PROTAC, an E3 ligase is recruited to the PROTAC-bound SLX4, inducing ubiquitin transfer from the E3 ligase complex to the target protein (in the case of the present disclosure, SLX4). Once the PROTAC has induced a sufficient degree of ubiquitination of the target, it is then recognised and degraded by the proteasome.

[0263] As a non-limiting example, a PROTAC may be produced by conjugating a ligand for an E3-ligase to a small molecule inhibitor as described herein or a nucleic acid such as Z-DNA (as described in Wang et al. (2024) J. Am. Chem. Soc. doi: 10.1021 / jacs.3cl3646, which is herein incorporated by reference) via a linker. Preferably, a PROTAC comprises a ligand for the E3 RING Cullin ligase von-Hippel Lindau protein (VHL) or cereblon - a part of a CRL4 E3 RING Cullin ligase complex, connected to a small molecule inhibitor of the disclosure via a linker. A PROTAC may comprise a ligand for the E3 RING Cullin ligase von-Hippel Lindau protein (VHL) connected to a small molecule inhibitor as described herein, connected via a linker. A PROTAC may comprise cereblon (a part of a CRL4 E3 RING Cullin ligase complex) and a small molecule inhibitor as described, connected via a linker.

[0264] Because of their mechanism of action, PROTACs simply need any ligand for the target protein. The functional pharmacology of the ligand, in the absence of the linker and E3 ligase ligand, is unimportant. Therefore, in some embodiments an SLX4 inhibitory PROTAC reagent of the present disclosure may comprise a small molecule SLX4 agonist as the ligand.

[0265] Aptamers

[0266] Another class of SLX4 inhibitors which may be used according to the present disclosure are aptamer.

[0267] Aptamers are generally nucleic acid molecules that bind a specific target molecule. Aptamers can be engineered completely in vitro, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications. These characteristics make them particularly useful in pharmaceutical and therapeutic utilities.

[0268] As used herein, "aptamer" refers in general to a single or double stranded oligonucleotide or a mixture of such oligonucleotides, wherein the oligonucleotide or mixture is capable of binding specifically to a target. Oligonucleotide aptamers will be discussed here, but the skilled reader will appreciate that other aptamers having equivalent binding characteristics can also be used, such as peptide aptamers.

[0269] In general, aptamers may comprise oligonucleotides that are at least 5, at least 10 or at least 15 nucleotides in length. Aptamers may comprise sequences that are up to 40, up to 60 or up to 100 or more nucleotides in length. For example, aptamers may be from 5 to 100 nucleotides, from 10 to 40 nucleotides, or from 15 to 40 nucleotides in length. Where possible, aptamers of shorter length are preferred as these will often lead to less interference by other molecules or materials.

[0270] Aptamers may be generated using routine methods such as the Systematic Evolution of Ligands by Exponential enrichment (SELEX) procedure. SELEX is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules. It is described in, for example, US 5,654, 151, US 5,503,978, US 5,567,588 and WO 96 / 38579.

[0271] The SELEX method involves the selection of nucleic acid aptamers and in particular single stranded nucleic acids capable of binding to a desired target, from a collection of oligonucleotides. A collection of single- stranded nucleic acids (e.g., DNA, RNA, or variants thereof) is contacted with a target, under conditions favourable for binding, those nucleic acids which are bound to targets in the mixture are separated from those which do not bind, the nucleic acid-target complexes are dissociated, those nucleic acids which had bound to the target are amplified to yield a collection or library which is enriched in nucleic acids having the desired binding activity, and then this series of steps is repeated as necessary to produce a library of nucleic acids (aptamers) having specific binding affinity for the relevant target.

[0272] Macrocyclic Molecules

[0273] In addition, the disclosure encompasses the use of macrocyclic molecules as inhibitors of SLX4. For example, the use of macrocyclic molecules that would directly or indirectly inhibit SLX4 is embraced by the present disclosure.

[0274] A macrocyclic molecule (also referred to as a macrocycle) is a molecule that contains a cyclic framework of at least twelve atoms. Although the size of naturally occurring macrocycles can reach 50+ atoms in the largest ring, a recent analysis of natural products suggested that 14-, 16-, and 18-membered frameworks are the most common naturally occurring macrocyclic scaffolds. Cyclization of a linear molecule into a macrocyclic ring constitutes a significant change in molecular shape, biological activity, and drug-like properties. Compared with corresponding acyclic linear molecules, cyclised molecules typically have better physicochemical properties, such as good solubility, lipophilicity, metabolic stability, bioavailability and overall pharmacokinetics.

[0275] Methods for producing s macrocyclic molecules are known in the art, as are the sequences of SLX4 and its ligands. Thus, it would be routine for one of skill in the art to produce suitable macrocyclic molecules which directly or indirectly inhibit SLX4 using known techniques and based on the known sequence and structures of SLX4 and SLX4 targets. By way of non-limiting example, high-dilution chemistry can be used to produce macrocycles, wherein large amounts of solvent and low concentrations are added (typically slowly), to allow ring formation to occur whilst preventing nascent macrocyclic molecules from reacting with each other and polymerising. Alternatively, template synthetic techniques, typically using transition metals, to organise components of the reaction and guide them towards the desired ring formation.

[0276] Molecular Glues

[0277] Molecular glues may be used to inhibit SLX4 activity as described herein. Like PROTAC reagents, molecular glues are small molecules. However, whereas PROTAC reagents simultaneously bind a target protein and ubiquitin ligase, molecular glues interact with only one of the target protein (SLX4 according to the disclosure) or the ubiquitin ligase. Typically molecular glues interact only with the ubiquitin ligase. This interaction stabilises the protein-protein interaction between the ubiquitin ligase and its target (SLX4), forming ternary complexes which induce ubiquitination and degradation of the target.

[0278] Molecular glues typically have lower molecular weight, higher cell permeability and better oral absorption compared with PROTAC reagents.

[0279] A molecular glue may bind the E3 ligase Cereblon (CRBN) or an aryl sulfonamides that engages DCAF15. Examples of such molecular glues include immunomodulatory imide drugs (ImiD) such as thalidomide, lenalidomide and pomalidomide. Other molecular glues that induce protein degradation through various non-E3 ligase mechanisms of action include autophagy-mediated protein degradation, protein-protein interaction stabilisation, KRAS mutant inhibition, microtubule stabilisation stabilization, and mTOR inhibition. In Non-limiting examples of naturally occurring compounds which can function as molecular glues include rapamycin, cyclosporin A, voclosporine and sanglifehrin A. Further examples of molecular glues are described in Geiger etal. Curr. Res. Chem. Biol.

[0280] 2(2022):100018, which is herein incorporated by reference in its entirety. T0PBP1 inhibitors

[0281] As discussed and exemplified herein, the present inventors have demonstrated that inhibiting the TOPBP1-SLX4 interaction is sufficient to impair proliferation in BRCA1 / 2 deficient cells and cells exhibiting heightened replication stress induced by aphidicolin. This indicates that targeting the TOPBP1-SLX4 interaction is a promising avenue for the development of anti-cancer therapies for a broader range of cancers having high levels of replication stress, including HRD cancers.

[0282] Whilst the above disclosure focuses on the use of SLX4 inhibitors to target the TOPBP1-SLX4 interaction, alternatively or in addition, TOPBP1 inhibitors may also be used to target this interaction.

[0283] Thus, the invention further relates to TOPBP1 inhibitors for targeting the TOPBP1-SLX4 interaction. All disclosure herein in relation to SLX4 inhibitors, and methods / applications / uses thereof applies equally and without reservation to the use of TOPBP1 inhibitors unless expressly stated to the contrary.

[0284] As exemplified herein, the inventors have identified a mitotic interaction between TOPBP1 and components of the SMX complex, and particularly that the TOPBP1 BRCT1 and 2 domains directly interact with CDK1 dependent phosphorylated threonine 1260 of SLX4. The Examples herein (including the fluorescence polarisation data, in vitro reconstitution, and mutant mapping coimmunoprecipitation data in Examples 1 and 3 and Figures 3 and 4) demonstrate the role of the N-terminus of TOPBP1, and specifically the TOPBP1 BRCT 1 and 2 domains to facilitate this interaction. The invention provides the use of inhibitors of this mitotic interaction between SLX4 and TOPBP1 in the treatment of cancers with high levels of replicative stress.

[0285] In the present disclosure, the term "TOPBP1 inhibitor" refers to any agent, compound or substance that inhibits the interaction of SLX4 and TOPBP1.

[0286] A TOPBP1 inhibitor according to the disclosure directly inhibits TOPBP1 as described herein. Unless explicitly stated, references herein to inhibition of TOPBP1 refer to direct inhibition of the interaction of TOPBP1 with SLX4.

[0287] "Direct inhibition of TOPBP1" as used herein means inhibition of the interaction of TOPBP1 with SLX4, i.e. without any intermediary step. By way of non-limiting example, direct inhibition of TOPBP1 may elicited by competitive or non-competitive inhibitors of TOPBP1.

[0288] A TOPBP1 inhibitor according to the disclosure may selectively inhibit TOPBP1. Selective inhibition is described above in the context of SLX4. This definition of selective inhibition (e.g. magnitude of inhibition and / or methods for quantifying inhibition) applies equally to selective inhibition of TOPBP1.

[0289] A TOPBP1 inhibitor according to the disclosure may inhibit the expression of TOPBP1. Such TOPBP1 inhibitors are still considered to inhibit the interaction of SLX4 and TOPBP1 according to the present invention. This is because a reduction in the expression of TOPBP1 will reduce the amount of TOPBP1 protein that is available to bind to SLX4, and hence the interaction between SLX4 and TOPBP1 will be reduced.

[0290] A TOPBP1 inhibitor of the disclosure may have off-target effects. An off-target effect is activity against a target other than TOPBP1. Typically compounds with off-target effects are encompassed by the present disclosure if the activity against the non-TOPBP1 target is not significant compared with the activity against TOPBP1.

[0291] A TOPBP1 inhibitor of the invention may be of any appropriate modality, such as those described in the context of SLX4 inhibitors. Examples of TOPBP1 inhibitors may be found by the application of screening technologies, examples of suitable screening methods are described herein.

[0292] By way of non-limiting example, a TOPBP1 inhibitor may be a peptide, a small molecule, a PROTAC, a macrocyclic molecule, a molecular glue, a nucleic acid molecule, an antibody or antigenbinding fragment thereof, an antibody-drug conjugate, a peptidomimetic, or an aptamer. Relevant disclosures of such modalities are provided herein in the context of SLX4 inhibitors, but apply equally and without reservation to TOPBP1 inhibitors.

[0293] A TOPBP1 inhibitor of the disclosure typically targets the SLX4 binding site of TOPBP1. The SLX4 binding site of TOPBP1 is the region of TOPBP1 which interacts with the TOPBP1 binding site of SLX4. The TOPBP1 binding site of SLX4 is defined in detail herein. Thus, a TOPBP1 inhibitor may inhibit the interaction of the BRCT1 and / or BRCT2 domains of TOPBP1 with SLX4, particularly the interaction of the BRCT1 and / or BRCT2 domains of TOPBP1 with the TOPBP1 binding site of SLX4 as defined herein.

[0294] In some preferred embodiments, a peptide TOPBP1 inhibitor of the disclosure may be a fragment of TOPBP1. Said fragment typically comprises or consists of the SLX4 binding site of TOPBP1 as defined herein. Thus, a peptide TOPBP1 inhibitor of the disclosure may comprise or consist of the BRCT1 and / or BRCT2 domains of TOPBP1.

[0295] A peptide TOPBP1 inhibitor of the disclosure may be a variant of TOPBP1 or a fragment of such a variant. Said variant typically comprises or consists of the SLX4 binding site of TOPBP1 as defined herein (e.g. the BRCT1 and / or BRCT2 domains), or a variant thereof.

[0296] Peptide inhibitors of TOPBP1 that are fragments and / or variants of TOPBP1 are not particularly limited provided that they retain the ability to inhibit TOPBP1 and / or activity as described herein.

[0297] A peptide TOPBP1 inhibitor may comprise or consist of the amino acid sequence of SEQ. ID NO: 16. A peptide TOPBP1 inhibitor may comprise or consist of an amino acid sequence with at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%) identity to SEQ ID NO: 16 (i.e. be a variant of SEQ ID NO: 16). A peptide TOPBP1 inhibitor may comprise or consist of a fragment of about 6, about 7, about 8 or about 9 amino acids of SEQ ID NO: 16 or said variant. Said fragment or variant will typically comprise an amino acid corresponding to Lysl55 and / or Lys250 of SEQ ID NO: 15, preferably amino acids corresponding to Lysl55 and Lys250 of SEQ ID NO: 15.

[0298] A peptide TOPBP1 inhibitor may comprise or consist of the amino acid sequence of SEQ ID NO: 17. A peptide TOPBP1 inhibitor may comprise or consist of an amino acid sequence with at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%) identity to SEQ ID NO: 17 (i.e. be a variant of SEQ ID NO: 17). A peptide TOPBP1 inhibitor may comprise or consist of a fragment of about 6, about 7, about 8 or about 9 amino acids of SEQ ID NO: 17 or said variant. Said fragment or variant will typically comprise an amino acid corresponding to Lysl55 and / or Lys250 of SEQ ID NO: 15, preferably amino acids corresponding to Lysl55 and Lys250 of SEQ ID NO: 15.

[0299] A peptide TOPBP1 inhibitor may comprise or consist of the amino acid sequence of SEQ ID NO: 50 or 51. A peptide TOPBP1 inhibitor may comprise or consist of an amino acid sequence with at least 80% (e.g. at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%) identity to SEQ ID NO: 50 or 51 (i.e. be a variant of SEQ ID NO: 50 or 51). A peptide TOPBP1 inhibitor may comprise or consist of a fragment of about 6, about 7, about 8 or about 9 amino acids of SEQ ID NO: 50 or 51, or said variant. Said fragment or variant will typically comprise an amino acid corresponding to Lysl55 and / or Lys250 of SEQ ID NO: 15, preferably amino acids corresponding to Lysl55 and Lys250 of SEQ ID NO: 15.

[0300] A peptide TOPBP1 inhibitor may comprise or consist of a fragment of from about 6 amino acids to about 300 amino acids in length, such as from about 6 amino acids to about 250 amino acids, from about 6 amino acids to about 200 amino acids, from about 6 amino acids to about 150 amino acids, from about 6 amino acids to about 100 amino acids, from about 10 amino acids to about 100 amino acids; from about 10 amino acids to about 80 amino acids; from about 10 amino acids to about 70 amino acids; from about 10 amino acids to about 60 amino acids; from about 10 amino acids to about 50 amino acids; from about 10 amino acids to about 40 amino acids; from about 10 amino acids to about 30 amino acids; from about 15 amino acids to about 100 amino acids; from about 15 amino acids to about 80 amino acids; from about 15 amino acids to about 70 amino acids; from about 15 amino acids to about 60 amino acids; from about 15 amino acids to about 50 amino acids; from about 15 amino acids to about 40 amino acids; from about 15 amino acids to about 30 amino acids; from about 20 amino acids to about 100 amino acids; from about 20 amino acids to about 80 amino acids; from about 20 amino acids to about 70 amino acids; from about 20 amino acids to about 60 amino acids; from about 20 amino acids to about 50 amino acids; from about 20 amino acids to about 40 amino acids; from about 20 amino acids to about 30 amino acids; from about 25 amino acids to about 100 amino acids; from about 25 amino acids to about 80 amino acids; from about 25 amino acids to about 70 amino acids; from about 25 amino acids to about 60 amino acids; from about 25 amino acids to about 50 amino acids; or from about 25 amino acids to about 40 amino acids of any of the above TOPBP1 inhibitors (e.g. SEQ ID NOs: 16, 17, 50, or 51, or variants thereof). Said fragment or variant will typically comprise an amino acid corresponding to Lysl55 and / or Lys250 of SEQ ID NO: 15, preferably amino acids corresponding to Lysl55 and Lys250 of SEQ ID NO: 15.

