Numa inhibitors for use in the treatment of cancer

Abstract

The present invention relates to nuclear mitotic apparatus (NuMA) protein serine 395 (S395) phosphorylation inhibitor for use in preventing and / or treating cancer in a subject. The invention also relates to a pharmaceutical composition comprising a NuMA S395 phosphorylation inhibitor. Furthermore, the invention provides a method of screening for a candidate compound for preventing and / or treating cancer, and a method of detecting a dormant cancer cell in a subject. Corresponding methods of treating and / or preventing cancer are also provided.

Description

[0001] METHODS FOR TREATING CANCER

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to nuclear mitotic apparatus (NuMA) protein serine 395 (S395) phosphorylation inhibitor for use in preventing and / or treating cancer in a subject. The invention also relates to a pharmaceutical composition comprising a NuMA S395 phosphorylation inhibitor. Furthermore, the invention provides a method of screening for a candidate compound for preventing and / or treating cancer, and a method of detecting a dormant cancer cell in a subject. Corresponding methods of treating and / or preventing cancer are also provided.

[0004] INTRODUCTION

[0005] Breast cancer remains the second leading cause of death. Despite the recent improved survival rate of patients, 20-30% of patients experience cancer recurrence either at the original tumour site or at distant organ, months to years’ post-treatment. This phenomenon is not peculiar to breast cancers but also with other cancers, including brain, prostate, lung, ovarian tumours, and melanoma.

[0006] This is because a cohort of disseminated and / or remnant cancer cells after treatment, may lie dormant for years to decades before re-emerging as clinically detectable metastases upon changes to their environment. Recurrent cancer remains a fatal disease and account for most of the deaths among breast cancer patients.

[0007] Dormant cancers cells are non-proliferating, and are resistant to most current therapeutics, which rely on inducing cell death during cell division. There is an urgent need for novel approach to induce cell death in these dormant cancer cells. It is an object of one or more of the aspects of the present invention to, at least, in part, address this need.

[0008] SUMMARY OF THE INVENTION

[0009] Currently, there is no approved treatment to eradicate dormant cancer cells. Maintenance therapy remains the solution available to keep dormant cancer cells at their dormant state for a prolonged period of time and preventing them from re-activating and re-proliferating. For breast cancer patients, clinicians employ a combination of chemotherapy, endocrine therapy, or anti-angiogenic therapy to suppress potential re-growth of residual dormant cancer cells after treatment. Although this therapy is evidenced to prolonged progression-free survival and overall survival of patients, the strategy is limited with combined toxicity, which affects patient quality of life, and the cancer remains un-cured. The present invention is based on the inventors’ development of a novel approach aimed at eradicating dormant cancer cells, and thereby preventing them from re-emerging as metastasised cancers months or years later.

[0010] Unlike the current palliative maintenance therapy, the present invention may provide a definitive approach to disrupt the adaptive survival mechanism of dormant cancers cell, leading to eradicating these cells, hence a curative therapy.

[0011] Specifically, the present inventors have identified a modification of a DNA repair protein called, nuclear mitotic apparatus (NuMA), which allows cancer cells to remain in a dormant stage and thereby evade anti-cancer therapy. More specifically, the inventors have identified that phosphorylation of serine at amino acid position 395 is a key regulator of the adaptative survival mechanism of dormant cancer cells and transcription silencing required to maintain dormancy.

[0012] As described in more detail in the Examples section of the present disclosure, the inventors surprisingly identified that dormant cancer cells exhibit much higher levels of phosphorylation at NuMA S395 than active cancer cells. For example, in the aggressive breast cancer cell line (MDA-MB-231), the inventors identified that S395 phosphorylation levels are 5-10-fold greater in dormancy conditions than in a control, i.e. in proliferating conditions. The phosphorylation was observed to be critical to the survival of the dormant cancer cells, both in their dormant cell and key for their awakened (re-activated) state.

[0013] Specifically, the inventors have shown that reducing S395 phosphorylation of NuMA resulted in a reduction of dormant cancer cell number. Additionally, the inventors have shown that reducing S395 phosphorylation of NuMA reduced the ability of the surviving dormant cancer cells to proliferate upon being re-activated. Moreover, the inventors believe that by exposing active cancer cells to NuMA S395 phosphorylation inhibitors, active cancer cells may be prevented from entering dormancy, rendering them more susceptible to cancer therapeutics.

[0014] Collectively these data identify S395 phosphorylation of NuMA as an attractive therapeutic target treating cancer (for example by preventing active cancer cells to escape therapy by becoming dormant), and / or preventing cancer by killing dormant cancer cells and / or reducing the ability of re-activated cancer cells from successfully reproliferating. Accordingly, in one aspect, the present invention provides a NuMA S395 phosphorylation inhibitor for use as a medicament.

[0015] In one aspect, the present invention provides a nuclear mitotic apparatus (NuMA) protein serine 395 (S395) phosphorylation inhibitor for use in preventing and / or treating cancer in a subject.

[0016] In one embodiment, the inhibitor may be a small molecule, a biological product, or a conjugate thereof.

[0017] In one embodiment, the small molecule may be a compound having the structure of:

[0018] [Formula I],

[0019] In one embodiment, the biological product may be selected from the group consisting of a peptide, a polypeptide, a protein, a dendrimer, and a nucleic acid; optionally wherein the protein is an antibody or a fragment thereof.

[0020] In one embodiment, the antibody may be a monoclonal antibody, and / or the antibody may be monospecific or multispecific.

[0021] In one embodiment, the cancer may be a solid cancer cell or a liquid cancer cell.

[0022] Suitably, the solid cancer cell may be selected from the group consisting of a breast cancer cell, a colorectal cancer cell, a prostate cancer cell, a glioblastoma cell, a lung cancer cell, an ovarian cancer cell, and a bone cancer cell or any other cancer whereby dormancy is known to underpin recurrence.

[0023] More suitably, the solid cancer cell may be a breast cancer cell, prostate cancer cell, or colorectal cancer cell.

[0024] More suitably, the solid cancer cell may be a breast cancer cell.

[0025] More suitably, the solid cancer cell may be a prostate cancer cell. More suitably, the solid cancer cell may be a colorectal cancer cell.

[0026] In one embodiment the preventing and / or treating of cancer may be by: i) reducing the number of dormant cancer cells in the subject; ii) reducing the number of re-activated cancer cells in the subject, optionally wherein the reduction of re-activated cancer cells is by reducing proliferation of re-activated cancer cells; and / or iii) inhibiting an active cancer cell becoming a dormant cancer cell.

[0027] In one embodiment, the dormant cancer cell may be selected from the group consisting of dormant breast cancer cell, dormant colorectal cancer cell, dormant prostate cancer cell, dormant glioblastoma cell, dormant lung cancer cell, dormant bone cancer cell, and dormant ovarian cancer cell.

[0028] Suitably, the dormant cancer cell may have upregulated expression of NR2F1 and / or AXOL.

[0029] Suitably, the dormant breast cancer cell may have upregulated expression of a marker selected from the group consisting of OGN, Gas6, CD13, and MME.

[0030] In one embodiment, the NuMA S395 phosphorylation inhibitor may be for use in combination with a cancer therapeutic.

[0031] Suitably, the cancer therapeutic may be selected from the group consisting of immunotherapy, chemotherapy, radiation therapy, hormonal therapy, an anti-cancer vaccine, an anti-cancer virus, and surgery.

[0032] Suitably, the NuMA S395 phosphorylation inhibitor and cancer therapeutic may be provided sequentially and / or simultaneously.

[0033] In one aspect, the present invention provides a pharmaceutical composition comprising a NuMA S395 phosphorylation inhibitor and a pharmaceutically acceptable excipient, diluent, and / or carrier.

[0034] In one aspect, the present invention provides a method of screening for a candidate compound for preventing and / or treating cancer, wherein the method comprises:

[0035] • contacting a cancer cell with a test compound; and • determining a level of phosphorylation on NuMA S395; wherein a reduction in the level of phosphorylation on NuMA S395 in the contacted cell as compared to a control is indicative of the test compound being a compound suitable for preventing and / or treating cancer.

[0036] In a further aspect, provided herein is a method of detecting a dormant cancer cell in a subject, the method comprising determining the level of phosphorylation on NuMA S395 in a cancer cell in a sample from the subject, wherein if the cancer cell has hyperphosphorylated NuMA S395 it is indicative of the cancer cell being a dormant cancer cell.

[0037] Suitably, the sample may be selected from the group consisting of a solid tissue biopsy sample, a blood sample, a faecal sample, and a urine sample.

[0038] In yet a further aspect, the present invention provides a method of preventing and / or treating cancer in a subject, the method comprising administering a therapeutically effective amount of an NuMA S395 phosphorylation inhibitor to the subject in need thereof.

[0039] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0040] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0041] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0042] Various aspects of the invention are described in further detail below.

[0043] BRIEF DESCRIPTION OF THE FIGURES

[0044] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1 : Determination of dormancy status of Hypoxia and low nutrient induced MDA- MB-231 cells. MDA-MB-231 cells demonstrated cell cycle arrest at GO (Fig 1A) with no significant signs of apoptosis / cell death either by the lack of the appearance of sub-GO population (Fig 1 B) or cleave PARP expression (Fig. 1 D &E). Induced dormant cells did not proliferate (Fig 1C). Lack of proliferation was confirmed with relatively low expression of Ki67 in DC cells (cells cultured in dormant conditions) compared to PC cells (cells cultured in proliferative conditions) (Fig 1 F) and the lack of Rb phosphorylation, a key regulator of cell proliferation (Fig. 1 D and E). In addition, DC cells retained the intensity of DiD dye (a membrane dye) compared to the loss of DiD intensity (about 70%) in control PC cells (Fig 1G). Consistently, high expression levels of dormancy-associated markers including OGN and NR2F1 was also observed both at transcript and protein levels in DC-induced dormant MDA- MB-231 (Fig 1 E and H).

[0045] Figure 2: In vitro induced dormant cancer cells demonstrate the ability to proliferate in favourable conditions. The DC-induced dormant cells in PC entered cell cycle after 24 hours, with the appearance of significant S and G2 / M populations (Fig 2A), which indicate cell division and proliferation. Consistently, proliferation-associated protein Rb was hyper-phosphorylated (Fig.2B) upon incubation in PC. Re-proliferation of dormant cells was also observed with increased in cell numbers after 96 hours (D4) in PC (Fig 2C). To further validate this in vitro observation, the inventors injected DC-induced CT-26 cells in mice to assess their ability to grow tumour. As shown in Fig 2D, the DC-induced dormant cells develop tumours in vivo at a rate comparable to PC cells, confirming that DC cells can be reactivated in vivo.

[0046] Fig 3: Site specific phosphorylation of NuMA in mitotic and dormant breast cancer cells. The expression of NuMA did not significantly change at the transcript level in induced dormant cancer cells (Fig. 3A), but a significant change at the protein level (Fig 3B) was observed. Surprisingly, a significant hyper-phosphorylation of serine residue (S395) of NuMA in dormant cancer cells (Fig 3B and C) was observed. The phosphorylation of NuMA at T2055 (C- terminal) appear specific to mitotic cells whilst S395 (N-terminal) is specific to dormant cells (Fig 3D). As shown in Fig 3E, 3D spheroids of MDA-MB-231 cells exhibited heterogeneous cell populations i.e high proliferative cells (DiD negative), medium proliferative cells, (DiD medium), and non- or slow-proliferative dormant cells (DiD positive). Strikingly, only the dormant population (DiD positive) of MDA-MDA-231 spheroids were observed with p-NuMA (S395) expression (Fig 3F).

[0047] Fig 4: S395 phosphorylation is critical for the survival of breast cancer dormant cells. Kinase activity of ATR but not ATM was observed to be key in the S395 phosphorylation of NuMA (Fig 4A). The blocking of p-NuMA (S395) with ATRi was observed to significantly decrease the viability of dormant cancer cells at DC (Fig 4B), and further inhibit the reproliferation of dormant cancer cells when re-incubated in PC conditions (Fig 4C). ATR has many other substrates and the use of ATRi may affect the functions of other ATR substrates other than NuMA (Fig 4D and E). This decrease in cell viability was rescued with either the transfection of WT NuMA or S395 mutant (A395) that cannot be phosphorylated (Fig 4F and G), demonstrating the dependence of these dormant cells on NuMA phosphorylation for survival in dormant condition. The transfection of NuMA S395 mutant did block the S395 phosphorylation in DC (Fig 4H, left) though this did not suppress the survival of dormant cancer cell in DC. Blocking NuMA S395 phosphorylation in DC significantly reduced the ability of dormant cancer cells to re-proliferate when introduced to PC conditions, contrary to reactivation and proliferation of NuMA WT-transfected, SiNuMA, and SiControl (Fig 4H, right).

[0048] Fig 5: p-NuMA (S395) is essential to transcription silencing of dormant breast cancer cells. S395 phosphorylation of NuMA was observed mainly in nucleoplasm and completely absent on chromatin in DC-induced dormant breast cancer cells (Figure 5A and B). Fig 5C shows that blocking of S395 phosphorylation of NuMA restored transcription level comparable to PC controls. In addition, using a proteomic approach the S395 phosphorylation of NuMA was observed to recruit transcription-associated proteins from the chromatin in induced dormant breast cancer cells (Fig. 5D).

[0049] Fig 6: p-NuMA (S395) modelling and ligand-binding screen. To ascertain the specificity and selectivity of observed p-NuMA (S395) to dormant cancer cells as compared to dormant normal tissues like the brain, the inventors investigated NuMA S395 phosphorylation in human brain lysate. Surprisingly, p-NuMA was absent in human brain tissue yet present in MDA-MB- 231 dormant cells and its derived xenograft, CDX (Fig. 6A). Fig 6B shows a homology model using the 1-900 sequence from Numal . One binding hotspot covering Ser395 was identified in this model using SiteMap study. In addition, further MD simulations reveal that the secondary structures of the identified binding site region appear reasonably stable (Fig 6C). This homology model was used in a virtual screening approach to identify compounds with the ligand binding ability toward the S395 region of NuMA (Fig 6D and E).

