WRN inhibitor for use in treating cancer

Abstract

A Werner helicase (WRN) inhibitor for use in the treatment of cancer is described. Also described herein are said WRN inhibitor for use in methods by which cancer patients susceptible to treatment with a WRN inhibitor can be identified and treated, and in methods by which cancer patients resistant to treatment with a WRN inhibitor can be identified; as well as combination treatments comprising said WRN inhibitor.

Description

[0001] WRN INHIBITOR FOR USE IN TREATING CANCER

[0002] Field of the Disclosure

[0003] The present invention relates to the use of a Werner helicase (WRN) inhibitor for the treatment of cancer. Also provided herein are methods by which cancer patients susceptible to treatment with a WRN inhibitor can be identified and treated, methods by which cancer patients resistant to treatment with a WRN inhibitor can be identified, and combination treatments comprising a WRN inhibitor.

[0004] Background

[0005] Exploiting synthetic lethality by targeting Werner helicase (WRN) represents a new paradigm for treating microsatellite instability (MSI) cancers (Behan et al. 2019; Chan et al. 2019; van Wietmarschen et al. 2020; Kategaya et al. 2019; Lieb et al. 2019; Picco et al. 2021). Recently, the discovery of three WRN inhibitors has been reported (Picco et al. 2024; Ferretti et al. 2024; Baltgalvis et al. 2024), including both covalent and non-covalent inhibitors.

[0006] As with all targeted therapies, the clinical benefit of WRN inhibitors is likely limited by the emergence of resistance. However, cellular adaptation to genetic or pharmacological inactivation of the WRN helicase in MSI cancer cells remains largely unexplored. Notably, the synthetic lethality mechanism characterising WRN helicase dependency, elicited by mismatch repair deficiency-associated aberrant TA dinucleotide repeat expansions (van Wietmarschen et al. 2020; Picco et al. 2021), is unique and unprecedented in cancer therapy, and potential escape mechanisms for WRN inhibitors remain unconfirmed.

[0007] Drug resistance is a major limitation to the long-term efficacy of cancer therapies. Cancer genome sequencing can delineate the genetic basis of drug resistance, but this retrospective approach requires sequencing of large numbers of post-treatment samples to distinguish causal variants from background mutational events.

[0008] Despite considerable advances in the development of molecularly-targeted therapies for cancer patients, resistance to anti-cancer treatments remains a major clinical challenge (Vasan 2019). Drug resistance is frequently caused by DNA single nucleotide variants (SNVs) in the cancer genome (Pao 2005), leading to point mutations in the drug target itself, or proteins within the same signalling pathway (van de Haar 2021). The study of drug resistance is crucial to understand drug mechanism of action, to generate second-generation inhibitors targeting drug-resistant proteins, for the development of combination therapies, and for effective patient stratification for second-line therapies.

[0009] The present invention has been devised in light of the above considerations.

[0010] Summary of the Disclosure

[0011] Here, the inventors systematically evaluated potential acquired resistance mechanisms to WRN genetic and pharmacological inhibition. The inventors employed a comprehensive approach, including CRISPR- based knockout screens, semi-saturation mutagenesis, pharmacogenomic analyses, and adaptive resistance evolution experiments. The data herein are consistent with there being no universal WRN resistance mechanisms to genetic inactivation, however the inventors identified biologically relevant modulators of WRN inhibitor sensitivity, unveiling new players in this novel synthetic lethality process in human MSI cancer cells.

[0012] Monitoring the spontaneous evolution of drug resistance after pharmacological WRN inhibition revealed the emergence of on-target WRN mutations that invariably cause secondary resistance to multiple WRN inhibitors, both in vitro and in vivo. Specifically, the inventors used semi-saturation mutagenesis of WRN and prolonged drug treatment to identify on-target WRN mutations driving acquired resistance to multiple WRN inhibitors in vitro and in vivo. Surprisingly, the inventors discovered that specific resistance mutations preserve sensitivity to alternative WRN inhibitors, whereas others induce cross- resista nee to multiple WRN inhibitors, both in vitro and in vivo.

[0013] The findings herein highlight potential clinical hurdles associated with WRN inhibitor treatment, offering a preemptive insight into tumor adaptation strategies and having immediate clinical implications. The results suggest that combination strategies have advantages over monotherapy in enhancing treatment success, for example by delaying the onset of resistance. The inventors’ discovery of WRN drug resistance mechanisms thus has implications for patient stratification, therapy combinations and the scheduling of drugs in cancer treatment.

[0014] In particular, the discovery of mutations linked to acquired resistance to some, but not all, WRN inhibitors reveals a key opportunity to switch subjects being treated with a first WRN inhibitor to treatment with a different WRN inhibitor with a distinct mechanism of action, potentially overcoming resistance to treatment with the first WRN inhibitor.

[0015] Thus, according to a first aspect, there is provided a Werner helicase (WRN) inhibitor for use in a method of treatment of cancer in a subject, wherein the cancer has been identified as expressing a mutated form of the WRN protein, wherein the WRN inhibitor is VVD-133214.

[0016] According to a second aspect, there is provided a Werner helicase (WRN) inhibitor for use in a method of treatment of cancer in a subject, wherein the WRN inhibitor is VVD-133214, and wherein the method comprises: i. determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein; and ii. when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, administering to the subject an effective amount of the WRN inhibitor.

[0017] According to a third aspect, there is provided a method of treatment of cancer in a subject in need thereof, wherein the cancer has been identified as expressing a mutated form of the WRN protein, wherein the method comprises the step of administering to the subject an effective amount of a WRN inhibitor, wherein the WRN inhibitor is VVD-133214. Also described herein is the use of a WRN inhibitor in the manufacture of a medicament for the treatment of cancer is a subject, wherein the cancer has been identified as expressing a mutated form of the WRN protein, wherein the WRN inhibitor is VVD-133214. According to a fourth aspect, there is provided a method of treatment of cancer in a subject in need thereof, wherein the method comprises the steps of: i. determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein; and ii. when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, administering to the subject an effective amount of a WRN inhibitor, wherein the WRN inhibitor is VVD-133214.

[0018] According to a fifth aspect, there is provided a method of selecting a subject with cancer for treatment with a Werner helicase (WRN) inhibitor, wherein the WRN inhibitor is VVD-133214, the method comprising determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, wherein the subject is selected for treatment when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein.

[0019] According to a sixth aspect, there is provided a method of predicting whether a subject with cancer is likely to respond to treatment with a Werner helicase (WRN) inhibitor, wherein the WRN inhibitor is VVD-133214, the method comprising determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, wherein the subject is considered likely to respond to treatment with the WRN inhibitor when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein.

[0020] According to a seventh aspect, there is provided a method for identifying a subject with cancer who is likely to respond to treatment with a Werner helicase (WRN) inhibitor, wherein the WRN inhibitor is VVD-133214, the method comprising determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, wherein the subject is considered likely to respond to treatment with the WRN inhibitor when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein.

[0021] According to a eighth aspect, there is provided a method for assessing the susceptibility of a cancer in a subject to treatment with a Werner helicase (WRN) inhibitor, wherein the WRN inhibitor is VVD-133214, comprising determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, wherein the cancer is considered susceptible to treatment with the WRN inhibitor when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein.

[0022] In embodiments of any one of the sixth to eighth aspects, when the subject is considered likely to respond to treatment with the WRN inhibitor or the cancer is considered susceptible to treatment with the WRN inhibitor, the subject is selected for treatment with the WRN inhibitor.

[0023] In embodiments of any one of the fifth to eighth aspects, when the subject is selected for treatment with the WRN inhibitor, the subject is considered likely to respond to treatment with the WRN inhibitor and / or the cancer is considered susceptible to treatment with the WRN inhibitor, the method further comprises administering the WRN inhibitor to the subject. Embodiments of any one of the first to eighth aspects may have any one or more of the following optional features.

[0024] In embodiments, the mutated form of the WRN protein comprises a mutation conferring resistance to treatment of the cancer with a WRN inhibitor other than VVD-133214. For example, the WRN inhibitor other than VVD-133214 may be HRO761.

[0025] In embodiments, the mutated form of the WRN protein comprises a mutation at position 852 of the amino acid sequence set out in SEQ ID NO: 25, optionally wherein the mutation is I852F.

[0026] In embodiments, the nucleic acid sequence encoding the mutated form of the WRN protein comprises a truncated exon 13. For example, the nucleic acid sequence may comprise an exon-truncating splice site mutation 1557-1 G>C at position chr8:31088889 or 1557-2 A>T at position chr8:31088888, wherein the position refers to a location in the GRCh38 reference genome assembly.

[0027] In a ninth aspect, there is provided a method of predicting whether a subject with cancer is unlikely to respond to treatment with a Werner helicase (WRN) inhibitor, the method comprising determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, wherein the subject is considered unlikely to respond to treatment with the WRN inhibitor when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein.

[0028] In a tenth aspect, there is provided a method for assessing the resistance of a cancer in a subject to treatment with a Werner helicase (WRN) inhibitor, wherein the subject has previously been administered the WRN inhibitor, the method comprising determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, wherein the cancer is considered resistant to treatment with the WRN inhibitor when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein.

[0029] In an eleventh aspect, there is provided a method for assessing the susceptibility of a cancer in a subject to treatment with a Werner helicase (WRN) inhibitor, comprising determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, wherein the cancer is considered not susceptible to treatment with the WRN inhibitor when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein.

[0030] Embodiments of any one of the ninth to eleventh aspects may have any one or more of the following optional features.

[0031] In embodiments, the mutated form of the WRN protein comprises a mutation at a position selected from the list consisting of positions 521 , 530, 725 to 731 , 852, and / or 975 of the amino acid sequence set out in SEQ ID NO: 25.

[0032] In embodiments, the mutated form of the WRN protein comprises a mutation at one or more of the following positions of the amino acid sequence set out in SEQ ID NO: 25: position 521; position 530; position 725; position 727; position 729, optionally wherein the mutation is G729D orG729S; position 730, optionally wherein the mutation is F730L or F730S; position 731 ; position 852, optionally wherein the mutation is I852F; and / or position 975.

[0033] In embodiments, the nucleic acid sequence encoding the mutated form of the WRN protein comprises a truncated exon 13. For example, the nucleic acid sequence may comprise an exon-truncating splice site mutation 1557-1 G>C at position chr8:31088889 or 1557-2 A>T at position chr8:31088888, wherein the position refers to a location in the GRCh38 reference genome assembly.

[0034] In embodiments, the WRN inhibitor is VVD-133214 or HRO761.

[0035] Alternatively, embodiments of any one of the ninth to eleventh aspects may have any one or more of the following optional features.

[0036] In embodiments, the mutated form of the WRN protein comprises a mutation at one or more of the following positions of the amino acid sequence set out in SEQ ID NO: 25: position 709, optionally wherein the mutation is S709P; position 730, optionally wherein the mutation is F730L or F730S; position 1207, optionally wherein the mutation is G1207D; and / or position 1209, optionally wherein the mutation is A1209D.

[0037] In embodiments, the WRN inhibitor is VVD-133214 and / or HRO761.Embodiments of any one of the first to eleventh aspects may have any one or more of the following optional features.

[0038] In embodiments, the sample is a tissue sample or a biological fluid sample. For example, the biological fluid sample may comprise a circulating tumour cell, or a cell-free sample comprising cell-free DNA, circulating tumour DNA (ctDNA) and / or circulating tumour RNA (ctRNA). For example, the biological fluid sample may be a blood sample, a urine sample, ora cerebrospinal fluid (CSF) sample. For example, the blood sample may be a whole blood sample or a plasma sample. For example, the tissue sample may be a tumour tissue sample, optionally a tumour biopsy sample.

[0039] In embodiments, determining whether the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein comprises nucleic acid sequencing of the sample. In a twelfth aspect, there is provided a combination of a first WRN inhibitor and a second WRN inhibitor for use in a method of treatment of cancer in a subject, wherein the first WRN inhibitor is VVD-133214 and the second WRN inhibitor is HRO761. Also described herein is a method of treatment of cancer in a subject, wherein the method comprises the step of administering a combination of an effective amount of a first WRN inhibitor together with an effective amount of a second WRN inhibitor to the subject, wherein the first WRN inhibitor is VVD-133214 and the second WRN inhibitor is HRO761. Also described herein is a use of a combination of a first WRN inhibitor together with a second WRN inhibitor in the manufacture of a medicament for the treatment of cancer in a subject, wherein the first WRN inhibitor is VVD-133214 and the second WRN inhibitor is HRO761.

[0040] In a thirteenth aspect, there is provided a WRN inhibitor for use in a method of treatment of cancer in a subject, wherein the WRN inhibitor is VVD-133214, and wherein VVD-133214 is administered to the subject in combination with a second WRN inhibitor, wherein the second WRN inhibitor is HRO761. Also described herein is a method of treatment of cancer in a subject, wherein the method comprises the step of administering an effective amount of a first WRN inhibitor to the subject in combination with an effective amount of a second WRN inhibitor, wherein the first WRN inhibitor is VVD-133214 and the second WRN inhibitor is HRO761. Also described herein is a use of a first WRN inhibitor in the manufacture of a medicament for the treatment of cancer in a subject in combination with a second WRN inhibitor, wherein the first WRN inhibitor is VVD-133214 and the second WRN inhibitor is HRO761.

[0041] In a fourteenth aspect, there is provided a WRN inhibitor for use in a method of treatment of cancer in a subject, wherein the WRN inhibitor is HRO761 , and wherein HRO76 is administered to the subject in combination with a second WRN inhibitor, wherein the second WRN inhibitor is VVD-133214. Also described herein is a method of treatment of cancer in a subject, wherein the method comprises the step of administering an effective amount of a first WRN inhibitor to the subject in combination with an effective amount of a second WRN inhibitor, wherein the first WRN inhibitor is HRO761 and the second WRN inhibitor is VVD-133214. Also described herein is a use of a first WRN inhibitor in the manufacture of a medicament for the treatment of cancer in a subject in combination with a second WRN inhibitor, wherein the first WRN inhibitor is HRO761 and the second WRN inhibitor is VVD-133214.

[0042] In embodiments of any one of the twelfth to fourteenth aspects, the first WRN inhibitor and the second WRN inhibitor are administered separately.

[0043] In a fifteenth aspect, there is provided a combination of a WRN inhibitor and an ataxia telangiectasia and Rad3 related kinase (ATR) inhibitor for use in a method of treatment of cancer in a subject, wherein the WRN inhibitor is VVD-133214 or HRO761. Also described herein is a method of treatment of cancer in a subject, wherein the method comprises the step of administering a combination of an effective amount of a WRN inhibitor together with an effective amount of an ATR inhibitor to the subject, wherein the WRN inhibitor is VVD-133214 or HRO761. Also described herein is a use of a combination of a WRN inhibitor together with an ATR inhibitor in the manufacture of a medicament for the treatment of cancer in a subject, wherein the WRN inhibitor is VVD-133214 or HRO761.