[0301] Modifications to peptide TOPBP1 inhibitors are also encompassed by the present disclosure. Non-limiting examples of such modifications are described herein in the context of SLX4, but apply equally to peptide TOPBP1 inhibitors.

[0302] A peptide TOPBP1 inhibitor may be a linear peptide or a cyclic peptide.

[0303] Other modalities of TOPBP1 inhibitors are also encompassed. By way of further non-limiting example, a TOPBP1 inhibitor may bind to the BRCT1 and / or BRCT2 domains of TOPBP1. Non-limiting examples of such TOPBP1 inhibitors may include antibodies or other modalities (as described herein) which bind to an epitope comprising or consisting of the BRCT1 and / or BRCT2 domains of TOPBP1.

[0304] Cancers with high levels of replication stress

[0305] The invention relates to the treatment of cancer with high levels of replication stress and / or defective DDR mechanisms, particularly to the treatment of cancer with high levels of replication stress. As described herein, the invention particularly relates to the treatment of cancer with high levels of replication stress by a mechanism of synthetic lethality. In particular, SLX4 inhibitors (or TOPBP1 inhibitors) elicit a selective therapeutic effect (also referred to herein as synthetic lethality) in such cancer cells, which may be described as SLX4 / replication stress synthetic lethality.

[0306] High levels of replication stress may result from a defect in one or more mechanism of DDR, such homologous recombination (HR), non-homologous end-joining (NHEJ), microhomology mediated end-joining (MMEJ), break-induced DNA replication (BIR, including mitotic DNA synthesis (MiDAS)), base excision repair (BER), DNA mismatch repair (MMR), nucleotide excision repair (NER), trans-lesion synthesis (TLS), the Fanconi anaemia pathway (FA) and / or single-strand annealing (SSA), which are described in more detail herein.

[0307] Homologous recombination (HR) is a process by which DNA lesions are repaired via the use of a homologous DNA sequence template, normally found on the homologous chromosome in meiotic cells or on sister chromatids in mitotic cells. The use of the homologous DNA sequence template in HR results in error-free, conservative, DNA repair.

[0308] Non-homologous end-joining (NHEJ) is a process for repair of DNA-breaks that does not require a DNA-template, involves synapsis, end-processing and ligation. Synapsis is initiated by assembly of the Ku-heterodimer on DNA broken ends, followed by the formation of DNA-dependent protein kinase (DNA-PK) — an assembly of the catalytic subunit (DNA-PKcs), the Ku-heterodimer and DNA.

[0309] Both HR and NJEJ are inactivated during mitosis. Mitotic cells instead rely on the mechanisms of microhomology mediated end-joining (MMEJ) and mitotic DNA synthesis (MiDAS) to repair DSB.

[0310] MMEJ relies on microhomologous sequences to align the broken ends of DNA strands before joining, frequently resulting in deletions and occasionally insertions. In MMEJ, repair of the DSB is initiated by end resection by the MRE nuclease, leaving single stranded overhangs. These single stranded overhangs anneal at microhomologies, which are short regions of complementarity, often 5-25 base pairs, between the two strands.

[0311] Break-induced DNA replication (BIR) repairs one-ended double-strand DNA breaks through invasion into a homologous template followed by DNA synthesis. BIR copies the template DNA in a migrating displacement loop (D-loop) and results in conservative inheritance of newly synthesised DNA.

[0312] Mitotic DNA synthesis (MiDAS) is a form of break-induced DNA replication (BIR), a DNA repair process based on homologous recombination. During MiDAS, parts of the genome that are not fully replicated when cells enter mitotic prophase complete their replicative cycle at that point. References herein to BIR will therefore, by definition encompass MiDAS.

[0313] DNA mismatch repair (MMR) is a highly conserved pathway that removes base-base mismatches and insertion-deletion loops that arise during DNA replication and recombination. Four principal steps are required for MMR: (1) mismatch recognition, (2) recruitment of additional MMR factors, (3) identification of the newly synthesised DNA strand containing the mismatched nucleotides, followed by their excision, and (4) resynthesis of the excised tract and ligation.

[0314] Base excision repair (BER) repairs non-distorting DNA lesions following oxidation, deamination or alkylation of bases in the DNA backbone. Initially, a damaged base is recognized and removed by a DNA glycosylase generating an AP site. The AP site is cleaved, and the resulting product processed to complete the repair.

[0315] Nucleotide excision repair (NER) removes a wide variety of bulky and helix-distorting lesions. In NER, two distinct mechanisms exist, global genome repair and transcription-coupled repair. After recognition of the lesion by a multi-subunit enzyme system, the DNA is unwound and dual single- strand incisions made to remove an oligodeoxynucleotide containing the lesion. DNA synthesis fills in the remaining gap and, then DNA ligation seals the nick, completing the repair.

[0316] Single-strand annealing (SSA) is a DSB repair pathway that uses homologous repeats to bridge DSB ends. SSA anneals tandem repeat sequences flanking the DSB, thereby deleting one of the repeats and the intervening sequence.

[0317] Trans-lesion synthesis (TLS) is a DNA damage tolerance pathway that allows the cell to overcome replication barriers, such as DNA adducts. In TLS, specialised low-fidelity polymerases utilise the damaged template to restart DNA synthesis.

[0318] The Fanconi anaemia (FA) pathway functions to remove DNA interstrand crosslinks (ICLs), which are known to interfere with DNA replication and genetic transcription. The FA pathway ensures fidelity in the repair of the ICL-derived double-strand break by blocking the error-prone non-homologous end-joining pathway and funneling the ICL-derived double-strand break to FA-pathway-dependent homologous recombination repair.

[0319] Replication stress is a cause of DNA damage that induces the DNA damage response (DDR). The level of replication stress can be determined and / or quantified by determining and / or quantifying the level of DNA damage within a cell. This can be determined and / or quantified directly, such as by quantifying DSBs or single-strand breaks (SSBs) by comet assay, pulsed field gel electrophoresis, measurement of hallmarks of CIN such as Micronuclei, anaphase abnormalities (DNA chromatin bridges, DNA laggards, ultra-fine anaphase bridges and chromatid gaps). Alternatively or in addition, this can be determined and / or quantified indirectly, such as by quantifying: markers of DSB (e.g. phospho-Histone H2A. X S139); phosphorylation of key components of the DNA damage repair (DDR) pathway (such as Chkl and RPA2), 53BP1 and FANCD2; and replication fork dynamics dysfunction. Suitable assays are known in the art and exemplified herein. For example, western blot, immunofluorescence microscopy, immunohistochemistry and / or flow cytometry may be used to detect markers such as Phospho-Histone H2A. X (Serl39), 53BP1 or FANCD2, phospho-S371 Chkl, phospho-S345 Chkl, phospho-S33 RPA2, phospho-S4 / 8 RPA2. Byway of further example, neutral and alkaline comet assays may be used to determine and / or quantify the presence of DSBs or SSBs respectively. By way of further example, DNA fibre assays may be used to determine and / or quantify replication fork dynamics- dysfunction, and BrdU assays may be used to detect excess single-stranded DNA generation in response to replication stress.

[0320] As used herein, the term "cancer with high levels of replication stress" is used to refer to any cancer which has high levels of replication stress, which may be determined and / or quantified as described herein. Cancers with high levels of replication stress typically exhibit an aggressive phenotype associated with elevated proliferation, etc. Alternatively or in addition, such cancers may be defined by the loss of one or more checkpoint activator or tumour suppressor gene; and / or by the overexpression or constitutive expression of one or more oncogene. Typically, the high levels of replication stress arise endogenously, rather than as a result of treatment with (non-SLX4) anti-cancer therapy. Thus, high levels of replication stress may result from a deficiency in one or more gene known to be associated with inhibiting replication stress, and / or upregulation of one or more gene involved in driving replication stress. Said deficiency in one or more gene known to be associated with inhibiting replication stress may be a loss-of-fu notion mutation, or deletion of said one or more gene, with loss-of-function mutations being more common.

[0321] Cancers with high levels of replication stress can exhibit increased expression of certain proteins, such as Myc, Cyclin D and / or Cyclin E. Overexpression of one or more of Myc, Cyclin D and / or Cyclin E may be observed. Cancers with high levels of replication stress can exhibit increased expression of certain proteins, such as Myc, Cyclin D, Cyclin E and / or a RAS protein (KRAS4A, KRAS4B, HRAS and / or NRAS). Overexpression of one or more of Myc, Cyclin D, Cyclin E and / or a RAS protein (KRAS4A, KRAS4B, HRAS and / or NRAS) may be observed. Overexpression may be defined as an increase in protein and / or mRNA expression of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 100%, at least about 150% or more. Said increase may be compared with an appropriate control, such as a corresponding cell type not experiencing replication stress, such as a healthy cell of the same cell type.

[0322] Alternatively or in addition, cancers with high levels of replication stress can exhibit a mutation and / or deficiency in one or more gene associated with replication stress or checkpoint regulation, such as one or more gene associated with any one or more of HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA. Genes with mutations and / or deficiencies known to be associated with replication stress are known in the art. Accordingly, a skilled person would readily be able to determine whether a cancer or a cancer cell is associated with a high level of replication stress. Non-limiting examples of genes where mutations and / or deficiencies are known to be associated with replication stress include BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, LIG1, and Liglll. Non-limiting examples of genes where mutations and / or deficiencies are known to be associated with replication stress include BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, LIG1, Liglll, FEN1, XRCC1, RBBP8 / CHP, REV1, MAD2L2, REV3L, RAD9a, RADI, RHNO1, HUS1, and RAD17. NCBI gene IDs and UniProt accession numbers for these genes and the proteins encoded thereby are set out in the Table below (all accessed on 27 October 2024 or 27 October 2025 for FEN1, XRCC1, RBBP8 / CHP, REV1, MAD2L2, REV3L, RAD9a, RADI, RHNO1, HUS1, and RAD17).

[0323]

[0324]

[0325] Typically, high levels of replication stress is associated with a mutation (e.g. a loss-of-fu notion mutation) or other deficiency such as epigenetic silencing in any gene associated with replication stress or checkpoint regulation, such as one or more gene associated with any one or more of HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA. Non-limiting examples of such genes include BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, LIG1, and Liglll. Thus, the invention relates to the treatment of cancers which have a mutation (e.g. a loss-of-function mutation) or other deficiency in one or more gene associated with replication stress or checkpoint regulation, such as one or more gene associated with any one or more of HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, including but not limited to BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, LIG1, and Liglll. In other words, according to the invention, SLX4 inhibitors may be used in the treatment of cancers with high levels of replication stress, wherein the high levels of replication stress may be associated with a mutation (e.g. a loss-of-fu notion mutation) or other deficiency (e.g. epigenetic silencing) in one or more gene associated with replication stress or checkpoint regulation, such as one or more gene associated with any one or more of HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, including but not limited to BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, LIG1, and Liglll. Preferably the invention relates to the treatment of cancers which have a mutation (e.g. a loss-of-function mutation) or other deficiency (e.g. epigenetic silencing) in BRCA1 and / or BRCA2, with the treatment of cancers which have a mutation (e.g. a loss-of-function mutation) or other deficiency (e.g. epigenetic silencing) in BRCA2 being particularly preferred.

[0326] The nature of the (loss-of-function) mutation or other deficiency (e.g. epigenetic silencing) in the one or more gene associated with replication stress or checkpoint regulation, such as one or more gene associated with any one or more of HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, including BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, LIG1, and Liglll is not particularly limited according to the invention, provided that the (loss-of-function) mutation or other deficiency (e.g. epigenetic silencing) results in a deficiency in replication stress or checkpoint regulation, such as in one or more gene associated with any one or more of HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA.

[0327] For the avoidance of doubt, any reference herein to cancer with high levels of replication stress applies equally and without reservation to cancer cells with high levels of replication stress. By way of non-limiting example, cancer cells with high levels of replication stress typically have a deficiency in one or more gene involved in replication stress or checkpoint regulation, such as one or more gene associated with any one or more of HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein.

[0328] Thus, a cancer or cancer cell to be treated according to the invention may be associated with one or more mutation and / or deficiency in BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, LIG1, and Liglll, or any combination thereof. In particular, a cancer or cancer cell to be treated according to the invention may have high levels of replication stress associated with a mutation and / or deficiency in BRCA2 and / or BRCA1. A cancer or cancer cell to be treated according to the invention may have high levels of replication stress associated with a mutation and / or deficiency in BRCA2. A cancer or cancer cell to be treated according to the invention may have high levels of replication stress associated with a mutation and / or deficiency in BRCA1. A cancer or cancer cell to be treated according to the invention may have high levels of replication stress associated with a mutation and / or deficiency in BRCA2 and BRCA1.

[0329] Thus, a cancer or cancer cell to be treated according to the invention may be associated with a mutation and / or deficiency in BRCA2 and / or BRCA1. A cancer or cancer cell to be treated according to the invention may be associated with a mutation and / or deficiency in BRCA2. A cancer or cancer cell to be treated according to the invention may be associated with a mutation and / or deficiency in BRCA1. A cancer or cancer cell to be treated according to the invention may be associated with a mutation and / or deficiency in in BRCA2 and BRCA1.

[0330] High replication stress is a common characteristic of many tumors. Non-limiting examples of types of cancer known to be associated with a (loss-of-function) mutation or other deficiency in one or more gene associated with high replication stress, including pathways such as replication stress or checkpoint regulation, such as one or more gene associated with any one or more of HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA include ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, adrenal cancer, uterine cancer, biliary cancer, cancer of the urinary tract, head and neck cancer, bone / soft tissue cancer, lymphoid cancer, liver cancers, mesothelioma, oeseophageal cancer, neuroendocrine tumours, lung cancer, colorectal cancer and skin cancer. Thus, the invention relates to the treatment of cancers with high replication stress including ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, adrenal cancer, uterine cancer, biliary cancer, cancer of the urinary tract, head and neck cancer, bone / soft tissue cancer, lymphoid cancer, liver cancers, mesothelioma, oeseophageal cancer, neuroendocrine tumours, lung cancer, colorectal cancer and skin cancer. Ovarian cancer, breast cancer, pancreatic cancer, and prostate cancer are particularly associated with (loss-of-function) mutations or other deficiencies in one or more gene associated with high replication stress, including pathways such as replication stress or checkpoint regulation, such as one or more gene associated with any one or more of HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA. Therefore, the treatment of high replication stress ovarian cancer, breast cancer, pancreatic cancer, biliary tract cancer and prostate cancer is preferred according to the invention. The invention also relates to the treatment of high replication stress ovarian cancer, pancreatic cancer, biliary tract cancer and prostate cancer. In some preferred embodiments, the invention relates to the treatment of cancer with high levels of replication stress, particularly ovarian cancer, breast cancer, pancreatic cancer, biliary tract cancer or prostate cancer, that is associated with a (loss-of-function) mutation in BRCA2 and / or BRCA1.