[0050] Fig 7: NP-26A blocks S395 phosphorylation in DC-induced dormant cancer cells. A) shows the structure of NP-26A identified by the inventors as a NuMA S395 phosphorylation inhibitor. B) NP-26A forms two H-bonds near to Ser395 of NuMA. C) Western blotting showing the effect of treatment with NP-26A on NuMA phosphorylation and DNA strand breaks as measured by gH2AX. NP-26A was found to block the NuMA S395 phosphorylation in DC- induced-breast cancer cells. D) Western blot showing the effect of treatment with NP-26A on the general phosphorylation of ATM / ATR substrates using an anti-phospho-ATM / ATR substrate antibody.

[0051] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0052] Various aspects of the invention are described in further detail below.

[0053] DETAILED DESCRIPTION

[0054] In one aspect, the present invention provides a nuclear mitotic apparatus protein (NuMA) serine 395 (S395) phosphorylation inhibitor is for use as a medicament.

[0055] Suitably, the medicament may be for preventing and / or treating a cancer. Such an embodiment gives rise to a further aspect of the invention which provides a NuMA S395 phosphorylation inhibitor for use in preventing and / or treating cancer in a subject.

[0056] Nuclear mitotic apparatus (NuMA) also in the art sometimes referred to as “NuMA1” is a Microtubule (MT)-binding protein that plays a role in the formation and maintenance of the spindle poles and the alignment and the segregation of chromosomes during mitotic cell division. Human NuMA is ~238 kDa in size, and has the amino acid sequence as shown in SEQ ID NO: 1.

[0057] NuMA is typically expressed in the nucleoplasm, cytoplasm, and on the chromatin of proliferating cells, including proliferating cancer cells.

[0058] NuMA is well conserved in vertebrates and has been found in, for example, humans, monkeys, rodents, companion animals, and livestock. Accordingly, the products and methods described herein can be applied to any vertebrate. Subjects suitable in the context of the present disclosure are described hereinbelow. As used herein the term “NuMA” refers to a protein according to SEQ ID NO: 1 and variants thereof. Suitably, such variants will comprise serine at a position corresponding to amino acid 395 of SEQ ID NO: 1. The variants may be naturally occurring, such as for example SNPs. Such naturally existing variants will be well known to those skilled in the art. Suitably, the NuMA variants may have substantially the same function as a NuMA protein according to SEQ ID NO: 1.

[0059] Suitably, the NuMA variant may share at least 70%, at least 75%, at least 80%, least 85%, or more sequence identity with SEQ ID NO: 1. Suitably, the NuMA variant may share at least 90%, at least 95%, at least 98%, least 99%, or more sequence identity with SEQ ID NO: 1 .

[0060] The term “sequence identity” as used herein refers to the degree of sequence matching between two nucleic acid sequences or two amino acid sequences as determined using the algorithm of for example Karlin & Attschul (1990) Proc. Natl. Acad. Sci. 87: 2264-2268, modified as in Karlin & Attschul (1993) Proc. Natl. Acad. Sci. 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Attschul et al. (1990) T. Mol. Biol. Q15: 403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a reference amino acid sequence. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Attschul et al. (1997) Nucl. Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g. XBLAST and NBLAST) are used. Other algorithms, programs and default settings may also be suitable such as, but not only, the GCG-Sequence Analysis Package of the U.K. Human Genome Mapping Project Resource Centre that includes programs for nucleotide or amino acid sequence comparisons.

[0061] Human NuMA having the sequence as shown in SEQ ID NO: 1 comprises a serine at amino acid position 395. Herein, this serine residue is referred to as S395, also referred to herein as “NuMA S395” or “S395 of NuMA”. S395 is capable of undergoing phosphorylation and dephosphorylation. As outlined in the examples herein, the inventors surprisingly identified that phosphorylation of NuMA S395 increased in dormant cancer cells and critical to their survival. As NuMA is well conserved in vertebrates, it would be clear to the person skilled in the art that phosphorylation of an amino acid corresponding to that of human NuMA S395 may be critical to the survival of dormant cancer cells in other vertebrates. The term “corresponding amino acid” refers to an amino acid which is present within a corresponding region and which is the counterpart of S395 of SEQ ID NO: 1 in a sequence alignment.

[0062] It will be appreciated that the corresponding amino acid does not have to be the same amino acid as in the reference polypeptide. Suitably, the corresponding amino acid may be a similar amino acid. Suitably, the corresponding amino acid may be one that can be phosphorylated (for example an amino acid that can be phosphorylated may be selected from the group consisting of: serine, threonine, tyrosine, histidine, arginine, lysine, aspartic acid, glutamic acid, and cysteine).

[0063] Methods for obtaining sequence alignments in order to determine corresponding amino acids are well known in the art. Merely by way of example, sequence alignment may be obtained by using bioinformatics tools such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). When the same position in the sequences to be compared is occupied by the same amino acid residue, then the respective molecules are identical at that very position. Accordingly, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100%. For instance, if 6 out of 10 sequence positions are identical, then the identity is 60%. The percent identity between two protein sequences can, e.g., be determined using the Needleman and Wunsch algorithm (NEEDLEMAN, S. B. and Wunsch, C. D. A general method applicable to the search for similarities in the amino acid sequence of two proteins. Journal of Molecular Biology 1970, vol. 48, p. 443-453) which has been incorporated into EMBOSS Needle. The % identity is typically determined over the entire length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence are identical irrespective of any chemical and / or biological modification. For example, two antibodies having the same primary amino acid sequence but different glycosylation patterns are identical by this definition. Similar protein sequences are those which, when aligned, share similar amino acid residues and most often, but not mandatorily, identical amino acid residues at the same positions of the sequences to be compared.

[0064] As used herein, the term “phosphorylation” refers to the addition of a phosphate group to a molecule. Suitably, the phosphate group may replace a side chain hydroxyl group of an amino acid. Accordingly, a molecule that is “phosphorylated” is one that comprises a phosphate group. Suitably, the molecule is a protein, for example an amino acid residue of a protein. In the context of the present disclosure, phosphorylation may be of S395 of NuMA.

[0065] In vitro, phosphorylation is a common reversible post-translational modification of one or more amino acid residues of a protein, wherein the one or more amino acid residue side chain hydroxyl group has been substituted for a phosphate group. Phosphorylation is typically catalysed by kinases. The most commonly phosphorylated amino acid residues are serine, threonine, tyrosine, and histidine; however, other amino acids can also be phosphorylated, including arginine, lysine, aspartic acid, glutamic acid and cysteine. For example, serine (Ser or S) is a polar amino acid that is used in the biosynthesis of proteins and comprises a side chain consisting of a hydroxymethyl group (-CH2-OH) which can undergo reversible phosphorylation to give -CH2-PO3 and subsequent dephosphorylation back to a hydroxymethyl group.

[0066] In vivo, phosphorylation of a protein is commonly associated with regulating protein activity and function and, in turn, the regulation of many cellular functions, including metabolism, proliferation, differentiation, motility, and survival. Accordingly, it may be favourable to regulate the levels of phosphorylation of proteins to control said cellular functions. Altered phosphorylation is strongly associated with cancer.

[0067] As outlined in the examples herein, the inventors’ surprisingly identified that NuMA S395 in dormant cancer cells has significantly increased levels of phosphorylation. As used herein, the term “levels of phosphorylation” refers to the proportion of cells within a population of cells comprising phosphorylated NuMA S395, proportion of NuMA proteins within a population of cells which are phosphorylated at S395, and / or proportion of NuMA proteins in a cell which is phosphorylated at S395. Suitably the cells and / or the population of cells may comprise cancer cells (for example active, re-activated, and / or dormant cancer cells). Methods for measuring the level of phosphorylation are well known in the art and include for example Western blotting as shown in the Examples section.

[0068] The present inventors have also surprisingly identified that by reducing the levels of phosphorylation of NuMA S395 in dormant cancer cells, or preventing phosphorylation at NuMA S395 of active cancer cells, cancer disease can be prevented and / or treated. Such a reduction of the levels of phosphorylation may be achieved by contacting active and / or dormant cancer cells with a phosphorylation inhibitor. As used herein, the term “phosphorylation inhibitor” refers to an agent capable of reducing the levels of phosphorylation.

[0069] Reducing the level of phosphorylation can be achieved through inhibiting phosphorylation and / or dephosphorylation. Accordingly, a phosphorylation inhibitor is an agent that inhibits phosphorylation and / or causes dephosphorylation. Suitably, in the context of the present disclosure, the phosphorylation inhibitor inhibits phosphorylation of S395 of NuMA and / or dephosphorylates S395 of NuMA.

[0070] The term “dephosphorylation” refers to the removal of a phosphate group from a molecule such as an amino acid. An amino acid can be dephosphorylated by the replacement of a phosphate group with a hydroxyl group. In vivo, dephosphorylation is typically catalysed by protein phosphatases. Protein kinases and phosphatases work independently and, in a balance, to regulate the function of proteins. Accordingly, a phosphorylation inhibitor may be a phosphatase.

[0071] As mentioned, a reduction in the level of phosphorylation can be achieved by inhibiting phosphorylation (i.e. preventing / blocking a phosphate group from being added to a molecule, such a serine 395 of NuMA).

[0072] Suitably, inhibiting phosphorylation may be achieved through inhibiting kinase activity of a relevant kinase (such as a kinase that phosphorylates serine 395 of NuMA). Suitably, inhibiting the action of a kinase may be achieved by an agent that binds the kinases, for example at its active site. An agent that inhibits kinase activity by binding the kinase, may be referred to as a kinase inhibitor.

[0073] Alternatively, or additionally, phosphorylation may be inhibited by an agent preventing the addition of a phosphate group to the target site, by binding the target (such as serine 395 of NuMA) or proximal residues, in a manner that blocks the phosphate group from being added.

[0074] Suitably, the phosphorylation inhibitor is a NuMA S395 phosphorylation inhibitor. The aim of the NuMA S395 phosphorylation inhibitor is to inhibit the phosphorylation of NuMA S395, thereby reducing the level of phosphorylation of NuMA S395. Suitably, the NuMA S395 phosphorylation inhibitor inhibits phosphorylation of NuMA S395.

[0075] Suitably, in the context of the present disclosure, the reduction of phosphorylation may be partial, substantially complete, or complete. As used herein, the terms “reduces”, “reduced”, or “reducing” generally means a decrease by a statistically significant amount; for the avoidance of any doubt, the terms “reduces”, “reduced”, or “reducing” means a decrease of at least about 5% as compared to a reference value, for example an decrease of at least about 10%, or at least about 20%, or at least 30%, or at least about 40%, or at least about 50% as compared to a reference value. For example, the decrease may be of at least about 60%, or at least about 70%, or at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more as compared to a reference value. The reference value, as it will be appreciated by the skilled person, refers to a value of an appropriate parameter in a suitable control. As it will be appreciated, the skilled person will be capable of determining what constitutes a suitable control. Merely by way of example, the control may comprise or consist of cancer cells (for example active cancer cells, re-activated cancer cells, dormant cancer cells, or a combination thereof) not exposed to a phosphorylation inhibitor. Suitably, such cancer cells may be obtained by a biopsy or a blood sample (for example circulating tumor cells).

[0076] As outlined in the examples herein, the inventors’ surprisingly identified that in dormant cancer cells (such as dormant breast cancer cells), increased levels of NuMA S395 phosphorylation was observed predominantly in the nucleoplasm. Accordingly, the NuMA S395 phosphorylation inhibitor may be a nucleoplasm NuMA S395 phosphorylation inhibitor. Such as inhibitor may have the ability to enter the nucleoplasm.

[0077] As understood by the person skilled in the art, when NuMA S395 is phosphorylated the NuMA S395 phosphorylation inhibitor may not be able to inhibit phosphorylation, although depending on the phosphorylation inhibitor it may be able to dephosphorylate NuMA S395. Nevertheless, in an embodiment where the phosphorylation inhibitor does not dephosphorylate NuMA S395 but only prevents NuMA S395 phosphorylation, such an inhibitor may also be able to reduce phosphorylation levels of NuMA S395. This is because the level of phosphorylation within a cancer cell and / or population of cancer cells is dynamic and therefore changes naturally over time. Therefore, when natural dephosphorylation occurs, the NuMA S395 phosphorylation inhibitor that blocks NuMA S395 phosphorylation may prevent further phosphorylation of that S395 residue, overtime reducing the steady state level of phosphorylation, as shown in the Figure 7.

[0078] As mentioned elsewhere herein, the inventors have shown that reducing S395 phosphorylation of NuMA resulted in a reduction of dormant cancer cell number. Additionally, the inventors have shown that the surviving dormant cancer cells ability to proliferate upon being re-activated was significantly reduced. Moreover, the inventors believe that by exposing active cancer cells to NuMA S395 phosphorylation inhibitors, active cancer cells may be prevented from entering dormancy, rendering them more susceptible to cancer therapeutics. Accordingly, as it will be appreciated by a person skilled in the art, NuMA S395 phosphorylation inhibitors may prevent and / or treat cancer.

[0079] As used herein, the term “cancer” refers to a disease caused by the presence of cells with abnormal cellular proliferation. Such cells may be an “active cancer cell” and / or a “re-activated cancer cell”. Abnormal cellular proliferation refers to uncontrolled cellular proliferation and / or excessive cell division. Uncontrolled and / or excessive cell division is typically caused by mutations in genes that control cell division, leading to altered expression and / or activity of cell cycle related proteins, and in turn unchecked cell division. For avoidance of doubt, a reactivated cancer cell is an active cancer that has been dormant in the past.

[0080] A cancer may be a solid cancer or a liquid cancer. As used herein, the term “solid cancer” refers to a plurality of active cancer cells and / or re-activated cancer cells manifesting as a cancerous mass or lump.

[0081] Different types of solid cancers are named for the type of cancerous cells that form them. Suitably, a solid cancer may be selected from the group consisting of: breast cancer, colorectal cancer, prostate cancer, glioblastoma cancer, lung cancer, bone cancer, and ovarian cancer, or any other cancer type whereby dormancy is known to cause recurrence.

[0082] More suitably, the solid cancer may be all subtypes of breast cancer as shown in the examples herein.

[0083] Suitably, the breast cancer may be selected from the group consisting of: adenocarcinoma, triple negative, hormone positive, hormone negative or triple negative adenocarcinoma breast cancer. Suitably, the breast cancer may be adenocarcinoma breast cancer. Suitably, the breast cancer may be triple negative breast cancer. Suitably, the breast cancer may be triple negative or hormone (Estrogen) positive adenocarcinoma breast cancer. However, the skilled person would appreciate that the aspects of the present invention may apply to other cancers and types of breast cancer.