[0044] In a sixteenth aspect, there is provided a WRN inhibitor for use in a method of treatment of cancer in a subject, wherein the WRN inhibitor is VVD-133214 or HRO761 , and wherein the WRN inhibitor is administered to the subject in combination with an ATR inhibitor. Also described herein is a method of treatment of cancer in a subject, wherein the method comprises the step of administering an effective amount of a WRN inhibitor to the subject in combination with an effective amount of an ATR inhibitor, wherein the WRN inhibitor is VVD-133214 or HRO761. Also described herein is a use of a WRN inhibitor in the manufacture of a medicament for the treatment of cancer in a subject in combination with an ATR inhibitor, wherein the WRN inhibitor is VVD-133214 or HRO761.

[0045] In a seventeenth aspect, there is provided an ATR inhibitor for use in a method of treatment of cancer in a subject, wherein the ATR inhibitor is administered to the subject in combination with a WRN inhibitor, wherein the WRN inhibitor is VVD-133214 or HRO761. Also described herein is a method of treatment of cancer in a subject, wherein the method comprises the step of administering an effective amount of an ATR inhibitor to the subject in combination with an effective amount of a WRN inhibitor, wherein the WRN inhibitor is VVD-133214 or HRO761. Also described herein is a use of an ATR inhibitor in the manufacture of a medicament for the treatment of cancer in a subject in combination with a WRN inhibitor, wherein the WRN inhibitor is VVD-133214 or HRO761.

[0046] In an eighteenth aspect, there is provided a method of treating a subject with a cancer wherein the subject has received VVD-133214 and / or HRO761 , wherein the method comprises administering to the subject an effective amount of GSK_WRN5.

[0047] In a nineteenth aspect, there is provided a method of treating a subject with a cancer with a mutated form of the WRN protein, wherein the subject has received VVD-133214 and / or HRO761 , wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

[0048] In a twentieth aspect, there is provided a method of treating a subject with cancer, wherein the subject is resistant to treatment with VVD-144214 and / or HRO761 , further wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

[0049] In a twenty-first aspect, there is provided a method of treating a subject with a cancer with a mutated form of the WRN protein, wherein the subject is resistant to treatment with VVD-133214 and / or HRO761 , wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

[0050] In a twenty-second aspect, there is provided a method of treating a subject with a cancer with a mutated form of the WRN protein, wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

[0051] In a twenty-third aspect, there is provided a method of treating a subject with a cancer, wherein the subject was previously treated with VVD-133214 and / or HRO761 , wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

[0052] In a twenty-fourth aspect, there is provided a method of treating a subject with a cancer with a mutated form of the WRN protein, wherein the subject was previously treated with VVD-133214 and / or HRO761 , wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5

[0053] In a twenty-fifth aspect of the invention, there is provided a method of treating a subject with cancer comprising: (a) selecting a subject who has received VVD-133214 and / or HRO761 ; and (b) administering an effective amount of GSK.WRN5.

[0054] In a twenty-sixth aspect of the invention, there is provided a method of treating a subject with cancer comprising: (a) selecting a subject who has received VVD-133214 and / or HRO761 and is resistant to therapy with VVD-133214 and / or HRO761 ; and (b) administering an effective amount of GSK_WRN5. In a twenty-seventh aspect of the invention, there is provided a method of treating a subject with cancer comprising: (a) selecting a subject who has received VVD-133214 and / or HRO761 and who has a mutated form of WRN protein; and (b) administering an effective amount of GSK.WRN5.

[0055] In a twenty-eighth aspect of the invention, there is provided a method of treating a WRN-mutant cancer in a subject the method comprising administering an effective amount of GSK_WRN5 In a In a twenty-ninth aspect of the invention, there is provided a method of treating a WRN-mutant cancer in a subject the method comprising administering an effective amount of GSK_WRN5, wherein the subject has been previously treated with an inhibitor of WRN that is not GSK_WRN5. For example, the inhibitor of WRN that is not GSK_WRN5 is VVD-133214, HRO716, ora combination thereof. The WRN-mutant cancer may affect positions of the amino acid sequence of SEQ ID NO: 25 selected from the group consisting of position 709, position 730, position 1207, and position 1209

[0056] In embodiments of any one of the twelfth to fourteenth aspects, the WRN inhibitor and the ATR inhibitor are administered separately. Embodiments of any of the eighteenth to twenty-nineth aspects may have any one or more of the following optional features.

[0057] In embodiments, the subject may have mutated form of the WRN protein.

[0058] In embodiments, the subject may have mutated form of the WRN protein comprising a mutation at any one or more (for example one, two, three, or each) of position 709, position 730, position 1207, and / or position 1209 in SEQ ID NO: 25; or any combination thereof.

[0059] In embodiments, the subject may have mutated form of the WRN protein comprising a mutation at one or more (for example, one, two, three, or four) of the following positions of the amino acid sequence set out in SEQ ID NO: 25: position 709, optionally wherein the mutation is S709P; position 730, optionally wherein the mutation is F730L or F730S; position 1207, optionally wherein the mutation is G1207D; and / or position 1209, optionally wherein the mutation is A1209D. For example, the mutated form of the WRN protein may comprises one or more mutation (for example, one, two, three or four) selected the group consisting of S709P; F730L or F730S, G1207D, A1209D, and combinations thereof.

[0060] In embodiments, the subject may have received VVD-33214 and / or HOR761.

[0061] In embodiments, the subject may have previously received treatment with VVD-33214 and / or HOR761. In embodiments, the cancer may be resistant to treatment with VVD-33214 and / or HRO761.

[0062] Embodiments of any one of the first to twenty-ninth aspects may have any one or more of the following optional features.

[0063] In embodiments, the subject has previously received treatment with HRO761. For example, the subject may have been identified as resistant to treatment with HRO761. In other embodiments, the subject has previously received treatment with VVD-133214. For example, the subject may have been identified as resistant to treatment with VVD-133214. In further embodiments, the subject has previously received treatment with HRO761 and VVD-133214. For example, the subject may have been identified as resistant to treatment with HRO761 and VVD-133214. In embodiments, the subject is a human. In embodiments, the cancer is a microsatellite instability - high (MSI-High) cancer. In embodiments, the cancer is defective DNA mismatch repair (dMMR) cancer. In embodiments, the cancer is colorectal cancer, gastric cancer, endometrial cancer, ovarian cancer, hepatobiliary tract cancer, urinary tract cancer, brain cancer, or skin cancer. For example, the cancer may be colorectal cancer, gastric cancer, endometrial cancer, or ovarian cancer. In embodiments, the cancer is colorectal cancer. Further aspects of the invention include a WRN inhibitor for use in a method as defined in any one of the eighteenth to twenty-nineth aspects, wherein the WRN inhibitor is GSK_WRN5; and the use of a WRN inhibitor in the manufacture of a medicament for treating a subject with cancer (for example a cancer with a mutated form of the WRN protein), or for treating cancer (for example a cancer with a mutated form of the WRN protein) in a subject, wherein the WRN inhibitor is GSK_WRN5, and wherein treating the cancer in the subject comprises a method as defined in any one of the eighteenth to twenty-nineth aspects.

[0064] The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

[0065] Summary of the Figures

[0066] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

[0067] Figure 1. Tumor volume of WRN sgRNA-expressing (di-WRN) HCT116 xenografts (clone a) treated with doxycycline (yellow and red lines) or vehicle (grey line). Data are mean ± s.e.m. The numbers of mice in each cohort are indicated.

[0068] Figure 2. Representative WRN and KI-67 immunohistochemistry of WRN sgRNA-expressing (di-WRN) HCT116 tumors (clone a) explanted after one week or at day 40, as indicated. Scale bar, 50 pm; 40x magnification.

[0069] Figure 3. Quantification of WRN and KI-67 staining. Data are mean ± s.d. of 10 fields from three samples (n = 30); P-values were calculated using a two-sided Welch’s t-test (*** = p < 0.001).

[0070] Figure 4. Schematic of the screening strategy

[0071] Figure 5. Representative images of resistant clones emerging after doxycycline administration in WRN sgRNA-expressing (di-WRN) lines transduced with a genome-wide CRISPR knockout library.

[0072] Figure 6. Gene-level quantification of sgRNAs enriched in doxycycline-treated cells versus controls. Resistance-associated genes, including TADA1, SUPT7L, TAF6L, and SUPT3H, are highlighted in color in the scatter plots.

[0073] Figure 7. Results of a whole-genome CRISPR / Cas9 chemogenomic dropout screen in SW48 cells treated with GSK_WRN3. The x-axis represents the Iog2 fold change (FC) of gene expression after WRNi treatment compared to control (CTRL), and the y-axis represents the -Iog10 p-value. Genes with significant negative fold changes (potential sensitizers) are highlighted in green, and those with significant positive fold changes (potential resistors) are highlighted in red.

[0074] Figure 8. Western blots for SMARCAL1 in SW48 and HCT116 parental cells and their corresponding SMARCAL1 knockout (KO) derivatives. Tubulin is used as a loading control. Figure 9. Clonogenic assays assessing the sensitivity of SW48 and HCT116 parental and SMARCAL1 KO cells to increasing concentrations of GSK_WRN3 (left) and HRO761 (right). Cells were treated with DMSO as a control.

[0075] Figure 10. Normalized Iog2 read counts of TrAEL-seq data at TA-rich peaks in SW48 parental and SMARCAL1 KO cells treated with GSK_WRN3 (left) or HRO761 (right).

[0076] Figure 11. Metaplots of normalized TrAEL-seq read counts centered around TA dinucleotide repeats in SW48 parental and SMARCAL1 KO cells treated with GSK_WRN3 (left) or HRO761 (right).

[0077] Figure 12. Scatter plots showing the comparison of Z-scores between CTRL vs. plasmid and GSK_WRN3 (left and middle) or HRO761 (right) vs. plasmid in RL95-2 and KM12 cells. Points are color-coded by base editor type (adenine base editor (ABE) or cytidine base editor (CBE)). Hit guide RNAs and the predicted installed mutations are labelled.

[0078] Figure 13. Time-to-progression (TTP) assay of SW48 colorectal cancer cells exposed to WRN inhibitors GSK_WRN3 (1 pM) and HRO761 (0.5 pM) shows rapid acquisition of resistance.

[0079] Figure 14. Dose-response curve demonstrating decreased sensitivity of GSK_WRN3-resistant SW48 cells compared to control cells.

[0080] Figure 15. Colony formation assay highlighting the difference in sensitivity between parental and GSK_WRN3-resistant SW48 cells to escalating doses of GSK_WRN3.

[0081] Figure 16. Liner plots displaying the median sequencing depth of broken and not-broken (TA)-repeat loci in WGS data from parental and WRNi-resistant counterparts across preclinical models from different tissue types, as indicated.

[0082] Figure 17. Immunoblot analysis of WRN protein levels in parental, GSK_WRN3-resistant, and HRO761-resistant SW48 cells. WRNi-resistant cells show impaired WRN downregulation, suggesting loss of drug binding.

[0083] Figure 18. Schematic of the WRN gene showing the exon-truncating splice site mutation (c.1557-1 G>C) identified in HRO761 -resistant SW48 cells, leading to aberrant splicing and production of a truncated WRN protein.

[0084] Figure 19. Scatter plot comparing Z-scores of non-exonic WRN-targeting sgRNAs between CTRL vs. plasmid and HRO761 vs. plasmid in KM12 cells. Points are color-coded by base editor type (adenine base editor (ABE) or cytidine base editor (CBE)), with the sgRNA predicted to target position 31 ,088,889 on chromosome 8 labelled.

[0085] Figure 20. Tumor volume measurements from SW48 MSI tumor xenografts treated with HRO761 show initial tumor regression followed by resistance and tumor regrowth, recapitulating in vivo resistance mechanisms observed in vitro.

[0086] Figure 21. Mapping of WRN resistance mutations onto the WRN 3D structure ATPase domain bound to HRO761 highlights the proximity of these mutations to the reactive cysteine at position 727, critical for drug binding. Figure 22. WRN mutations identified across MSI cancer cell lines and xenografts following resistance to WRN inhibitors

[0087] Figure 23. Structural models of WRN in complex with HRO761 and VVD-133214. HRO761 stabilize WRN in an open conformation, preventing DNA binding, while VVD-133214 irreversibly locks WRN in a closed, inactive state. The hinge region is shown in red (residues 728-732), highlighting the G729D mutation's impact on binding pocket architecture. The recurrent I852F mutation creates a hydrophobic cage, affecting HRO761 binding but not VVD-133214.

[0088] Figure 24. Structural models of the WRN complex associated with different ligands. The left panel shows the WRN complex with HRO761 (PDB ID: 8PF0). The highlighted components include the ligand HRO761 in pink. G729D mutation is highlighted in red, indicating its significance in the context of ligand binding. The right panel depicts the WRN complex with VVD-133214 (PDB ID: 7GQU), with the ligand highlighted ingreen. The use of distinct colors and labels emphasises the interaction between WRN and the respective ligands and the potential implications of the G729D mutation on these interactions.

[0089] Figure 25. Close-up views of the binding sites for HRO761, and VVD-133214, showing the altered conformation due to resistance mutation I852F. This mutant residue impairs HRO761 binding but does not affect VVD-133214.

[0090] Figure 26. WRN screening heatmap (ongoing) comparing parental and WRNi-resistant MSI cells treated with the three WRN inhibitors, ATR inhibitors, and irinotecan. Heatmap refers to compound natural log micromolar IC50 value.

[0091] Figure 27. Effect of VVD-133214 on SW48 parental and HRO761 -resistant cell lines. Clonogenic assay showing the sensitivity of SW48 parental (SW48 Par.) and HRO761 -resistant (SW48 HRO761 Res) cell lines with mutations I852F and c.1577-1 G>C to VVD-133214 treatment. Cells were treated with increasing concentrations of VVD-133214 (0 to 100 nM) or DMSO (control) for a specified period, followed by staining to visualize surviving colonies. SW48 parental and HRO761 -resistant cells ( carrying I852F and c.1577-1 G>C mutations) retain sensitivity to VVD-133214.

[0092] Figure 28. Western blot analysis showing WRN, tubulin, and y-H2AX expression levels in SW48 cells with HRO761 -resistant mutations treated with VVD-133214.

[0093] Figure 29. Schematic of derivation and time-to-progression (TTP) assay in patient-derived organoids (PDOs) from sporadic MSI colorectal cancer (CRC-14a) treated with HRO761 shows rapid emergence of resistance, similar to cell line models.