[0331] The invention may relate to the treatment of cancers that are sensitive to treatment with one or more inhibitor of a component of the DNA damage response (DDR) pathway. Alternatively, the invention may relate to the treatment of cancers that are insensitive to treatment with one or more inhibitor of a DDR pathway component, as described herein (e.g. Pol0 inhibitors). When the invention relates to the treatment of cancers that are insensitive to treatment with one or more inhibitor of a DDR pathway component, said cancers may have previously been sensitive to treatment with one or more inhibitor of a DDR pathway component, but have developed resistance to the one or more inhibitor of a DDR pathway component following treatment therewith.

[0332] The invention may relate to the treatment of breast cancer that (i) has a (loss-of-function) mutation in BRCA1 and / or BRCA2; (ii) has a (loss-of-function) mutation in BRCA1 and / or BRCA2 and is human epidermal growth factor 2 negative (HER2 ); or (ill) is triple-negative (Estrogen Receptor negative (ER ), Progesterone Receptor negative (PR ) and HER2 ).

[0333] Homologous Recombination Defective (HRD) Cancer

[0334] In particular, the disclosure relates to the treatment of homologous recombination defective (HRD) cancer. HRD cancers exhibit high levels of replication stress as described herein. As described herein, the invention particularly relates to the treatment of HRD cancer by a mechanism of synthetic lethality. In particular, SLX4 inhibitors (or TOPBP1 inhibitors) elicit synthetic lethality in HRD cancer cells, which may be described as SLX4 / HR deficiency synthetic lethality, with SLX4 / BRCA (BRCA1 and / or BRCA2) synthetic lethality being a particular focus.

[0335] HR is a process by which DNA lesions are repaired via the use of a homologous DNA sequence template, normally found on the homologous chromosome in meiotic cells or on sister chromatids in mitotic cells. The use of the homologous DNA sequence template in HR results in error-free, conservative, DNA repair. Additionally, during DNA replication, HR repair (HRR) pathways support the recovery of stalled replication forks. Successful HRR depends on several properly functioning proteins, with BRCA1 and BRCA2 proteins playing critical roles. BRCA1 is a tumor suppressor protein central to several macromolecular complexes which drive HRR and cell cycle progression. MRN and CtIP are involved in DNA resection, after which BRCA1 travels to sites of DSBs where it participates in DNA damage signaling and coordinates DNA damage repair. During the synthesis (S) phase of normal cell cycle progression, if DNA becomes damaged, BRCA1 protein complexes recruit BRCA2 protein complexes to initiate strand invasion and / or homology-directed repair. The HR capacity of cells and cancers can be defined in several ways including the presence of deleterious BRCA1 or BRCA2 mutations; the presence of deleterious mutations in other genes that control HR; the presence of hypermethylation of the promoter region of one or more genethat controls HR, such as BRCA1, PALB2, RAD51, RAD51C, and ATR, preferably the promoter region of BRCA1; the inability to form RAD51 nuclear foci; a clinical response to platinum-based chemotherapy or PARP inhibitor; or the presence of a genomic DNA scar reflective of a HR defect in the lineage of a cancer.

[0336] The HR pathway is important for high-fidelity DSB repair. In HRD cancers, loss of function of one or more gene associated with HR leads HR deficiency, resulting in increased levels of genomic alterations.

[0337] As used herein, the term "homologous recombination defective cancer" ("HRD cancer") is used to refer to any cancer which has a deficiency (complete or partial) in HR or HRR. A deficiency in HR or HRR may result from a deficiency in one or more gene involved in HRR, and / or upregulation of one or more miRNA which target a gene involved in HRR. Typically, an HRD cancer has a deficiency in in one or more gene involved in HRR. Said deficiency may be a loss-of-function mutation, or deletion of said one or more gene, with loss-of-function mutations being more common.

[0338] Non-limiting examples of miRNA which may be upregulated and result in a deficiency in HR or HRR include miR-182 and miR-1255b (which targetsBRCAl), miR-148b (which targets BRCA2) and miR-193b (which targets RAD51).

[0339] Typically, HR deficiency is associated with a mutation (e.g. a loss-of-function mutation) or other deficiency such as epigenetic silencing in any gene associated with HR or HRR. Non-limiting examples of such genes include BRCA1, BRCA2, ATM, BARD1, PALB2, BRIP1, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, CHEK2, FAN1, FANCE, FANCM, and POLQ. Thus, SLX4 inhibitors may be used in the treatment of HRD cancers, wherein the HR deficiency may be associated with a mutation (e.g. a loss-of-function mutation) or other deficiency (e.g. epigenetic silencing) in one or more gene associated with HR or HRR, including but not limited to BRCA1, BRCA2, ATM, BARD1, PALB2, BRIP1, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, CHEK2, FAN1, FANCE, FANCM, and POLQ. Preferably the invention relates to the treatment of HRD cancers which have a mutation (e.g. a loss-of-function mutation) or other deficiency (e.g. epigenetic silencing) in BRCA1 and / or BRCA2, with the treatment of HRD cancers which have a mutation (e.g. a loss-of-function mutation) or other deficiency (e.g. epigenetic silencing) in BRCA2 being particularly preferred.

[0340] The nature of the (loss-of-function) mutation or other deficiency (e.g. epigenetic silencing) in the one or more gene associated with HR or HRR, including BRCA1, BRCA2, ATM, BARD1, PALB2, BRIP1, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, CHEK2, FAN1, FANCE, FANCM, and POLQ is not particularly limited according to the invention, provided that the (loss-of-function) mutation or other deficiency (e.g. epigenetic silencing) results in a deficiency in HR or HRR.

[0341] For the avoidance of doubt, any reference herein to HRD cancer applies equally and without reservation to HDR cancer cells. By way of non-limiting example, HRD cancer cells typically have a deficiency in one or more gene involved in HRR, such as those described herein.

[0342] HRD is a common characteristic of many tumors. Non-limiting examples of types of cancer known to be associated with a (loss-of-function) mutation or other deficiency in one or more gene associated with HR or HRR include ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, adrenal cancer, uterine cancer, biliary cancer, cancer of the urinary tract, head and neck cancer, bone / soft tissue cancer, lymphoid cancer, liver cancers, mesothelioma, oeseophageal cancer, neuroendocrine tumours, lung cancer, colorectal cancer and skin cancer. Thus, the invention relates to the treatment of HRD cancers including ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, adrenal cancer, uterine cancer, biliary cancer, cancer of the urinary tract, head and neck cancer, bone / soft tissue cancer, lymphoid cancer, liver cancers, mesothelioma, oeseophageal cancer, neuroendocrine tumours, lung cancer, colorectal cancer and skin cancer. Ovarian cancer, breast cancer, pancreatic cancer, and prostate cancer are particularly associated with (loss-of-function) mutations or other deficiencies in one or more gene associated with HR or HRR. Therefore, the treatment of HRD ovarian cancer, breast cancer, pancreatic cancer, biliary tract cancer and prostate cancer is preferred according to the invention. The invention also relates to the treatment of HRD ovarian cancer, pancreatic cancer, biliary tract cancer and prostate cancer.

[0343] In some preferred embodiments, the invention relates to the treatment of HRD cancer, particularly ovarian cancer, breast cancer, pancreatic cancer, biliary tract cancer or prostate cancer, that is associated with a (loss-of-function) mutation in BRCA2 and / or BRCA1.

[0344] Treatment of Cancers with high levels of replication stress

[0345] The present invention provides methods and medical uses for the treatment of cancers with high levels of replication stress and / or defective DDR mechanisms with SLX4 inhibitors (or TOPBP1 inhibitors). In particular, the present invention provides methods and medical uses for the treatment of cancers with high levels of replication stress with SLX4 inhibitors (or TOPBP1 inhibitors).

[0346] Thus, the invention provides SLX4 inhibitors (or TOPBP1 inhibitors) for use in a method of treating cancers with high levels of replication stress and / or defective DDR mechanisms. In particular, the invention provides SLX4 inhibitors (or TOPBP1 inhibitors) for use in a method of treating cancers with high levels of replication stress. Such cancers can exhibit a mutation and / or deficiency in one or more gene associated with replication stress or checkpoint regulation, such as one or more gene associated with any one or more of HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA as described herein. For example, such cancers may be associated with one or more mutation and / or deficiency in one or more gene selected from BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, LIG1, and Liglll, as described herein. For the avoidance of doubt, treatment of cancer according to the present disclosure relates to the treatment of any and all such cancers, with HRD cancers (such as BRCA2 and / or BRCA1 deficient cancers) being used merely as non-limiting examples. Therefore, references to the treatment of with HRD cancers (such as BRCA2 and / or BRCA1 deficient cancers) applies equally and without reservation to the treatment of cancers exhibiting a mutation and / or deficiency in one or more gene associated with replication stress or checkpoint regulation, such as one or more gene associated with any one or more of HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA as described herein. For example, such cancers may be associated with one or more mutation and / or deficiency in one or more gene selected from BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, LIG1, and Liglll.

[0347] Thus, the invention provides SLX4 inhibitors (or TOPBP1 inhibitors) for use in a method of treating cancers with high levels of replication stress and / or defective DDR mechanisms. In particular, the invention provides SLX4 inhibitors (or TOPBP1 inhibitors) for use in a method of treating cancers with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer). The invention also provides a method of treating a cancer with high levels of replication stress and / or defective DDR mechanisms, said method comprising administering a therapeutically effective amount of an SLX4 inhibitor (or a TOPBP1 inhibitor) to an individual in need thereof. In particular, the invention also provides a method of treating a cancer with high levels of replication stress (such as an HRD cancer, e.g. a BRCA2 and / or BRCA1 deficient cancer), said method comprising administering a therapeutically effective amount of an SLX4 inhibitor (or a TOPBP1 inhibitor) to an individual in need thereof. The invention also relates to the use of an SLX4 inhibitor in the manufacture of a medicament for treating a cancer with high levels of replication stress and / or defective DDR mechanisms. In particular, the invention also relates to the use of an SLX4 inhibitor (or a TOPBP1 inhibitor) in the manufacture of a medicament for treating a cancer with high levels of replication stress (such as an HRD cancer, e.g. a BRCA2 and / or BRCA1 deficient cancer). These medical uses and methods exploit the SLX4 / high replication stress synthetic lethality identified by the present inventors, particularly SLX4 / BRCA (BRCA1 and / or BRCA2, particularly BRCA1) synthetic lethality. All disclosure herein in relation to SLX4 inhibitors (or TOPBP1 inhibitors) for the treatment of cancer with high levels of replication stress and / or defective DDR mechanisms (such as an HRD cancer, e.g. a BRCA2 and / or BRCA1 deficient cancer), and in particular all disclosure herein in relation to SLX4 inhibitors (or TOPBP1 inhibitors) for the treatment of cancer with high levels of replication stress, apply equally and without reservation to all medical uses and methods of treatments described herein unless expressly stated to the contrary.

[0348] A method of treating a cancer with high levels of replication stress (such as an HRD cancer, e.g. a BRCA2 and / or BRCA1 deficient cancer) according to the invention may comprise determining whether the cancer to be treated is a cancer with high levels of replication stress (such as an HRD cancer, e.g. a BRCA2 and / or BRCA1 deficient cancer). If the cancer is determined to be a cancer with high levels of replication stress (such as an HRD cancer, e.g. a BRCA2 and / or BRCA1 deficient cancer), then a therapeutically effective amount of an SLX4 inhibitor may be administered to the individual.

[0349] Any appropriate means or method may be used to determine whether the cancer is a cancer with high levels of replication stress (such as an HRD cancer, e.g. a BRCA2 and / or BRCA1 deficient cancer). By way of non-limiting example, a cancer with high levels of replication stress (such as an HRD cancer, e.g. a BRCA2 and / or BRCA1 deficient cancer) may be identified as such by testing a sample comprising cancer cells from an individual, for example to determine whether one or more cancer cells in said sample comprise a (loss-of-function) mutation or other deficiency in one or more gene associated with high levels of replication stress, such as a deficiency in one or more gene associated with any one or more of checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those genes identified herein (such as any one or more of BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, LIG1, and Liglll).

[0350] Non-limiting examples of types of cancer known to be associated associated with high levels of replication stress, such as a deficiency in one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA are set out above. Thus, nonlimiting examples of cancer include ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, adrenal cancer, uterine cancer, biliary cancer, cancer of the urinary tract, head and neck cancer, bone / soft tissue cancer, lymphoid cancer, liver cancers, mesothelioma, oeseophageal cancer, neuroendocrine tumours, lung cancer, colorectal cancer and skin cancer. Thus, the invention relates to the treatment of such cancers including ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, adrenal cancer, uterine cancer, biliary cancer, cancer of the urinary tract, head and neck cancer, bone / soft tissue cancer, lymphoid cancer, liver cancers, mesothelioma, oeseophageal cancer, neuroendocrine tumours, lung cancer, colorectal cancer and skin cancer. Ovarian cancer, breast cancer, pancreatic cancer, prostate cancer are particularly associated with high levels of replication stress, such as a deficiency in one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA. Therefore, the treatment of such ovarian cancer, breast cancer, pancreatic cancer, biliary tract cancer and prostate cancer is preferred according to the invention. The invention also relates to the treatment of ovarian cancer, pancreatic cancer, biliary tract cancer and prostate cancer.

[0351] The cancer may be characterised by one or more mutation (e.g. a loss-of-function mutation) or other deficiency (e.g. epigenetic silencing) in one or more gene associated with high levels of replication stress, particularly by one or more mutation (e.g. a loss-of-function mutation) or other deficiency (e.g. epigenetic silencing) in one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, including but not limited to BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, LIG1, and Liglll. Preferably the cancer may be characterised by one or more mutation (e.g. a loss-of-function mutation) or other deficiency (e.g. epigenetic silencing) in BRCA1 and / or BRCA2, BRCA2 being particularly preferred.

[0352] Said one or more mutation (e.g. a loss-of-function mutation) or other deficiency (e.g. epigenetic silencing) in one or more gene associated with high levels of replication stress, particularly by one or more mutation (e.g. a loss-of-function mutation) or other deficiency (e.g. epigenetic silencing) in one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA may occur in somatic pre-cancerous or cancerous cells. While such mutations are mostly believed to be somatic, any of the one or more mutation (e.g. a loss-of-function mutation) or other deficiency (e.g. epigenetic silencing) in one or more gene associated with high levels of replication stress, particularly by one or more mutation (e.g. a loss-of-function mutation) or other deficiency (e.g. epigenetic silencing) in one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, mutations may be associated with clonal haematopoiesis, e.g. as a result of ageing, and there may also be cancers characterised by one or more mutation or other deficiency in one or more gene associated with high levels of replication stress, particularly by one or more mutation (e.g. a loss-of-function mutation) or other deficiency (e.g. epigenetic silencing) in one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA occurring in the germ line of the individual patient. Thus, an SLX4 inhibitor (or a TOPBP1 inhibitor) may be used in a method of treating a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) according to the disclosure wherein said method comprises determining whether the cancer has high levels of replication stress or HRD by determining the presence of a deficiency and / or mutation in one or more gene associated with high levels of replication stress, particularly by one or more mutation (e.g. a loss-of-function mutation) or other deficiency (e.g. epigenetic silencing) in one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA optionally a deficiency and / or mutation in one or more gene selected from BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, LIG1, and / or Liglll.