[0084] As used herein, the term “liquid cancer” refers to conditions associated with the presence of active cancer cells and / or re-activated cancer cells in body fluids, such as blood, lymph, and / or bone marrow. Examples of liquid cancers are leukaemia, myeloma, myelodysplastic syndrome (MDS), and liquid lymphomas.

[0085] As the person skilled in the art would understand, a subject that has cancer will typically have active cancer cells, and re-activated and / or dormant cancer cells.

[0086] Suitably, an active cancer cell or re-activated cancer cell may be in the G1 , S, G2, or M phase of the cell cycle. As it will be understood by a person skilled in the art, when more than one active or reactivated cancer cell is present, some or all of the cells may be in the same or different phase of the cell cycle.

[0087] The G1 phase is the first phase of the cell cycle that takes place in eukaryotic cell division. The S phase (synthesis phase) is the phase of the cell cycle in which DNA is replicated. The G2 phase is the third subphase of interphase in the cell cycle directly preceding mitosis. The M phase (mitosis) is the phase where the mother cell divides into two genetically identical daughter cells.

[0088] In contrast to an active (or re-activated) cancer cell, a “dormant cancer cell” is a nonproliferating cell, that has the potential to exhibit uncontrolled and / or excessive cell proliferation. Suitably, a dormant cancer cell is in a state of quiescence, senescence, or dormancy. Suitably, a dormant cancer cell may identified by being in the GO phase of the cell cycle.

[0089] Suitably, a dormant cancer cell may have upregulated expression of nuclear receptor subfamily 2 group F member 1 (NR2F1). NR2F1 is a nuclear hormone receptor and transcriptional regulator. It may be capable of acting as a homodimer and binding to 5'- AGGTCA-3' repeats. NR2F1 is a known biomarker of dormant cancer cells.

[0090] As used herein, the term “upregulated” generally means an increase by a statistically significant amount; for the avoidance of any doubt, the terms upregulated means an increase of at least about 5% as compared to a reference value, for example an increase of at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50% as compared to a reference value. For example, the increase may be of at least about 60%, or at least about 70%, or at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more as compared to a reference value. The reference value, as it will be appreciated by the skilled person, refers to value of an appropriate parameter (such as the level of expression) of a control, such as NR2F1 expression in a (known) active cancer cell.

[0091] Suitably, the dormant cancer cell may be selected from the group consisting of dormant breast cancer cell, dormant colorectal cancer cell, dormant prostate cancer cell, dormant glioblastoma cell, dormant lung cancer cell, and dormant ovarian cancer cell.

[0092] More suitably, the dormant cancer cell may be a dormant breast cancer cell. Suitably, the dormant breast cancer cell may have upregulated expression of a marker selected from the group consisting of osteoglycin (OGN), growth arrest specific 6 (Gas6), aminopeptidase N (CD13), and membrane metallo-endopeptidase / neprilysin (MME).

[0093] Suitably, the dormant breast cancer cell may have upregulated expression of OGN.

[0094] Suitably, the dormant breast cancer cell may have upregulated expression of Gas6.

[0095] Suitably, the dormant breast cancer cell may have upregulated expression of CD13.

[0096] Suitably, the dormant breast cancer cell may have upregulated expression of MME.

[0097] Suitably, a dormant breast cancer cell may have reduced Rb phosphorylation and / or downregulated Ki67 expression.

[0098] As used herein, the term “downregulated” generally means a decrease by a statistically significant amount; for the avoidance of any doubt, the terms downregulated means a decrease of at least about 5% as compared to a reference value, for example a decrease of at least about 10%, or at least about 20%, or at least 30%, or at least about 40%, or at least about 50% as compared to a reference value. For example, the decrease may be of at least about 60%, or at least about 70%, or at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more as compared to a reference value. The reference value, as it will be appreciated by the skilled person, refers to value of an appropriate parameter (such as the level of expression) of a control, such as Ki67 expression in an active breast cancer cell.

[0099] Suitably, a dormant breast cancer cell may have reduced Rb phosphorylation.

[0100] Suitably, a dormant breast cancer cell may have downregulated Ki67 expression. Suitably, the dormant cancer cell is a dormant colorectal cancer cell. Suitably, the dormant cancer cell is a dormant prostate cancer cell.

[0101] As outlined in the examples herein, the inventors’ surprisingly identified that treating a dormant cell population with a phosphorylation inhibitor that inhibits phosphorylation of S395 NuMA resulted in a decrease in the number of dormant cancer cells in a dormant cancer cell population. Accordingly, the NuMA S395 phosphorylation inhibitor of the invention may find use in preventing cancer in a subject due to its ability to reduce the number of dormant cancer cells which have the potential to become re-activated cancer cells. As used herein, the term “preventing cancer” refers to delaying or inhibiting the development of cancer disease. Suitably, preventing cancer is by reducing the number of dormant cancer cells in a subject. Suitably, preventing cancer is delaying or inhibiting cancer recurrence in a subject.

[0102] As outlined in the examples herein, the inventors’ surprisingly also identified that by decreasing NuMA S395 phosphorylation levels in dormant cancer cells, such cells upon their re-activation have significantly reduced ability to re-proliferate thereby treating cancer. Such treatment may be as a result of a decrease in the number of re-activated cancer cells (i.e. reduced active cancer cell load).

[0103] Additionally, the present inventors believe that by contacting active cancer cells with a NuMA S395 phosphorylation inhibitor may fully or partially prevented active cancer cells from becoming dormant. As cancer dormancy is a key mechanism for resistance to anti-cancer therapy, by providing a NuMA S395 phosphorylation inhibitor to a subject with cancer, the inventors believe that the cancer may be more effectively treated. This is because most cancer therapeutics act on active (or re-activated) cancer cells.

[0104] The NuMA S395 phosphorylation inhibitor of the invention may find use in treating cancer in a subject. As used herein, the term “treating cancer” refers to reducing the number of active cancer cells.

[0105] As used herein the terms “treat”, “treating” or “treatment” refer to a clinical improvement of cancer in a subject with this disease. Such a clinical improvement may be demonstrated by an improvement of the pathology and / or symptoms associated with the cancer.

[0106] In one embodiment, effective treatment may be demonstrated by slowing or halting the progression of the disease in the subject, or reversing the disease. Suitably, the disease may be reversed partially, or completely. In some embodiments, complete reversal of the diseases may be sufficient to result in curing of the disease.

[0107] Clinical improvement of the pathology may be, but is not limited to anti-tumour effects, reduced biomarker levels in the subject, increased time to regrowth of cancer upon stopping of treatment, lack of regrowth of cancer upon stopping treatment, decreased tumour invasiveness, reduction of metastasis, increased cancer cell differentiation, or increased survival rate. In some embodiments, anti-tumour effects may be demonstrated by inhibition of tumour growth, reduced speed of tumour growth, or a partial or complete reduction in tumour mass / lump.

[0108] Other suitable indications of clinical improvement in the pathology will be known to the skilled person. It will be appreciated that indications of clinical improvement of the pathology will vary depending on the type of cancer.

[0109] In the context of the present disclosure, the clinical improvement may be as a result of a reduction of active (or re-activated) cancer cells. Such a reduction may be as a result of a cancer therapeutic provided in combination with a NuMA S395 phosphorylation inhibitor.

[0110] Suitably, the cancer therapeutic may be provided sequentially and / or simultaneously to the NuMA S395 phosphorylation inhibitor. By “sequentially”, it is meant that the cancer therapeutic may be provided before and / or after the NuMA S395 phosphorylation inhibitor. Merely by way of example the cancer therapeutic may be provided at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months or at least 12 months before the NuMA S395 phosphorylation inhibitor. Alternatively or additionally the cancer therapeutic may be provided at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 9 months or at least 12 months after the NuMA S395 phosphorylation inhibitor. By “simultaneously” it is mean that the cancer therapeutic may be provided around the same time as the NuMA S395 phosphorylation inhibitor. It will be appreciated that simultaneous administration need not to occur at exactly the same time. Accordingly, simultaneous administration encompasses administration of the cancer therapeutic and NuMA S395 phosphorylation inhibitor separated by a time frame that is less than 1 month, for example 2 weeks, 1 week, 3 days, 1 day, 12 hours, 6 hours, 3 hours or less. Suitably, the cancer therapeutic and NuMA S395 phosphorylation inhibitor may be provided at the same time. Suitably, the cancer therapeutic may be selected from the group consisting of immunotherapy, chemotherapy, radiation therapy, hormonal therapy, an anti-cancer vaccine, an anti-cancer virus, and surgery. The term “immunotherapy” relates to the treatment of disease by inducing or enhancing an immune response, in particular a T cell mediated immune response. Different types of immunotherapies for cancer are known in the art. These include for example immune checkpoint inhibitors, monoclonal antibodies, and / or cancer vaccines.

[0111] Immune checkpoint inhibitors are agents that inhibit proteins or peptides (e.g. immune checkpoint proteins) which are blocking the immune system, e.g., from attacking cancer cells. In some examples, the immune checkpoint protein blocking the immune system prevents the production and / or activation of T cells. An immune checkpoint inhibitor can be an antibody or antigen-binding fragment thereof, a protein, a peptide, a small molecule, or combination thereof. Typically, the inhibitor interacts directly to a target immune checkpoint protein (or its ligand, where appropriate) and thereby disrupts its function / biological activity. For example, it may bind directly to a target immune checkpoint protein (or its ligand, where appropriate). In one example, direct binding to a target immune checkpoint protein (or its ligand, where appropriate) inhibits, prevents or reduces the formation of protein complexes which are needed for immune checkpoint protein function / biological activity.

[0112] PD-1 inhibitors, PD-L1 , and PD-L2 inhibitors are a group of checkpoint inhibitors that block or reduce the activity of PD-1 , PD-L1 and PD-L2 immune checkpoint proteins. A review describing immune checkpoint pathways and the blockade of such pathways with immune checkpoint inhibitor compounds is provided by Pardoll in Nature Reviews Cancer (April, 2012). Immune check point inhibitor compounds display anti-tumour activity by blocking one or more of the endogenous immune checkpoint pathways that downregulate an anti-tumour immune response. The inhibition or blockade of an immune checkpoint pathway typically involves inhibiting a checkpoint receptor and ligand interaction with an immune checkpoint inhibitor compound to reduce or eliminate the signal and resulting diminishment of the anti-tumour response.

[0113] The immune checkpoint inhibitor compound may inhibit the signaling interaction between an immune checkpoint receptor and the corresponding ligand of the immune checkpoint receptor. The immune checkpoint inhibitor compound can act by blocking activation of the immune checkpoint pathway by inhibition (antagonism) of an immune checkpoint receptor (some examples of receptors include CTLA-4, PD-1 , and NKG2A) or by inhibition of a ligand of an immune checkpoint receptor (some examples of ligands include PD-L1 and PD-L2). In such examples, the effect of the immune checkpoint inhibitor compound is to reduce or eliminate down regulation of certain aspects of the immune system anti-tumour response in the tumour microenvironment.

[0114] The immune checkpoint receptor programmed death 1 (PD-1) is expressed by activated T- cells upon extended exposure to antigen. Engagement of PD-1 with its known binding ligands, PD-L1 and PD-L2, occurs primarily within the tumour microenvironment and results in downregulation of anti-tumour specific T-cell responses. Both PD-L1 and PD-L2 are known to be expressed on tumour cells. The expression of PD-L1 and PD-L2 on tumours has been correlated with decreased survival outcomes.

[0115] Many PD-1 inhibitors and / or PD-L1 inhibitors are known in the art. In some examples, the PD- 1 inhibitor and / or PD-L1 inhibitor is a small organic molecule (molecular weight less than 1000 daltons), a peptide, a polypeptide, a protein, an antibody, an antibody fragment, or an antibody derivative. In some embodiments, the inhibitor compound is an antibody. In some embodiments, the antibody is a monoclonal antibody, specifically a human or a humanized monoclonal antibody.

[0116] In some examples, the PD-1 inhibitor is an anti-PD-1 antibody or derivative or antigen-binding fragment thereof. In some embodiments, the anti-PD-1 antibody selectively binds a PD-1 protein or fragment thereof. In some embodiments, the anti-PD1 antibody is nivolumab, pembrolizumab, or pidilizumab.

[0117] In some examples, the PD-L1 inhibitor is an anti-PDL-1 antibody or derivative or antigenbinding fragment thereof. In some examples, the anti-PD-L1 antibody or derivative or antigenbinding fragment thereof selectively binds a PD-L1 protein or fragment thereof. Examples of anti-PD-L1 antibodies and derivatives and fragments thereof are described in, e.g., WO 01 / 14556, WO 2007 / 005874, WO 2009 / 089149, WO 2011 / 066389, WO 2012 / 145493; US 8,217,149, US 8,779,108; US 2012 / 0039906, US 2013 / 0034559, US 2014 / 0044738, and US 2014 / 0356353. In some embodiments, the anti-PD-L1 antibody is MEDI4736 (durvalumab), MDPL3280A, 2.7A4, AMP-814, MDX-1105, atezolizumab (MPDL3280A), or BMS-936559.

[0118] In some examples, the anti-PD-L1 antibody is MEDI4736, also known as durvalumab. MEDI4736 is an anti-PD-L1 antibody that is selective for a PD-L1 polypeptide and blocks the binding of PD-L1 to the PD-1 and CD80 receptors. MEDI4736 can relieve PD-L1 -mediated suppression of human T-cell activation in vitro and can further inhibit tumour growth in a xenograft model via a T-cell dependent mechanism. MEDI4736 is further described in, e.g., US 8,779,108. The fragment crystallizable (Fc) domain of MEDI4736 contains a triple mutation in the constant domain of the lgG1 heavy chain that reduces binding to the complement component C1q and the Fey receptors responsible for mediating antibody-dependent cell- mediated cytotoxicity (ADCC).

[0119] CTLA4 inhibitors are inhibitors that block or reduce the activity of CTLA4. The immune checkpoint receptor cytotoxic T-lymphocyte associated antigen 4 (CTLA4 or CTLA-4) is expressed on T-cells and is involved in signaling pathways that reduce the level of T-cell activation. It is believed that CTLA4 can downregulate T-cell activation through competitive binding and sequestration of CD80 and CD86. In addition, CTLA4 has been shown to be involved in enhancing the immunosuppressive activity of TReg cells.

