WO2018158297A1

WO2018158297A1 - Chk1 inhibition, synthetic lethality and cancer treatment

Info

Publication number: WO2018158297A1
Application number: PCT/EP2018/054896
Authority: WO
Inventors: Paul Workman; Rebecca Florence ROGERS; Paul Andrew Clarke
Original assignee: The Institute Of Cancer Research: Royal Cancer Hospital
Priority date: 2017-02-28
Filing date: 2018-02-28
Publication date: 2018-09-07
Also published as: GB201703248D0

Abstract

The present invention provides a composition comprising a Checkpoint (Chk1) inhibitor for use in a method of treatment of a cancer in a mammalian subject, wherein the cancer comprises one or more cells that carry mutations in or are deficient in DNA polymerase alpha and/or DNA polymerase epsilon. Also provided is a composition comprising a Checkpoint 1 (Chk1) inhibitor for use in a method of treatment of a cancer in a mammalian subject, wherein the Chk inhibitor is administered simultaneously, separately or sequentially with an inhibitor of DNA polymerase epsilon. Related methods of treatment are also provided.

Description

CHKl Inhibition, Synthetic Lethality and Cancer Treatment

This application claims priority from GB1703248.3 filed 28 February 2017, the contents and elements of which are herein incorporated by reference for all purposes.

Field of the invention

The present invention relates to materials and methods for

sensitising and treating cancers, including with combination therapy, and relates to methods for selecting and treating cancer patients expected to benefit from Checkpoint kinase 1 (Chkl) inhibition .

Background to the invention

Genomic instability is a feature of many human cancers and can arise from intracellular and extracellular sources of DNA damage. To ensure genomic integrity is maintained, cells respond to DNA damage by activating the DNA damage response (DDR) . Cell-cycle checkpoints are activated as part of the DDR, one of which is the ATR-CHK1 checkpoint which responds primarily to the presence of single- stranded DNA (ssDNA) . ssDNA accumulates at stalled replication forks as a result of the functional uncoupling of the MCM helicase and DNA polymerase activity. ssDNA is coated and protected by replication protein A (RPA) which is able to recruit a number of DDR proteins including ATR-CHK1. ATR phosphorylates CHKl, a Ser/Thr kinase, which then undergoes autophosphorylation resulting in active CHKl which then phosphorylates a number of down-stream targets ultimately resulting in cell cycle arrest and replication fork stabilisation. Several studies have shown that CHKl inhibitors have single agent activity in certain cancer types with high levels of replication stress (Brooks et al . , Oncogene, 2013, Vol. 32(6), pp. 788-796; Cole et al., PNAS, 2011, Vol. 108(8), pp. 3336-3341; Walton et al . , Oncotarget, 2016, Vol. 7(3), pp. 2329-2342; and Ferrao et al., Oncogene, 2012, Vol. 31, pp. 1661-1672) . However, there are currently no reported biomarkers of sensitivity to single agent CHK1 inhibition .

Synthetic lethality occurs when the simultaneous loss of two gene products results in cell death whereas loss of either of the genes alone is compatible with cell viability.

Hocke et al . , Oncotarget, 2016, Vol. 7, No. 6, pp. 7080-7095, describes a synthetic lethal screen identification of ATR-inhibition as a novel therapeutic approach for POLDl-deficient cancers.

Taricani et al . , Cell Cycle, 2009, Vol. 8, No. 3, pp. 482-489, reports that replication stress activates DNA polymerase alpha- associated Chkl . It is further suggested that DNA polymerase alpha is a key target of DNA antimetabolites and that the Chkl/DNA Pol replication complex plays a major role during replication checkpoint activation .

Han et al., Nature Chemical Biology, 2016, Vol. 12, pp. 511-515, reports that the antitumor toxin CD437 is a direct inhibitor of DNA polymerase alpha.

US2010/ 0143332 describes combination therapy for proliferative disorders. In particular, therapy that combines a CHK1 inhibitor with a DNA polymerase alpha inhibitor is suggested. It is further stated that the DNA polymerase inhibitor should be relatively selective for DNA polymerase alpha, e.g. exhibiting 10-fold or greater inhibition of DNA polymerase alpha compared with another DNA polymerase such as DNA polymerase epsilon. US2010/ 0143332 also describes treatment of a proliferative disorder with a DNA

polymerase alpha inhibitor and a Chkl inhibitor of subjects in whom the proliferative disorder to be treated involves reduction or loss of function of at least one of the p53 or Rb gene products. WO2013/171470 describes compounds that inhibit checkpoint kinase 1

(CHK1) inhibitors, including CCT245737, and their use in the treatment of proliferative disorders such as cancer. While CHKl inhibition shows promise in the treatment of various cancers, there remains an unmet need for further agents to sensitise cancer cells to CHKl inhibition therapy and for biomarkers of sensitivity to CHKl inhibition therapy. The present invention seeks to fulfil these needs and provides further related advantages.

Brief Description of the Invention

The present inventors have performed a large synthetic lethal siRNA screen of the druggable genome to identify those gene products whose loss is synthetically lethal with CHKl inhibition in cancer cells. With the aims of identifying patient populations who are sensitive to single agent CHKl inhibition and also to identify novel molecular targets for use in combination with CHKl inhibitors. As described in detail herein, reduction in POLA1, POLE or POLE2 activity sensitises NSCLC and colon cancer cell lines to CHKl inhibition. This is in contrast to the findings reported in US2010/0143332 where no synergy was seen between Chkl inhibition and Pol ε or Pol δ inhibition.