[0353] A cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) may be identified as such by testing a sample comprising cancer cells from an individual to determine the expression of one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) to evaluate whether expression of the protein is absent or at a reduced level compared to normal.

[0354] Alternatively or in addition, a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) may be characterised by the cancer cells having a defect in or the cancer cells exhibiting epigenetic inactivation of one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2), or loss of protein function.

[0355] More generally, a cancer may be identified as a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) by determining the activity of the proteins encoded by one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) in a sample of cells from an individual. The sample may be of normal cells from the individual where the individual has a mutation in one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) or the sample may be of cancer cells, e.g. where the cells forming a tumour exhibit defects in the activity of the protein encoded by one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2). Activity may be determined relative to a control, for example in the case of defects in cancer cells, a relative to non-cancerous cells, preferably from the same tissue. The activity of the one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) may be determined by using techniques well known in the art such as Western blot analysis, immunoprecipitation, immunohistology, chromosomal abnormalities, enzymatic or DNA binding assays, and plasmid-based assays.

[0356] The sample may comprise or consist of normal cells from the individual where the individual has a mutation in one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) or the sample may comprise or consist of cancer cells, e.g. where the cells forming a tumour contain one or more mutation in one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2). Activity may be determined relative to a control, for example in the case of defects in cancer cells, relative to non-cancerous cells, preferably from the same tissue.

[0357] The determination of expression of one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) may involve determining the presence or amount of said one or more gene mRNA in a sample. Methods for doing this are well known to the skilled person. By way of example, they include determining the presence of mRNA of one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) (i) using a labelled probe that is capable of hybridising to the nucleic acid of said one or more gene; and / or (ii) using PCR involving one or more primers based on a nucleic acid sequence of said one or more gene to determine whetherthe transcript of said one or more gene is present in a sample. The probe may also be immobilised as a sequence included in a microarray. It is also possible to use quantitative PCR or nanostring nCounter technology to assess the downstream consequences of mutation.

[0358] Detecting mRNA of one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) may be carried out by extracting RNA from a sample of the tumour and measuring expression of said one or more gene specifically using quantitative real time RT-PCR. Alternatively or additionally, the expression of said one or more gene could be assessed using RNA extracted from a tumour sample using microarray analysis, which measures the levels of mRNA for a group of genes using a plurality of probes immobilised on a substrate to form the array.

[0359] A cancer may be identified as a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) by determining the presence in a cell sample from an individual's tumour of one or more chromosomal abnormalities, for example deletions in part or loss of entire chromosomes, corresponding to gene loss. Chromosomal abnormalities may be visualised through any karyotyping technique known in the art, including but not limited to Giemesa staining, quinacrine staining, Hoechst 33258 staining, DAPI (4'-6-diamidino-2-phenylindole) staining, daunomycin staining, and fluorescence in situ hybridization.

[0360] A cancer may be identified as a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) by determining the presence in a cell sample from the individual of one or more variations, for example, polymorphisms or mutations, in the nucleic acid sequence of one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2).

[0361] Alternatively or in addition, a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) may be identified by determining the presence in the circulating tumour DNA (ctDNA) and / or cell-free DNA (cfDNA) in a patient. ctDNA and / or cfDNA may be extracted from a blood sample from the patient. The presence of one or more variations, for example, polymorphisms or mutations, in the nucleic acid sequence of one or more gene associated with high levels of replication stress, particularly by one or more gene with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) in ctDNA and / or cfDNA from the patient may be used to identify a cancer as a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer). Thus, analysis of ctDNA and / or cfDNA of a patient for variations, for example, polymorphisms or mutations, in the nucleic acid sequence of one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) may be used to identify a cancer as a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer).

[0362] Sequence variations such as mutations and polymorphisms may include a deletion, insertion or substitution of one or more nucleotides, relative to the wild-type nucleotide sequence. The one or more variations may be in a coding or non-coding region of the nucleic acid sequence and may reduce or abolish the expression or function of the one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2). In other words, the variant nucleic acid may encode a variant polypeptide which has reduced or abolished activity or may encode a wild-type polypeptide which has little or no expression within the cell, for example through the altered activity of a regulatory element. A variant nucleic acid may have one or more mutations or polymorphisms relative to the wild-type sequence.

[0363] Alternatively or additionally, in the present invention the determination of whether a patient has a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) can be carried out by analysis of expression of the protein encoded by one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2), for example by examining whether levels of said protein (e.g. BRCA1 and / or BRCA2) are supressed.

[0364] In some aspects, the presence or amount of protein encoded by one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) may be determined using a binding agent capable of specifically binding to said protein, or fragments thereof. A preferred type of protein binding agent is an antibody capable of specifically binding to said protein or fragment thereof (e.g. BRCA1 and / or BRCA2). The antibody may be labelled to enable it to be detected or capable of detection following reaction with one or more further species, for example using a secondary antibody that is labelled or capable of producing a detectable result, e.g. in an ELISA type assay. As an alternative, a labelled binding agent may be employed in a western blot to detect said protein (e.g. BRCA1 and / or BRCA2). Alternatively, or additionally, the method for determining the presence of a protein encoded by one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) may be carried out on tumour samples, for example using immunohistochemical (IHC) analysis or in situ RNA-hybridisation. IHC analysis can be carried out using paraffin fixed samples or frozen tissue samples, and generally involves staining the samples to highlight the presence and location of said protein (e.g. BRCA1 and / or BRCA2).

[0365] As described herein, an SLX4 inhibitor may be used in a method of treating a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) according to the invention wherein sad method comprises determining whether the cancer is HRD by determining the presence of a deficiency and / or mutation in one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, optionally a deficiency and / or mutation in one or more gene selected from BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, LIG1, and Liglll, preferably BRCA2 and / or BRCA1.

[0366] The step of determining the presence of a deficiency and / or mutation in said one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA (e.g. BRCA1 and / or BRCA2) may be performed on nucleic acid sequences obtained from an individual's cancerous and / or noncancerous cells, using any standard technique known in the art, examples of which are described herein. Non-limiting examples of suitable techniques for determining the presence of a deficiency and / or mutation in one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA (e.g. BRCA1 and / or BRCA2) using nucleic acid sequences obtained from an individual's cancerous and / or noncancerous cells include direct sequencing, hybridisation to a probe, restriction fragment length polymorphism (RFLP) analysis, single-stranded conformation polymorphism (SSCP), PCR amplification of specific alleles, amplification of DNA target by PCR followed by a mini-sequencing assay, allelic discrimination during PCR, Genetic Bit Analysis, pyrosequencing, oligonucleotide ligation assay, analysis of melting curves, testing for a loss of heterozygosity (LOH) or next generation sequencing (NGS) techniques, single molecule sequencing techniques or nanostring nCounter technology. Alternatively or additionally, the step of determining the presence of a deficiency and / or mutation in said one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA (e.g. BRCA1 and / or BRCA2) may comprise measuring / quantifying protein expression of the one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA (e.g. BRCA1 and / or BRCA2) in a sample obtained from the individual (which may comprise or consist of cancerous and / or non-cancerous cells). Any standard technique known in the art, may be used to measure / quantify the protein expression of the one or more gene associated with homologous recombination deficiency, examples of which are described herein. Non-limiting examples of suitable techniques for measuring / quantifying protein expression of the one or more gene associated with homologous recombination deficiency in a sample obtained from the individual (which may comprise or consist of cancerous and / or non-cancerous cells) include immunohistochemistry, determining protein levels in a cell lysate by ELISA or Western blotting, and / or determining protein expression using a binding agent capable of specifically binding to a protein, or a fragment thereof.

[0367] Alternatively or additionally, the step of determining the presence of a deficiency and / or mutation in said one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA (e.g. BRCA1 and / or BRCA2) may comprise extracting RNA from a sample of an individual's cancerous and / or noncancerous cells and measuring / quantifying the RNA of the one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA (e.g. BRCA1 and / or BRCA2). Any standard technique known in the art may be used, examples of which are described herein. Non-limiting examples of suitable techniques for measuring / quantifying the RNA of the one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA (e.g. BRCA1 and / or BRCA2) include real time PCR and / or by using a probe capable of hybridising to the RNA of one or more gene associated with homologous recombination deficiency RNA. Said probe may be immobilised in a microarray.

[0368] Alternatively or additionally, the step of determining the presence of a deficiency and / or mutation in said one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA (e.g. BRCA1 and / or BRCA2) may comprise identifying gene loss resulting from chromosomal instability through karyotype analysis of a sample obtained from the individual.

[0369] Alternatively or additionally, the step of determining the presence of a deficiency and / or mutation in said one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA (e.g. BRCA1 and / or BRCA2) may comprise identifying specific signatures ("genomic scars") through whole genome sequencing (e.g. HRDetect) or shallow whole genome sequencing.

[0370] As described herein, an SLX4 inhibitor (or a TOPBP1 inhibitor) may be used in a method of treating a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) according to the invention wherein sad method comprises determining whether the cancer is a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer). Additional techniques may be used to determine whether a cancer is a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer), in combination with or as an alternative to determining the presence of a deficiency and / or mutation in one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, as described herein.

[0371] Thus, an SLX4 inhibitor (or a TOPBP1 inhibitor) may be used in a method of treating a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) according to the invention wherein sad method comprises determining whether the cancer is a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) by (a) use of a companion diagnostic for high levels of replication stress or homologous recombination deficiency; (b) detecting and / or quantifying RAD51 foci within a sample of cancerous and / or non-cancerous cells from an individual, wherein reduced RAD51 signal is associated with high levels of replication stress or homologous recombination deficiency; (c) detecting and / or quantifying levels of replication stress, optionally a transcriptomic signature and / or mutational scar associated with replication stress such as, but not exclusive to, the Myriad MyChoice assay; (d) detecting and / or quantifying a homologous recombination deficiency defect, optionally a transcriptomic signature and / or mutational scar associated with homologous recombination deficiency such as, but not exclusive to, the Myriad MyChoice assay; and / or (e) detecting and / or quantifying platinum sensitivity of the cancer, wherein platinum sensitivity is associated with homologous recombination deficiency. A companion diagnostic is a diagnostic test, which provides information that facilitates the safe and effective use of a corresponding drug, in this case the use of an SLX4 inhibitor (or a TOPBP1 inhibitor) according to the disclosure. A number of companion diagnostics tests are available for HRD cancers. For example, companion diagnostics for homologous recombination deficiency may determine and / or quantify loss of heterozygosity (LOH), typically genome-wide LOH, telomeric allelic imbalance (LAI) and / or large-scale state transitions (LST), or any combination thereof.

[0372] The term "loss of heterozygosity" or "LOH" refers to a common form of allelic imbalance by which a heterozygous somatic cell becomes homozygous because one of the two alleles of a gene gets lost. There are two types of LOH: (i) copy number neutral LOH which refers to a change in the gene without a change in the chromosomal copy number; and (ii) deletion LOH, which occurs as a result of copy number loss.

[0373] The term "telomeric allelic imbalance" or "LAI" refers to a chromosomal aberration where the telomeric and subtelomeric regions of a chromosome does not have the expected 1:1 ratio for the alleles and telomeric sequences inherited from the two parental chromosomes. TAI is similar to LOH, but the difference is that the structural change occurs specifically at the telomere.

[0374] The term "large-scale state transitions" or "LST" refers to chromosomal breaks that generate fragments of 10Mb or larger. These LST can be caused by transfer of DNA from one chromosome to another chromosome, but can also be caused by inversions, deletions and duplications of DNA. Quantification of LSTs can be used as a surrogate measure for genomic instability.

[0375] Other markers for DNA damage can also be used, such as mitotic abnormalities, micronuclei, replication fork dynamic dysfunction, DSBs and SSBs. Examples of suitable assays are known in the art and described herein, such as DNA fibre assays for determining and / or quantifying replication fork dynamics- dysfunction, comet assays for determining and / or quantifying DSBs and SSBs, and antibodybased assays.

[0376] Any suitable companion diagnostic may be used to determine whether a cancer has high levels of replication stress (e.g. is an HRD cancer) according to the present disclosure. Suitable companion diagnostics, including those which determine one or more of LOH, LAI and / or LST are known in the art. Companion diagnostics for HRD cancer are also commercially available, and include MyChoice® CDx (from Myriad), FoundationOne CDx (from Foundation Medicine), Tempus xT (from Tempus), and tests from Caris Molecular Intelligence.

[0377] RAD51 protein forms subnuclear complexes that are microscopically detectible as foci, which contain many of the enzymatic activities required for efficient repair of DSBs. RAD51 foci may be used as a surrogate marker of HRR functionality. Thus, reduced number of RAD51 foci and / or RAD51 signal is associated with homologous recombination deficiency and hence replication stress. A genomic scar (also referred to as a mutational scar) can be defined as a genomic aberration with a known origin, and may include aberrations such as LOH, TAI, LST, total number of somatic, synonymous, and non-synonymous coding mutations (Nmut). Genomic scar assays may be used to determine whether a cancer has high levels of replication stress (e.g. is an HRD cancer), as described in Watkins et al. (2014) Breast Cancer Res. 16(3):211, which is herein incorporated by reference. Alternatively or in addition, specific transcriptome abnormalities which create a unique transcriptomic signature may be determined / quantified using techniques such as gene expression, allele-specific expression, and alternative splicing from RNA-sequencing data, and used to determine whether a cancer has high levels of replication stress (e.g. is an HRD cancer).

[0378] Cancers with high levels of replication stress, particularly those associated with deficiencies in HR or HRR, are known to be associated with sensitivity to platinum-based chemotherapeutic agents. Consequently, detecting and / or quantifying platinum sensitivity of cancerous and / or non-cancerous cells from an individual may be used as determine whether a cancer has high levels of replication stress (e.g. is an HRD cancer). Typically platinum sensitivity is assessed at the level of a clinical response, i.e. whether a cancer or cells thereof respond fully or partially to platinum chemotherapy assessed using normal clinical tests and / or parameters.

[0379] The present invention provides an assay comprising: measuring or quantifying a mutation or deficiency in one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) in a biological sample obtained from an individual with cancer; and comparing the measured or quantified amount of the one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) with a reference value, and if the one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as those described herein (e.g. BRCA1 and / or BRCA2) is mutated or deficient relative to the reference value, identifying the individual as having an increased probability of being responsive to treatment with an SLX4 inhibitor.

[0380] Combination therapies

[0381] An SLX4 inhibitor (or a TOPBP1 inhibitor) as described herein may be used in combination with one or more further anti-cancer therapy. The further anti-cancer therapy is not particularly limited. By way of example, an SLX4 inhibitor (or a TOPBP1 inhibitor) as described herein may be used in combination with one or more further chemotherapeutic agent. Non-limiting examples of such chemotherapeutic agents are described below. Alternatively or in addition, an SLX4 inhibitor (or a TOPBP1 inhibitor) as described herein may be used in combination with one or more immunotherapeutic agent. Further alternatively or in addition, an SLX4 inhibitor (or a TOPBP1 inhibitor) may be used in conjunction with radiotherapy and / or surgery.