[0094] Figure 30. Validation of WRN mutations in microsatellite instability (MSI) colorectal cancer (CRC) patient-derived organoids. The organoids (CRC14a) were derived from a sporadic MSI CRC tumor (left panel). The central graph tracks the cell number overtime for CRC14a organoids treated with the WRN inhibitor HRO761 , which carries a WRN 1577-2 A>T splice site mutation (WRN exon 13 truncation). The mutation affects the same codon and splice site alteration as the WRN 1577-1 G>C mutation. The images on the right show the effect of VVD-133214 (62.5 nM) and HRO761 (62.5 nM) compared to the control (DMSO) on organoid growth. Organoids treated with VVD-133214 show significantly reduced growth, while those treated with HRO761 continue to proliferate, suggesting resistance to HRO761 but sensitivity to VVD-13314. This highlights the selective pressure of VVD-133214 on WRN-mutated cells.

[0095] Figure 31. Tumor volume of WRN sgRNA-expressing (di-WRN) HCT116 xenografts (clone b) treated with doxycycline (yellow and red lines) or vehicle (grey line). Data are mean ± s.e.m. The numbers of mice in each cohort are indicated.

[0096] Figure 32. Representative WRN and KI-67 immunohistochemistry of WRN sgRNA-expressing (di-WRN) HCT116 tumors (clone b) explanted after one week or at day 40, as indicated. Scale bar, 50 pm; 40x magnification.

[0097] Figure 33. Data are mean ± s.d. of 10 fields from three samples (n = 30); P-values were calculated using a two-sided Welch’s t-test (*** = p < 0.001).

[0098] Figure 34. Schematic representation of the models exploited to study acquired resistance to WRN knockout in vitro and outline of the experimental design.

[0099] Figure 35. WRN knockout induced by doxycycline treatment in HCT116 WRN sgRNA-expressing (di-WRN) and SW48 di-WRN subclonal cell lines measured by Western blot. Data are representative of two independent experiments.

[0100] Figure 36. Growth curves of HCT116 WRN sgRNA-expressing (di-WRN) and SW48 di-WRN subclonal cell lines grown in the absence (black line) or presence of doxycycline (2 pg ml-1 ; yellow line). Data are mean ± s.d. of 10 technical replicate wells for each condition (1 image per well) and representative of two independent experiments.

[0101] Figure 37. Representative images showing reduced viability in HCT116 c9 cells transduced with inducible NonEss and WRN sgRNAs after 96h and one week of exposure to Doxycycline.

[0102] Figure 38. WRN knockout induced by doxycycline treatment in HCT116 WRN sgRNA-expressing (di-WRN) cells (bulk) measured by Western blot after 96h and one week of exposure to Doxycycline. Data are representative of two independent experiments.

[0103] Figure 39. Cas9 activity was evaluated in inducible WRN knockout models before and after the emergence of secondary resistance in vitro. Cas9 activity was evaluated by flow cytometry. Data are mean ± s.d. of two separate transductions with the reporter plasmid.

[0104] Figure 40. Normalized viability upon siRNA-mediated WRN depletion in inducible WRN knockout models (SW48, left and HCT116, right) before and after secondary resistance emergence in vitro. Non-targeting siRNA (siNT) and siPLKI were used as negative and positive controls, respectively. Data are mean ± SD of 2 independent experiments with four technical replicates each.

[0105] Figure 41. Bar plot displaying the sum of all sgRNA reads for each sample, indicating consistent library representation across replicates.

[0106] Figure 42. Bar plot illustrating the distribution of sgRNAs with counts greater than 10 in each sample, ensuring sufficient coverage and representation in the genetic screens Figure 43. Rank-ordered gene-level Iog2 fold change comparisons between Dox-induced and control conditions in RKO, HCT116, and SW48 cells. The graphs depict the distribution of gene knockouts with specific genes annotated. Significant hits such as SAGA complex members (e.g., SUPT7H, TAF6) are highlighted.

[0107] Figure 44. Growth curves of Sw48 WRN sgRNA-expressing (di-WRN) (clone a) and SW48 di-WRN SUPT3H KO or SUPT7L KO grown in the absence (black line) or presence of doxycycline (2 pg ml-1 ; yellow line). Data are mean ± s.d. of 10 technical replicate wells for each condition (1 image per well) and representative of two independent experiments.

[0108] Figure 45. Western blot analysis shows the expression levels of SUPT7L and WRN in SW48 WRN sgRNA-expressing (di-WRN) parental and SUPT7L knockout (KO) cells. Tubulin is used as a loading control. The first blot demonstrates SUPT7L protein levels in parental and SUPT7L KO cells, while the second blot shows WRN induction upon doxycycline (Dox) treatment in both parental and SUPT7L KO cells.

[0109] Figure 46. Western blot analysis of WRN and TADA1 protein levels in HCT116 WRN sgRNA-expressing (di-WRN) cells. Parental and TADA1 knockout (KO) cells were treated with doxycycline (Dox) to induce WRN expression. Tubulin was used as a loading control.

[0110] Figure 47. Log2 fold-change in TP53 and MUS81 median values across SW48, HCT116, and RKO cell lines upon WRN depletion, with SAGA complex members included for comparison.

[0111] Figure 48. Western blot analysis showing WRN and MUS81 protein levels in HCT116 and SW48 WRN sgRNA-expressing (di-WRN) cells. Cells were treated with doxycycline (Dox) to induce WRN knockdown, and MUS81 knockout (KO) was confirmed in the corresponding lanes. Tubulin is shown as a loading control.

[0112] Figure 49. Dose-response curves for HCT116 and SW48 WRN sgRNA-expressing (di-WRN) cells with or without MUS81 knockout. The data are plotted as a percentage of viability relative to control, with the log concentration of the inhibitor on the x-axis.

[0113] Figure 50. Gene set enrichment analysis (GSEA) of pathways differentially enriched based on Iog2 foldchange ranked values from the chemogenomic screen in SW48 cells treated with GSK_WRN3.

[0114] Figure 51. Quantification of TrAEL-seq read counts at random non-cut TA peaks in SW48 cells under various conditions, as indicated.

[0115] Figure 52. Metaplot analysis of TrAEL-seq data centred around TA dinucleotide repeats, comparing parental c9 and SMARCAL1 knockout SW48 cells treated with GSK_WRN3 or HRO761.

[0116] Figure 53. Viability assays were performed on SW48 and HCT116 parental and SMARCAL1 knockout cells treated with GSK_WRN3, indicating the level of resistance conferred by SMARCAL1 knockout. Figure 54. Schematic of the experimental setup: KM12 and RL95-2 cell lines were subjected to CRISPR / Cas9 base editing using a library containing 3759 sgRNAs with either CBE-NGN (C>T) or ABE-NGN (A>G) editors. Cells were transduced with the base editing library, selected with puromycin for three days, followed by doxycycline induction for three days to activate base editing. Cells were treated with either GSK_WRN3 (3 pM for KM12 and 2 pM for RL95-2 ), HRO761 (120 nM), or left untreated as a control for ten days before sequencing.

[0117] Figure 55. Density scatter plots comparing the z-scores for GSK_WRN3 and HRO761 treatment across two biological replicates forCBE (top row) and ABE (bottom row) in KM12 and RL95-2 cells. High correlation (R-values) indicates reproducibility across experiments. While KM12 cells were screened with GSK_WRN3 and HRO761 , RL95-2 cells were only screened with HRO761.

[0118] Figure 56. Western blot analysis showing WRN protein levels in KM12 and RL95-2 base-edited (BE) cells. Cells were treated with either GSK_WRN3 or HRO761 , with or without sgRNA targeting Cys727 (sgRNA C727). Tubulin is used as a loading control. In the presence of the sgRNA C727, WRN degradation is prevented, demonstrating resistance to both WRN inhibitors in the edited cells.

[0119] Figure 57. Dose-response curves for BE-expressing KM12 and RL95-2 were edited to introduce mutation in position 727. The data are plotted as a percentage of viability relative to control, with the log concentration of the two WRN inhibitors on the x-axis.

[0120] Figure 58. The dose-response curve showed decreased sensitivity of HRO761 -resistant SW48 cells compared to control cells.

[0121] Figure 59. Colony formation assay highlighting the difference in sensitivity between parental and HRO761 -resistant SW48 cells to escalating doses of HRO761.

[0122] Figure 60. Time-to-progression (TTP) assay in additional MSI cancer cell lines (KM12, RL95-2, OER2, IM-95) treated with WRN inhibitors GSK_WRN3 and HRO761 shows similar resistance patterns to SW48 cells. The tissue origin of the models is indicated.

[0123] Figure 61. RNAi knockdown of WRN in GSK_WRN3- and HRO761 -resistant SW48 cells shows persistent sensitivity to genetic WRN inactivation, indicating continued reliance on WRN function despite pharmacologic resistance. Data represent the average of two independent experiments performed with four technical replicates.

[0124] Figure 62. Tumor volume measurements from SW48 MSI tumor xenografts in individual mice treated with 60 mpk of HRO761 (right) or 120 mpk of HRO761 (left) show initial tumor regression followed by resistance and tumor regrowth. Each curve represents a single mouse, highlighting the variability in resistance development and tumor regrowth under different dosage conditions (60 mpk left, 120 mpk right), mirroring in vitro resistance mechanisms. The WRN mutations shown in the graph were identified post-explant via deep-targeted sequencing, confirming their role in resistance.

[0125] Figure 63. Time-to-progression (TTP) assay in additional MSI cancer cell lines (top SW48, bottom KM12) treated with WRN inhibitors VVD-133214 or VVD-133214 plus HRO761 acquired rapid resistance.

[0126] Genome sequencing identified WRN mutations enriched in respective resistant cells, as indicated in the labels.

[0127] Figure 64. The dose-response curve showed decreased sensitivity of WD-133214-resistant KM12 cells or VVD-133214 plus HRO761 KM12 cells double resistant cells to HRO761 (middle panel) and VVD- 133214 (bottom panel) compared to control (parental) cells. Resistant KM12 cells with WRN G1207D mutation remain sensitive to HRO761. WD-133214-resistant KM12 cells or VVD-133214 plus HRO761 KM12 double resistant cells retained sensitivity to GSK_WRN5 (top panel) similar to the parental KM12 cells.

[0128] Figure 65. The dose-response curve showed decreased sensitivity of WD-133214-resistant SW48 cells to HRO761 (middle panel) and VVD-133214 (bottom panel) compared to control (parental) cells. The resistant cells showed partial sensitivity to GSK_WRN5 (top panel) compared to the parental SW48 cells.

[0129] Figure 66. Colony formation assay showed loss of activity of HRO761 (250 nM, left) and VVD-133214 (250nM, middle) to WD-133214-resistant SW48 cells. GSK_WRN5 (right) remain active to VVD-133214-resistant SW48 cells at 250 nM.

[0130] Figure 67. Tumor volume measurements from SW48 MSI tumor xenografts a group of mice treated with 60 mpk of HRO761 show initial tumor regression followed by resistance and tumor regrowth. HRO761 was switched to GSK_WRN5 (100 mpk, twice daily) led to regression of HRO761 -resistant tumors. Figure 68. Tumor volume measurements from SW48 MSI tumor xenografts a group of mice treated with 60 mpk of HRO761 show initial tumor regression followed by resistance and tumor regrowth. HRO761 was switched to VVD-133214 (100 mpk, q.d.) which did not cause tumor regression.

[0131] Detailed Description of the Invention

[0132] As used herein, the term “resistance”, also referred to as “drug resistance”, “multiple drug resistance”, “cancer drug resistance”, “canonical drug resistance”, “antineoplastic resistance”, or “chemotherapy resistance”, refers to the resistance of cancerous cells to a particular drug or class of drugs, or the ability of cancer cells to survive and grow despite the presence of a particular drug or class of drugs. In the context of a patient or subject with a cancer or tumour, the term “resistance” refers to a lack of response ora lower response of the patient’s cancer or tumour to a drug or class of drugs. In some embodiments, the cancer cells, cancer or tumour is resistant to multiple drugs, resulting in multiple drug resistance. In some embodiments, resistance is the result of inherent genetic characteristics in the cancer cells. In some embodiments, resistance is acquired after drug exposure. For example, tumour heterogeneity, wherein tumours are made up of different populations of cancer cells that are morphologically, phenotypically, genetically and / or functionally different, can result in the effective selection of certain populations of cancer cells which possess characteristics that confer drug resistance, when the tumour is treated with said drug.

[0133] According to the present disclosure, resistance or sensitivity to a drug or class of drugs is associated with specific mutations (also referred to as “variants”) present in the cancerous cells or a proportion thereof in a tumour or cancer. The term “variant” (also referred to herein as “mutant” or “mutated form of’) refers to a protein that differs from the wild type protein present in normal I healthy cell, to the particular mutation or change in protein sequence that differs between the variant protein and the wild type I normal protein, and / or to the underlying genetic sequence change that caused the change in the protein. Thus, mutants described herein are all associated with a different protein compared to the wild type protein (i.e. no silent mutation). A variant may be a single or multiple amino acid substitution, a splice variant or a truncated sequence variant. A variant may be associated with a position, which may be expressed as an amino acid number (referring to the position of the amino acid(s) that are varied, in the reference protein sequence) or as a position number in the gene sequence encoding the protein. Unless indicated otherwise, all variant positions are indicated by reference to the amino acid sequence of the corresponding reference protein sequence (for WRN, Uniprot identifier Q14191 (SEQ ID NO: 25)).

[0134] Werner syndrome helicase (WRN)

[0135] Werner syndrome helicase (WRN) (also known as RecQ-like type 3 / RECQ3) is a bifunctional 3'-5' exonuclease / ATP-dependent helicase. WRN is a multifunctional enzyme with both helicase and exonuclease activities and has roles in various cellular processes that are crucial for the maintenance of genome stability, including DNA replication, transcription, DNA repair and telomere maintenance (Ferretti et al., 2024)

[0136] The human gene encoding WRN (Gene ID: 7486) is present on 8p12 and consists of 37 exons. The genomic sequence of human WRN is available under NCBI GenelD 7486, and is incorporated herein in its entirety. Genomic locations referred to herein refer to a location in the GRCh38 reference genome assembly (NCBI RefSeq Assembly GCF_000001405.50 (GRCh38.p14)).

[0137] The sequence of human WRN is available under Uniprot identifier Q14191 and (SEQ ID NO: 25) and is incorporated herein in its entirety. WRN is a 1 ,432-amino acid, 162-kD polypeptide that contains a central helicase core of two domains (D1 and D2) (residues 528-730 and 731-868, respectively), together with three additional helicase associated domains in the C terminus: a zinc-binding subdomain (869-994), a winged helix (WH) domain (956-1,064) and a helicase and RNase D C-terminal (HRDC) domain (1,140-1,239) (Newman et al., 2020). WRN has a hinge region (residues 728-732) between the D1 and D2 domains.