[0120] A CTLA4 inhibitor may prevent or reduce binding to CD80 and / or CD86. In some embodiments, a CTLA-4 inhibitor comprises an antibody binding compound, such as an antibody or an antigen-binding fragment thereof. U.S. Pat. Nos. 5,855,887; 5,811 ,097; 6,682,736; 7,452,535 disclose antibodies specific for human CTLA-4, including antibodies specific for the extracellular domain of CTLA-4 and which are capable of blocking its binding to CD80 or CD86; methods of making such antibodies, and methods of using such antibodies as anti-cancer agents. In some examples, the anti-CTLA-4 antibody is Tremelimumab, Ipilimumab, or Pembrolizumab.

[0121] TIGIT (T-cell immunoreceptor containing Ig and ITIM domains) belongs to the immunoglobulin superfamily, also known as Wucam, Vstm3 or Vsig9. TIGIT has an extracellular immunoglobulin domain, type I transmembrane domain and two Immune receptor tyrosine inhibition motif (ITIM). TIGIT is mainly distributed in regulatory T cells (Tregs), activated T cells and natural killer cells (NK), etc. It is a co-suppressive receptor protein, which can be combined with the positive proteins CD226 (Dnam-1) and APC on T cells The expressed ligands CD155 (Pvr or Necl-5) and CD112 (Pvrl-2 or Nectin2) constitute a costimulatory network. Among them, TIGIT competes with CD226 to bind CD155 and CD112, and TIGIT binds its ligand with a higher affinity than CD226. The connection between TIGIT and CD155 or CD112 is mediated by its cytoplasmic ITIM or ITT-like motif, recruiting phosphatase SHIP- 1 to the tail of TIGIT to trigger inhibitory signaling. In addition, the ITIM domain is also responsible for the inhibitory ability of mouse TIGIT.

[0122] Suitably, TIGIT inhibitors (such as anti-TIGIT antibodies) can inhibit, reduce, or neutralize one or more activities of TIGIT, for example, result in the blocking or reduction of immune checkpoints on T cells or NK cells, or The immune response is reactivated by adjusting antigen presenting cells. Examples of anti-TIGIT antibodies include Vibostolimab, Etigilimab, Tiragolumab, and Domvanalimab.

[0123] The term “LAG-3”, “LAG3”, or “Lymphocyte Activation Gene-3” refers to Lymphocyte Activation Gene-3. LAG-3's main ligand is MHC class II, to which it binds with higher affinity than CD4. The protein negatively regulates cellular proliferation, activation, and homeostasis of T cells, in a similar fashion to CTLA-4 and PD-1and has been reported to play a role in Treg suppressive function. LAG3 is known to be involved in the maturation and activation of dendritic cells. A LAG-3 inhibitor can reduce or block the binding of LAG-3 to the MHC class II molecule, and thereby reduce or block its activity. Suitably, the LAG-3 inhibitor may be an anti-LAG-3 antibody, for example Favezelimab or Relatlimab.

[0124] TIM-3 is an immune checkpoint receptor that suppresses antitumor responses by negatively regulating the activity of CD8 T cells and antigen-presenting cells. A TIM-3 inhibitor may reduce or block the activity of TIM-3. Suitably, the TIM-3 inhibitor may be an anti-TIM-3 antibody, for example, Cobolimab.

[0125] B and T lymphocyte attenuator (BTLA) is an important co-signaling molecule. It belongs to the CD28 superfamily and is similar to programmed cell death-1 (PD-1) and cytotoxic T lymphocyte associated antigen-4 (CTLA-4) in terms of its structure and function. BTLA can be detected in most lymphocytes and induces immunosuppression by inhibiting B and T cell activation and proliferation. BTLA is found to be expressed in tumor-infiltrating lymphocytes (TILs) and is often associated with impaired anti-tumor immune response. A BTLA inhibitor may reduce or block the activity of BTLA. Such a reduction or blockage may increase B and T cell activation and proliferation. Suitably, the BTLA inhibitor may be an anti-BTLA antibody, for example, Tifcemalimab.

[0126] Killer immunoglobulin-like receptors (KI Rs), are a family of cell surface proteins found on natural killer (NK) cells. They inhibit the killing function of these cells by interacting with MHC class I molecules. KIR inhibitors may reduce or block the activity of KIR. Such a reduction or blockage may increase the killing ability of NK cells. Suitably, a KIR inhibitor may be an anti- KIR antibody, for example, Lirilumab.

[0127] The term “chemotherapy” as used herein refers to the treatment of cancer using specific chemical agents or drugs that are destructive of malignant cells and tissues. Such agents or drugs can be destructive by killing and / or controlling the growth (for example by preventing the growth) of malignant cells. Many chemotherapeutic agents are known in the art. As it will be clear to a person of skill in the art the type of chemotherapeutic used may depend upon the type of cancer. For example, in the context of breast cancer, the chemotherapy may be paclitaxel, docetaxel, epirubicin, carboplatin, capecitabine, eribulin epirubicin and cyclophosphamide (EC), doxorubicin and cyclophosphamide (AC), and / pr docetaxel and cyclophosphamide (TC).

[0128] As used herein, the term radiotherapy should be broadly construed and is intended to include various techniques used to irradiate a patient, including use of photons (such as high energy x-rays and gamma rays), particles (such as electron and proton beams), and radio surgical techniques. In the context of cancer, radiotherapy typically kills malignant cancer cells.

[0129] The term “hormonal therapy” as used herein refers to a type of therapy that reduces the amount of a hormone in the body. A reduction of hormone levels may slow down or stop the growth of a cancer. Hormone therapy may be especially relevant in the context of breast, prostate, or womb cancer. Examples of hormone therapy that may be used in combination with the NuMA S395 phosphorylation inhibitor in the context of breast cancer in line with the uses and methods of the invention include tamoxifen, aromatase inhibitors (such as anastrozole (Arimidex), exemestane (Aromasin), letrozole (Femara)), Luteinising hormone releasing hormone (LHRH) agonists or LH blockers (such as goserelin (Zoladex), leuprorelin (Prostap, Lutrate)), and / or Fulvestrant. Examples of hormone therapy that may be used in combination with the NuMA S395 phosphorylation inhibitor in the context of prostate cancer in line with the uses and methods of the invention include Luteinising hormone releasing hormone (LHRH) agonists or LH blockers (such as goserelin (Zoladex), leuprorelin (Prostap), triptorelin (Decapetyl), buserelin (Suprefact)), anti-androgens (bicalutamide (Casodex), cyproterone acetate (Cyprostat), flutamide (Drogenil), enzalutamide (Xtandi), apalutamide (Erleada), darolutamide (Nubeqa), abiraterone (Zytiga)), Gonadotrophin releasing hormone (GnRH) blocker (degarelix (Firmagon)). Examples of hormone therapy that may be used in combination with the NuMA S395 phosphorylation inhibitor in the context of womb cancer in line with the uses and methods of the invention include medroxyprogesterone acetate (Provera) and megestrol (Megace).

[0130] Surgery, in the context of the present disclosure, is an operation or procedure to remove some or all cancer tissue from the subject’s body.

[0131] As used herein, the terms “provided”, “providing” or “provision” refer to giving the NuMA S395 phosphorylation inhibitor and / or cancer therapeutic to a subject by any suitable route of administration to the subject. Suitably, administration may be by oral, injection, intravenous, intramuscular, or transdermal administration. Suitable methods of administration will depend upon the nature of the drug to be given and will be known to those with skill in the art.

[0132] Suitably, the NuMA S395 phosphorylation inhibitor may be provided to a subject for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 12 months. As mentioned, the NuMA S395 phosphorylation inhibitor may be provided as a monotherapy, or combination therapy. When the NuMA S395 phosphorylation inhibitor is provided as a combination therapy, it may be provided with at least one anti-cancer therapeutic. For avoidance of doubt, when provided as a combination therapy, the NuMA S395 phosphorylation inhibitor can be provided simultaneously or sequentially with at least one at anti-cancer therapeutic.

[0133] Suitably, the NuMA S395 phosphorylation inhibitor may be a small molecule, a biological product, or a conjugate thereof. The term “small molecule” refers to any molecule with a molecular weight of 2000 Daltons or less. The small molecule may exist naturally or be synthetically made.

[0134] Suitably, the NuMA S395 phosphorylation inhibitor may be a small molecule. Suitably, the small molecule is a compound having the structure of Formula I:

[0135] [Formula I],

[0136] Herein, the compound according to Formula I may be referred to as NP-26A.

[0137] As shown in the examples section below, as NP-26A is a small molecule that is capable of inhibiting the phosphorylation of NuMA S395.

[0138] Suitably, the NuMA S395 phosphorylation inhibitor may be a biological product. The term “biological product” refers to a product that is produced from living organisms or comprises components found within living organisms, such as nucleotides or amino acid residues. Suitably, the biological products may be produced through biotechnology in a living system, such as a microorganism, plant cell, or animal cell. Suitably, biological products may be produced through a biological process such as fermentation, cell cultivation, enzymatic processes or the like.

[0139] Suitably, the NuMA S395 phosphorylation inhibitor may bind to NuMA S395 or epitope comprising NuMA S395 and thereby block phosphorylation of NuMA serine 395. Suitably, the NuMA S395 phosphorylation inhibitor may be capable of selective binding an epitope to NuMA S395 or epitope comprising NuMA S395.

[0140] The term "capable of specifically binding" as used herein shall mean that the phosphorylation inhibitor is under certain conditions able to selectively bind its target (e.g. NuMA S395 or epitope comprising NuMA S395). Selective binding means that the binding agent, under certain conditions, will not bind in a significant amount to other targets. Depending on the kind of binding agent used, these conditions may vary.

[0141] Non-limiting examples of biological NuMA S395 phosphorylation inhibitors described herein include, but are not limited to nucleic acids, such as aptamers (e.g. nucleic acid aptamers, peptide aptamers, aptabodies, affimers); proteins (e.g. antibodies, antibody mimetics, camelid antibodies, duobodies etc, peptides, polypeptides), and dendrimers. Other appropriate binding agents are also well known and readily identifiable to a person of skill in the art using routine experimental procedures.

[0142] The term “nucleic acid” as referred to herein comprises all forms of DNA (e.g. genomic DNA, mtDNA) or RNA (mRNA, tRNA, rRNA, small RNA, siRNA, miRNA, non-coding RNA, animal RNA, plant RNA, viral RNA or bacterial RNA), as well as recombinant RNA and DNA molecules or analogues of DNA or RNA generated using nucleotide analogues. The nucleic acids may be single stranded or double stranded. The nucleic acids may include the coding or non-coding strands. The term also comprises fragments of nucleic acids, such as naturally occurring RNA or DNA which may be recovered using the extraction methods disclosed. Nucleic acid may also refer to a portion of a nucleic acid (e.g., RNA or DNA). The extracted nucleic acids may further comprise peptide nucleic acids (PNA).

[0143] As used herein, “aptamer” refers to nucleic acid aptamers and / or peptide aptamers. Examples of aptamers include affimers (an evolution of peptide aptamers) and aptabodies (formed by hybridisation of two DNA aptamers), which are also well known and readily identifiable to a person of skill in the art using routine experimental procedures. In one example, the NuMA S395 phosphorylation inhibitor may be a protein. Examples of appropriate proteins include antibodies and antibody mimetics. Examples of antibodies are provided elsewhere herein. Examples of appropriate antibody mimetics include affibody molecules (including affimabs) affilins, peptide aptamers (including affimers), affitins, alphabodies, anticalins, avimers, DARPins, Fynomers, Kunitz domain peptides, monobodies, nanoCLAMPs etc., which are also well known and readily identifiable to a person of skill in the art using routine experimental procedures.

[0144] The terms “antibody” or “antibodies” as used herein refer to molecules or active fragments of molecules that bind to known antigens, particularly it refers to immunoglobulin molecules and to immunologically active portions of immunoglobulin molecules, i.e. molecules that contain a binding site that specifically binds an antigen. The immunoglobulin described herein can be of any class (IgG, IgM, I g D, IgE, IgA and IgY) or subclass (e.g. I gG 1 , 1 gG2, 1 gG3, 1 gG4, 1 gA1 and lgA2) of immunoglobulin molecule and based on heavy chain sequences from any species. For example, the species may be, but not limited to dogs, cats, horses, cows, pigs, guinea pigs, mice, rats and the like. The species may be a primate (e.g. a non-human primate). In a preferred example, the species is a human.

[0145] The term “antibody” or “antibodies” include monoclonal, polyclonal, chimeric, single chain, bispecific, human and humanized antibodies as well as active fragments thereof. Examples of active fragments of molecules that bind to known antigens and are useful include Fab, bispecific Fab2, tri-specific Fab3, scFv, bi-specific di-scFv, bi-specific scFv-Fc, bi-specific diabody, a duobody, a tri-specific triabody, a single domain antibody and a bi-specific minibody, including the products of an Fab immunoglobulin expression library and epitopebinding fragments of any of the antibodies and fragments mentioned above.

[0146] Suitably, the antibody may be “monospecific”. Such an antibody has one or more binding sites that each bind to the same epitope on the same antigen. Suitably, the antibody may be "multispecific”, i.e. comprising two or more different epitopes (e.g., two, three, four, or more different epitopes). The epitopes can be on the same or different antigens. The multispecific antigen binding protein may be, for example, bi-specific, tri-specific, tetra-specific, or pentaspecific.

[0147] In a particular example, the antibody may be a monoclonal antibody. As used herein, the term “monoclonal antibody” refers to an antibody that can be mass produced in the laboratory from a single clone and that recognizes only one antigen. Monoclonal antibodies may be generated by any appropriate technique known in the art (e.g. by production in HEK or insect cells, or by generation of B cell hybridomas).