[0382] An immunotherapeutic for use with an SLX4 inhibitor (or a TOPBP1 inhibitor) as described herein may be selected from an immune checkpoint inhibitor, a monoclonal antibody, a cytokine, a cell therapy, a cancer vaccine, an oncolytic virus, an antisense oligodeoxynucleotide, an antibody-drug conjugate and / or any modulator of the cytosolic nucleic acid sensing pathways.

[0383] SLX4 inhibitors (or TOPBP1 inhibitors) and compositions comprising SLX4 inhibitors (or TOPBP1 inhibitors) disclosed herein for the treatment of a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) may be used in the methods described herein in combination with standard chemotherapeutic regimes and / or in conjunction with radiotherapy. As radiotherapy also leads to DNA strand breaks, causing severe DNA damage and leading to cell death, the combination of radiotherapy with SLX4 inhibitors (or TOPBP1 inhibitors) offers the potential to lead to formation of double strand breaks from the single-strand breaks generated by the radiotherapy in tumour tissue. This combination could therefore lead to either more powerful therapy with the same radiation dose or similarly powerful therapy with a lower radiation dose, potentially avoiding some of the side effects with radiotherapy.

[0384] Non-limiting examples of additional agents that might be employed in combination with SLX4 inhibitors (or TOPBP1 inhibitors) as disclosed herein include one or more spliceosomal inhibitors, for example agents that target components of the spliceosome, such as SF3B1, e.g. using a SF3BI inhibitor, particularly small molecule inhibitors of SF3B1.

[0385] An SLX4 inhibitor (or a TOPBP1 inhibitor) may be administered alone or in combination with other treatments, either separately, sequentially or simultaneously, dependent upon the condition to be treated.

[0386] Preferably, an SLX4 inhibitor (or a TOPBP1 inhibitor) as described herein may be used in combination with one or more inhibitor of a component of a DNA damage repair (DDR) component; and / or an inhibitor of microhomology mediated end-joining (MMEJ). DDR and the various components of the DDR pathway are described further herein.

[0387] DDR inhibitors As disclosed and exemplified herein, the inventors have demonstrated that pharmacological inhibition of Pol0, combined with loss of the TOPBP1-SLX4 interaction, exacerbates genome instability (particularly chromosomal instability (CIN)) and reduces cellular proliferation under replication stress. Notably, SLX4, Pol0, and CIP2A are essential for cellular proliferation in BRCAl / 2-deficient cells. Therefore, the data provided herein supports the clinical potential of combination therapies comprising agents targeting SLX4 (or TOPBP1), and particularly the TOPBP1-SLX4 interaction, and agents targeting the DNA damage response (DDR) pathway, such as Pol0 inhibitors.

[0388] Accordingly, an SLX4 inhibitor (or a TOPBP1 inhibitor) may be used in combination with one or more inhibitor of a component of a DNA damage repair (DDR) component; and / or an inhibitor of microhomology mediated end-joining (MMEJ).

[0389] DDR refers to the cellular mechanisms of signalling and repair in response to damage caused by all types of damage, including that by DNA replication stress. Thus, DDR components include all proteins involved in the mechanisms of DNA repair, such as those that directly interact and functionally repair DNA, (e.g. MRE11, NBS1, RAD50, Pol0 etc.), and also those that facilitate signalling to coordinate these process (e.g. ATR, ATM, CHK1 etc.). As used herein the term "DDR pathway component" encompasses both those which directly interact and functionally repair DNA, and those which signal to coordinate the process.

[0390] In particular, SLX4 inhibitors (or TOPBP1 inhibitors) may be used in combination with an inhibitor of any DDR component selected from Pol0, CHK1, ATR, WEE1, PARP, ATM, DNApk and / or RAD51. Alternatively or in addition, SLX4 inhibitors (orTOPBPl inhibitors) may be used in combination with a mitotic poison. In some preferred embodiments, SLX4 inhibitors (orTOPBPl inhibitors) may be used in combination with one or more inhibitor of Pol0, CHK1, ATR, WEE1, PARP, and / or ATM. In some particularly preferred embodiments, SLX4 inhibitors (or TOPBP1 inhibitors) may be used in combination with one or more inhibitor of Pol0.

[0391] The particular inhibitor for a DDR component of interest is not particularly limited, and selection of such an inhibitor is within the routine practice of one of ordinary skill in the art. Examples of such inhibitors are known in the art. By way of non-limiting example, AZD6738 is an inhibitor of ATR (as described in Wilson et al. (2022) Cancer Res 82(6):1140-1152. doi: 10.1158 / 0008-5472. CAN-21-2997); KU-55933 is an inhibitor of ATM (as described in Li and Yang (2010) Mol Cancer Ther 9(1):113-25. doi: 10.1158 / 1535-7163. MCT-08-1189); AZD7762 is an inhibitor of CHK1 (as described in Zabludoff et al. (2008) Mol Cancer Ther 7(9):2955-66. doi: 10.1158 / 1535-7163. MCT-08-0492); AZD1775 is an inhibitor of WEE1 (as described in Lallo et al. (2018) Clin Cancer Res 24(20):5153-5164. doi: 10.1158 / 1078-0432. CCR-17-2805); AZD2281 is an inhibitor of PARP (as described in Fong et al. (2008) J Clin Oncol 26( 15su ppi) doi: 10.1200 / jco.2008.26.15_suppl.55100); ART558 is an inhibitor of Pol0 (as described in Zatreanu et al. (2021) Nat Commun 12(1):3636. doi: 10.1038 / s41467-021-23463-8); B02 is an inhibitor of RAD51 (as described in Alapaluisa et al. (2014) Front Oncol 30:4:289. doi: 10.3389 / fonc.2014.00289. eCollection 2014); AZD7648 is an inhibitor of DNAPK (as described in Fok et al. (2019) Nat Commun 10:5065. doi: 10.1038 / s41467-019-12836-9); and paclitaxel is an example of a mitotic poison (as described in Rowinsky and Donehower (1993) Semin Oncol 20(4 Soppl 3): 16-25), each of which is herein incorporated by reference. Thus, an SLX4 inhibitor may be used in combination with any one or more of ART558; AZD6738; KU-55933; AZD7762; AZD1775; AZD2281; B02; AZD7648; or paclitaxel.

[0392] SLX4 inhibitors (or TOPBP1 inhibitors) may be used in combination with inhibitors of any two or more DDR components, such as inhibitors of any two or more DDR components selected from Pol0, CHK1, ATR, WEE1, PARP, ATM, DNApk and / or RAD51, particularly any two or more of Pol0, CHK1, ATR, WEE1, PARP, and / or ATM.

[0393] Without being bound by theory, it is believed that inhibition of SLX4 (or TOPBP1) results in inhibition of the MiDAS pathway, such that inhibition of other DDR pathways (e.g. MMEJ, HR, etc.) removes redundancy in DNA repair and can lead to the accumulation of fatal levels of DNA damage within cancer cells.

[0394] Thus, the use of SLX4 inhibitors (or TOPBP1 inhibitors) in combination with one or more inhibitor of a component of the DDR pathway can lead to increased clinical efficacy. Said increase in clinical efficacy may be determined by reference to any suitable control. By way of non-limiting example, said increase in clinical efficacy may be compared with treatment of the same cancer or same cancer type (in the same individual or a different individual) with the SLX4 inhibitor (or TOPBP1 inhibitor) alone and / or compared with treatment with the inhibitor of a component of the DDR pathway alone.

[0395] It is within the routine practice of one of ordinary skill in the art to determine whether a combination treatment leads to increased clinical efficacy. Any appropriate measure of clinical efficacy may be used. Measures of clinical efficacy may vary between cancer types and / or cancer stages.

[0396] By way of non-limiting example, clinical efficacy may be quantified in terms of tumour regression (i.e. a sustained shrinkage and reduction of tumour burden over time, which can be measured by any appropriate technique, particularly by imaging techniques such as CT or MRI scans); progression-free survival (PFS), which is defined as the length of time during and after treatment that the patient lives without the disease worsening; overall survival (OS); reduction in metastasis; complete or partial response rates, which can be quantified in terms of tumour shrinkage (i.e. a decrease in tumour size due to treatment, typically measured against the initial size at diagnosis or start of treatment), disease-Free Survival (DFS); time to Progression (TTP); objective response rate (ORR); biomarker reduction, i.e. a decreases in specific biomarkers that correlate with progression of a specific cancer, such as PSA in prostate cancer or CA-125 in ovarian cancer; and / or symptom improvement. These may be quantified by increases or decreases (as appropriate) with values as defined herein.

[0397] In some preferred embodiments, SLX4 inhibitors (or TOPBP1 inhibitors) may be used in combination with one or more inhibitor of Pol0 and may increase the clinical efficacy of the one or more inhibitor of Pol0.

[0398] As described above it is believed that inhibition of SLX4 (orTOPBPl) results in inhibition of the MiDAS pathway, such that inhibition of other DDR pathways (e.g. MMEJ, HR, etc.) removes redundancy in DNA repair and can lead to the accumulation of fatal levels of DNA damage within cancer cells. Again, without being bound by theory, it is believed that as a consequence of this, combined treatment with an SLX4 inhibitor (or a TOPBP1 inhibitor) and one or more inhibitor of a component of the DDR pathway may also reduce resistance to the inhibitor of the component of the DDR pathway, as the cancer is less able to adapt given the greater levels of DNA damage which accumulate in the cells.

[0399] Thus, the use of SLX4 inhibitors (or TOPBP1 inhibitors) in combination with one or more inhibitor of a component of the DDR pathway can lead to reduced resistance to an inhibitor of a DDR pathway component. Said decrease in resistance may be determined by reference to any suitable control. By way of non-limiting example, said increase in decrease in resistance may be compared with treatment of the same cancer or same cancer type (in the same individual or a different individual) with the inhibitor of the component of the DDR pathway alone.

[0400] It is within the routine practice of one of ordinary skill in the art to determine whether a combination treatment leads to a decrease in resistance. Any appropriate measure of a decrease in resistance may be used. Measures of a decrease in resistance may vary between cancer types and / or cancer stages. For example, a decrease in resistance may be quantified in terms of increased clinical efficacy of the inhibitor of the component of the DDR pathway. Non-limiting examples of parameters for determining and / or quantifying increased clinical efficacy are given above, and may thus be used to determine and / or quantify resistance to an inhibitor of the component of the DDR pathway.

[0401] In some preferred embodiments, SLX4 inhibitors (or TOPBP1 inhibitors) may be used in combination with one or more inhibitor of Pol0 and may reduce resistance to the one or more inhibitor of Pol0.

[0402] Selecting Individuals with HRD Cancer for Treatment with an SLX4 Inhibitor (or a TOPBP1 Inhibitor) The invention also provides a method of selecting an individual having cancer for treatment with an SLX4 inhibitor (or a TOPBP1 inhibitor). Said method typically comprises (a) determining whether the cancer is a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer); and (b) selecting the individual for treatment with the SLX4 inhibitor (or TOPBP1 inhibitor) where the cancer is a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer). Said method may further comprise providing an SLX4 inhibitor (or a TOPBP1 inhibitor) suitable for administration to the individual, and may yet further comprise administering a therapeutically effective amount of the SLX4 inhibitor (or TOPBP1 inhibitor) to the individual. Determining whether the cancer is a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) may comprise the use of a sample comprising or consisting of cancerous and / or non-cancerous cells from the individual.

[0403] Any and all disclosure herein in relation to determining whether a cancer is a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) in the context of treating an individual with cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) applies equally and without reservation to methods for screening or selecting an individual with HRD cancer for treatment according to the invention.

[0404] By way of non-limiting example, in a method of selecting an individual having a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) for treatment with an SLX4 inhibitor (or a TOPBP1 inhibitor), determining whether the cancer is a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) may comprise: (a) determining whether the cancer is mutated or deficient in one or more gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA; (b) use of a companion diagnostic for high levels of replication stress or homologous recombination deficiency; (c) detecting and / or quantifying RAD51 foci within a sample of cancerous and / or non-cancerous cells from an individual, wherein reduced RAD51 signal is associated with high levels of replication stress or homologous recombination deficiency; (d) detecting and / or quantifying levels of replication stress, optionally a transcriptomic signature and / or mutational scar associated with replication stress such as, but not exclusive to, the Myriad MyChoice assay; (e) detecting and / or quantifying a homologous recombination deficiency defect, optionally a transcriptomic signature and / or mutational scar associated with homologous recombination deficiency such as, but not exclusive to, the Myriad MyChoice assay; and / or (f) detecting and / or quantifying platinum sensitivity of the cancer, wherein platinum sensitivity is associated with homologous recombination deficiency. Any and all of the disclosure herein in relation to the determination of whether a cancer is a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer), e.g. in relation to particular genes associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA and / or techniques for determining the presence of a deficiency and / or mutation in one or more gene associated with with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, applies equally and without reservation to the selection methods of the invention.

[0405] Preferably the invention provides a method of selecting an individual having cancer for treatment with an SLX4 inhibitor (or aTOPBPl inhibitor), the method comprising: (a) determining in a sample obtained from the individual whether the cancer is a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer); (b) selecting the individual for treatment with the SLX4 inhibitor (or TOPBP1 inhibitor) where the cancer is a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) cancer; and optionally providing a SLX4 inhibitor (or a TOPBP1 inhibitor) suitable for administration to the individual; wherein determining whether the cancer is cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) comprises determining whether the cancer is mutated or deficient in a gene associated with high levels of replication stress, particularly by one or more gene associated with checkpoint regulation, HR, NHEJ, MMEJ, BIR (including MiDAS), BER, NER, TLS, FA and / or SSA, such as BRCA2 and / or BRCA1, preferably BRCA2.

[0406] Preferably the invention provides a method of selecting an individual having a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) for treatment with an SLX4 inhibitor (or a TOPBP1 inhibitor), wherein the cancer is breast cancer, ovarian cancer, pancreatic cancer, biliary tract cancer or prostate cancer.

[0407] Methods of Screening for SLX4 Inhibitors (or TOPBP1 Inhibitors)

[0408] The present invention also includes methods of screening that employ SLX4 (or TOPBP1) as a protein target for the screening of candidate compounds to find SLX4 inhibitors (orTOPBPl inhibitors). Accordingly, methods of screening may be carried out for identifying candidate agents that are capable of inhibiting SLX4 (or TOPBP1), for subsequent use of development as agents for the treatment of cancers with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer). Conveniently, this may be done in an assay buffer to help the components of the assay interact, and in a multiple well format to test a plurality of candidate agents. The activity of SLX4 (or TOPBP1) can then be determined in the presence and absence of the one or more candidate compounds to determine whether a given candidate is a SLX4 inhibitor (or a TOPBP1 inhibitor).

[0409] By way of example, the candidate agent may be a known inhibitor of one of the protein targets disclosed herein, an antibody, a peptide, a nucleic acid molecule or a small molecule (e.g. an organic or inorganic compound), typically of molecular weight of less than 500 Da. In some instances, the use of candidate agents that are small molecules is preferred. However, for any type of candidate agent, combinatorial library technology provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a target protein. Such libraries and their use are known in the art. The present invention also specifically envisages screening candidate agents known for the treatment of other conditions, and especially other forms of cancer. This has the advantage that the patient or disease profile of known therapeutic agents might be expanded or modified using the screening techniques disclosed herein, or for therapeutic agents in development, patient or disease profiles established that are relevant for the treatment of cancers with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer).