[0138] However, as used herein, the term “WRN” encompasses truncations, derivatives, and variants of the sequence of WRN provided herein (SEQ ID NO:25), and may refer to any protein with at least 80%, at least 90%, or at least 95% sequence identity.

[0139] Reference non-human WRN amino acid and coding sequences are available in public databases The present disclosure provides a plurality of variants in the WRN protein sequence that are associated with altered response to one or more WRN inhibitors. These include variants that are associated with a truncation of WRN, and in particular a partial truncation of the region of WRN corresponding to exon 13.

[0140] WRN variants of the disclosure further include variants in which substitutions occur at any one or more of the following amino acid positions: position 521; position 530; position 725; position 727; position 729, optionally wherein the mutation is G729D or G729S; position 730, optionally wherein the mutation is F730L or F730S; position 731; position 852, optionally wherein the mutation is I852F; position 975. Werner Helicase (WRN) Inhibitors

[0141] VVD-133214

[0142] The term “VVD-133214” refers to the covalent allosteric inhibitor of WRN helicase, with formula PubChem CID 170717998 and IUPAC nomenclature N-[(E,1S)-1-cyclopropyl-3-methylsulfonylprop-2-enyl]-2-(1 ,1-difluoroethyl)-4-phenoxypyrimidine-5-carboxamide. It has the following structure:

[0143]

[0144] -

[0145] VVD-133214 is also known as RO7589831. VVD-133214 binds to WRN's cysteine 727 (C727) residue and locks the enzyme in a rigid conformation that abbrogates helicase function (Baltgalvis et al., 2024).

[0146] HRO761

[0147] The term “HRO761 ” refers to the allosteric inhibitor of WRN helicase, with formula PubChem CID 166140536 and IUPAC nomenclature A / -[2-chloro-4-(trifluoromethyl)phenyl]-2-[2-(3,6-dihydro-2 / 7-pyran-4-yl)-5-ethyl-6-[4-(5-hydroxy-6-methylpyrimidine-4-carbonyl)piperazin-1-yl]-7-oxo-[1 ,2,4]triazolo[1 ,5-a]pyrimidin-4-yl]acetamide. It has the following structure:

[0148]

[0149] HRO761 is a potent, selective, allosteric non-covalent WRN inhibitor that binds WRN at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation (Ferretti et al., 2024).

[0150] GSK WRN3

[0151] The term “GSK_WRN3” refers to the covalent allosteric inhibitor of WRN helicase with the following structure:

[0152]

[0153] GSK_WRN3 binds to cysteine 727 (C727) of WRN (Picco et al., 2024).

[0154] GSK WRN5

[0155] The term “GSK_WRN5” refers to the inhibitor of WRN helicase with the following nomenclature (1R,2R,6S)-2-((2-fluoro-4-(trifluoromethyl)phenyl)carbamoyl)-6-(4-((A / ,1 ,2-trimethyl-1 / 7- benzo[cf]imidazole)-5-sulfonamido)phenyl)cyclohexane-1 -carboxylic acid. It has the following structure:

[0156] CH3

[0157] I

[0158]

[0159] Pharmaceutically acceptable salts

[0160] As described herein, any compound (e.g. any WRNi) may be provided as a pharmaceutically acceptable salt, hydrate or solvate. Suitable pharmaceutically acceptable salts are known in the art and are described in, for example, in Berge etal., J Pharm Sci, 197766(1) p 1.

[0161] ATR inhibitors

[0162] The term “ATR inhibitor” refers to an inhibitor of ataxia-telangiectasia and RAD-3-related protein kinase. ATR inhibitors (ATRi) elicit cell death in rapidly growing tumor cells by exacerbating endogenous replication stress and replication fork collapse, as well as by disabling cell cycle checkpoints. An ATR inhibitor is a compound that upon contacting the enzyme ATR kinase, whether in vitro, in cell culture, or in an animal, reduces the activity of ATR kinase, such that the measured ATR kinase IC50 is 10 pM or less (e.g., 5 pM or less or 1 pM or less). For certain ATR inhibitors, the ATR kinase IC50 may be 100 nM or less (e.g., 10 nM or less, or 1 nM or less) and could be as low as 100 pM or 10 pM.

[0163] Preferably, the ATR kinase IC50 is 0.1 nM to 1 pM (e.g., 0.1 nM to 750 nM, 0.1 nM to 500 nM, or 0.1 nM to 250 nM).

[0164] Non-limiting examples of ATR inhibitors include those described in Yano et al., 2023, e.g Berzosertib, Ceralasertib (AZD6738), Elimusertib, IMP9064, ATG-018, ATRN-119, ART0380.

[0165] Subject and cancer type

[0166] The term “patient” or “subject” are used herein interchangeably. A patient may refer to a subject that has been identified as having or being likely to have cancer. The patient may be any animal or human. The patient may be a non-human mammal (e.g. a model animal such as a mouse or rat, a pet such as a dog, cat or horse). The patient may be a human patient. The patient may be male orfemale. Unless indicated otherwise, all references to proteins and variants refer to human proteins. References to proteins and variants encompass the human protein variant and a homologue thereof in a different organism, such as e.g. a non-human mammal.

[0167] The invention provides methods, including methods of treatment of cancer in a subject, as defined herein. The invention further provides a WRN inhibitor for use in the methods defined herein, for example for use in the methods of treatment of cancer in a subject as defined herein. The invention also provides the use of a WRN inhibitor in the manufacture of a medicament for the treatment of cancer is a subject as defined herein.

[0168] The invention relates to methods for the treatment of cancer in patients, and in particular the treatment of cancer classified as Microsatellite Instability - High (MSI-High).

[0169] That is, the tumour is classified as MSI-High by genomic profiling and / or immunohistochemistry. Tumours which are not MSI-High are termed Microsatellite Stable (MSS). Genomic profiling to determine MSI / MSS status is described in Kawakami etal., Curr. Treat. Options Oncol., 2015 Jul;16(7):30 (PMID: 26031544) and testing systems are commercially available from, for example, Promega® (OncoMate™ MSI Dx Analysis System).

[0170] A "cancer" can comprise any one or more of the following: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical cancer, anal cancer, bladder cancer, blood cancer, bone cancer, brain tumor, breast cancer, cancer of the female genital system, cancer of the male genital system, central nervous system lymphoma, cervical cancer, childhood rhabdomyosarcoma, childhood sarcoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), colon and rectal cancer, colon cancer, endometrial cancer, endometrial sarcoma, esophageal cancer, eye cancer, gallbladder cancer, gastric cancer, gastrointestinal tract cancer, hairy cell leukemia, head and neck cancer, hepatocellular cancer, Hodgkin's disease, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, leukemia, liver cancer, lung cancer, malignant fibrous histiocytoma, malignant thymoma, melanoma, mesothelioma, multiple myeloma, myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nervous system cancer, neuroblastoma, non-Hodgkin's lymphoma, oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, plasma cell neoplasm, primary CNS lymphoma, prostate cancer, rectal cancer, respiratory system, retinoblastoma, salivary gland cancer, skin cancer, small intestine cancer, soft tissue sarcoma, stomach cancer, stomach cancer, testicular cancer, thyroid cancer, urinary system cancer, uterine sarcoma, vaginal cancer, vascular system, Waldenstrom's macroglobulinemia and Wilms' tumor.

[0171] In embodiments, the cancer is colorectal cancer, gastric cancer, endometrial cancer, ovarian cancer, hepatobiliary tract cancer, urinary tract cancer, brain cancer, or skin cancer. For example, the cancer may be endometrial cancer or colorectal cancer. In embodiments, the cancer is colorectal cancer. In embodiments, the cancer is endometrial cancer. In embodiments, the cancer is gastric cancer.

[0172] Combinations with other agents

[0173] Compounds and combinations used in the methods of the invention may be administered together with one or more other active agents, such as chemotherapeutic agents or targeted immunotherapies, for the treatment of cancer. For example, the therapies as described herein may be used in combination with chemotherapeutic agents such as an anti-PD1 antibody such as pembrolizumab. In such cases, the therapy (i.e. WRN inhibitor) and the additional active agent(s) may be given together or separately, e.g. as constituents in the same pharmaceutical composition or formulation, or as separate formulations. For example, the therapies as described herein may be used in combination with chemotherapeutic agents such as a TOP1 inhibitor.

[0174] DNA topoisomerase I is commonly referred to in the literature as TOP1. It controls and alters the topologic states of DNA during transcription and is an interesting target for oncology.

[0175] TOP1 inhibitors previously used for clinical treatment include irinotecan and camptothecin. These are members of the class of compounds based on camptothecin, collectively referred to as the camptothecins or camptothecin derivatives. That is, a camptothecin derivative refers to camptothecin itself or a derivative thereof based on the core camptothecin motif. Camptothecin derivatives include irinotecan, topotecan, belotecan, lurtotecan, exatecan, gimatecan, and sinotecan. Alkylated, alkoxylated and hydroxylated derivatives have been reported in the literature. Accordingly, the TOP1 inhibitor may be selected from irinotecan, topotecan, belotecan, lurtotecan, exatecan, gimatecan, and sinotecan, and alkylated, alkoxylated and hydroxylated derivatives thereof.

[0176] Suitably, the TOP1 inhibitor administered in the methods or combinations of the present invention is a campothecin derivative, for example selected from irinotecan, topotecan, and camptothecin, or an active metabolite therefore such as SN-38 (a metabolite of irinotecan). Preferably, the TOP1 inhibitor is irinotecan. Administration of active ingredients

[0177] Compounds used in the methods of the invention may be administered by any suitable route, including oral and intravenous routes. It will be understood that oral administration may be preferred. The compounds may be provided in pharmaceutical compositions comprising the compound and one or more pharmaceutically acceptable excipients. Formulation for oral administration may be in the form of a tablet or a capsule comprising a powder or liquid.

[0178] Administration is preferably in a "therapeutically effective amount" or an "effective amount” (used interchangeably), 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 the disease 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, pub. Lippincott, Williams & Wilkins.

[0179] Dosage regimens known in the art for the active ingredients described herein may also be used in the present invention.

[0180] The active ingredients described herein may be administered simultaneously or sequentially.

[0181] In some embodiments of the combination therapies described herein, the WRN inhibitors and ATR inhibitors are administered sequentially. In some embodiments, the WRN inhibitor (such as VVD-133214, such as HRO761) would be administered first, followed by the ATR inhibitor.

[0182] In some embodiments of the combination therapies described herein, the first and second WRN inhibitors administered sequentially. In some embodiments, the first WRN inhibitor (such as VVD-133214, such as HRO761) would be administered first, followed by the second WRN inhibitor (such as HRO761 , such as VVD-133214).

[0183] The active ingredients described herein may be administered at different times within a prescribed dosing cycle. The active ingredients described herein may be administered daily, such as once daily (QD), twice daily (BID), three times daily (TID), or four times daily (QID), or on a less frequent or intermittent schedule.

[0184] Prognostic methods, and patient selection

[0185] Reference to “determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein” refers to determination of the presence or absence of a nucleic acid sequence, for example mRNA or DNA, optionally circulating tumour RNA or DNA (ctRNA orctDNA), encoding a mutated form of the WRN protein.

[0186] The methods may be performed in vitro on a sample obtained from a subject, or following processing of a sample obtained from a subject. Once the sample is collected, the subject is not required to be present for the in vitro method to be performed, and therefore the method may be one which is not practised on the human or animal body.

[0187] Detection of a variant in a subject may be performed using any method known in the art, including genomic and transcriptomic detection methods applied to a sample obtained from the patient.

[0188] A “sample” as used herein may be a cell or tissue sample, a biological fluid, an extract (e.g. a DNA extract obtained from the subject), from which genomic and / or transcriptomic and / or proteomic material can be obtained for genomic and / or transcriptomic and / or proteomic analysis, such as genomic sequencing (e.g. whole genome sequencing, whole exome sequencing, targeted I panel sequencing), RNA sequencing (also referred to as “RNAseq” or “RNA-seq”), molecular counting, PCR, RT-PCR, microarrays, protein detection assays (e.g. using mass spectrometry, affinity based tests, protein arrays etc.). The sample may be a cell, tissue or biological fluid sample obtained from a subject (e.g. a biopsy). Such samples may be referred to as “subject samples”. In particular, the sample may be a blood sample, or a tumour sample, or a sample derived therefrom. For the purposes of obtaining RNA sequence data, a “sample” as used herein may be a cell or tissue sample, or an extract (e.g. a RNA extract obtained from a subject) from which transcriptomic material can be obtained. For the purposes of obtaining DNA / genomic sequence data, a “sample” as used herein may be a cell or tissue sample, a biological fluid, an extract (e.g. a DNA extract obtained from the subject), from which genomic material can be obtained for genomic analysis, such as genomic sequencing (e.g. whole genome sequencing, whole exome sequencing). The sample may be one which has been freshly obtained from a subject or may be one which has been processed and / or stored prior to genomic / transcriptomic / proteomic analysis (e.g. frozen, fixed or subjected to one or more purification, enrichment or extraction steps). The sample may be a cell or tissue culture sample. As such, a sample as described herein may refer to any type of sample comprising cells or genomic and / or transcriptomic and / or proteomic material derived therefrom, whether from a biological sample obtained from a subject, or from a sample obtained from e.g. a cell line. Further, the sample may be transported and / or stored, and collection may take place at a location remote from the sequence data acquisition (e.g. sequencing) location, and / or any computer-implemented method steps described herein may take place at a location remote from the sample collection location and / or remote from the sequence data acquisition (e.g. sequencing) location (e.g. the computer-implemented method steps may be performed by means of a networked computer, such as by means of a “cloud” provider).The samples used in methods of the present disclosure are typically samples comprising tumour cells (e.g. a tumour sample or sample comprising circulating tumour cells) or genetic material derived from tumour cells (such as e.g. cell free DNA or cell DNA and / or RNA extracted from a sample comprising cells). Such as sample may be a “mixed sample”. A “mixed sample” refers to a sample that is assumed to comprise multiple cell types or genetic material derived from multiple cell types. Within the context of the present disclosure, a mixed sample is typically one that comprises tumour cells or is assumed (expected) to comprise tumour cells, or genetic material derived from tumour cells, and normal cells or genetic material derived from normal cells. Genetic material can comprise genomic material (e.g. DNA) or transcriptomic material (e.g. RNA). Samples obtained from subjects, such as e.g. tumour samples, are typically mixed samples (unless they are subject to one or more purification and / or separation steps). Typically, the sample comprises tumour cells and at least one non-tumour cell type (and / or genetic material derived therefrom). A “tumour sample” refers to a sample derived from or obtained from a tumour. Such samples may comprise tumour cells and normal (non-tumour) cells. The normal cells may comprise immune cells (such as e.g. lymphocytes), and / or other normal (non-tumour) cells (e.g. stromal cells). A tumour may be a solid tumour or a non-solid or haematological tumour. A tumour sample may be a primary tumour sample, tumour-associated lymph node sample, or a sample from a metastatic site from the subject. A sample comprising tumour cells or genetic material derived from tumour cells may be a bodily fluid sample. Thus, the genetic material derived from tumour cells may be circulating tumour DNA or tumour DNA in exosomes. Instead or in addition to this, the sample may comprise circulating tumour cells. A mixed sample may be a sample of cells, tissue or bodily fluid that has been processed to extract genetic material (e.g. DNA or RNA) and / or proteromic material. Methods for extracting genetic material from biological samples are known in the art. A mixed sample may have been subject to one or more processing steps that may modify the proportion of the multiple cell types or genetic material derived from the multiple cell types in the sample. For example, a mixed sample comprising tumour cells may have been processed to enrich the sample in tumour cells. Thus, a sample of purified tumour cells may be referred to as a “mixed sample” on the basis that small amounts of other types of cells may be present, even if the sample may be assumed, for a particular purpose, to be pure (i.e. to have a tumour fraction of 1 or 100%).