[0148] As used herein, the term “chimeric antibody” refers to a monoclonal antibody comprising a variable region, i.e., binding region, from one source or species, (i.e. non-human primates, humans, dogs, cats, horses, cows, pigs, guinea pigs, mice, rats and the like) and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a mouse variable region and a human constant region are examples. Such chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding mouse immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions. Other forms of “chimeric antibodies” encompassed by the present disclosure are those in which the class or subclass has been modified or changed from that of the original antibody, for example to alter them so that they are complement and Fc receptor binding deficient. Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now well known in the art. See, e.g., Morrison, S. L., et al., Proc. Natl. Acad Sci. USA 81 (1984) 6851-6855; U.S. Pat. No. 5,202,238 and U.S. Pat. No. 5,204,244.

[0149] Suitably, the antibody may be a human antibody or a humanized antibody.

[0150] As used herein the term “humanized antibody” or “humanized version of an antibody” refers to antibodies in which the framework or “complementarity determining regions” (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin. In some examples, the CDRs of the VH and VL are grafted into the framework region of human antibody to prepare the “humanized antibody.” See e.g. Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, M. S., et al., Nature 314 (1985) 268-270. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. Both the heavy chain and the light chain may be required for effective antigen binding. The human antibody sequences can be the sequences of naturally occurring human antibodies. Human heavy and light chain variable framework regions are listed e.g. in Lefranc, M.-P., Current Protocols in Immunology (2000) — Appendix 1 P A.1 P.1-A.1 P.37 and are accessible via IMGT, the international ImMunoGeneTics information System® (http: / / imgt.cines.fr) or via http: / / vbase.mrc- cpe.cam.ac.uk, for example. Optionally the framework region can be modified by further mutations. Exemplary CDRs correspond to those representing sequences recognizing the antigens noted above for chimeric antibodies. In some examples, such humanized version is chimerized with a human constant region. As used herein the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germ line immunoglobulin sequences. Human antibodies are well-known in the state of the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or a selection of human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Brueggemann, M. D., et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., et al., J. Mol. Biol. 222 (1991) 581- 597). The techniques of Cole, A., et al. and Boerner, P., et al. are also available for the preparation of human monoclonal antibodies (Cole, A., et al., Monoclonal Antibodies and Cancer Therapy, Liss, A. R. (1985) p. 77; and Boerner, P., et al., J. Immunol. 147 (1991) 86- 95).

[0151] In a particular example, the antibody may be selected from a Fab, bi-specific Fab2, tri-specific Fab3, scFv, bi-specific di-scFv, bi-specific scFv-Fc, bi-specific diabody, a tri-specific triabody, a single domain antibody or a bi-specific minibody.

[0152] Other examples include single domain antibodies such as those found in camelids including, but not limited to, llamas and alpacas; and cartilaginous fish including, but not limited to, sharks which are widely known in the art.

[0153] As used herein “single chain antibody” refers to single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883 or a bi-specific single chain Fv (WO 03 / 11161). Typical scFv linkers are well known in the art, are generally 10 to 25 amino acids in length and include glycines and serines.

[0154] Single domain antibody fragments (VHH or nanobodies) e.g. the functional antibodies produced by camelids that are devoid of light chains and wherein a single N-terminal domain is fully capable of antigen binding are also an example of a binding agent described herein. Such fragments can also form bivalent VHH, or pentabodies (i.e. with 5 VHH domains). As used herein, a “di-ScFv” refers to a dimerized scFV.

[0155] As used herein, “minibodies” are minimized antibody-like proteins comprising a scFv joined to a CH3 domain. See Hu et al., 1996, Cancer Res. 56:3055-3061. In some cases, the scFv can be joined to the Fc region, and may include some or the entire hinge region.

[0156] By "Fab" or "Fab region" as used herein is meant the polypeptides that comprise the VH, CH1 , VH, and CL immunoglobulin domains. Fab may refer to this region in isolation, or this region in the context of a full length antibody or antibody fragment or fab fusion protein.

[0157] The terms “Fab”, “Fab region”, “Fab portion” or “Fab fragment” are understood to define a polypeptide that includes a VH, a CH1 , a VL, and a CL immunoglobulin domain. Fab may refer to this region in isolation, or this region in the context of an antibody molecule described herein, as well as a full length immunoglobulin or immunoglobulin fragment. Typically a Fab region contains an entire light chain of an antibody. A Fab region can be taken to define “an arm” of an immunoglobulin molecule. It contains the epitope-binding portion of that Ig. The Fab region of a naturally occurring immunoglobulin can be obtained as a proteolytic fragment by a papaindigestion. A “F(ab')2 portion” is the proteolytic fragment of a pepsin-digested immunoglobulin. A “Fab' portion” is the product resulting from reducing the disulfide bonds of an F(ab')2 portion. As used herein the terms “Fab”, “Fab region”, “Fab portion” or “Fab fragment” may further include a hinge region that defines the C-terminal end of the antibody arm (cf. above). This hinge region corresponds to the hinge region found C-terminally of the CH1 domain within a full length immunoglobulin at which the arms of the antibody molecule can be taken to define a Y. The term hinge region is used in the art because an immunoglobulin has some flexibility at this region.

[0158] By "Fc fusion" as used herein is meant a protein wherein one or more polypeptides is operably linked to Fc. Fc fusion is herein meant to be synonymous with the terms "immunoadhesin", "Ig fusion", "Ig chimera", and "receptor globulin" (sometimes with dashes) as used in the prior art (Chamow et al., 1996, Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200). An Fc fusion combines the Fc region of an immunoglobulin with a fusion partner, which in general may be any protein, polypeptide or small molecule. The role of the non-Fc part of an Fc fusion, i.e., the fusion partner, is to mediate target binding, and thus it is functionally analogous to the variable regions of an antibody. Virtually any protein or small molecule may be linked to Fc to generate an Fc fusion. Protein fusion partners may include, but are not limited to, the target-binding region of a receptor, an adhesion molecule, a ligand, an enzyme, a cytokine, a chemokine, or some other protein or protein domain. Small molecule fusion partners may include any therapeutic agent that directs the Fc fusion to a therapeutic target. Such targets may be any molecule, e.g., an extracellular receptor that is implicated in disease.

[0159] As used herein the term “antibody fragments” refers to a portion of a full length antibody, for example possibly a variable domain thereof, or at least an antigen binding site thereof. Examples of antibody fragments include diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. scFv antibodies are, e.g., described in Huston, J. S., Methods in Enzymol. 203 (1991) 46-88. Antibody fragments can be derived from an antibody described herein by a number of art-known techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme, such as pepsin, and subjected to HPLC gel filtration. The appropriate fraction containing Fab fragments can then be collected and concentrated by membrane filtration and the like. For further description of general techniques for the isolation of active fragments of antibodies, see for example, Khaw, B. A. et al. J. Nucl. Med. 23:1011-1019 (1982); Rousseaux et al. Methods Enzymology, 121 :663-69, Academic Press, 1986.

[0160] The term “dendrimer” as used herein is to be understood in its broadest sense, and to include within its scope all forms and compositions of these dendrimers as disclosed in Patent Publications Nos. WO 88 / 01178, WO 88 / 01179 and WO 88 / 01180. The term also includes linked or bridged dendrimers as disclosed in these patent publications.

[0161] Suitably, the dendrimer may comprise a polyvalent core covalently bonded to at least two dendritic branches, and preferably extend through at least two generations. Suitably, the dendrimers are polyamidoamine (PAMAM) dendrimers, PAMAM (EDA) dendrimers and polylysine dendrimers.

[0162] Suitably, the NuMA S395 phosphorylation inhibitor may be a conjugate of a small molecule and a biological product. For example, the NuMA S395 phosphorylation inhibitor may be an antibody conjugated to a small molecule.

[0163] As outlined in the examples herein, the inventors’ surprisingly identified that in dormant breast cancer, NuMA phosphorylated at S395 was observed predominantly in the nucleoplasm. Suitably, the NuMA S395 phosphorylation inhibitor may be capable of entering the nucleoplasm. Methods for facilitating therapeutic entry into the nucleoplasm are known in the art. For example, the NuMA S395 phosphorylation inhibitor (such as a protein or small molecule) may comprise a nuclear localisation signal NLS and / or packaged in a suitable formulation to promote nuclear entry.

[0164] Suitably, the NuMA S395 phosphorylation inhibitor may be stable in the nucleoplasm.

[0165] As it will be understood by the person skilled in the art, phosphorylation and dephosphorylation occurs in cycles in response to upstream signalling. Therefore, the level of phosphorylation within a population of cancer cells and / or the level of phosphorylation in a cancer cell changes naturally over time. Accordingly, the NuMA S395 phosphorylation inhibitor may be provided to a subject over a period. The provision of a NuMA S395 phosphorylation inhibitor to a subject over a period allows the inhibitor to inhibit phosphorylation of NuMA S395 post natural dephosphorylation of NuMA S395. Suitably, the NuMA S395 phosphorylation inhibitor may be provided to a subject for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or at least 12 months.

[0166] In the context of the present invention, the term “subject” includes humans and other mammals (e.g., mice, rats, pigs, cats, dogs, and horses) which express NuMA. Suitably, the term “other mammals” encompasses primates, livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. Suitably, (e.g., particularly in research contexts) subject mammals will be, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like. Suitably, the subject may be have or be suspected of having cancer. Suitably, the subject may have or be suspected of having cancer comprising dormant cancer cells. Suitably, the subject may comprise or be suspected of comprising dormant cancer cells.

[0167] Also provided by the present invention is a pharmaceutical composition comprising a NuMA S395 phosphorylation inhibitor. Suitably, the composition is a composition comprising the NuMA S395 phosphorylation inhibitor and a pharmaceutically acceptable diluent, carrier or excipient. Such compositions may further routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.

[0168] The compositions may also include antioxidants and / or preservatives. As antioxidants may be mentioned thiol derivatives (e.g. thioglycerol, cysteine, acetylcysteine, cystine, dithioerythreitol, dithiothreitol, glutathione), tocopherols, butylated hydroxyanisole, butylated hydroxytoluene, sulfurous acid salts (e.g. sodium sulfate, sodium bisulfite, acetone sodium bisulfite, sodium metabisulfite, sodium sulfite, sodium formaldehyde sulfoxylate, sodium thiosulfate) and nordihydroguaiareticacid. Suitable preservatives may for instance be phenol, chlorobutanol, benzylalcohol, methyl paraben, propyl paraben, benzalkonium chloride and cetylpyridinium chloride.

[0169] The NuMA S395 phosphorylation inhibitor may be presented as solids in finely divided solid form, for example they may be micronised. Powders or finely divided solids may be encapsulated.

[0170] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings or animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0171] It will be appreciated that the pharmaceutical compositions described above may be suitable for use in preventing and / or treating cancer, and particularly those various forms of cancer described herein.

[0172] The NuMA S395 phosphorylation inhibitor may be for administration to the subject by any suitable route by which a therapeutically effective amount of the NuMA S395 phosphorylation inhibitor may be provided.

[0173] Suitably, the NuMA S395 phosphorylation inhibitor is for oral administration to treat cancer. Suitable oral administration forms that may be used in such embodiments include solid dosage forms. Solid dosage forms for oral administration include capsules, tablets (also called pills), powders and granules. In such solid dosage forms, the NuMA S395 phosphorylation inhibitor is typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or one or more: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules and tablets, the dosage form may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycol, for example.

[0174] Suitably, oral formulations may contain a dissolution aid. The dissolution aid is not limited as to its identity so long as it is pharmaceutically acceptable. Examples include nonionic surface agents, such as sucrose fatty acid esters, glycerol fatty acid esters, sorbitan fatty acid esters (e.g., sorbitan trioleate), polyethylene glycol, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, methoxypolyoxyethylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyethylene glycol fatty acid esters, polyoxyethylene alkylamines, polyoxyethylene alkyl thioethers, polyoxyethylene polyoxypropylene copolymers, polyoxyethylene glycerol fatty acid esters, pentaerythritol fatty acid esters, propylene glycol monofatty acid esters, polyoxyethylene propylene glycol monofatty acid esters, polyoxyethylene sorbitol fatty acid esters, fatty acid alkylolamides, and alkylamine oxides; bile acid and salts thereof (e.g., chenodeoxycholic acid, cholic acid, deoxycholic acid, dehydrocholic acid and salts thereof, and glycine or taurine conjugate thereof); ionic surface agents, such as sodium laurylsulfate, fatty acid soaps, alkylsulfonates, alkylphosphates, ether phosphates, fatty acid salts of basic amino acids; triethanolamine soap, and alkyl quaternary ammonium salts; and amphoteric surface agents, such as betaines and aminocarboxylic acid salts. Pharmaceutical compositions of the invention, comprising NuMA S395 phosphorylation inhibitor, may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0175] Suitably, the NuMA S395 phosphorylation inhibitor is for administration in liquid dosage form.

[0176] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the NuMA S395 phosphorylation inhibitor, the liquid dosage forms may contain inert diluents commonly used in the art such as water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3- butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavouring and perfuming agents. Suspensions, in addition to the inactivated NuMA S395 phosphorylation inhibitor, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar, and tragacanth and mixtures thereof.

[0177] Suitably, the NuMA S395 phosphorylation inhibitor may be for administration to the subject by intravenous route. Suitably, a sterile pharmaceutical composition may be especially desirable.

[0178] A sterile pharmaceutical composition may be created, for example, by filtration through sterile filtration membranes, prior to or following lyophilisation and reconstitution of the NuMA S395 phosphorylation inhibitor. The NuMA S395 phosphorylation inhibitor may be stored in lyophilised form or in solution.

[0179] A pharmaceutical composition comprising the NuMA S395 phosphorylation inhibitor may be placed into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierce-able by a hypodermic injection needle.

[0180] A sterile pharmaceutical composition comprising the NuMA S395 phosphorylation inhibitor suitable for intravenous delivery may be formulated according to conventional pharmaceutical practice as described in Remington: The Science and Practice of Pharmacy (20th ed, Lippincott Williams & Wilkens Publishers (2003)). For example, dissolution or suspension of the active compound in a vehicle such as water or naturally occurring vegetable oil like sesame, peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or the like may be desired. Buffers, preservatives, antioxidants and the like can be incorporated according to accepted pharmaceutical practice.