[0410] Following identification of a candidate agent for further investigation, the agent in question may be tested to determine whether it is not lethal to normal cells or otherwise is suited to therapeutic use. Following these studies, the agent may be manufactured and / or used in the preparation of a medicament, pharmaceutical composition, or dosage form.

[0411] The development of lead agents or compounds from an initial hit in screening assays might be desirable where the agent in question is difficult or expensive to synthesise or where it is unsuitable for a particular method of administration, e.g. peptides are unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis and testing is generally used to avoid randomly screening large number of molecules for a target property.

[0412] There are several steps commonly taken in the design of a mimetic from a compound having a given target property. Firstly, the particular parts of the compound that are critical and / or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide, e.g. by substituting each residue in turn. These parts or residues constituting the active region of the compound are known as a "pharmacophore". Once the pharmacophore has been found, its structure is modelled to according to its physical properties, e.g. stereochemistry, bonding, size and / or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and NMR. Computational analysis, similarity mapping (which models the charge and / or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process. In a variant of this approach, the three-dimensional structure of the ligand and its binding partner are modelled. This can be especially useful where the ligand and / or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.

[0413] A template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound. The mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

[0414] Formulations, Compositions and Administration

[0415] The SLX4 inhibitors (or TOPBP1 inhibitors) herein for the treatment of cancers with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) may be administered alone, but it is generally preferable to provide them in pharmaceutical compositions that additionally comprise with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents. Examples of components of pharmaceutical compositions are provided in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0416] These compounds or derivatives of them may be used in the present invention for the treatment of cancers with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer).

[0417] As used herein "derivatives" of the SLX4 inhibitors (or TOPBP1 inhibitors) includes salts, coordination complexes, esters such as in vivo hydrolysable esters, free acids or bases, hydrates, prodrugs or lipids, coupling partners.

[0418] Salts of the SLX4 inhibitors (or TOPBP1 inhibitors) of the invention are preferably physiologically well tolerated and non-toxic. Many examples of salts are known to those skilled in the art. Compounds having acidic groups, such as phosphates or sulfates, can form salts with alkaline or alkaline earth metals such as Na, K, Mg and Ca, and with organic amines such as triethylamine and Tris (2-hydroxyethyl)amine. Salts can be formed between compounds with basic groups, e.g., amines, with inorganic acids such as hydrochloric acid, phosphoric acid or sulfuric acid, or organic acids such as acetic acid, citric acid, benzoic acid, fumaric acid, or tartaric acid. Compounds having both acidic and basic groups can form internal salts.

[0419] Esters can be formed between hydroxyl or carboxylic acid groups present in an SLX4 inhibitor (or a TOPBP1 inhibitor) and an appropriate carboxylic acid or alcohol reaction partner, using techniques well known in the art.

[0420] Derivatives include prodrugs of the SLX4 inhibitors (orTOPBPl inhibitors) which are convertible in vivo or in vitro into an active SLX4 inhibitor (or an active TOPBP1 inhibitor). Typically, at least one of the biological activities of an SLX4 inhibitor (or a TOPBP1 inhibitor) will be reduced in the prodrug form of the SLX4 inhibitor (or TOPBP1 inhibitor), and can be activated by conversion of the prodrug to release the SLX4 inhibitor (or TOPBP1 inhibitor) or a metabolite of it.

[0421] Other derivatives include coupling partners of the SLX4 inhibitors (or TOPBP1 inhibitors) in which an SLX4 inhibitor (or a TOPBP1 inhibitor) is linked to a coupling partner, e.g. by being chemically coupled to the SLX4 inhibitor (or TOPBP1 inhibitor) or physically associated with it. Examples of coupling partners include a label or reporter molecule, a supporting substrate, a carrier or transport molecule, an effector, a drug, an antibody or an inhibitor. Coupling partners can be covalently linked to SLX4 inhibitors (or TOPBP1 inhibitors) of the invention via an appropriate functional group on the SLX4 inhibitor (or TOPBP1 inhibitor), such as a hydroxyl group, a carboxyl group or an amino group. Other derivatives include formulating the SLX4 inhibitors (or TOPBP1 inhibitors) with liposomes.

[0422] The term "pharmaceutically acceptable" as used herein includes compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

[0423] The SLX4 inhibitors (or TOPBP1 inhibitors) disclosed herein for the treatment of cancers with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) according to the present invention are preferably for administration to an individual in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, Lippincott, Williams & Wilkins. By way of non-limiting example, ttherapeutic efficacy and toxicity of the compound can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50 / ED50. It will be appreciated that the dosage range required depends on the precise nature of the SLX4 inhibitor (orTOPBPl inhibitor), the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgement of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation.

[0424] Any two or more SLX4 inhibitors (or TOPBP1 inhibitors) of the invention may be administered separately, sequentially or simultaneously. The two or more SLX4 inhibitors (or TOPBP1 inhibitors) may be administered in the same or different compositions. In a preferred instance, the two or more SLX4 inhibitors (orTOPBPl inhibitors) may be delivered in the same composition.

[0425] An SLX4 inhibitor (or a TOPBP1 inhibitor) may be formulated using standard techniques and additional components as described herein. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing the SLX4 inhibitor (or TOPBP1 inhibitor) into association with a carrier which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the SLX4 inhibitor (or TOPBP1 inhibitor) with liquid carriers or finely divided solid carriers or both, and then if necessary, shaping the product.

[0426] The SLX4 inhibitors (or TOPBP1 inhibitors) may be administered to an individual by any convenient route of administration, whether systemically / peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly. It may be desired to direct the compositions of the present invention (as described above) to the tissue or organ comprising a tumour to be treated according to the invention. Formulations suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the SLX4 inhibitor (or TOPBP1 inhibitor); as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

[0427] Formulations suitable for parenteral administration (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.

[0428] The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the SLX4 inhibitor to blood components or one or more organs.

[0429] Liquid compositions may be sterilised by filtration through a sterile filter using aseptic techniques before filling into suitable sterile containers (e.g. vials or ampoules) and sealing. Alternatively, if solution stability is adequate, the solution in its sealed containers may be sterilised by autoclaving. Additives such as preservative or bactericidal, suspending or emulsifying agents and or local anaesthetic agents may be dissolved in the vehicle.

[0430] Solid formulations (e.g. dry powders), which are dissolved or suspended in a suitable vehicle prior to use, may be prepared by filling pre-sterilised ingredients into a sterile container using aseptic technique in a sterile area. Alternatively the ingredients may be dissolved into suitable containers using aseptic technique in a sterile area. The product is then lyophilised and the containers are sealed aseptically.

[0431] Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal injection, are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be accomplished by filtration. The components may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma irradiation.

[0432] The composition may be in lyophilized form, in which case it may include a stabilizer, such as bovine serum albumin (BSA). In some embodiments, it may be desirable to formulate the composition with a preservative, such as thiomersal or sodium azide, to facilitate long-term storage.

[0433] Administration in accordance with the present invention may take advantage of a variety of delivery technologies including microparticle encapsulation, viral delivery systems or high-pressure aerosol impingement.

[0434] SLX4 inhibitors (or TOPBP1 inhibitors) may be used in combination with one or more further anti-cancer therapies according to the invention. In particular, SLX4 inhibitors (or TOPBP1 inhibitors) may be used in combination with (i) one or more further chemotherapeutic agent; (ii) one or more immunotherapeutic agent; and / or (ill) radiotherapy, or a combination thereof.

[0435] Thus, SLX4 inhibitors (or TOPBP1 inhibitors) and compositions comprising SLX4 inhibitors (or TOPBP1 inhibitors) disclosed herein for the treatment of cancers with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer) may be used in the methods described herein in combination with standard chemotherapeutic regimes and / or in conjunction with radiotherapy.

[0436] As described herein, by way of example, additional agents that might be employed in combination with the use of SLX4 inhibitors (orTOPBPl inhibitors) as disclosed herein include one or more spliceosomal inhibitors, for example agents that target components of the spliceosome, such as SF3B1, e.g. using a SF3BI inhibitor, particularly small molecule inhibitors of SF3B1.

[0437] Alternatively or in addition, SLX4 inhibitors (orTOPBPl inhibitors) may be used in combination with one or more chemotherapeutic agent according to the invention. Non-limiting examples of chemotherapeutic agents which may be used in combination with one or more SLX4 inhibitor (or TOPBP1 inhibitor) include Amsacrine (Amsidine), Bevacizumab (Avastin), Bleomycin, Busulfan, Capecitabine (Xeloda), Carboplatin, Carmustine (BCNU), Chlorambucil (Leukeran), Cisplatin, Cladribine (Leustat), Clofarabine (Evoltra), Crisantaspase (Erwinase), Cyclophosphamide, Cytarabine (ARA-C), Dacarbazine (DTIC), Dactinomycin (Actinomycin D), Daunorubicin, Docetaxel (Taxotere), Doxorubicin, Epirubicin, Etoposide (Vepesid, VP-16), Fludarabine (Fludara), Fluorouracil (5-FU), Gemcitabine (Gemzar), Hydroxyurea (Hydroxycarbamide, Hydrea), Idarubicin (Zavedos), Ifosfamide (Mitoxana), Irinotecan (CPT-11, Campto), Leucovorin (folinic acid), Liposomal doxorubicin (Caelyx, Myocet), Liposomal daunorubicin (DaunoXome®) Lomustine, Melphalan, Mercaptopurine, Mesna, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin (Eloxatin), Paclitaxel (Taxol), Pemetrexed (Alimta), Pentostatin (Nipent), Procarbazine, Raltitrexed (Tomudex®), Streptozocin (Zanosar®), Tegafur-uracil (Uftoral), Temozolomide (Temodal), Teniposide (Vumon), Thiotepa, Tioguanine (6-TG) (Lanvis), Topotecan (Hycamtin), Treosulfan, Vinblastine (Velbe), Vincristine (Oncovin), Vindesine (Eldisine) or Vinorelbine (Navelbine), orthe PARP inhibitors (Olaparib (Lynparza), Rucaparib, Niraparib, Veliparib and Talazoparib), ATR inhibitors, Pol0 (also known as PolQ) inhibitors, or any other DNA repair inhibitor. Further non-limiting examples of chemotherapeutic agents which may be used in combination with one or more SLX4 inhibitor include Cisplatin, Carboplatin, Oxaliplatin, Camptothecin, Doxirubicin, etoposide, hydrocyurea, 5-FU, Methotrexate, and mitomycin-C. In particular, SLX4 inhibitors (or TOPBP1 inhibitors) may be used in combination with one or more Pol0 inhibitor.

[0438] Alternatively or in addition, SLX4 inhibitors (orTOPBPl inhibitors) may be used in combination with one or more immunotherapeutic agent, such as an immune checkpoint inhibitor, monoclonal antibody (including bi-, tri- and multi-specific antibodies), cytokine, cell therapy, cancer vaccines, oncolytic viruses, antisense oligodeoxynucleotides, antibody-drug conjugates and / or any modulator of the cytosolic nucleic acid sensing pathways (e.g. agonist of the cGAS / stimulator of interferon genes (STING) pathway, i.e. a STING agonist).

[0439] Preferably, an SLX4 inhibitor (or a TOPBP1 inhibitor) of the invention may be used in combination with one or more anti-cancer therapy for a cancer with high levels of replication stress, such as HRD cancers as described herein (e.g. a BRCA2 and / or BRCA1 deficient cancer), for example platinum-based chemotherapeutics, PARP inhibitors, Pol0 inhibitors, RS-response kinase ataxia telangiectasia and Rad3-related protein (ATR) inhibitors and / or VEGF inhibitors (e.g. anti-VEGF monoclonal antibodies and tyrosine kinase inhibitors). Non-limiting examples of platinum-based chemotherapeutics include cisplatin, carboplatin, oxaliplatin and nedaplatin. Non-limiting examples of PARP inhibitors include niraparib (Zejula), olaparib (Lynparza), talazoparib (Talzenna) and rucaparib (Rubraca). Non-limiting examples of Pol0 inhibitors include novobiocin, ART4215, ART0380 and ART558. Non-limiting examples of ATR inhibitors include AZD6738, M6620 (VX-970), BAY1895344, and M4344 (VX-803). Non-limiting examples of VEGF inhibitors include bevacizumab, aflibercept, ramucirumab, axitinib, cabozantinib, lapatinib, lenvatinib, pazopanib, ponatinib, regorafenib, sorafenib, sunitinib, and vandetanib.

[0440] Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. In some cases after an initial administration of an SLX4 inhibitor (or a TOPBP1 inhibitor), a subsequent administration of an SLX4 inhibitor (or a TOPBP1 inhibitor) may be performed. The administration may, for instance, be at least one week, two weeks, three weeks, four weeks, six weeks, two months, four months, six months or more after the initial administration. The SLX4 inhibitor (or TOPBP1 inhibitor) may, for instance, be administered at intervals dictated by when the effects of the previous administration are decreasing, and when an additional administration will not exceed the therapeutic window. Where the SLX4 inhibitor (or TOPBP1 inhibitor) is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent SLX4 inhibitor (or TOPBP1 inhibitor), and so the actual weight to be used is increased proportionately.

[0441] The invention also provides a kit comprising a (solid or liquid) composition as described herein and instructions for therapeutic administration of said SLX4 inhibitor (or TOPBP1 inhibitor) to an individual in need thereof. More precisely, the invention relates to a kit comprising one or more SLX4 inhibitor (or TOPBPl inhibitor) of the invention, or a pharmaceutical composition comprising one or more SLX4 inhibitor (or TOPBP1 inhibitor) of the invention, and instructions for therapeutic administration of said one or more SLX4 inhibitor (or TOPBP1 inhibitor) to an individual in need thereof. The one or more SLX4 inhibitor (or TOPBP1 inhibitor) may be provided in any suitable composition or formulation, such as those described herein. By way of non-limiting example, the one or more SLX4 inhibitor (or TOPBP1 inhibitor) may be provided in lyophilised form. As used herein, the term "instructions" refers to a publication, a recording, a diagram, or any other medium of expression which can be used to communicate how to perform a method or use of the invention, such as therapeutic or cosmetic administration of said composition to an individual in need thereof Said instructions can, for example, be affixed to a container which comprises said composition or said kit.