[0189] Thus, any method described herein may comprise one or more of: obtaining a sample from a subject, processing said sample to extract DNA, and / or RNA, and detecting the presence of one or more nucleic acid sequences encoding a mutated form of the WRN protein in said sample or extract using any method known in the art (such as e.g. sequencing, PCR, digital PCR, RT-PCR, microarrays, etc.).

[0190] Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0191] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof. While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations. Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Throughout this specification, including the clauses and claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. It must be noted that, as used in the specification and the clauses and appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / - 10%.

[0192] All references cited herein are incorporated herein by reference in their entirety. For standard molecular biology techniques, see Sambrook, J., Russel, D.W. Molecular Cloning, A Laboratory Manual. 3 ed. 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

[0193] Some methods of the present disclosure involve a sample containing cells. The sample may be a culture of cells grown in vitro. For example, the culture may comprise a suspension of cells or cells cultured in a culture plate or dish. Methods according to the present disclosure may be performed, or products may be present, in vitro, ex vivo, or in vivo. The term “in vitro" is intended to encompass experiments with materials, biological substances, cells and / or tissues in laboratory conditions or in culture whereas the term “in vivo" is intended to encompass experiments and procedures with intact multi-cellular organisms. “Ex vivo" refers to something present or taking place outside an organism, e.g. outside the human or animal body, which may be on tissue (e.g. whole organs) or cells taken from the organism.

[0194] Sequences

[0195] Sequences described herein are listed in the table below.

[0196] SEQ Description Sequence

[0197] ID NO

[0198] 1 Nucleotide sequence of GTCCTGTGGAACATACCATG

[0199] WRN sgRNA 4 (Behan et

[0200] al. 2019) used to generate

[0201] di-WRN cells

[0202] 2 Nucleotide sequence of ATGTTGGAAGGATGAGGAAA

[0203] gRNA targeting the CLYBL

[0204] locus

[0205] 3 sgRNA against TP53 GCATTGTTCAATATCGTCCG

[0206] 4 sgRNA against TP53 GAACAAGA I G I I I I GCCAAC

[0207]

[0208] sgRNA against SMARCAL1 GCGCTGTCGAGCAGCTATGC

[0209] sgRNA against SMARCAL1 GGCAGCTATGCCGGTCCTAA

[0210] sgRNA against MUS81 GGTGCTGTATCGATCCACCA

[0211] sgRNA against MUS81 GAGCGGCACCGAACATCGGG

[0212] sgRNA against SUPT7L GCACAGTCAAAGCCCGCGT

[0213] sgRNA against SUPT7L GCACAGTCAAAGCCCGCGT

[0214] sgRNA against TADA1 G I I I I GC I GCGGAACCCCC

[0215] sgRNA against TADA1 ACAACGCGTGAGAATGGCC

[0216] Nontargeting siRNA UGGUUUACAUGUCGACUAA

[0217] Nontargeting siRNA UGGUUUACAUGUUGUGUGA

[0218] Nontargeting siRNA UGGUUUACAUGUUUUCUGA

[0219] Nontargeting siRNA UGGUUUACAUGUUUUCCUA

[0220] siRNA targeting PLK1 GCACAUACCGCCUGAGUCU

[0221] siRNA targeting PLK1 CCACCAAGGUUUUCGAUUG

[0222] siRNA targeting PLK1 GCUCUUCAAUGACUCAACA

[0223] siRNA targeting PLK1 UCUCAAGGCCUCCUAAUAG

[0224] siRNA targeting WRN GAUCCAUUGUGUAUAGUUA

[0225] siRNA targeting WRN GCACCAAAGAGCAUUGUUA

[0226] siRNA targeting WRN AUACGUAACUCCAGAAUAC

[0227] siRNA targeting WRN GAGGGUUUCUAUCUUACUA

[0228] Amino acid sequence of MSEKKLETTAQQRKCPEWMNVQNKRCAVEERKACVRKSVFEDD human WRN (UniProt: LPFLEFTGSIVYSYDASDCSFLSEDISMSLSDGDVVGFDMEWPPL Q14191) YNRGKLGKVALIQLCVSESKCYLFHVSSMSVFPQGLKMLLENKAV KKAGVGIEGDQWKLLRDFDIKLKNFVELTDVANKKLKCTETWSLN SLVKHLLGKQLLKDKSIRCSNWSKFPLTEDQKLYAATDAYAGFIIY RNLEILDDTVQRFAINKEEEILLSDMNKQLTSISEEVMDLAKHLPHA FSKLENPRRVSILLKDISENLYSLRRMIIGSTNIETELRPSNNLNLLS FEDSTTGGVQQKQIREHEVLIHVEDETWDPTLDHLAKHDGEDVL

[0229]

[0230] GNKVERKEDGFEDGVEDNKLKENMERACLMSLDITEHELQILEQ QSQEEYLSDIAYKSTEHLSPNDNENDTSYVIESDEDLEMEMLKHL SPNDNENDTSYVIESDEDLEMEMLKSLENLNSGTVEPTHSKCLKM ERNLGLPTKEEEEDDENEANEGEEDDDKDFLWPAPNEEQVTCLK MYFGHSSFKPVQWKVIHSVLEERRDNVAVMATGYGKSLCFQYPP VYVGKIGLVISPLISLMEDQVLQLKMSNIPACFLGSAQSENVLTDIK LGKYRIVYVTPEYCSGNMGLLQQLEADIGITLIAVDEAHCISEWGH DFRDSFRKLGSLKTALPMVPIVALTATASSSIREDIVRCLNLRNPQI TCTGFDRPNLYLEVRRKTGNILQDLQPFLVKTSSHWEFEGPTIIYC PSRKMTQQVTGELRKLNLSCGTYHAGMSFSTRKDIHHRFVRDEI QCVIATIAFGMGINKADIRQVIHYGAPKDMESYYQEIGRAGRDGLQ SSCHVLWAPADINLNRHLLTEIRNEKFRLYKLKMMAKMEKYLHSS RCRRQIILSHFEDKQVQKASLGIMGTEKCCDNCRSRLDHCYSMD DSEDTSWDFGPQAFKLLSAVDILGEKFGIGLPILFLRGSNSQRLAD QYRRHSLFGTGKDQTESWWKAFSRQLITEGFLVEVSRYNKFMKI CALTKKGRNWLHKANTESQSLILQANEELCPKKLLLPSSKTVSSG TKEHCYNQVPVELSTEKKSNLEKLYSYKPCDKISSGSNISKKSIMV QSPEKAYSSSQPVISAQEQETQIVLYGKLVEARQKHANKMDVPPA ILATNKILVDMAKMRPTTVENVKRIDGVSEGKAAMLAPLLEVIKHF CQTNSVQTDLFSSTKPQEEQKTSLVAKNKICTLSQSMAITYSLFQE KKMPLKSIAESRILPLMTIGMHLSQAVKAGCPLDLERAGLTPEVQK IIADVIRNPPVNSDMSKISLIRMLVPENIDTYLIHMAIEILKHGPDSGL QPSCDVNKRRCFPGSEEICSSSKRSKEEVGINTETSSAERKRRLP VWFAKGSDTSKKLMDKTKRGGLFS

[0231]

[0232] Certain embodiments of the invention are set out in the following clauses:

[0233] §1. A Werner helicase (WRN) inhibitor for use in a method of treatment of cancer in a subject, wherein the cancer has been identified as expressing a mutated form of the WRN protein, wherein the WRN inhibitor is VVD-133214.

[0234] §2. A Werner helicase (WRN) inhibitor for use in a method of treatment of cancer in a subject, wherein the WRN inhibitor is VVD-133214, and wherein the method comprises:

[0235] i. determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein; and

[0236] ii. when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, administering to the subject an effective amount of the WRN inhibitor.

[0237] §3. A method of treatment of cancer in a subject in need thereof, wherein the cancer has been identified as expressing a mutated form of the WRN protein, wherein the method comprises the step of administering to the subject an effective amount of a WRN inhibitor, wherein the WRN inhibitor is VVD-133214.

[0238] §4. A method of treatment of cancer in a subject in need thereof, wherein the method comprises the steps of:

[0239] i. determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein; and

[0240] ii.when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, administering to the subject an effective amount of a WRN inhibitor, wherein the WRN inhibitor is VVD-133214.

[0241] §5. A method of selecting a subject with cancer for treatment with a Werner helicase (WRN) inhibitor, wherein the WRN inhibitor is VVD-133214, the method comprising determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, wherein the subject is selected for treatment when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein.

[0242] §6. A method of predicting whether a subject with cancer is likely to respond to treatment with a Werner helicase (WRN) inhibitor, wherein the WRN inhibitor is VVD-133214, the method comprising determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein,

[0243] wherein the subject is considered likely to respond to treatment with the WRN inhibitor when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein.

[0244] §7. A method for identifying a subject with cancer who is likely to respond to treatment with a Werner helicase (WRN) inhibitor, wherein the WRN inhibitor is VVD-133214, the method comprising determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein,

[0245] wherein the subject is considered likely to respond to treatment with the WRN inhibitor when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein.

[0246] §8. A method for assessing the susceptibility of a cancer in a subject to treatment with a Werner helicase (WRN) inhibitor, wherein the WRN inhibitor is VVD-133214, comprising determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, wherein the cancer is considered susceptible to treatment with the WRN inhibitor when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein. §9. The method of any one of clauses §6 to §8, wherein when the subject is considered likely to respond to treatment with the WRN inhibitor or the cancer is considered susceptible to treatment with the WRN inhibitor, the subject is selected for treatment with the WRN inhibitor.

[0247] §10. The method of any one of clauses §5 to §9, wherein when the subject is selected for treatment with the WRN inhibitor, the subject is considered likely to respond to treatment with the WRN inhibitor and / or the cancer is considered susceptible to treatment with the WRN inhibitor, the method further comprises administering the WRN inhibitor to the subject.

[0248] §11. The WRN inhibitor for use or the method according to any one of the preceding clauses, wherein the mutated form of the WRN protein comprises a mutation conferring resistance to treatment of the cancer with a WRN inhibitor other than VVD-133214, optionally wherein the WRN inhibitor other than VVD-133214 is HRO761.

[0249] §12. The WRN inhibitor for use or the method according to any one of the preceding clauses, wherein the mutated form of the WRN protein comprises a mutation at position 852 of the amino acid sequence set out in SEQ ID NO: 25, optionally wherein the mutation is I852F.

[0250] §13. The WRN inhibitor for use or the method according to any one of the preceding clauses, wherein the nucleic acid sequence encoding the mutated form of the WRN protein comprises a truncated exon 13, optionally wherein the nucleic acid sequence comprises exon-truncating splice site mutation 1557-1 G>C at position chr8:31088889 or 1557-2 A>T at position chr8:31088888, wherein the position refers to a location in the GRCh38 reference genome assembly.

[0251] §14. A method of predicting whether a subject with cancer is unlikely to respond to treatment with a Werner helicase (WRN) inhibitor, the method comprising determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, wherein the subject is considered unlikely to respond to treatment with the WRN inhibitor when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein.

[0252] §15. A method for assessing the resistance of a cancer in a subject to treatment with a Werner helicase (WRN) inhibitor, wherein the subject has previously been administered the WRN inhibitor, the method comprising determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, wherein the cancer is considered resistant to treatment with the WRN inhibitor when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein.

[0253] §16. A method for assessing the susceptibility of a cancer in a subject to treatment with a Werner helicase (WRN) inhibitor, comprising determining whether a sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein, wherein the cancer is considered not susceptible to treatment with the WRN inhibitor when the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein. §17. The method according to any one of clauses §14 to §16, wherein the mutated form of the WRN protein comprises a mutation at a position selected from the list consisting of positions 521 , 530, 725 to 731 , 852, and / or 975 of the amino acid sequence set out in SEQ ID NO: 25.

[0254] §18. The method according to clause §17, wherein the mutated form of the WRN protein comprises a mutation at one or more of the following positions of the amino acid sequence set out in SEQ ID NO: 25:

[0255] position 521 ;

[0256] position 530;

[0257] position 725;

[0258] position 727;

[0259] position 729, optionally wherein the mutation is G729D orG729S;

[0260] position 730, optionally wherein the mutation is F730L or F730S;

[0261] position 731 ;

[0262] position 852, optionally wherein the mutation is I852F; and / or

[0263] position 975.

[0264] §19. The method according to any one of clauses §14 to §18, wherein the nucleic acid sequence encoding the mutated form of the WRN protein comprises a truncated exon 13, optionally wherein the nucleic acid sequence comprises exon-truncating splice site mutation 1557-1 G>C at position chr8:31088889 or 1557-2 A>T at position chr8:31088888, wherein the position refers to a location in the GRCh38 reference genome assembly.

[0265] §20. The method according to any one of clauses §14 to §19, wherein the WRN inhibitor is VVD-133214 or HRO761.

[0266] §21. The WRN inhibitor for use or the method according to any one of the preceding clauses, wherein the sample is a tissue sample or a biological fluid sample.

[0267] §22. The WRN inhibitor for use or the method according to clause §21, wherein the biological fluid sample comprises a circulating tumour cell, or a cell-free sample comprising cell-free DNA, circulating tumour DNA (ctDNA) and / or circulating tumour RNA (ctRNA).