[0181] Suitably, the pharmaceutical composition comprising the NuMA S395 phosphorylation inhibitor may be for the sustained release of the NuMA S395 phosphorylation inhibitor. Such a pharmaceutical composition may comprise semipermeable matrices of solid hydrophobic polymers containing the NuMA S395 phosphorylation inhibitor, which matrices are in the form of shaped articles, films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels, copolymers of L-glutamic acid and gamma ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LLIPRON Depot™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Suitably, pharmaceutical compositions for sustained release of NuMA S395 phosphorylation inhibitor, may comprise crystals of the NuMA S395 phosphorylation inhibitor suspended in suitable formulations capable of maintaining crystals in suspension. Such pharmaceutical compositions, when injected intravenously, subcutaneously or intraperitoneally may produce a sustained release effect.

[0182] In another aspect, the present invention provides a method of screening for a candidate compound for preventing and / or treating cancer, wherein the method comprises:

[0183] • contacting a cancer cell with a test compound; and

[0184] • determining a level of phosphorylation of NuMA S395; wherein a reduction in the level of phosphorylation of NuMA S395 in the contacted cell as compared to a control is indicative of the test compound being a compound for preventing and / or treating cancer. A compound that is identified as capable of reducing the level of phosphorylation of NuMA S395 may be referred to as a “candidate compound”.

[0185] As used herein, the term “screening” refers to a method of evaluating test compounds in order to discover compounds and / or confirm whether compounds are capable of reducing the level of phosphorylation of NuMA S395 and thereby are capable of preventing and / or treating cancer.

[0186] As used herein, the term “test compound” refers to a molecule which has unknown effects on NuMA S395 phosphorylation, although it may be suspected of having the ability to reduce the level of phosphorylation on NuMA S395. Merely by way of example, such a test compound may have been identified as potentially capable of reducing NuMA S395 phosphorylation by in silico screening. Suitably, the test compound may be a small molecule, a biological product, or a conjugate thereof. Suitably, the test compound may be a biological product selected from the group consisting of a protein, a dendrimer, and a nucleic acid.

[0187] The term "contacting" as used herein is to be understood broadly and can be any means that enables a chemical reaction of at least two reactants, for example cells (such as cancer cells and / or dormant cancer cells), with an agent (such as a test compound). Appropriate conditions that allow specific interactions are well known to those skilled in the art. It will be appreciated that the conditions will depend on the test agent used in the method, however the skilled person will routinely be able to adapt these conditions. Furthermore, sufficient time to allow interaction can be readily determined by the skilled person. Suitably, the cancer cells may be contacted with the test compound by being cultured in a cell culture medium comprising said test compound. As used herein, the term “determining a level of phosphorylation of NuMA S395” refers to quantitatively or qualitatively measuring the amount of phosphorylation on NuMA S395. Methods for measuring the level of phosphorylation are described elsewhere herein. Merely byway of example, levels of phosphorylation may be determined by a Western blot. Antibodies that specifically binding phosphorylated NuMA S395 and un phosphorylated NuMA S395 are known in the art. Suitably, phosphorylation of NuMA S395 may be measured on a single cell and / or on a plurality of cells. The cell and / or population of cells may be dormant cancer cells, active cancer cells, re-activated cancer cells, or a mixture thereof. More suitably the level of phosphorylation it may be measured on a plurality of cells (for example 10 cells, 100 cells, 10,000 cells, 100,000 cells, 1 ,000,000 cells or more).

[0188] As it will be clear to a person skilled in the art, the control in the context of the screening assay described herein may be the level of phosphorylation of NuMA S395 in a cancer cell and / or plurality of cancer cells prior to the step of contacting a cancer cell with a test compound. Thus suitably, the screening method may comprise the steps of: a) determining a level of phosphorylation of NuMA S395 of a cancer cell prior to contacting the cancer cell with a test compound; b) contacting the cancer cell with the test compound; and c) determining the level of phosphorylation of NuMA S395 after contacting the cancer cell with the test compound; wherein a reduction in the level of phosphorylation of NuMA S395 in c) as compared to a) is indicative of the test compound being a compound for preventing and / or treating cancer.

[0189] However, in suitable embodiments, the control may be a reference value. In such embodiment, the method does not necessarily require step a). Instead, the reference value may be obtained from a database, and / or previously acquired by determining the level of phosphorylation on corresponding cancer cells. In this context, corresponding cancer cells are cancer cells that would be expected to have a level of phosphorylation of NuMA S395 comparative (for example the same, or very similar, for example no more than 10% different) to the level of phosphorylation of NuMA S395 of the cancer cell prior to it being contacted with a NuMA S395 phosphorylation inhibitor. Suitably, the corresponding cancer cells may be of the same type, and optionally obtained from the same subject.

[0190] It will be appreciated that the cancer cell in step a) and c) need not to be the same, single cancer cell. For example the cells in step a) and c) may be corresponding cells, obtained for example from the same cancer cell population. In another aspect, the present invention provides a method of detecting a dormant cancer cell in a subject, the method comprising determining the level of phosphorylation on NuMA S395 in a cancer cell in a sample from the subject, wherein if the cancer cell has hyperphosphorylated NuMA S395 it is indicative of the cancer cell being a dormant cancer cell.

[0191] As used herein, the term “detecting” refers identifying the presence of a dormant cancer cell. Suitably, the dormant cancer cell may be detected in a sample from a subject (such as a subject diagnosed with cancer). The method of detecting a dormant cancer cell in a subject may be useful for determining whether a subject may benefit from treatment with a NuMA S395 phosphorylation inhibitor.

[0192] As used herein, the term “sample” is intended to include biological matter from the subject. Suitably, the sample may be selected from the list consisting of a solid tissue biopsy sample, a blood sample, a fecal sample, and a urine sample.

[0193] Suitably, the solid tissue biopsy sample may be obtained via surgery. Suitably, the solid tissue biopsy sample may be fresh. In this context, fresh means the solid tissue biopsy sample has been removed from the subject less than 48 hours prior to being used in a method described herein. Suitably, the sample may have been refrigerated and / or frozen for some or all of the time between being obtained from the subject and being used in the method described herein.

[0194] Suitably, the blood sample may be arterial blood, capillary blood, venous blood or a mixture thereof. In some examples, the blood sample may be selected from the group consisting of whole blood, blood plasma, and blood serum. The term “whole blood” as used herein refers to blood containing all its natural constituents, components, or elements or a substantial amount of the natural constituents, components, or elements. Accordingly, whole blood will comprise plasma, buffy coat (white cells and platelets), and red blood cells. The term “plasma” or “blood plasma” refers to the complete soluble fraction of the blood, whilst the term “serum” or “blood serum” refers to plasma devoid of coagulation factors, i.e. obtained after coagulation of blood.

[0195] The term “hyperphosphorylated” as used herein refers to increased levels of NuMA S395 phosphorylation. Suitably, the term hyperphosphorylated means that the levels of phosphorylated NuMA S395 is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than the phosphorylation levels of NuMA S395 typically found in active cancer cells. Suitably, the term hyperphosphorylated means that the levels of phosphorylated NuMA S395 are at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 500%, or at least 1000% or more greater than the phosphorylation levels of NuMA S395 typically found in active cancer cells. Methods to determine hyperphosphorylation are known by the skilled person, for example immunofluorescence staining with a p-NuMA specific antibody.

[0196] In a further aspect, the present invention provides method of preventing and / or treating cancer in a subject, the method comprising administering a therapeutically effective amount of an NuMA S395 phosphorylation inhibitor to the subject in need thereof.

[0197] As used herein, the term “therapeutically effective amount” means a dose of the NuMA S395 phosphorylation inhibitor that is effective in exerting a therapeutic effect, particularly a dose of which, after administration to the subject, yields a concentration of the NuMA S395 phosphorylation inhibitor in the blood effective in exerting a therapeutic effect on a target organ. Persons of ordinary skill in the art will understand that the amounts of the drug presented in the composition vary with the particular situation, including but not limited to, the species and dosage form of the drug and the size, age and condition of the subject, for example. In this context, the therapeutic effect may be preventing cancer and / or treating cancer. As mentioned elsewhere herein “treating” may include slowing down the progression of cancer or halting the progression of cancer.

[0198] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0199] Aspects of the invention are demonstrated by the following non-limiting examples.

[0200] EXAMPLES

[0201] Materials and methods

[0202] Cell culture

[0203] Human breast adenocarcinoma cell lines; MDA-MB-231 and MCF-7 and mouse colorectal cancer cell line, CT-26, were obtained from American Type Culture Collection (ATCC). Cells were cultured in DM EM media supplemented with 10% (v / v) foetal bovine serum, sodium pyruvate (1 mM), and L-glutamine (2 mM) in a humidified incubator at 37 °C with 5% of carbon dioxide. Cell lines were used at low passage (< 12 passages) for less than 6 months. Hypoxic condition (0.1 02%) conditions was achieved using Whitley H35 Hypoxystation (DW Scientific, UK). In-vitro engineered dormant cancer cells was done by incubating cancer cells in serum- free RPMI-1640 supplemented with sodium pyruvate (1 mM), and L-glutamine (2 mM) in hypoxic conditions.

[0204] Cell cycle analysis

[0205] Conditioned cells and controls were trypsinised, washed with PBS, and fixed with 66.6% ethanol overnight. The fixed cells were rinsed with PBS, labelled with FxCycle™ PI / RNase staining solution (Thermo-Fisher Scientific, UK), following the manufacturer’s instructions, and analysed by flow cytometry (BD Accuri C6 Plus Flow Cytometer).

[0206] For 2D DiD staining, cells were labelled with Vybrant DiD Dye (ThemoFisher Scientific, UK) following the manufacturer instructions and incubated for 30 mins. Cells were washed 3X with PBS. Cells were transferred to dormancy conditions or left at normal conditions for 48 hours, after which cell were trypsinised, washed and analysed by flow cytometry (BD Accuri C6 Plus Flow Cytometer).

[0207] Western Blotting

[0208] Cell pellets or tumour xenograft tissues were lysed or homogenised in Pierce™ IP lysis buffer with protease and phosphatase inhibitors, incubated on ice for 30 min, sonicated and centrifuged (10,000 g, 10 min, 4 °C). Subcellular fractionation was achieved using subcellular protein fractionation kit for cultured cells (ThermoFisher Scientific UK). Total protein concentrations of lysates were determined using a BCA protein assay kit (ThermoFisher Scientific, UK). Protein (40 pg) of total cell lysate was separated by 4-15% Mini-PROTEAN TGX Gels (Bio-Rad, UK) and then transferred onto nitrocellulose membranes. Membranes were blocked with 5% dry milk or 5% BSA in PBST. Membranes were probed for specific protein expression with respective antibodies, followed by horseradish peroxidase (HRP)- conjugated secondary antibodies. These immunoblots were visualised and analysed using ChemiDoc Imaging System with Image Lab Software 6.1.

[0209] Immunoprecipitation (IP)

[0210] In vitro engineered dormant MDA-MB-231 cells and proliferative control were collected and lysed with IP lysis buffer with protease and phosphatase inhibitors. Cell lysates (1 mg) were pre-cleared with either protein G agarose (CST, UK) or protein A / G PLUS-Agarose (Santa Cruz, UK), by incubating for 30 mins and centrifuged. Supernatant (precleared lysate) were incubated on a rotator at 4 °C overnight with p-NuMA antibody or NuMA antibody, or with appropriate IgG controls. Agarose beads were added and incubated on a rotator at 4 °C for 2 h. Beads were washed three times with IP lysis buffers before boiling in 2X SDS loading buffer. All immunoprecipitated eluates and inputs were then run on a 4-15% Mini-Protean TGX Precast Protein Gels (Bio-Rad) then analysed by immunoblotting.

[0211] IP-Mass spectrometry

[0212] Immunoprecipitated samples on beads samples were reduced (10mM TCEP, 55°C for 1 h), alkylated (18.75 mM iodoacetamide, room temperature for 30min.) and then digested from the beads with trypsin (1.25pg trypsin; 37°C, overnight). The resulting peptides were then labelled with Tandem Mass Tag (TMTpro) sixteen plex reagents according to the manufacturer’s protocol (Thermo Fisher Scientific, Loughborough, LE11 5RG, UK) and the labelled samples pooled. The pooled sample was desalted using a SepPak cartridge according to the manufacturer’s instructions (Waters, Milford, Massachusetts, USA). Eluate from the SepPak cartridge was evaporated to dryness and resuspended in buffer A (20 mM ammonium hydroxide, pH 10) prior to fractionation by high pH reversed-phase chromatography using an Ultimate 3000 liquid chromatography system (Thermo Fisher Scientific). In brief, the sample was loaded onto an XBridge BEH C18 Column (130A, 3.5 pm, 2.1 mm X 150 mm, Waters, UK) in buffer A and peptides eluted with an increasing gradient of buffer B (20 mM Ammonium Hydroxide in acetonitrile, pH 10) from 0-95% over 60 minutes. The resulting fractions (6 in total) were evaporated to dryness and resuspended in 1 % formic acid prior to analysis by nano-LC MSMS using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific). Nano-LC Mass Spectrometry: High pH RP fractions were further fractionated using an Ultimate 3000 nano-LC system in line with an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific). In brief, peptides in 1 % (vol / vol) formic acid were injected onto an Acclaim PepMap C18 nano-trap column (Thermo Scientific). After washing with 0.5% (vol / vol) acetonitrile 0.1% (vol / vol) formic acid peptides were resolved on a 250 mm x 75 pm Acclaim PepMap C18 reverse phase analytical column (Thermo Scientific) over a 150 min organic gradient, using 7 gradient segments (1-6% solvent B over 1min., 6-15% B over 58min., 15- 32%B over 58min., 32-40%B over 5min., 40-90%B over 1min., held at 90%B for 6min and then reduced to 1%B over 1 min.) with a flow rate of 300 nl min-1. Solvent A was 0.1 % formic acid and Solvent B was aqueous 80% acetonitrile in 0.1% formic acid. Peptides were ionized by nano-electrospray ionization at 2.0kV using a stainless-steel emitter with an internal diameter of 30 pm (Thermo Scientific) and a capillary temperature of 300°C. All spectra were acquired using an Orbitrap Fusion Lumos mass spectrometer controlled by Xcalibur 3.0 software (Thermo Scientific) and operated in data-dependent acquisition mode using an SPS- MS3 workflow. FTMS1 spectra were collected at a resolution of 120 000, with an automatic gain control (AGO) target of 200000 and a max injection time of 50ms. Precursors were filtered with an intensity threshold of 5000, according to charge state (to include charge states 2-7) and with monoisotopic peak determination set to Peptide. Previously interrogated precursors were excluded using a dynamic window (60s + / -10ppm). The MS2 precursors were isolated with a quadrupole isolation window of 0.7m / z. ITMS2 spectra were collected with an AGC target of 10 000, max injection time of 70ms and CID collision energy of 35%.