[0442] SEQUENCE INFORMATION

[0443] Sequence key

[0444] SEQ ID NO: 1 SLX4 amino acid sequence (UniProt Accession No. Q8IY92)

[0445] SEQ ID NO: 2 SLX4 nucleic acid sequence (NCBI Accession No: NM_032444.4)

[0446] SEQ ID NO: 3 TOPBP1 BRCT1 binding motif of SLX4 (amnio acid residues 1254-1263 of SEQ ID NO:

[0447] 1)

[0448] SEQ ID NO: 4 TOPBP1 binding site of SLX4 (amnio acid residues 1175-1375 of SEQ ID NO: 1) SEQ ID NO: 5 TOPBP1 binding site of SLX4 (amnio acid residues 1235-1296 of SEQ ID NO: 1) SEQ ID NO: 6 TOPBP1 binding site of SLX4 (amnio acid residues 1253-1270 of SEQ ID NO: 1) SEQ ID NO: 7 78mer SLX4 peptide inhibitor SEQ ID NO 8 17mer SLX4 peptide inhibitor

[0449] SEQ ID NO 9 SLX4 siRNA antisense strand 1

[0450] SEQ ID NO 10 SLX4 siRNA antisense strand 2

[0451] SEQ ID NO 11 SLX4 siRNA sense strand 1

[0452] SEQ ID NO 12 SLX4 siRNA sense strand 2

[0453] SEQ ID NO 13 epitope recognised by exemplified anti-SLX4 antibody (with phosphorylated T1260) SEQ ID NO 14 corresponding unphosphorylated epitope (without phosphorylated T1260) SEQ ID NO 15 TOPBP1 amino acid sequence (UniProt Accession No. Q92547)

[0454] SEQ ID NO 16 TOPBP1 protein BRCT1 domain (amino acid residues 101-189 of SEQ ID NO: 15) SEQ ID NO 17 TOPBP1 protein BRCT2 domain (amino acid residues 195-284 of SEQ ID NO: 15) SEQ ID NO 18 SLX4gRNAlF Sequence

[0455] SEQ ID NO 19 SLX4gRNAlR Sequence

[0456] SEQ ID NO 20 SLX4gRNA2F Sequence

[0457] SEQ ID NO 21 SLX4gRNA2R Sequence

[0458] SEQ ID NO 22 AIQseq sequencing primer

[0459] SEQ ID NO 23 forward primer

[0460] SEQ ID NO 24 reverse primer

[0461] SEQ ID NO 25 Guide RNA 1 forward

[0462] SEQ ID NO 26 Guide RNA 1 reverse

[0463] SEQ ID NO 27 Guide RNA 2 forward

[0464] SEQ ID NO 28 Guide RNA 2 reverse

[0465] SEQ ID NO 29 T1260A repair template

[0466] SEQ ID NO 30 ATP1A1 repair template

[0467] SEQ ID NO 31 forward primer

[0468] SEQ ID NO 32 reverse primer

[0469] SEQ ID NO 33 siCTRL / siLUC

[0470] SEQ ID NO 34 CIP2A (1) siRNA

[0471] SEQ ID NO 35 CIP2A (2) siRNA

[0472] SEQ ID NO 36 CIP2A (3) siRNA

[0473] SEQ ID NO 37 CIP2A (4) siRNA

[0474] SEQ ID NO 38 TOPBP1 siRNA

[0475] SEQ ID NO 39 Biotin-SLX4_T1260

[0476] SEQ ID NO 40 Biotin-SLX4_pT1260

[0477] SEQ ID NO 41 Flu-SLX4_pT1260 SEQ ID NO: 42 Flu-SLX4_pT1476

[0478] SEQ ID NO: 43 crRNA EJ2

[0479] SEQ ID NO: 44 crRNA BIR

[0480] SEQ ID NO: 45 BRCT1 binding consensus motif

[0481] SEQ ID NO: 46 forward sequence from Figure 6E

[0482] SEQ ID NO: 47 reverse sequence from Figure 6E

[0483] SEQ ID NO: 48 forward sequence from Figure 6G

[0484] SEQ ID NO: 49 reverse sequence from Figure 6G

[0485] SEQ ID NO: 50 TOPBP1 protein fragment comprising BRCT1 and BRCT2 domains (amino acid residues 101-284 of SEQ ID NO: 15)

[0486] SEQ ID NO: 51 TOPBP1 protein fragment comprising BRCT1 and BRCT2 domains (amino acid residues 1-284 of SEQ ID NO: 15)

[0487] Sequences

[0488] Where an initial Met amino acid residue or a corresponding initial codon is indicated in any of the following SEQ ID NOs, said residue / codon is optional.

[0489] SEQ ID NO: 1 UniProt Accession No. Q8IY92 MKLSVNEAQLGFYLGSLSHLSACPGIDPRSSEDQPESLKTGQMMDESDEDFKELCASFFQRVKKHGIKEVSGERKTQKAASNG TQIRSKLKRTKQTATKTKTLQGPAEKKPPSGSQAPRTKKQRVTKWQASEPAHSVNGEGGVLASAPDPPVLRETAQNTQTGNQQ EPSPNLSREKTRENVPNSDSQPPPSCLTTAVPSPSKPRTAQLVLQRMQQFKRADPERLRHASEECSLEAAREENVPKDPQEEM MAGNVYGLGPPAPESDAAVALTLQQEFARVGASAHDDSLEEKGLFFCQICQKNLSAMNVTRREQHVNRCLDEAEKTLRPSVPQ IPECPICGKPFLTLKSRTSHLKQCAVKMEVGPQLLLQAVRLQTAQPEGSSSPPMFSFSDHSRGLKRRGPTSKKEPRKRRKVDE APSEDLLVAMALSRSEMEPGAAVPALRLESAFSERIRPEAENKSRKKKPPVSPPLLLVQDSETTGRQIEDRVALLLSEEVELS STPPLPASRILKEGWERAGQCPPPPERKQSFLWEGSALTGAWAMEDFYTARLVPPLVPQRPAQGLMQEPVPPLVPPEHSELSE RRSPALHGTPTAGCGSRGPSPSASQREHQALQDLVDLAREGLSASPWPGSGGLAGSEGTAGLDWPGGLPLTGFWPSQDKHP DRGGRTLLSLGLLVADFGAMVNNPHLSDVQFQTDSGEVLYAHKFVLYARCPLLIQYVNNEGFSAVEDGVLTQRVLLGDVSTEA ARTFLHYLYTADTGLPPGLSSELSSLAHRFGVSELVHLCEQVPIATDSEGKPWEEKEAENCESRAENFQELLRSMWADEEEEA ETLLKSKDHEEDQENVNEAEMEEIYEFAATQRKLLQEERAAGAGEDADWLEGGSPVSGQLLAGVQVQKQWDKVEEMEPLEPGR DEAATTWEKMGQCALPPPQGQHSGARGAEAPEQEAPEEALGHSSCSSPSRDCQAERKEGSLPHSDDAGDYEQLFSSTQGEISE PSQITSEPEEQSGAVRERGLEVSHRLAPWQASPPHPCRFLLGPPQGGSPRGSHHTSGSSLSTPRSRGGTSQVGSPTLLSPAVP SKQKRDRSILTLSKEPGHQKGKERRSVLECRNKGVLMFPEKSPSIDLTQSNPDHSSSRSQKSSSKLNEEDEVILLLDSDEELE LEQTKMKSISSDPLEEKKALEISPRSCELFSIIDVDADQEPSQSPPRSEAVLQQEDEGALPENRGSLGRRGAPWLFCDRESSP SEASTTDTSWLVPATPLASRSRDCSSQTQISSLRSGLAVQAVTQHTPRASVGNREGNEVAQKFSVIRPQTPPPQTPSSCLTPV SPGTSDGRRQGHRSPSRPHPGGHPHSSPLAPHPISGDRAHFSRRFLKHSPPGPSFLNQTPAGEWEVGDSDDEQEVASHQANR SPPLDSDPPIPIDDCCWHMEPLSPIPIDHWNLERTGPLSTSSPSRRMNEAADSRDCRSPGLLDTTPIRGSCTTQRKLQEKSSG AGSLGNSRPSFLNSALWDVWDGEEQRPPETPPPAQMPSAGGAQKPEGLETPKGANRKKNLPPKVPITPMPQYSIMETPVLKKE LDRFGVRPLPKRQMVLKLKEIFQYTHQTLDSDSEDESQSSQPLLQAPHCQTLASQTYKPSRAGVHAQQEATTGPGAHRPKGPA KTKGPRHQRKHHESITPPSRSPTKEAPPGLNDDAQIPASQESVATSVDGSDSSLSSQSSSSCEFGAAFESAGEEEGEGEVSAS QAAVQAADTDEALRCYIRSKPALYQKVLLYQPFELRELQAELRQNGLRVSSRRLLDFLDTHCITFTTAATRREKLQGRRRQPR GKKKVERN SEQ ID NO: 2 NCBI Accession No: NM_032444.4 gcaggcgcgcgagtctccgttaagaaggtgccgcggcggcgccggagatgtgtaattaagtgaaccatatatgtttcatcatc atggagatcttggagaattatctgagcaccaggttcatatgtattcgatctcagaggcatctattggacaacaaaacactctt tcagttgtgaactttatttatttattattattattttttgagacagagttttgctcttgttgcccaggttagagtgcagtggc acgatctcggctcactgcaatctccgcctcccaggttcaagcgattctcttgcctctgcctcccgagtagctgggattacagg catctgctaccacgcctggccaattttttgtattttcagttgaaacgaggtttcaccatattggccaggctggtctcgaactt ctgacctcaggtgatccaccccccgcctcgtcctccaaaagagctgggattacaagtgtgagccaccgcgcccggcccagttg tggactttaacagagggaagctttaaacatgtttaaccacaggcccaatttgaacaaagatacttcaatcattatagagagga aaacagtactttttgttcaattgtgcaaactctccaagtatctaatggagaagtagagaagaaccctaatgaaactgagtgtg aatgaggct cage taggcttctacttgggttcactttct cat ctgtctgcctgtcctgggattgaccctcgctcctctgaaga ccagcctgaaagccttaaaactgg cagatgatggatgagtctgatgaggactttaaagaactctgcgctagc tttccaaa gggtgaaaaaaca ggaa caaggaagtg caggagaaaggaagacacaaaaggc gcc caaacggcac cagataagaagc aaa gaaaaggaccaaacaaactgctaccaagaccaaaaccc caaggccctgcagagaagaaacc ccg c ggcagcca ggcccc aggac aaaaagcaaagggtaaccaaatggcaagcaagtgaaccggcccac c gtgaatggggaggggggtgtgc gcc c gc ccaga ccacctgtgc ccgggaaacagcacaaaacacccagacgggtaaccagcaagaacca cgccaaac c ccagagagaaaaccagagagaatgtgcccaacagcgac cccagcc cc cc cc g gacaacagcagtgccaag ccc ccaaaccccgcacagcacaa gg cctacagcgaatgcagcag caagagagcagaccccgagcg gagacacg c cagaagagtgc ccc cgaggc gcgcgggaagaaaatg cccaaagga cc caagaggagatgatggcggggaatgtg a gggc gggccccctgccccagagagcgacgctgcggtggcc gaccctgcagcaggag gcacggg aggagca c ggcacatgatgatagcctggaggaaaaggg tgttcttctgccagat gtcaaaagaacctctcagccatgaacgtgaccc gaagggaacagca gtgaacaggtgc gga gaagc gaaaagacac aagacc ctgtgcc caga ccctgagtgcccg atttgtgggaaaccgtttcttacc aaagagcagaaccagtcacttgaagcagtgtgctgtgaagatggagg ggccccca gc cctgc caggctgtgcggctgcagacagcacagcctgagggtagcagcagcccacccatg cagc cagtga caca gtagagg ctgaaacggagaggacccaccagcaagaaggagccacggaagaggcggaaggtggacgaggcaccg ccgaggac ctgctggtggccatggctctgtcccggtcggagatggagccgggtgcggctgtaccagcgctcaggctggaaagtgcct tc tgagaggataagaccagaagcagagaataaaag cgcaagaagaaacccccgg a ccccccca g g ag ccaggact ctgaaaccacaggccgacagatagaggaccgtgtggccctgc cc c c gaggaagtggaa g c agcacgccaccac cctgccagcaggattttaaaggaagggtgggaaagagcgggccagtgtcctcctccacctgaacgcaagcagagctttctgtg ggagggcagcgcactgactggggcc gggccatggaggac c acacggccaggctgg ccc cc c cgtgccccagcggc ctgcccagggccttatgcaggagcccgtgccgcctctggtgccacctgagcactcagagctgagcgagcgaaggtcacccgct ctccacggcacccccactgcaggctgtggctccaggggcccgtcgccttcggccagccagagggagcaccaggccctgcagga cctcgtggacctggcgagggagggactgagcgccagcccgtggcccggcagtgggggcctggctggctcggaagggactgcag ggttggacgtggtgcccggcggccttcctctgactgggtttgtggtgccatcgcaggacaagcacccggacaggggcggccgc accttgctctccctcgggctgctggttgctgactttggcgccatggtcaataacccacacctgagtgatgtccagtttcagac ggacagcggggaggtgctttacgcccacaagttcgtgctttatgcccgatgcccgctcctcatccagtatgtgaacaatgaag gcttctccgctgtagaggacggggttctgacccagcgtgtcctgctgggtgacgtgagcaccgaggccgcccgcacgttcctg cactatctctacactgcggacactggccttcctcctggccttagctctgagctgagctccctggcccacaggtttggcgtgag tgagctcgttcacctgtgcgaacaggtgcctattgccactgactcagagggcaaaccatgggaggagaaggaagcagagaatt gcgaaagcagggccgagaatttccaggaactcttgaggtcaatgtgggcagatgaagaggaggaagcggagactttgttgaaa tccaaggaccacgaagaagatcaagaaaacgtgaatgaagcagaaatggaagaaatttatgaatttgcagctactcagcgaaa gcttctccaggaagaaagggcagcgggtgccggcgaggacgctgactggctggagggtggcagtccggtttctgggcaactcc tagcaggtgtccaggtgcagaaacagtgggacaaggtggaggagatggagccgttggagccaggaagagatgaggccgccacc acctgggagaagatgggacagtgcgctctcccgccaccccagggccagcactcaggggcacggggagcagaggcccctgagca ggaggcgccagaggaggcgcttggccattccagctgctccagcccttccagggactgccaggcagagagaaaagaaggctctc ttccgcactcagatgatgccggggattacgaacagctcttctcatcaactcagggagagatctcagagccgtcccaaataaca agtgagcccgaggaacaaagtggcgctgtcagggaaagggggctggaggtttctcatcgcctggctccctggcaggcatctcc accgcacccgtgccgcttcctattggggcctccccagggcgggagtccccgcgggtctcatcacacaagtgggtcgtccctgt caacaccccggtcccgtggcggaacttcccaggtgggctccccaaccttgctgtctccagctgtgccatcaaagcagaaaagg gacaggagcatcctcacgctgtctaaagagccagggcaccagaaaggcaaagagcgtcggtccgtgctggagtgcagaaataa gggggtcctgatgttcccagaaaaatctccgtctattgacctaacccagtcaaatcctgaccattcgagctccagatctcaga aatcttcatccaaactgaacgaagaagatgaggtcatcctcttactggactcggatgaggagctggagctagaacaaaccaaa atgaagtccatttctagtgatcctctggaagaaaagaaagctctagaaattagccctaggtcctgtgagctgttttccatcat tgatgttgatgcagatcaggaaccttcccagagcccaccaagaagcgaagctgtgctgcagcaggaggatgagggggcgctgc cggagaatcggggctctttgggcaggagaggggctccctggctgttctgtgaccgtgagagcagccccagcgaggccagcacc acagacacctcgtggctggtgcccgccaccccgctggccagcagaagccgtgactgttcttcccagacccaaatcagcagcct caggagcgggctggccgtgcaggcggtgactcagcacacgcccagggcctcagtaggaaacagggaagggaacgaagtcgcac agaagttttctgtcatcaggccccagacaccaccgccccagacaccgtcctcatgcctcactcccgtctctccaggaacttct gacggcagaaggcaaggccacagaagcccttcccgtccccaccccgggggccacccgcactcctctccgctggctccacatcc catctcaggggaccgcgcccacttcagcaggcggttcctgaaacactcgccgcctgggccaagcttcctgaaccagaccccag cgggtgaagtggtggaagtcggagacagtgacgatgagcaggaggtggcctcccatcaggccaacagaagccccccactggac agtgaccccccaattccaattgacgactgctgctggcacatggagcccctctcgccaattcccattgaccactggaacctgga gcggaccggccccctgagcaccagcagccccagccgcaggatgaacgaggccgccgacagccgtgactgtcgctccccgggac tcctggacaccacccccatccgaggaagctgcactacccagaggaaattgcaagagaagtcctcgggcgcgggctccctgggg aatagcaggccgagctttctgaattcggctctgtgggacgtttgggacggggaagagcagaggcctccagagacccctcctcc ggcccagatgccaagcgctggtggagctcagaagcccgaagggttagagacacccaaaggtgctaatcggaagaagaacttgc cccccaaagtgcccataacgccgatgccacagtattccattatggagacgccggtgctgaagaaggaactggataggtttgga gtccgccctctgcctaaacgccagatggttctgaagctgaaggagatattccagtacactcaccagaccctggactcagactc cgaggacgagagccagtcctcacagccgctgttgcaggcgcctcactgccagaccctcgcctcccagacctacaagccttcaa gggcaggggtccatgcccagcaggaggccaccacaggacctggggcccataggcccaagggacctgctaagaccaagggcccc cgacatcaaaggaagcatcatgaaagcatcacacccccaagcaggtcgcccaccaaggaggcacctccaggcctcaatgatga cgcccagatcccagcctctcaagaatccgtggccacctctgtggatggcagtgacagctccttgagctcacagagttcttcct cctgtgagtttggagcggcatttgagtctgcaggtgaagaggagggcgagggggaggtcagtgcctcgcaggcagccgtgcag gcggcggacacagacgaggcgctgaggtgctacatccgctccaagccggccctgtaccagaaggtgctgctgtaccagccctt tgagctgcgggagctgcaggcagagctgaggcagaacggcctccgtgtgtcctcgcgcaggctgttggacttcctggacaccc actgtatcaccttcaccactgccgccacccgcagggagaagctccagggcaggaggcggcagcctcggggcaagaagaaggtg gagcggaactgatggggccatcccgaccccaccccaacctgccatcagcagcccccacccccgccatttgcagggaggacctg ggacacccagcgtgggtcaggcctccacaggcatttctgggcctggggaccacatcagctctgcgctgtgatgatgaccacag cccaatccagggcttcctcctctgggctctgctttctagggtggcatttggagcatgtcacccactggatttacagactccag ccccttcctctgtccgtgctcacagtgtgtctccctttttggttttctttttttttttctttttgagacagtcgtgctgtgtc acctaggctggagtgcagtggcacaatctcggctcactgcaacctccgcctcccgggttcaagcaattctcctgcctcagcct cccagatagctgggactacaggcacacgctgccacgcccagctgattttttatattttagtagaaacggggtttcaccatgtt gctcaggctggtcacaaactccagagctcaggcaatccgcctgctgcggcctcccaaagtgctgggatcacaggcgccagcca ctgcgcccggcccagtgtgtctcccttaacccaagagggccctcagctgtcccagggggcagtgggccatcaccagctggcca gggcatggcctattctgccacatttgccaccctctgagcccaccagtcctgggcacagctgccctacatgtctgtcctgagat ggacgtcaggtccagcctgccccggcagcccgggcccgtcctcctcagcactcaggccaaccccagccaccgccagcctgaga ccaggtgtcctgaggctccctgcactgccacagcccagatgcagttctcctgacccagccgtgctacccggacacttgtcatt gttaccagcagtctccaaactggacagtgcacaagggcccagaacaactctgatgccaccacaaaacaaacatgttcactagc ggattccattctttgggttaaagctgcctccagcctcaggagcagtgtggaggaagatgagggccaggaaagaaggaaacctt ggtttctccatccttgtgaatgtcctcgtctgtttcaaatacagtgcagtcagttttatatgatgtgcaataaaccaaaaagg ctttattaaaa