[0268] §23. The WRN inhibitor for use or the method according to clause §21 or clause §22, wherein the biological fluid sample is a blood sample, a urine sample, or a cerebrospinal fluid (CSF) sample.

[0269] §24. The WRN inhibitor for use or the method according to clause §23, wherein the biological fluid sample is a blood sample, optionally wherein the blood sample is a whole blood sample or a plasma sample. §25. The WRN inhibitor for use or the method according to clause §21 , wherein the tissue sample is a tumour tissue sample, optionally a tumour biopsy sample.

[0270] §26. The WRN inhibitor for use or the method according to any one of the preceding clauses, wherein determining whether the sample obtained from the subject comprises a nucleic acid sequence encoding a mutated form of the WRN protein comprises nucleic acid sequencing of the sample.

[0271] §27. A combination of a first WRN inhibitor and a second WRN inhibitor for use in a method of treatment of cancer in a subject, wherein the first WRN inhibitor is VVD-133214 and the second WRN inhibitor is HRO761.

[0272] §28. A WRN inhibitor for use in a method of treatment of cancer in a subject, wherein the WRN inhibitor is VVD-133214, and wherein VVD-133214 is administered to the subject in combination with a second WRN inhibitor, wherein the second WRN inhibitor is HRO761.

[0273] §29. A WRN inhibitor for use in a method of treatment of cancer in a subject, wherein the WRN inhibitor is HRO761 , and wherein HRO76 is administered to the subject in combination with a second WRN inhibitor, wherein the second WRN inhibitor is VVD-133214.

[0274] §30. The combination, or the WRN inhibitor, for use according to any one of clauses §27 to §29, wherein the first WRN inhibitor and the second WRN inhibitor are administered separately.

[0275] §31. A combination of a WRN inhibitor and an ataxia telangiectasia and Rad3 related kinase (ATR) inhibitor for use in a method of treatment of cancer in a subject, wherein the WRN inhibitor is VVD-133214 or HRQ761.

[0276] §32. A WRN inhibitor for use in a method of treatment of cancer in a subject, wherein the WRN inhibitor is VVD-133214 or HRO761 , and wherein the WRN inhibitor is administered to the subject in combination with an ATR inhibitor.

[0277] §33. An ATR inhibitor for use in a method of treatment of cancer in a subject, wherein the ATR inhibitor is administered to the subject in combination with a WRN inhibitor, wherein the WRN inhibitor is VVD-133214 or HRO761.

[0278] §34. The combination, the WRN inhibitor, or the ATR inhibitor for use according to any one of clauses 31 to 33, wherein the WRN inhibitor and the ATR inhibitor are administered separately.

[0279] §35. The WRN inhibitor for use, the combination for use, the method, or the ATR inhibitor for use according to any one of the preceding clauses, wherein the subject has previously received treatment with HRO761. §36. The WRN inhibitor for use, the combination for use, the method, or the ATR inhibitor for use according to any one of the preceding clauses, wherein the subject is a human.

[0280] §37. The WRN inhibitor for use, the combination for use, the method, or the ATR inhibitor for use according to any one of the preceding clauses, wherein the cancer is a microsatellite instability - high (MSI-High) cancer.

[0281] §39. The WRN inhibitor for use, the combination for use, the method, or the ATR inhibitor for use according to any one of the preceding clauses, wherein the cancer is colorectal cancer, gastric cancer, endometrial cancer, ovarian cancer, hepatobiliary tract cancer, urinary tract cancer, brain cancer, or skin cancer.

[0282] §39. The WRN inhibitor for use, the combination for use, the method, or the ATR inhibitor for use according to clause §38, wherein the cancer is colorectal cancer, gastric cancer, endometrial cancer, or ovarian cancer.

[0283] Examples

[0284] Materials and Methods

[0285] Cell models

[0286] The cell lines used for the experiments described herein were sourced from the Genomics of Drug Sensitivity 1000 cell line collection and are listed in the Cell Model Passports database (https: / / cellmodelpassports.sanger.ac.uk / ) (van der Meer et al. 2019). Doxycycline inducible WRN sgRNA-expressing cells (di-WRN) were established as described in Behan et al., 2019. Briefly, the inventors cloned WRN sgRNA 4 (SEQ ID NO: 1) into the pRSGT16H-U6Tet-(sg)-CMV-TetRep-TagRFP-2A-Hygro vector (Cellecta) and transduced Cas9-expressing HCT116, RKO or SW48 cells, followed by selection with 500 pg / ml hygromycin (Thermo Fisher Scientific). Single-cell clones were generated via serial dilution to ensure uniform Cas9 expression and inducible WRN-targeting sgRNA. For growth rate measurement after conditional WRN knockout induction, cells were grown in flasks with or without 2 pg / ml doxycycline for 24 hours, then seeded in 96-well plates with the same doxycycline conditions. Cell growth was monitored every 6 hours using an automated IncuCyte-FLR 4X phase-contrast microscope (Essen Instruments), and average object-summed intensity was calculated with IncuCyte software. Cas9 activity was assessed as described in Tzelepis et al. 2016.

[0287] All cell lines were maintained under their original culture conditions according to supplier guidelines, with supplementation of 10% FBS, 2 mM L-glutamine, and antibiotics (100 U / mL penicillin and 100 mg / mL streptomycin) at 37 °C in a 5% CO2 incubator. Mycoplasma testing was conducted using complementary methods (MycoAlert, Lonza; EZ-PCR, Biological Industries), confirming that all cell models were Mycoplasma-free. Cell line authentication was performed using a panel of 94 single-nucleotide polymorphisms (SNPs) on a Fluidigm 96.96 Dynamic Array IFC, requiring at least a 75% match to the reference profile for positive authentication. Short tandem repeat (STR) profiles were also compared with those from cell line repositories. Cell models were kept in culture for an average of 36 days (up to 60 days), and all experiments were conducted within this timeframe.

[0288] Compounds, drug screening and dose-response curve fitting

[0289] DMSO-solubilized compounds were stored at room temperature in low humidity (<12% relative humidity) and low oxygen (<2.5%) environments using storage pods (Roylan Developments). Screening of cancer cell lines and organoids was performed using single treatments and combinations. Compounds were screened at twelve concentrations spanning a 2,048-fold range with a 2-fold dilution series. Cells were transferred into 384-well assay plates in 40 pl (cell lines) or 60 pl (organoids) of their respective growth medium using Multidrop Combi (Thermo Fisher Scientific) dispensers. Six cell densities were tested with a two-fold dilution step; each density was dispensed into 48 wells of a single 384-well assay plate and incubated for 96 h. Cell number was quantified using CellTiter-Glo 2.0 (Promega). The maximum density tested was 3200 cells per well. Assay plates were incubated at 37 °C in a humidified atmosphere at 5% CO2 for 24 hours and then dosed with the test compounds using an Echo555 (Labcyte). The final DMSO concentration was typically 0.1%. The assay plates were incubated after compound dosing, and the drug treatment duration was 72 h. CellTiter-Glo 2.0 (Promega) was added, 13.5 pl for cell lines or 20 pl for organoids, to measure cell viability. Each assay plate was incubated at room temperature for 10 min before quantification of luminescence using a Paradigm (Molecular Devices) plate reader. To estimate cell growth throughout drug treatment, an additional undrugged control plate was generated, and cell viability was measured at the time of drug treatment. These plates are referred to as a ‘day = 1 ’ and were repeated each time a cell line was screened. All screening plates contained negative control wells (untreated wells, n = 6; DMSO-treated wells, n = 62) and positive control wells (medium-only wells, n = 12; Staurosporine-treated wells, n = 8; and MG-132 treated wells, n = 8) distributed across the plates. These control wells were used to evaluate defined quality control criteria, including the coefficient of variation (CV) and Z-factor calculated as described in Jaaks et al., 2022. A maximum threshold of 0.23 was applied to the coefficient of variation (CV), and Z-factors were required to exceed a minimum threshold of 0.3. Where a cell line was sensitive to both positive controls, it had to pass Z-factor thresholds for both positive controls. Plates that did not meet these requirements were excluded from the study.

[0290] Luminescence readings were converted to cell viabilities by normalizing with reference to the DMSO-treated wells and the positive controls (viabilities of 1 and 0 respectively). Dose response curves were then fitted to the drug-treated wells using a non-linear mixed effect model described in (Vis et al. 2016) to obtain IC50 estimates. Curves with a root mean squared error of greater than 0.3 were excluded from further analysis.

[0291] Gene Editing

[0292] Base editing experiments were performed as described in Picco et al. 2024. Briefly, base editing machinery was introduced into cells through co-transfection with FuGENE HD (Promega), utilizing a plasmid encoding Cas9 and a gRNA targeting the CLYBL locus (5'-ATGTTGGAAGGATGAGGAAA-3'; SEQ ID NO: 2). This was paired with a plasmid containing a tet-ON base editor, blasticidin resistance, and mApple expression cassettes within CLYBL homology arms, following the method described in Picco et al., 2024. To boost homologous recombination (HR) rates, cells were pre-treated overnight with 1 pM DNA-PK inhibitor AZD7648. Post-transfection, cells were selected with 10 pg / mL blasticidin (Thermo Fisher Scientific) for four days, then maintained with 5 pg / mL. Cell pools were further refined via FACS for mApple expression. Base editing efficiency was assessed using BE-FLARE (Coelho et al., 2018). Clonal lines of BE3.9max NGN and ABE8e NGN were employed for detailed screening.

[0293] To genetically inactivate TP53, SMARCAL1 , SUPT7L, MUS81 and TADA1 , di-WRN or cas9-expressing cell lines were transduced overnight in the presence of polybrene (8 pg / mL) with lentiviral constructs containing sgRNAs against TP53 (SEQ ID Nos: 3 and 4), SMARCAL1 (SEQ ID Nos: 5 and 6), MUS81 (SEQ ID Nos: 7 and 8), SUPT7L (SEQ ID Nos: 9 and 10), and TADA1 (SEQ ID Nos: 11 and 12). Medium was refreshed with fresh complete medium the following day, and cells were treated with blasticidin (20 pg / mL) and puromycin (2 pg / mL, Thermo Fisher Scientific, A1113803), hygromycin or neomycin, depending on the respective selective agent, to select for Cas9-expressing cells carrying the sgRNAs.

[0294] RNA Interference-Based Sensitivity Assay

[0295] Approximately 1.5-3.5 x 103cells were seeded per well in a 96-well plate, followed by reverse transfection with ON-TARGETplus siRNA at a final concentration of 20 nmol / L, using RNAiMAX (Invitrogen) according to the manufacturer's protocol. Each experiment included controls: transfection reagent only (mock control), a nontargeting pool (negative control, Dharmacon, D-001810-10-05), a polo-like kinase 1 (PLK1) pool (positive control, Dharmacon, L-003290-00-0010), and a WRN-targeting pool (Dharmacon, L-010378-00-0005). The siRNA sequences were: nontargeting (SEQ ID Nos: 13-16), PLK1 (SEQ ID Nos: 17-20), and WRN (SEQ ID Nos: 21-24). Cells were cultured for 5-7 days, and viability was measured using the CellTiter-Glo 2.0 Assay (Promega, G9241).

[0296] Western blot

[0297] Western blotting confirmed WRN depletion and assessed DNA damage in cells treated with varying concentrations of multiple WRN inhibitors in different drug-resistant models. For the blots, 4-8 x 1OA6 cells were cultured in 10 cm dishes and treated with either 1uM of GSK_WRN3, 0.5uM of HRO761 or DMSO. Cells were lysed 48 hours post-treatment using 100-150 pL of RIPA buffer containing protease inhibitors. Lysate concentrations were determined using the BCA Assay. Each sample, containing 20-30 pg of lysate, was run on a 4-12% Bis-Tris gel (Invitrogen) and transferred to a PVDF membrane.

[0298] Membranes were blocked with 5% milk in TBST and incubated overnight with primary antibodies: anti-WRN (ABcam , AB124673, 1:1000), anti-P-Histone H2AX (NB100-384, 1:1000), and anti-p-tubulin (Sigma-Aldrich, T4026, 1 :5000) as a loading control. Blots were washed and incubated with an antiRabbit IgG HRP-linked secondary antibody (GE Healthcare, #NA931 V-ECL HPR) for 1 hour at room temperature. After a final wash in TBST, signals were detected using Super Signal Dura. Precision All Blue Plus Protein Standards (BioRad, cat. 1610373) were used as molecular weight markers.

[0299] Mouse Xenograft Studies

[0300] In vivo experiments with inducible di-WRN HCT116 were performed as described in Behan et al. 2019. Female NOD / SCID mice (Charles River Laboratories) were used for all in vivo studies. Mice were maintained in hyperventilated, pathogen-free conditions in individually sterilised cages with up to seven mice per cage and provided with sterilised food, water, and bedding. Inducible WRN sgRNA-expressing HCT 116 cells xenografts were established by subcutaneously injecting 2 x 1 oA6 cells into the right posterior flank of 5- to 6-week-old mice. Tumor size was measured with calipers, and volume was calculated using the formula 4 / 3TT X (d / 2)A2 x (D / 2), where d is the minor axis, and D is the major axis. When tumors averaged 250-300 mmA3, animals were randomized by tumor size. Doxycycline (Sigma-Aldrich, D9891) was administered daily by oral gavage at 50 mg / kg. Each experimental group consisted of 8-10 mice to estimate within-group variability. Allocation to treatment groups was done during randomization, with measurements taken blind. The maximum tumor volume allowed was 3,500 mmA3, which was not exceeded. In vivo procedures and data were managed using the Laboratory Assistant Suite for automated data tracking.

[0301] For the xenografting process, 5 x 106 cells each of SW48 were mixed in a 1:1 ratio with RPMI and Matrigel (100 pL) and then xenografted into the right flank of Crl:Nu-Foxn1 nu (Strain, #088, Charles River Laboratory) female mice, aged 6 to 8 weeks and weighing 20 to 25 g. Once the tumors reached approximately 80 to 140 mm3, animals were randomly assigned into groups based on individual tumor size. Body weight and tumor volume were measured 2 times per week using an electric caliper (Fowler Ultra-Cal V) and an Ohaus electronic scale (STX421), with data automatically recorded in the Study Log. Tumor volume was calculated using the formula 0.5 x L x W-2, in which L is the tumor length (the longest dimension) and Wis the tumor width (perpendicular to L). Tumor growth inhibition was calculated using the equation Mean % A Inhibition = [(mean (C)-mean (C0))-(mean (T)-mean (T0))] / [mean (C)-mean (CO)] x 100%. For acquired resistance studies, tumors were collected at different time point 24 hours after the final dose and snap-freezing. Growth curves were visualized using Prism GraphPad Prism 9 (GraphPad Software, Inc.). All data are presented as mean ± standard error. For pharmacokinetic studies, HRO761 was orally administered in the male BALB / c mouse as a suspension formulation. Two mice were used for the pharmacokinetic study underfed conditions. The compound was formulated in 1% aqueous methyl cellulose, in a white fluid suspension and administered through oral gavage.