[0213] For FTMS3 analysis, the Orbitrap was operated at 50 000 resolution with an AGC target of 50 000 and a max injection time of 105ms. Precursors were fragmented by high energy collision dissociation (HCD) at a normalised collision energy of 60% to ensure maximal TMT reporter ion yield. Synchronous Precursor Selection (SPS) was enabled to include up to 10 MS2 fragment ions in the FTMS3 scan.

[0214] IP-MS Data Analysis: The raw data files were processed and quantified using Proteome Discoverer software v2.4 (Thermo Scientific) and searched against the UniProt Human database (downloaded January 2023: 81579 entries) using the SEQUEST HT algorithm. Peptide precursor mass tolerance was set at 10ppm, and MS / MS tolerance was set at 0.6Da. Search criteria included oxidation of methionine (+15.995Da), acetylation of the protein N- terminus (+42.011 Da) and Methionine loss plus acetylation of the protein N-terminus (- 89.03Da) as variable modifications and carbamidomethylation of cysteine (+57.0214) and the addition of the TMTpro mass tag (+304.207) to peptide N-termini and lysine as fixed modifications. Searches were performed with full tryptic digestion and a maximum of 2 missed cleavages were allowed. The reverse database search option was enabled and all data was filtered to satisfy false discovery rate (FDR) of 5%.

[0215] Quantitative RT-PCR

[0216] Gene expression was determined by quantitative reverse transcription-PCR. RNA was extracted from cells after treatment using RNeasy Micro Kit (Qiagen). Complementary DNAs (cDNAs) were produced using High-Capacity cDNA Reverse Transcription Kit (ThermoFisher, UK). qPCR was performed using SYBR Green PCR Master Mix (Primer Design, UK). The B2M gene and b-actin was used as an endogenous control.

[0217] Gene silencing and siRNA-resistant NuMA Mutant transfection siRNA-mediated gene silencing was carried out using Metafectene (T020-2, Biontex) or Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher Scientific, UK) and either a scrambled (siSCR) or a pool of specific siRNA sequences (sc-43978, Santa Cruz; Silencer pre-designed NUMA1 siRNA (42851), ThermoFisher Scientific, UK). 50 nM siRNA and 3 pl transfection reagent were each incubated in 100 pl Opti-MEM reduced serum media for 5 min before combining and incubating further for 20 min at room temperature. Transfection mix was then added dropwise to cells seeded at 2 x 105cells / well in complete medium. After 48 h incubation, cells were collected, and immunoblotting was performed to check the efficiency of knockdown.

[0218] For NuMA-S395A mutant or WT transfection, siRNA-transfected MDA-MB-231 were transfected with GFP-NuMA-S395A or GFP-NuMA-WT plasmids using PEI or TransfeX™ Transfection Reagent. 1 pg plasmid and 2 pl PEI (1 mg / ml) were each incubated in 100 pl Opti-MEM reduced serum media for 5 min before combining and incubating further for 20 min at room temperature. Transfection mix was then added dropwise to cells in complete medium. NuMA gene silencing as describe above was repeated and cells harvested after 24 h. cells were collected. Transfection efficiency was assessed by cell imaging and immunoblotting.

[0219] Measurement of nascent RNA transcription

[0220] MDA-MB-231 were grown on coverslips and incubated in dormancy condition [hypoxia (0.1% 02) and low nutrients] for 48 h. Click-iT RNA Alexa Flour 594 Imaging Kit (Invitrogen) was used according to manufacturer’s instructions to quantify nascent RNA transcription. Briefly, cells were incubated with 1 mM 5-ethynyl uridine (EU) for 1 h to label newly synthesized RNA. Cells were washed with PBS, fixed with 3.7% formaldehyde for 15 min, and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Cells were then incubated with Click- iT reaction cocktail containing Click-iT additive and Alexa Flour azide for 30 min. Following washing with PBS, cells were incubated with 1 :1 ,000 Hoechst dye in PBS for 15 min, rinsed with PBS then mounted on a glass slide with Immu-Mount. RNA labelled with Ell was then subjected to immunofluorescence analyses using Leica Fluorescence microscope.

[0221] Immunofluorescence

[0222] MDA-MB-231 were grown on coverslips and transferred to dormancy conditions with or without treatment for 48 h. Coverslips were rinsed in PBS, fixed with 3.7% formaldehyde for 15 min, and permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Permeabilized cells were rinsed in PBS, blocked in 1 % goat serum albumin, and incubated for 2 h at room temperature with the primary antibody of choice in PBS supplemented 1% goat serum albumin. Cells were then washed in PBS and incubated with either Alexa Fluor 488 or 594 secondary antibodies (1 :1000) in PBS supplemented 1 h at room temperature. Nuclei were counterstained with DAPI. Cells were subjected to immunofluorescence analyses using Leica fluorescence microscope.

[0223] Cytotoxicity assay

[0224] For 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, MDA-MB-231 cells (5000 / well) were seeded in 96-well plates and incubated overnight at 37°C with 5% CO2. Cells were transferred to dormancy condition and treated with compounds or solvent (DMSO) for 48 h. After compound treatment, the chemosensitivity of the cells was assessed, and cell survival post-treatment determined. Survival curves were obtained and IC50 values calculated using GraphPad Prism 8.

[0225] Tryphan blue exclusion assay

[0226] MDA-MB-231 cells (1 x 105 / well) were seeded in 6-well plate incubated overnight at 37°C with 5% CO2. Cells were transferred to dormancy condition and treated with compounds or solvent (DMSO) for 48 h. After treatment cells trypsinised and viable cells counted using tryphan blue. 3D tumour spheroids: Spheroids were formed using the aggregation method. MDA-MB-231 were stained with Vybrant DiD dye (ThermoFischer) according to manufacturer instruction. DiD-stained cell and control (2000 cells / well) were seeded per well into Biofloat-Flex-coated 96-well plate (faCellitate, UK) and centrifuged at 500 g for 5mins. Cells were allowed to aggregate forming a single mass of cells under standard cell culture conditions (37 °C, 5% CO2) for 10 days. Spheriods were imaged using MuviCyte Live-Cell Imaging Kit.

[0227] For p-NuMA antibody staining, spheroids were collected together and washed in PBS. T rypsin was added to spheroids to fragment spheroids into cells. Cells were washed with PBS, fixed with 3.7% formaldehyde for 15 min, and permeabilized with 0.3% Tween-20 for 15 min. Cells were then washed 3X with 1% BSA and incubated with p-NuMA antibody (1 in 100) in 1 % BSA for 1 hours. Following washing with PBS, cells were incubated with 1 :2,000 Alexa Flour 594 anti-rabbit secondary antibody for 30 minutes. Cells were washed in PBS and analysed using flow cytometry (BD Accuri C6 Plus Flow Cytometer).

[0228] Quantification and statistical analysis

[0229] All results were expressed as mean ± standard error of measurement (SEM) from at least 3 independent experiments. The statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). P < 0.05 was considered statistically significant

[0230] In vivo experiment

[0231] Female Balb / C nude mice (Harlan, UK) aged 6-8 weeks were used for all animal studies. All animal procedures were carried out under a project license issued by the UK Home Office and following UKCCCR guidelines. Mice were injected subcutaneously with in vitro engineered dormant MDA-MB-231 or CT-26 cells. Tumour growth was monitored and measured for 8 weeks.

[0232] Results

[0233] Establishing in vitro model of dormant breast cancer cells

[0234] First, the inventors engineered an in vitro breast cancer dormancy model using an aggressive breast cancer cell (MDA-MB-231) in hypoxia (0.1% O2) and low nutrients for 48 hrs (dormancy condition, DC). This mimics the microenvironment of in vivo cellular and tumour dormancy. Dormancy status of this model was determined using well-defined dormancy metrics including GO cell cycle arrest, lack of proliferation and cell death, and the expression of established dormancy markers.

[0235] The engineered DC-induced dormant breast cancer cells (MDA-MB-231) demonstrated cell cycle arrest at GO (Fig 1A) with no significant signs of apoptosis / cell death either by the lack of the appearance of sub-GO population (Fig 1 B) or cleave PARP expression (Fig. 1 D &E). Also, contrary to the increased cell number in proliferation conditions upon 48 hours incubation (PC; normoxia and optimum nutrients), cell numbers of the induced dormant cells remained significantly the same demonstrating a lack of proliferation and induction of dormancy (Fig 1 C). This lack of proliferation was confirmed with relatively low expression of Ki67 in DC cells compared to PC cells (Fig 1 F) and the lack of Rb phosphorylation, a key regulator of cell proliferation (Fig. 1 D and E). In addition, DC cells retained the intensity of DiD dye compared to the loss of DiD intensity (about 70%) in control PC cells (Fig 1G). Consistently, high expression levels of dormancy-associated markers including OGN and NR2F1 was also observed both at transcript and protein levels in DC-induced dormant MDA-MB-231 (Fig 1 E and H). NR2F1 is an established dormancy marker dormant cancer cells and in bone metastatic cells, a phenomenon associated with the breast cancer disease.

[0236] Re-activation of established dormant breast cancer cells

[0237] One major feature of dormant cancer cells is their ability to re-activate and proliferate again under favourable conditions to remerge as clinically detectable metastases, which are the main cause of mortality. To validate this feature of the engineered induced dormant breast cancer cells, the inventors induced dormancy in DC for 48 hours and re-introduced the induced dormant cells to PC for another 48 hours. The DC-induced dormant cells in PC entered cell cycle after 24 hours, with the appearance of significant S and G2 / M populations (Fig 2A), which indicate cell division and proliferation. Consistently, proliferation-associated protein Rb was hyper-phosphorylated (Fig.2B) upon incubation in PC. Re-proliferation of dormant cells was also observed with increased in cell numbers after 96 hours (D4) in PC (Figure C). To further validate this in vitro observation, the inventors injected DC-induced CT-26 cells in mice to assess their ability to grow tumour. As shown in Fig 2D, the DC-induced dormant cells develop tumours in vivo at a rate comparable to PC cells. Collectively, these observations confirm the validity of this engineered in vitro breast cancer dormancy model and demonstrates its reflection to the in vivo setting.

[0238] Identification of the S395 phosphorylation of NuMA as a key biomarker for dormant breast cancer cells

[0239] NuMA is a structural nuclear protein with well-reported functions, including mitosis / cell division, apoptosis, chromatin re-organisation, and DNA repair, in proliferating cells. However, contrary to its well-established association with dividing cells, high level of NuMA has recently been reported in cerebellum (non-dividing cells). To study the role NuMA in dormant cancer cells, the inventors assessed the expression of NuMA and its phosphorylation in the DC- induced dormant breast cancer cells.

[0240] The expression of NuMA did not significantly change at the transcript level in induced dormant cancer cells (Fig. 3A), but the inventors observed a modest but significant change at the protein level (Fig 3B). Surprisingly, the inventors observed a significant hyper-phosphorylation of serine residue (S395) of NuMA in dormant cancer cells (Fig 3B). Also, using proliferation marker, Ki67, the S395 phosphorylation of NuMA was again observed to occur in dormancy specific manner (Fig 3C). The phosphorylation of NuMA, particularly at its C-terminal (T2055) has been widely reported to be critical for spindle assembly and chromatin organization in dividing cells. To again confirm that the observed S395 phosphorylation is specific to dormant cells, the inventors assessed the expression of S395 and T2055 phosphorylation of NuMA in both DC-induced dormant and synchronised G2 / M cells. As shown in Fig 3D, the phosphorylation of NuMA at T2055 (C-terminal) appear specific to mitotic cells whilst S395 (N-terminal) is specific to dormant cells. It is worth noting that S395 phosphorylation of NuMA has been reported in response to DNA damage in dividing cells. On the contrary, the inventors observed p-S395 of NuMA in absence of DNA damage in non-dividing cells (DC-induced dormant cells). In addition, the expression of total NuMA increase in mitotic cells contrary to decreased expression in dormancy. To further confirm the observed relationship between p- S395 of NuMA and cancer dormancy in a tumour-like model, MDA-MB-231 cells were labelled with the lipophilic membrane dye DiD and grown into 3D tumour spheroids in normal culture conditions for 12 days. As shown in Fig 3E, 3D spheroids of MDA-MB-231 cells exhibited heterogeneous cell populations i.e high proliferative cells (DiD negative), medium proliferative cells, (DiD medium), and non- or slow-proliferative dormant cells (DiD positive). Strikingly, only the dormant population (DiD positive) of MDA-MDA-231 spheroids were observed with p- NuMA (S395) expression (Fig 3F). This further supports the inventors’ observation in 2D dormant MDA-MB-231 cells and suggests the relevance of p-NuMA (S395) in tumour dormancy and its subsequent recurrence. This observation led us to suggest that interfering with NuMA S395 phosphorylation could provide a new means to disrupt cancer cell survival during dormancy. p-NuMA (S395) expression is critical for the survival of dormant breast cancer cells.

[0241] NuMA is an identified substrate of ATM / ATR and its S395 phosphorylation is reported to occur in ATM / ATR dependent manner. To ascertain which of these kinases is responsible for the observed S395 phosphorylation, the inventors pharmacologically inhibited the activities of these kinase in DC-induced dormant breast cancer cells. Kinase activity of ATR but not ATM was observed to be key in the S395 phosphorylation of NuMA in the situation (Fig 4A). This is contrary to the reported phosphorylation at S395 of NuMA in response to I R in proliferating cells. The blocking of p-NuMA (S395) with ATRi was observed to significantly decreased the viability of dormant cancer cells at DC (Fig 4B), and further inhibit the re-proliferation of dormant cancer cells when re-incubated in PC conditions (Fig 4C),

[0242] The inventors acknowledge that ATR has many other substrates and the use of ATRi may affect the functions of other ATR substrates other than NuMA (Fig 4D and E), which may contribute to this observation. The inventors depleted endogenous NuMA and transfected cells with S395 mutant (A395) to assess the direct effect S395 NuMA phosphorylation to the survival of dormant breast cancer cells. Knockdown of NuMA (-50% of total protein) significantly decrease the viability (about 90% compared to control) of dormant cancer cells. This decrease in cell viability was rescued with either the transfection of WT NuMA or S395 mutant (A395) (Fig 4F and G), demonstrating the dependence of these dormant cells on NuMA for survival in dormant condition. The transfection of NuMA S395 mutant did block the S395 phosphorylation in DC (Fig 4H, left) though this did not suppress the survival of dormant cancer cell in DC. Importantly however, blocking NuMA S395 phosphorylation in DC significantly reduced the ability dormant cancer cells to re- proliferate when introduce to PC conditions, contrary to re-activation and proliferation of NuMA WT-transfected, SiNuMA. and SiControl (Fig 4H, right). These data demonstrate a critical role of S395 phosphorylation NuMA in the re-activation and survival of dormant cancer cells and re-activated cancer cells. p-NuMA (S395) is critical in transcription silencing of dormant breast cancer cells.