[0490] SEQ ID NO: 3 SLX4TOPBP1 BRCT1 binding motif (amnio acid residues 1254-1263 of SEQ ID NO: 1) SWLVPATPLA SEQ ID NO: 4 TOPBP1 binding site of SLX4 (amnio acid residues 1175-1375 of SEQ ID NO: 1) PLEEKKALEISPRSCELFSIIDVDADQEPSQSPPRSEAVLQQEDEGALPENRGSLGRRGAPWLFCDRESSPSEASTTDTSWLV PATPLASRSRDCSSQTQISSLRSGLAVQAVTQHTPRASVGNREGNEVAQKFSVIRPQTPPPQTPSSCLTPVSPGTSDGRRQGH RSPSRPHPGGHPHSSPLAPHPISGDRAHFSRRFLK SEQ ID NO: 5 TOPBP1 binding site of SLX4 (amnio acid residues 1235-1296 of SEQ ID NO: 1) PWLFCDRESSPSEASTTDTSWLVPATPLASRSRDCSSQTQISSLRSGLAVQAVTQHTPRASV SEQ ID NO: 6 TOPBP1 binding site of SLX4 (amnio acid residues 1253-1270 of SEQ ID NO: 1) TSWLVPATPLASRSRDCS SEQ ID NO: 7 78mer SLX4 peptide inhibitor ENRGSLGRRGAPWLFCDRESSPSEASTTDTSWLVPATPLASRSRDCSSQTQISSLRSGLAVQAVTQHTPRASVGNREG SEQ ID NO: 8 17mer SLX4 peptide inhibitor

[0491] TSWLVPATPLASRSRDC SEQ ID NO: 9 SLX4 siRNA antisense strand 1

[0492] TTGTTCTGGGCCCTTGTGC SEQ. ID NO: 10 SLX4 siRNA antisense strand 2

[0493] TACATATGAACCTGGTGC SEQ ID NO: 11 SLX4 siRNA sense strand 1

[0494] GCACAAGGGCCCAGAACAA SEQ ID NO: 12 SLX4 siRNA sense strand 2

[0495] GCACCAGGUUCAUAUGUA SEQ ID NO: 13 epitope recognised by exemplified anti-SLX4 antibody (with phosphorylated T1260) CTTDTSWLVPATPLASR

[0496] wherein the bold and underlined T is phosphorylated and corresponds to T1260 of SEQ ID NO: 1 SEQ ID NO: 14 corresponding unphosphorylated epitope (without phosphorylated T1260) CTTDTSWLVPATPLASR SEQ ID NO: 15 TOPBP1 amino acid sequence (UniProt Accession No. Q92547) MSRNDKEPFFVKFLKSSDNSKCFFKALESIKEFQSEEYLQIITEEEALKIKENDRSLYICDPFSGWFDHLKKLGCRIVGPQV VIFCMHHQRCVPRAEHPVYNMVMSDVTISCTSLEKEKREEVHKYVQMMGGRVYRDLNVSVTHLIAGEVGSKKYLVAANLKKPI LLPSWIKTLWEKSQEKKITRYTDINMEDFKCPIFLGCIICVTGLCGLDRKEVQQLTVKHGGQYMGQLKMNECTHLIVQEPK...

Claims

1. CLAIMS1. An inhibitor of the structure-specific endonuclease subunit 4 (SLX4) - Topoisomerase beta binding protein 1 (TOPBP1) interaction for use in a method of treating an individual with a cancer with high levels of replication stress.

2. The inhibitor for use according to claim 1, wherein the inhibitor is: (i) an SLX4 inhibitor; and / or (ii) a TOPBP1 inhibitor.

3. The inhibitor for use according to claim 1 or 2, wherein the replication stress is associated with a mutation and / or deficiency in one or more gene associated with replication stress, optionally wherein said one or more gene associated with replication stress is selected from BRCA2, BRCA1, POLQ, ATM, BARD1, PALB2, BRIP1, RAD51, ARID1A, RAD51B, RAD51C, RAD51D, CDK12, FAAP20, FAAP100, CHEK2, FAN1, FANCE, FANCM, ATR, WEE1, CHK1, CDK2, APEX1, APEX2, MRE11, RAD50, NBS1, LIG1, and Liglll.

4. The inhibitor for use according to any one of the preceding claims, wherein the high levels of replication stress is associated with a mutation and / or deficiency in BRCA2 and / or BRCA1.

5. The inhibitor for use according to any one of the preceding claims, wherein the inhibitor is a peptide, a small molecule, a proteolysis-targeting chimeric molecule (PROTAC), a macrocyclic molecule, a molecular glue, a nucleic acid inhibitor, an antibody, or an antibody-drug conjugate.

6. The inhibitor for use according to any one of the preceding claims, wherein the inhibitor is an SLX4 inhibitor which targets the TOPBP1 binding site of SLX4, which site comprises an amino acid corresponding to Thr1260 of SEQ ID NO: 1.

7. The inhibitor for use according to claim 6, wherein the SLX4 inhibitor:9.(a) inhibits the phosphorylation of the amino acid corresponding to Thr1260 of SEQ ID NO: 1;10.(b) inhibits the interaction of the amino acid corresponding to Thr1260 of SEQ ID NO: 1 with BRCT1 and / or BRCT2 of TOPBP1; or11.(c) inhibits the expression of SLX4.

8. The inhibitor for use according to any one of the preceding claims, wherein the inhibitor is an SLX4 inhibitor and is a peptide between about 6 to about 100 amino acids in length which comprises or consists of an SLX4 fragment comprising a threonine at a position corresponding to Thr1260 of SEQ ID NO: 1, wherein optionally said peptide:12.(a) comprises or consists of the amino acid sequence of SEQ ID NO: 7 ( E N RGS LG R RG AP W LFCD R ESS PS EASTTDTSW LVPATP LAS RS RDCSSQTQISS LRSG LAVQAVTQHTP R ASVGNREG), or an amino acid sequence with at least 90% identity to SEQ ID NO: 7, or a fragment thereof, provided that said peptide retains a threonine at a position corresponding to Thr1260 of SEQ ID NO: 1; or13.(b) comprises or consists of the amino acid sequence of SEQ ID NO: 8 (TSWLVPATPLASRSRDC), or an amino acid sequence with at least 90% identity to SEQ ID NO: 8, or a fragment thereof, provided that said peptide retains a threonine at a position corresponding to Thr1260 of SEQ ID NO: 1.

9. The inhibitor for use according to any one of claims 1 to 7, wherein the inhibitor is an SLX4 inhibitor and is an siRNA, optionally wherein said siRNA has an antisense strand that is at least 80% reverse complementary to an equal length portion of at least 12 contiguous nucleobases within the nucleobase sequence of SEQ ID NO: 2.

10. The inhibitor for use according to claim 9, wherein said siRNA has an antisense strand which comprises or consists of a nucleobase sequence that is at least 80% identical to SEQ ID NO: 9 (TTGTTCTGGGCCCTTGTGC) or 10 (TACATATGAACCTGGTGC), and optionally said siRNA has a sense strand which comprises or consists of a nucleobase sequence that is at least 80% identical to SEQ ID NO: 11 (GCACAAGGGCCCAGAACAA) or 12 (GCACCAGGUUCAUAUGUA).

11. The inhibitor for use according to any one of claims 1 to 7, wherein the inhibitor is an SLX4 inhibitor and is an antibody or antigen-binding fragment thereof that specifically binds to SLX4; wherein optionally:17.(a) said antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of the TOPBP1 binding site of SLX4, which site comprises an amino acid corresponding to Thr1260 of SEQ ID NO: 1; (b) said antibody or antigen-binding fragment thereof specifically binds to SLX4 that is phosphorylated at a position corresponding to Thr1260 of SEQ ID NO: 1; and / or (c) said antibody or antigen-binding fragment thereof specifically binds to an epitope comprising or consisting of SEQ ID NO: 13 (CTTDTSWLVPA{pT}PLASR), or which competes for binding to SLX4 with an antibody that binds to such an epitope.

12. The inhibitor for use according to any one of the preceding claims, wherein treatment specifically inhibits mitotic DNA synthesis (MiDAS) and / or break induced replication (BIR) in a cancer cell.

13. The inhibitor for use according to any one of the preceding claims, wherein treatment with the inhibitor is combined with one or more further anti-cancer therapies.

14. The inhibitor for use according to claim 13, wherein treatment with the inhibitor is used in conjunction with:21.(a) one or more further chemotherapeutic agent(s); and / or22.(b) one or more immunotherapeutic agent, which is optionally selected from immune checkpoint inhibitors, monoclonal antibodies, cytokines, cell therapy, cancer vaccines, oncolytic viruses, antisense oligodeoxynucleotides, antibody-drug conjugates and / or any modulator of the cytosolic nucleic acid sensing pathways.

15. The inhibitor for use according to claim 13 or 14, wherein the one or more further anti-cancer therapy is:24.(a) an inhibitor of a component of the DNA damage repair (DDR) pathway; and / or (b) an inhibitor of microhomology mediated end-joining (MMEJ).

16. The inhibitor for use according to any one of claims 13 to 15, wherein the one or more further anti-cancer therapy is an inhibitor of any one of Pol0, CH KI, ATR, WEE1, PARP, RAD51, or ATM.

17. The inhibitor for use according to any one of claims 13 to 16, wherein the one or more further anti-cancer therapy is an inhibitor of a component of the DDR pathway, and wherein thetreatment has increased clinical efficacy compared with treatment with the inhibitor of a component of the DDR pathway alone.

18. The inhibitor for use according to any one of claims 13 to 17, wherein the one or more further anti-cancer therapy is an inhibitor of a component of the DDR pathway, and wherein the treatment reduces resistance to the inhibitor of a component of the DDR pathway compared with treatment with the inhibitor of component of the DDR pathway alone.

19. The inhibitor for use according to any one of the preceding claims, wherein treatment with the inhibitor is used in conjunction with radiotherapy.

20. A method of reducing resistance to an anti-cancer therapy with an inhibitor of a component of the DDR pathway, said method comprising administering an inhibitor of the SLX4-TOPBP1 interaction in conjunction with the inhibitor of a component of the DDR pathway, wherein optionally the inhibitor of the SLX4-TOPBP1 interaction is an SLX4 inhibitor.

21. The inhibitor for use according to any one of claims 13 to 18 or the method of claim 19 or 20, wherein the inhibitor and the one or more further anti-cancer therapy are administered separately, sequentially or simultaneously.

22. A method of treating a cancer with high levels of replication stress, said method comprising administering a therapeutically effective amount of an inhibitor of the SLX4 - TOPBP1 interaction, optionally an SLX4 inhibitor, to an individual in need thereof.

23. Use of an inhibitor of the SLX4 - TOPBP1 interaction, optionally an SLX4 inhibitor, in the manufacture of a medicament for treating a cancer with high levels of replication stress.

24. A method of selecting an individual having cancer for treatment with a inhibitor of the SLX4 - TOPBP1 interaction, optionally an SLX4 inhibitor, the method comprising:34.(a) determining in a sample obtained from the individual whether the cancer is a cancer with high levels of replication stress;35.(b) selecting the individual for treatment with the inhibitor where the cancer is a cancer with high levels of replication stress; and (c) providing an inhibitor suitable for administration to the individual;36.wherein said method optionally further comprises administering a therapeutically effective amount of the inhibitor to the individual.

25. The method according to claim 24, wherein treatment with the inhibitor is combined with one or more further anti-cancer therapies.

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