[0302] Immunohistochemistry

[0303] Formalin-fixed, paraffin-embedded tissues from xenografts were sectioned (10-pm thick), with 4-pm sections dried overnight at 37 °C. Slides were de paraffinized in xylene and rehydrated through graded alcohol to water. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 30 min. Antigen retrieval was performed in 10 mmol / l citrate buffer (pH 6.0) using a microwave (750 W for 10 min). Slides were incubated with monoclonal mouse anti-human KI-67 (1:100; DAKO) or polyclonal WRN antibody NBP1 -31895(1 : 100, Novus) overnight at 4 °C. After washing in TBS, slides were treated with anti-mouse secondary antibody (DAKO Envision+System HRP-labelled polymer) for 1 h at room temperature.

[0304] Immunoreactivity was detected using DAB chromogen (DakoCytomation Liquid DAB Substrate Chromogen System, DAKO) for 10 min. Slides were counterstained with Mayer’s haematoxylin, dehydrated, cleared in xylene, and mounted with DPX (Sigma-Aldrich). Negative controls omitted the primary antibody. Stained slides were scanned at 40x magnification. For ki-67, ten images from three cases were analyzed with Imaged (NIH) to segment cells with positive and negative nuclei. The percentage of positively stained cells was calculated, verified by visual inspection for accuracy.

[0305] TrAEL-seg for DSB mapping

[0306] For TrAEL-seq, 2-3 million SW48 cells were seeded in T150 flasks. Two days later, these cells were treated with GSK_WRN3 or HRO761 for 24h. Cells were harvested and counted 24 hours post-treatment.

[0307] 1-2 x 106cells per treatment were washed with PBS, centrifuged, and resuspended in 1mL of cold L Buffer (100mM EDTA pH8, 10mM Tris pH 7.5, 20mM NaCI), before embedding in agarose for DNA extraction. DNA ends were tailed with ATP and ligated to adaptors carrying in-line indexes, then pooled and processed into a single TrAEL-seq library as described in Kara et al., 2021. Libraries were sequenced on an Illumina NextSeq 500 as High Output 75 bp Single End, then read were trimmed, deduplicated, de-multiplexed into individual libraries and mapped to GRCh38 using scripts available at https: / / github.com / FelixKrueger / TrAEL-seq.

[0308] Analysis of TrAEL-seq data

[0309] Mapped reads were imported into SeqMonk v1.48 (https: / / www.bioinformatics.babraham.ac.uk / projects / seqmonk / ) and truncated to 1 nucleotide at the 5' end, representing the last nucleotide 5' of the strand break. Peak finding was performed in each sample using MACS implemented in SeqMonk (macs2 --nomodel --tsize 100 --pvalue 1e-15 --keep-dup), and distances from MACS peaks to nearest TA tract or random sites calculated using Distance to Feature Quantitation. For the metaplot, strand specific read counts were performed at 1 nt resolution over TA tracts defined in van Wietmarschen et al. 2020 - / + 100 bp using the Quantitation Trend Plot function. Final graphs were plotted using GraphPad Prism 10.1.1.

[0310] Data analysis

[0311] The Jaccard Index was calculated to assess the similarity between each sample's copy number profile and the parental model. For each sample, the number of altered regions (amplification, deletion, loss, soft gain) was compared to the neutral regions. The Jaccard Index was computed as the ratio of the number of shared neutral regions (intersection) to the total number of regions (union of altered and neutral). A higher Jaccard Index indicates greater similarity to the parental model. The plots and graphs were generated using GraphPad and Spotfire software.

[0312] EXAMPLE 1

[0313] MSI Cancer Cells Lack Evolutionary Paths to Bypass WRN Genetic Inhibition

[0314] In an HCT116 CRC MSI xenograft model with doxycycline-inducible CRISPR-mediated WRN knockdown (di-WRN), the inventors showed striking xenograft growth suppression upon WRN inactivation, highlighting its critical role. However, this anti-tumour effect was transient, with tumour regrowth after ~25 days (Fig. 1 and Fig. 31). At relapse, resistant tumours universally re-expressed WRN and Ki67, indicating that WRN-positive cells drove tumour regrowth (Fig. 2-3 and Fig. 32-33).

[0315] To evaluate resistance to WRN synthetic-lethality, the inventors used doxycycline inducible WRN sgRNA-expressing (di-WRN) colorectal cancer (CRC) microsatellite instability (MSI) cell lines, either maintaining parental heterogeneity or isolating efficient inducible WRN knockout clones, that is, after transduction with the plasmid carrying the doxycycline-inducible sgRNA, some experiments were conducted on the bulk cell population, while others focused on specific clones with high KO efficacy after doxycycline administration (Fig. 34-36). After initial growth inhibition, resistant subpopulations emerged within weeks across all models and conditions. HCT116 di-WRN bulk cells developed resistance rapidly (7 days), losing Cas9 activity and re-expressing WRN (Fig. 37-39). HCT116 di-WRN and SW48 di-WRN subclonal lines initially retained Cas9 activity, suggesting cloning prevents rapid selection of a population with low-Cas9 activity. The doxycycline- resista nt SW48 subclones died when exposed to a different WRN-targeting sgRNA. At the same time, HCT116 produced resistant cells with reduced Cas9 activity, indicating persistent WRN dependence and strong selection pressure (Fig. 39). All models remained sensitive to WRN knockdown with siRNA (Fig. 40). Ultimately, resistance in these models arose from inherent technical limitations of the CRISPR / Cas9 knockout system that led to WRN re-expression. To further investigate genetic mechanisms of resistance, the inventors performed genome-wide CRISPR / Cas9 genetic screens in three MSI models (HCT116, SW48 and RKO) using our WRN knockout (KO) doxy-inducible system (Fig. 4-5). With varying degrees of selection intensity (Fig. 4 and Fig. 41-43), the only consistent hits enriched in all models were knockouts of SAGA transcriptional coactivator complex members TADA1, SUPT3H, TAF6L and SUPT7L (Fig. 6). However, validation studies indicated these were technical artefacts rather than bona fide bypasses, as resistance in SAGA member knockouts was due to blocking WRN sgRNA expression and efficient KO (Fig. 44-45), consistent with the SAGA complex being crucial for RNA polymerase II transcription (Antonova et al. 2018). The TP53-targeting sgRNAs were depleted rather than enriched in our screens (Fig. 47), highlighting the limited predictive power of TP53 mutations in rescuing WRN dependency (Zong et al. 2023; Picco et al. 2024). Similarly, although MUS81 has been linked to DSB formation at TAs in the absence of WRN (van Wietmarschen et al. 2020), MUS81 -targeting sgRNA were not enriched in our screen (Fig. 47) and MUS81 KO did not affect sensitivity to WRNi (Fig. 48-49).

[0316] In summary, extensive efforts across multiple cell line models did not identify genetic bypass mechanisms that confer resistance to WRN synthetic lethality in MSI cancer cells. This is consistent with WRN having a functionally non-redundant role in MSI cells.

[0317] EXAMPLE 2

[0318] Pharmacogenomic Screens Identify Modulators of WRNi Sensitivity

[0319] To identify modulators of sensitivity to WRN inhibitors (WRNi), whole-genome CRISPR / Cas9-based chemogenomic dropout screens in SW48 cells treated with GSK_WRN3, a covalent WRN inhibitor (Picco et al. 2024) (Fig. 7). No major sensitivity enhancers were identified, likely due to the high inherent sensitivity to WRNi in MSI cells. However, DNA repair, G2M checkpoint and PI3K / AKT / MTOR pathway components were significantly enriched among genes with negative fold changes (NES = -1.58, FDR q-val = 0.043; NES = -1.77, FDR q-val = 0.013; NES = -1.40, FDR q-val = 0.21 , respectively) (Fig. 50). This suggests these pathways act as adaptive response mechanisms to mitigate the effect of WRNi treatment in MSI cells, and their inhibition reduces cell viability. Conversely, several sgRNAs were enriched following WRNi treatment, indicating potential resistance mechanisms (Fig. 7). The top hit,

[0320] C1orf115 / RDD1 , is a regulator of ABCB1 localisation and drug efflux (Masud et al. 2022) and is associated with resistance to cancer drugs (Lau et al. 2020), suggesting that it may confer resistance by enhancing WRN inhibitor efflux. Several double-strand break repair pathway members, including the MRN complex (MRE11 , RAD50, NBN) and ATM, emerged as significant hits, with the ATM pathway also showing enrichment (NES = 2.25, FDR q-val = 0.001) (Fig. 50). In addition, HELLS, a chromatin remodeler indirectly supporting double-strand break repair (Kollarovic et al. 2020), was identified among the top hits.

[0321] Amongst the strongest resistance genes in the study was SMARCAL1 , a DNA annealing helicase crucial for replication fork maintenance and DNA damage response. Clonogenic assays performed in two independent MSI cancer cell lines with SMARCAL1 knockout confirmed a modest resistance effect, showing a small (~2-fold) reduction in sensitivity to both GSK_WRN3 and HRO761 (Fig. 8-9).

[0322] SMARCAL1 has been reported to suppress DSBs formation at TA-rich repeats (Fielden et al. 2023; Feng et al. 2024). Thus, to gain mechanistic insights into the role of SMARCAL1 in mediating WRNi sensitivity, the inventors compared DSB signal at TA-repeat expansions using TrAEL-seq in SMARCAL1 KO and parental SW48 cells after acute (24-hour) treatment with GSK_WRN3 and HRO761 (Picco et al. 2024; Kara et al. 2021). In parental lines, a strong DSB signal localised to TAs was detected after treatment with both WRN inhibitors, whereas this signal was reduced approximately 2-fold in the absence of SMARCAL1 (Fig. 10-11 and Fig. 51). However, SMARCAL1 knockout cells retained sensitivity in additional viability assays, consistent with SMARCAL1 having a significant but modest suppressor effect (Fig. 52). This acute reduction in DSB accumulation indicates a role for SMARCAL1 in modulating DSB accumulation at TA dinucleotide repeats following WRN pharmacologic inhibition and provides a rationale for the modest but reproducible resistance effect observed. Notably, no identified genes conferred robust resistance to WRN inhibition, consistent with previous WRN genetic knockout data.

[0323] EXAMPLE 3

[0324] Base Editing Screens Identify On-target WRN Mutations Conferring Drug Resistance

[0325] To take an unbiased and systematic approach to identifying on-target resistance variants, the inventors performed a base editing semi-saturation mutagenesis approach to identify residues on the WRN protein required for WRN-MSI synthetic lethal interaction. Clones of the colorectal KM12 and endometrial RL95-2 MSI lines, engineered to express adenine base editor (ABE) or cytidine base editor (CBE), were transduced with a library of 3,735 sgRNAs predicted to mutagenise ~75% of WRN amino acid positions. Both lines were treated with GSK-WRN3 to assess if induced mutations modulate sensitivity to WRN inhibitors, with an additional screen in KM12 cells using HRO761 (Fig. 54).

[0326] The correlation between replicate screens was high, with R values ranging from 0.79 to 0.97 across CBE and ABE screens in KM12 and RL95-2 models (Fig. 55). Very few sgRNAs were differentially depleted in treated versus untreated conditions, indicating that no mutations strongly sensitized MSI cells to WRN inhibitors (Fig. 12).

[0327] Strikingly, sgRNAs predicted to induce mutations in Cys727 and nearby residues (residues 725, 729) showed the strongest enrichment after drug selection with both WRN inhibitors across both models (Fig.

[0328] 12). WRN inhibitors (GSK-WRN3, HRO761) failed to degrade WRN in BE-engineered KM12 and RL95 isogenic cells carrying a mutation at Cys727, consistent with their observed resistance (Fig. 56-57).

[0329] Additional sgRNAs showed enrichment across both screens. The WRN-GSK screen also revealed enrichment of sgRNAs installing mutations at positions 521 and 852, while the HRO761 screen showed enrichment of sgRNAs 729, 731 , 852, 530, and 975.

[0330] In summary, the semi-saturation mutagenesis approach validated mutations in Cys727 and neighbouring residues near the drug binding site of WRN inhibitors as potentially conferring resistance and identified additional critical residues that may also be involved in resistance. EXAMPLE 4

[0331] MSI Cancer Cells Develop WRNi Resistance Through On-Target Mutations In Vitro and In Vivo

[0332] To identify on- and off-target resistance mechanisms developed in response to treatment of MSI cancer cells with WRN inhibitors, the inventors performed an in vitro time-to-progression (TTP) assay, treating SW48 colorectal cancer cell lines with the WRN inhibitors GSK_WRN3 and HRO761 , to model how dMMR cancer cells evolve resistance to WRN inhibition.

[0333] SW48 cells rapidly acquired resistance to the WRN inhibitors (Fig. 13-15 and Fig. 58-59). Similar patterns were observed in additional cancer cell lines from colorectal (KM12), endometrial (RL95-2, OER2) and stomach (IM-95) cancers (Fig. 60), indicating that MSI cancer cells, regardless of their tissue of origin, possess an intrinsic ability to rapidly evolve resistance to WRN pharmacologic inhibition.

[0334] Resistant cells were analyzed using whole-genome sequencing (WGS). The inventors found that WRN inhibitor (WRNi) resistant cells maintained similar mutational load without significant changes in global copy number, with Jaccard Index values of 0.978 and 0.999, respectively. However, several hundred to thousands of novel mutations were detected in the resistant cells, likely reflecting the expansion of subclonal populations. Selection fora subpopulation of cells not presenting expanded TA repeats has been proposed as a potential mechanism of acquired resistance to WRNi (Morales-Juarez and Jackson 2022). To test this hypothesis, the inventors inferred the length of TA repeat expansions in WRNi-resistant cells and found no difference compared to the parental unselected counterparts (Fig. 16), suggesting that heterogeneity in TA repeat expansions was not a major determinant in the emergence of acquired resistance.

[0335] Notably, sensitivity to WRN genetic inactivation by RNAi persists in WRNi-resistant SW48 cells, indicating continued reliance on WRN and suggesting that WRNi has lost its ability to inhibit WRN function and suppress cancer cell growth (Fig. 61). Indeed, WRNi could no longer downregulate WRN protein, suggesting a binding deficiency (Fig. 17).