[0243] To explore the mechanism behind the observed critical role of NuMA’s S395 phosphorylation) in the survival of dormant cancer cells, the inventors first ascertained the cellular location of this hyper S395 phosphorylation considering that NuMA expression is found in nucleus as well as in cytoplasm. Using sub-cellular fractionation, the inventors observed NuMA expression in the cytoplasm, nucleoplasm and on chromatin of proliferating cells. However, in DC induced dormant breast cancer, NuMA was observed predominately in the nucleoplasm with significantly reduced expression in cytoplasm, and on chromatin. In addition, S395 phosphorylation of NuMA was observed mainly in nucleoplasm and completely absent on chromatin in DC-induced dormant breast cancer cells (Figure 5A and B). Dormancy of cancer cells is associated with a global reduction of rate of transcription, and the absence of NuMA on the chromatin in DC-induced dormant cancer cells suggested a possible role p- NuMA(S395) in dormancy associated transcription silencing. NuMA has recently been shown to promotes transcription and critical in the transcription machinery. To test this, the inventors pharmacologically blocked S395 phosphorylation in induced dormant breast cancer cells and assessed the level of transcription by labelling newly synthesized RNA with 5-ethynyl uridine (EU). As expected, transcription of induced dormant breast cancer cells was significantly silenced compared proliferating cells control. However, blocking of S395 phosphorylation of NuMA restored transcription level comparable to PC controls (Fig 5C). In addition, using proteomic approach the S395 phosphorylation of NuMA was observed recruit transcription- associated proteins from the chromatin in induced dormant breast cancer cells (Fig. 5D). Collectively these observations suggest S395 phosphorylation of NuMA as a key regulator of the adaptative survival mechanism of dormant cancer cells, hence blocking this event would disrupt these survival mechanisms.

[0244] Modelling of NuMA identifies S395 phosphorylation blocking ligands in silico

[0245] Collectively these in vitro data strongly suggest S395 phosphorylation of NuMA as a potential therapeutic target for the development of novel treatment option for dormant cancer cells. To ascertain the specificity and selectivity of observed p-NuMA (S395) to dormant cancer cells as compared to dormant normal tissues like the brain, the inventors investigated NuMA S395 phosphorylation in human brain lysate. Surprisingly, p-NuMA was absent in human brain tissue yet present in MDA-MB-231 dormant cells and its derived xenograft, CDX (Fig. 6A). This demonstrates the selectivity of NuMA (p-S395) in dormant cancer cells.

[0246] With this, the inventors further explored the therapeutic potential of p-S395 of NuMA in dormant cancer cells. The inventors investigated the ligand binding potential of the S395 region of NuMA. There is no available crystal structure of the full structure or the N-terminal of NuMA. The inventors therefore built a homology model using the 1-900 sequence from Numal (Fig 6B). One binding hotspot covering Ser395 was identified in this model using SiteMap study. In addition, further MD simulations reveal that the secondary structures of the identified binding site region appear reasonably stable (Fig 6C).

[0247] This homology model was used in a virtual screening approach to identify compounds with the ligand binding ability toward the S395 region of NuMA (Fig 6D and E).

[0248] NP-26A blocks S395 phosphorylation but do not affect A TR kinase activity

[0249] The inventors identified and tested compound NP-26A (compound according to formula I). NP-26A was found to block the NuMA S395 phosphorylation in DC- induced-breast cancer cells (Fig 7C). In addition, despite comparable inhibition of S395 phosphorylation by NP-26A at 50 pM, it demonstrates less effect on the phosphorylation of other ATR / ATM substrates which is contrary to earlier observations with the use of ATRi (Fig 4). This data demonstrates the promising potential of selectively targeting S395 of NuMA as new therapy for cancer dormancy.

[0250] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0251] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0252] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0253] Sequences

[0254] Homo sapiens NuMA protein sequence highlighting the N-terminal S395 residue (SEQ ID NO: 1):

[0255] TLHATRGAALLSWVNSLHVADPVEAVLQLQDCSI Fl KI I DRI HGTEEGQQI LKQPVSERLDFV CSFLQKNRKHPSSPECLVSAQKVLEGSELELAKMTMLLLYHSTMSSKSPRDWEQFEYKIQA ELAVILKFVLDHEDGLNLNEDLENFLQKAPVPSTCSSTFPEELSPPSHQAKREIRFLELQKVA SSSSGNNFLSGSPASPMGDILQTPQFQMRRLKKQLADERSNRDELELELAENRKLLTEKDA QIAMMQQRIDRLALLNEKQAASPLEPKELEELRDKNESLTMRLHETLKQCQDLKTEKSQMD RKINQLSEENGDLSFKLREFASHLQQLQDALNELTEEHSKATQEWLEKQAQLEKELSAALQ DKKCLEEKNEILQGKLSQLEEHLSQLQDNPPQEKGEVLGDVLQLETLKQEAATLAANNTQL QARVEMLETERGQQEAKLLAERGHFEEEKQQLSSLITDLQSSISNLSQAKEELEQASQAHG ARLTAQVASLTSELTTLNATIQQQDQELAGLKQQAKEKQAQLAQTLQQQEQASQGLRHQV EQLSSSLKQKEQQLKEVAEKQEATRQDHAQQLATAAEEREASLRERDAALKQLEALEKEK AAKLEILQQQLQVANEARDSAQTSVTQAQREKAELSRKVEELQACVETARQEQHEAQAQV AELELQLRSEQQKATEKERVAQEKDQLQEQLQALKESLKVTKGSLEEEKRRAADALEEQQ RCISELKAETRSLVEQHKRERKELEEERAGRKGLEARLQQLGEAHQAETEVLRRELAEAMA AQHTAESECEQLVKEVAAWRERYEDSQQEEAQYGAMFQEQLMTLKEECEKARQELQEAK EKVAGIESHSELQISRQQNELAELHANLARALQQVQEKEVRAQKLADDLSTLQEKMAATSK EVARLETLVRKAGEQQETASRELVKEPARAGDRQPEWLEEQQGRQFCSTQAALQAMERE AEQMGNELERLRAALMESQGQQQEERGQQEREVARLTQERGRAQADLALEKAARAELEM RLQNALNEQRVEFATLQEALAHALTEKEGKDQELAKLRGLEAAQIKELEELRQTVKQLKEQL AKKEKEHASGSGAQSEAAGRTEPTGPKLEALRAEVSKLEQQCQKQQEQADSLERSLEAER ASRAERDSALETLQGQLEEKAQELGHSQSALASAQRELAAFRTKVQDHSKAEDEWKAQVA RGRQEAERKNSLISSLEEEVSILNRQVLEKEGESKELKRLVMAESEKSQKLEERLRLLQAET ASNSARAAERSSALREEVQSLREEAEKQRVASENLRQELTSQAERAEELGQELKAWQEKF FQKEQALSTLQLEHTSTQALVSELLPAKHLCQQLQAEQAAAEKRHREELEQSKQAAGGLR AELLRAQRELGELIPLRQKVAEQERTAQQLRAEKASYAEQLSMLKKAHGLLAEENRGLGER ANLGRQFLEVELDQAREKYVQELAAVRADAETRLAEVQREAQSTARELEVMTAKYEGAKV KVLEERQRFQEERQKLTAQVEQLEVFQREQTKQVEELSKKLADSDQASKVQQQKLKAVQA QGGESQQEAQRLQAQLNELQAQLSQKEQAAEHYKLQMEKAKTHYDAKKQQNQELQEQL

[0256] RSLEQLQKENKELRAEAERLGHELQQAGLKTKEAEQTCRHLTAQVRSLEAQVAHADQQLR

[0257] DLGKFQVATDALKSREPQAKPQLDLSIDSLDLSCEEGTPLSITSKLPRTQPDGTSVPGEPAS

[0258] PISQRLPPKVESLESLYFTPIPARSQAPLESSLDSLGDVFLDSGRKTRSARRRTTQIINITMTK KLDVEEPDSANSSFYSTRSAPASQASLRATSSTQSLARLGSPDYGNSALLSLPGYRPTTRS

[0259] SARRSQAGVSSGAPPGRNSFYMGTCQDEPEQLDDWNRIAELQQRNRVCPPHLKTCYPLE

[0260] SRPSLSLGTITDEEMKTGDPQETLRRASMQPIQIAEGTGITTRQQRKRVSLEPHQGPGTPE

[0261] SKKATSCFPRPMTPRDRHEGRKQSTTEAQKKAAPASTKQADRRQSMAFSILNTPKKLGNS

[0262] LLRRGASKKALSKASPNTRSGTRRSPRIATTTASAATAAAIGATPRAKGKAKH

Claims

CLAIMS1. A nuclear mitotic apparatus (NuMA) protein serine 395 (S395) phosphorylation inhibitor for use in preventing and / or treating cancer in a subject.

2. The NuMA S395 phosphorylation inhibitor for use according to claim 1, wherein the inhibitor is a small molecule, a biological product, or a conjugate thereof.

3. The NuMA S395 phosphorylation inhibitor for use according to claim 2, wherein the small molecule is a compound having the structure of:[Formula I],4. The NuMA S395 phosphorylation inhibitor for use according to claim 2, wherein the biological product is selected from the group consisting of a peptide, a polypeptide, a protein, a dendrimer, and a nucleic acid; optionally wherein the protein is an antibody or a fragment thereof.

5. The NuMA S395 phosphorylation inhibitor for use according to claim 4, wherein the antibody is a monoclonal antibody, and / or the antibody is monospecific or multispecific.

6. The NuMA S395 phosphorylation inhibitor for use according to any one of claims 1-5, wherein the cancer cell is a solid cancer cell or a liquid cancer cell.

7. The NuMA S395 phosphorylation inhibitor for use according to claim 6, wherein the solid cancer cell is selected from the group consisting of a breast cancer cell, a colorectal cancer cell, a prostate cancer cell, a glioblastoma cell, a lung cancer cell, bone cancer cell, and an ovarian cancer cell, or any other cancer whereby cancer dormancy underpins recurrence.

8. The NuMA S395 phosphorylation inhibitor for use according to claim 7, wherein the solid cancer cell is a breast cancer cell or prostate cancer cell or colorectal cancer cell.

9. The NuMA S395 phosphorylation inhibitor for use according to any one of claims 1-8, wherein preventing and / or treating of cancer is by i) reducing the number of dormant cancer cells in the subject; ii) reducing the number of re-activated cancer cells in the subject, optionally wherein the reduction of re-activated cancer cells is by reducing proliferation of re-activated cancer cells; and / or iii) inhibiting an active cancer cell becoming a dormant cancer cell10. The NuMA S395 phosphorylation inhibitor for use according to claim 9, wherein the dormant cancer cell is selected from the group consisting of dormant breast cancer cell, dormant colorectal cancer cell, dormant prostate cancer cell, dormant glioblastoma cell, dormant lung cancer cell, and dormant ovarian cancer cell.11 . The NuMA S395 phosphorylation inhibitor for use according to claim 9 or 10, wherein the dormant cancer cell has upregulated expression of NR2F1 and / or AXOL.

12. The NuMA S395 phosphorylation inhibitor for use according to any one of claims 10 or 11 , wherein the dormant breast cancer cell has upregulated expression of a marker selected from the group consisting of OGN, Gas6, CD13, and MME.

13. The NuMA S395 phosphorylation inhibitor for use according to claims 10-12, wherein the dormant breast cancer cell has reduced Rb phosphorylation and / or downregulated Ki67 expression.

14. The NuMA S395 phosphorylation inhibitor of any one of claims 1 to 13, wherein the NuMA S395 phosphorylation inhibitor is provided in combination with a cancer therapeutic.

15. The NuMA S395 phosphorylation inhibitor for use according to claim 14, wherein the cancer therapeutic is selected from the group consisting of immunotherapy, chemotherapy, radiation therapy, hormonal therapy, an anti-cancer vaccine, an anti-cancer virus, and surgery.

16. The NuMA S395 phosphorylation inhibitor for use according to claim 16, wherein the NuMA S395 phosphorylation inhibitor and cancer therapeutic are provided sequentially and / or simultaneously.

17. A NuMA S395 phosphorylation inhibitor for use as a medicament, optionally wherein the NuMA S395 phosphorylation inhibitor is as defined in any one of claims 2-5.

18. A pharmaceutical composition comprising a NuMA S395 phosphorylation inhibitor and a pharmaceutically acceptable excipient, diluent, and / or carrier, optionally wherein the NuMA S395 phosphorylation inhibitor is as defined in any one of claims 2-5.

19. A method of screening for a candidate compound for preventing and / or treating cancer, wherein the method comprises:• contacting a cancer cell with a test compound; and• determining a level of phosphorylation on NuMA S395; wherein a reduction in the level of phosphorylation on NuMA S395 in the contacted cell as compared to a control is indicative of the test compound being a compound for preventing and / or treating cancer.

20. A method of detecting a dormant cancer cell in a subject, the method comprising determining the level of phosphorylation on NuMA S395 in a cancer cell in a sample from the subject, wherein if the cancer cell has hyperphosphorylated NuMA S395 it is indicative of the cancer cell being a dormant cancer cell.

21. The method according to claim 20, wherein the sample is selected from the list consisting of a solid tissue biopsy sample, a blood sample, a fecal sample, and a urine sample.

22. A method of preventing and / or treating cancer in a subject, the method comprising administering a therapeutically effective amount of an NuMA S395 phosphorylation inhibitor to the subject in need thereof.

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