[0336] The inventors identified a missense mutation (I852F) in the WRN gene in GSK_WRN3-resistant SW48 cells and an exon-truncating splice site mutation (1557-1 G>C) in HRO761 -resistant SW48 cells, both absent in the parental line. Additionally, recurrent somatic WRN mutations (G729D, F730L, F730S, G729S) were found in other resistant cell models, sitting adjacent to the reactive cysteine at position 727 involved in drug binding. Despite thousands of sample-specific mutations detected in resistant cells, the WRN gene was the only gene recurrently mutated across all models. The exon-truncating splice site mutation (1557-1 G>C; chr8:31088889) is notable as it generates a cryptic splice site, causing partial truncation of exon 13 up to position 31088904, and producing an aberrant WRN protein with a portion of the exon excluded (Fig. 18). Examination of non-exonic WRN-targeting sgRNAs in the base editing screen (Example 3) revealed a sgRNAs editing near position 31088889 was the sole resistance significant hit in HRO761 -treated KM12 cells (Fig. 19). This finding confirms that engineering this variant engenders HRO761 resistance, aligning with its spontaneous emergence in resistant cells. The inventors additionally assessed resistance mechanisms to prolonged drug treatment in vivo. When SW48 MSI tumor xenografts were treated long-term with two doses of HRO761 , after initial regression, a few tumors rebounded and resumed growth (Fig. 62). Deep-targeted sequencing of tumor explants from relapsed mice revealed I852F and G729S mutations in WRN, identical to those identified in WRNi-resistant cells in vitro.

[0337] These results indicate that MSI cells from different lineages follow a consistent evolutionary trajectory both in vitro and in vivo, rapidly developing resistance to WRN inhibitors through recurrent, on-target mutations in the WRN ATPase domain that disrupt WRNi binding (Fig. 21-22).

[0338] To further explore strategies for overcoming WRNi resistance, the inventors established additional MSI cancer cell line models under alternative WRN inhibitor treatments. In particular, SW48 and KM12 cells were continuously treated with the second WRN inhibitor, VVD-133214, either alone or in combination with HRO761. Despite the distinct mechanisms of these inhibitors, both treatment regimens led to the rapid emergence of drug-resistant cell populations, mirroring the dynamics observed with single-agent HRO761 in Example 4. As shown in Fig. 63, SW48 and KM12 cells acquired resistance under VVD-133214 monotherapy as well as dual therapy (VVD-133214 plus HRO761). Whole-genome sequencing of the resistant clones confirmed on-target WRN mutations enriched in each case (including a representative G1207D mutation in a VVD-133214-resistant KM12 clone), consistent with a shared mechanism of resistance through alterations in the WRN gene (Fig. 63).

[0339] EXAMPLE 5

[0340] MSI Cells with WRN Resistance Mutations Retain Sensitivity to Alternative WRNi

[0341] Two WRN helicase inhibitors, HRO761 and VVD-133214, have advanced to Phase I clinical trials and are actively recruiting patients (Clinical Trial Identifier: NCT06004245; VVD-133215 / RO7589831 ; Clinical Trial Identifier NCT05838768; HRO761). Each disrupt WRN helicase activity in MSI cancer cells through distinct mechanisms: HRO761 stabilizes WRN in an open conformation, preventing DNA binding; instead, VVD-133214 irreversibly locks WRN in a closed, inactive state, inhibiting ATPase function (Fig.23).

[0342] The inventors performed molecular modelling to predict the effects of these resistance variants on the binding of these two WRN inhibitors. The WRN G729D mutation is positioned within the hinge region (residues 728-732) of the protein. The substitution of glycine with a negatively charged, larger aspartate alters the hinge's structure and dynamics. Molecular modelling and local energy minimization (orange ribbon) revealed significant rearrangement of residues compared to the original structure, suggesting that this alteration disrupts the architecture of the binding pocket, likely impacting WRN’s ligand binding capacity for both HRO761 and VVD-133214 (Fig. 24). Interestingly, the inventors observed that recurrent I852F mutation identified in resistant models creates a protruding, smaller hydrophobic cage that hinders HRO761 binding, while not predicted to affect the binding pocket of VVD-133214 (Fig. 25).

[0343] To test this hypothesis, the inventors profiled both parental and WRNi-resistant MSI cells against both WRN inhibitors. In addition, the inventors tested an ATR inhibitor (ceralasertib; AZD6738), and irinotecan. The inventors found that WRN mutations emerging in inhibitor-resistant cells did not alter sensitivity to ATR inhibitors, or chemotherapy (irinotecan) (Fig. 26). HRO761 -resistant SW48 cells carrying mutations in positions G729 / F730 mutation exhibited cross- resista nee to VVD-133214, while, interestingly, those with the I852F mutation showed resistance to HRO761 , but retained notable sensitivity to VVD-133214 (Fig. 26).

[0344] WRNi resistant cell lines carrying 1557-1 G>C mutation also displayed sensitivity to VVD-133214, suggesting that this alteration may also not affect binding and activity of VVD-133214 (Fig. 26). These observations were confirmed by independent viability assays in these resistant cell line models (Fig. 27). Accordingly, WRN degradation and induction of DNA damage was retained in WRNi resistant models carrying 1557-1 G>C and I852F mutations when treated with VVD-133214 (Fig. 28).

[0345] To assess the cross-sensitivity of additional resistant lines to different WRN inhibitors, the inventors performed comparative viability assays. Fig. 64 illustrates that KM12 cells with acquired resistance to VVD-133214 (alone or in combination with HRO761) remained sensitive to HRO761 itself and to a third WRN inhibitor, GSK_WRN5. In particular, the KM12 line harboring the WRN G1207D mutation retained sensitivity to HRO761 , and both the single-agent and dual WRNi-resistant KM12 cells responded to GSK_WRN5 comparably to parental cells (Fig. 64). In contrast, SW48 cells that developed resistance to VVD-133214 displayed reduced sensitivity to both HRO761 and VVD-133214, indicating a degree of cross- resista nee in this background (Fig. 65). Notably, however, treatment with GSK_WRN5 still inhibited the growth of the VVD-133214-resistant SW48 cells (Fig. 65), demonstrating that this alternative WRN inhibitor retains substantial activity even against cells carrying resistance mutations that compromise HRO761 and VVD-133214 efficacy.

[0346] Confirmatory in vitro colony formation assays were conducted on the resistant SW48 line to visualize the differential effectiveness of the WRN inhibitors. As shown in Fig. 66, VVD-133214-resistant SW48 cells, carrying a WRN F730S mutation, formed robust colonies despite continued treatment with HRO761 (250 nM) or VVD-133214 (250 nM), whereas exposure to GSK_WRN5 at the same concentration completely abrogated colony formation. These findings reinforce that the resistant cells had lost responsiveness to the first-generation WRN inhibitors, yet remained vulnerable to inhibition by GSK_WRN5.

[0347] Additionally, the inventors validated their findings using a patient-derived organoid (PDO) derived from a patient with sporadic MSI colorectal cancer (CRC-14a)(Picco et al. 2021). The PDOs were treated with HRO761 in vitro, and resistance rapidly emerged (Fig. 29). Whole-genome sequencing of the resistant PDOs revealed the splice mutation (1557-2 A>T), affecting the same codon and inducing the same splice alteration previously observed in cell lines, further supporting the role of these on-target mutations in HRO761 resistance in a more clinically relevant context. Remarkably, HRO761 -resistant organoids maintained exquisite sensitivity to VVD-133214 (Fig. 30).

[0348] Finally, it was evaluated whether switching to an alternative WRN inhibitor could overcome established resistance in an in vivo setting. SW48 MSI tumor xenografts that had developed resistance to HRO761 (following prolonged treatment at 60 mg / kg) were divided into cohorts receiving a secondary WRN inhibitor. Strikingly, switching treatment to GSK_WRN5 (100 mg / kg, twice daily) induced renewed tumor regression in HRO761 -resistant tumors (Fig. 67). In contrast, a parallel group of HRO761 -resistant xenografts switched to VVD-133214 (100 mg / kg, once daily) showed no tumor regression (Fig. 68).

[0349] These results indicate that tumors harboring on-target WRN mutations which confer resistance to HRO761 can still respond to a mechanistically distinct WRN inhibitor like GSK_WRN5, whereas a second inhibitor with an overlapping resistance profile (such as VVD-133214 in the case of certain mutations) may not be effective. Together, the data from these additional studies demonstrate that utilizing multiple WRN inhibitors — either concurrently or sequentially — can, in certain cases, overcome on-target resistance or prolong the efficacy of WRN-targeted therapy. In particular, the observed regression of HRO761 -resistant tumors upon introduction of GSK_WRN5 (Fig. 67) highlights the potential of nextgeneration WRN inhibitors to counteract acquired resistance that renders first-line inhibitors ineffective (compare Fig. 68). Accordingly, combination or sequential treatment strategies employing mechanistically distinct WRN inhibitors may help to overcome or delay resistance development in MSI cancers. These findings further suggest that identifying the specific WRN mutation(s) present in resistant tumors can inform the selection of an alternative WRN inhibitor most likely to remain effective, thereby guiding a precision medicine approach to managing WRNi resistance.

[0350] Together, these findings suggest that specific WRN mutations may serve as predictive biomarkers for selecting alternative WRN inhibitors, and combination strategies targeting multiple pathways or combining multiple WRN inhibitors may help overcome or delay resistance development.

[0351] References

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Claims

Claims:

1. A method of treating a subject with cancer, wherein the subject has received VVD-144214 and / or HRO761 , further wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

2. A method of treating a subject with a cancer with a mutated form of the WRN protein, wherein the subject has received VVD-133214 and / or HRO761 , wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

3. A method of treating a subject with a cancer with a mutated form of the WRN protein comprising a mutation at any one or more of position 709, position 730, position 1207, and / or position 1209 in SEQ ID NO: 25, wherein the subject has received VVD-133214 and / or HRO761 , wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

4. A method of treating a subject with cancer, wherein the subject is resistant to treatment with VVD-144214 and / or HRO761 , further wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

5. A method of treating a subject with a cancer with a mutated form of the WRN protein, wherein the subject is resistant to treatment with VVD-133214 and / or HRO761 , wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

6. A method of treating a subject with a cancer with a mutated form of the WRN protein comprising a mutation at any one or more of position 709, position 730, position 1207, and / or position 1209 in SEQ ID NO: 25, wherein the cancer is considered resistant to treatment with VVD-133214, wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

7. A method of treating a subject with a cancer with a mutated form of the WRN protein comprising a mutation at any one or more of position 709, position 730, position 1207, and / or position 1209 in SEQ ID NO: 25, wherein the cancer is considered resistant to treatment with HRO761 , wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

8. A method of treating a subject with a cancer with a mutated form of the WRN protein comprising a mutation at any one or more of position 709, position 730, position 1207, and / or position 1209 in SEQ ID NO: 25, wherein the subject is resistant to treatment with VVD-133214 and HRO761 , wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

9. A method of treating a subject with a cancer with a mutated form of the WRN protein, wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

10. The method of claim 9 wherein the mutated form of the WRN protein comprises a mutation at one or more of the following positions of the amino acid sequence set out in SEQ ID NO: 25: position 709; position 730; position 1207; and / or position 1209.

11. A method of treating a subject with a cancer, wherein the subject was previously treated with VVD-133214 and / or HRO761 , wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

12. A method of treating a subject with a cancer with a mutated form of the WRN protein, wherein the subject was previously treated with VVD-133214 and / or HRO761 , wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

13. A method of treating a subject with a cancer with a mutated form of the WRN protein comprising a mutation at any one or more of position 709, position 730, position 1207, and / or position 1209 in SEQ ID NO: 25, wherein the subject was previously treated with VVD-133214 and / or HRO761 , wherein the method of treating comprises administering to the subject an effective amount of GSK_WRN5.

14. A method of any one of claims 1 - 13, wherein the cancer is microsatellite instability - high cancer.

15. A method of treating cancer in a subject, the method comprising: (a) selecting a subject who has received VVD-133214 and / or HRO761 ; and (b) administering an effective amount of GSK_WRN5.

16. A method of treating a subject with cancer, the method comprising: (a) selecting a subject who has received VVD-133214 and / or HRO761 and is resistant to therapy with VVD-133214 and / or HRO761; and (b) administering an effective amount of GSK_WRN5.

17. A method of treating cancer in a subject, the method comprising: (a) selecting a subject who has received VVD-133214 and / or HRO761 and who has a mutated form of WRN protein; and (b) administering an effective amount of GSK_WRN5.

18. A method of treating cancer in a subject, the method comprising: (a) selecting a subject who has received VVD-133214 and / or HRO761 and who has a mutated form of WRN protein comprising a mutation at any one or more of position 709, position 730, position 1207, and / or position 1209 in SEQ ID NO: 25; and (b) administering an effective amount of GSK_WRN5.

19. The method of any one of claims 1 - 18 wherein the cancer is endometrial cancer or colorectal cancer.

20. A method of any one of claims 2, 6 to 8, 13 to 14 or 17, wherein the mutated form of the WRN protein comprises a mutation at one or more of the following positions of the amino acid sequence in SEQ ID NO: 25:position 709, optionally wherein the mutation is S709P;position 730, optionally wherein the mutation is F730L or F730S;position 1207, optionally wherein the mutation is G1207D; and / orposition 1209, optionally wherein the mutation is A1209D.

21. A method of any one of claims 2, 9, 10, 12, 14 or 18 wherein the mutated form of the WRN protein comprises one or more mutation selected the group consisting of S709P; F730L or F730S, G1207D, A1209D, and combinations thereof.

22. A method of treating a WRN-mutant cancer in a human patient the method comprising administering an effective amount of GSK_WRN5.

23. The method of claim 22 wherein the human patient has been previously treated with an inhibitor of WRN that is not GSK_WRN5.

24. The method of claim 23 wherein the inhibitor of WRN that is not GSK_WRN5 is VVD-133214, HRO716, ora combination thereof.

25. The method of any one of claims 22 to 24 wherein the WRN-mutant cancer affects positions of the amino acid sequence of SEQ ID NO: 25 selected from the group consisting of position 709, position 730, position 1207, and position 1209.

26. A WRN inhibitor for use in a method as defined in any one of claims 1 to 25, wherein the WRN inhibitor is GSK_WRN5.

27. The use of a WRN inhibitor in the manufacture of a medicament for treating a subject with cancer (for example a cancer with a mutated form of the WRN protein), wherein the WRN inhibitor is GSK_WRN5, and wherein treating the subject with cancer comprises a method as defined in any one of claims 1 to 16, 19 or 25.

28. The use of a WRN inhibitor in the manufacture of a medicament for treating cancer (for example a cancer with a mutated form of the WRN protein) in a subject, wherein the WRN inhibitor is GSK_WRN5, and wherein treating the cancer in the subject comprises a method as defined in any one of claims 17 to 25.

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