Accordingly, in a first aspect the present invention provides a composition comprising a Checkpoint 1 (Chkl) inhibitor for use in a method of treatment of a cancer in a mammalian subject, wherein the Chkl inhibitor is administered simultaneously, separately or sequentially with an inhibitor of DNA polymerase epsilon.

In a second aspect, the present invention provides a composition comprising a DNA polymerase epsilon inhibitor for use in a method of treatment of a cancer in a mammalian subject, wherein the DNA polymerase epsilon inhibitor is administered simultaneously, separately or sequentially with an inhibitor of Checkpoint 1 (Chkl) .

In a third aspect, the present invention provides a composition (e.g. a pharmaceutical composition) comprising a Checkpoint 1 (Chkl) inhibitor and a DNA polymerase epsilon inhibitor for use in a method of treatment of a cancer in a mammalian subject. The composition may further comprise one or more pharmaceutically acceptable carriers in addition to the Chkl inhibitor and the DNA polymerase epsilon inhibitor. While it is envisaged that the Chkl inhibitor and the DNA polymerase epsilon inhibitor may be combined in a mixed composition (i.e. two actives in a single carrier), it is also specifically contemplated herein that the Chkl inhibitor and the DNA polymerase epsilon inhibitor may be provided as a co-formulation in which the two actives are kept physically separate, such as in a dual chamber vial or syringe. In accordance with the first, second and third aspects of the invention, the Chkl inhibitor may inhibit Chkl with an IC50 of ≤ 1 μΜ, for example < 100 nM, < 10 nM, or < 1 nM. Additionally or alternatively, the Chkl inhibitor may exhibit at least 10-fold (e.g. at least 100-fold or at least 1000-fold) selectivity for Chkl relative to Chk2. Selectivity may be determined by measuring the

IC50 for inhibition of Chkl and the IC50 for inhibition of Chk2. An inhibitor that is found to have an IC50 for inhibition of Chkl that is 10 times lower (i.e. more potent) than the corresponding IC50 for inhibition of Chk2 may be said to exhibit 10-fold selectivity for Chkl. The IC50 for Chkl may be measured using techniques known in the art, such as an in vitro human Chkl kinase assay described in Jiang et al . , Molecular Cancer Therapeutics, 2004, Vol. 3(10), pp. 1221-1227 (the content of which is expressly incorporated herein by reference) .

In accordance with the first, second and third aspects of the invention, the Chkl inhibitor may in some cases be selected from the group consisting of: CCT245737, LY-2606368, MK-8776, GDC-0575, V158411, LY2603618, GDC-0425, AZD7762, PF-477736, XL844, CHIR-124 and CEP-3891. Further details of these compounds are provided in Table 1 below. In some cases, the Chkl inhibitor may comprise one or more siRNAs directed against the CHEK1 gene (i.e. the gene encoding Chkl) . In some cases, the Chkl inhibitor may comprise an antibody molecule that selectively binds the Chkl protein or a portion thereof. For example, the antibody molecule may be a monoclonal antibody or fragment thereof (e.g. a Fab or an scFv) . In accordance with the first, second and third aspects of the invention, the DNA polymerase epsilon inhibitor may inhibit DNA polymerase epsilon with an IC50 of ≤ 1 μΜ. Additionally or

alternatively, the DNA polymerase epsilon inhibitor may exhibit at least 10-fold selectivity for DNA polymerase epsilon relative to DNA polymerase alpha and/or DNA polymerase delta. Selectivity may be determined by measuring the IC50 for inhibition of DNA polymerase epsilon and the IC50 for inhibition of Chk2. An inhibitor that is found to have an IC50 for inhibition of DNA polymerase epsilon that is 10 times lower (i.e. more potent) than the corresponding IC50 for inhibition of DNA polymerase alpha and/or of DNA polymerase delta may be said to exhibit 10-fold selectivity for DNA polymerase epsilon. The IC50 for DNA polymerase epsilon may be measured using techniques known in the art, such as an in vitro human DNA

polymerase epsilon, alpha and delta assay, respectively, which are described in Mizushina et al . , Biochem. J., 2003, Vol. 370, pp. 299- 305 (and references 25-27 cited therein) - the contents of which are expressly incorporated herein by reference. In accordance with the first, second and third aspects of the invention, the DNA polymerase epsilon inhibitor may inhibit the POLE subunit (e.g. the proof-reading domain thereof or the exonuclease domain thereof) and/or the POLE2 subunit of DNA polymerase epsilon. In accordance with the first, second and third aspects of the invention, the DNA polymerase epsilon inhibitor may be selected from the group consisting of: HO-CH₂-CH₂-CH (NH₂) -CO-NH-CH₂-CH₂-OH, L- homoserylaminoethanol , sulphoquinovosyl diacylglycerol and

carbonyldiphosphonate . Details of these compounds are found in Table 2 below. In some cases, the DNA polymerase epsilon inhibitor may comprise one or more siRNAs directed against the POLE gene and/or the P0LE2 gene. In some cases, the DNA polymerase epsilon inhibitor may comprise an antibody molecule that selectively binds the DNA polymerase epsilon complex or the POLE subunit thereof or the POLE2 subunit thereof. For example, the antibody molecule may be a monoclonal antibody or fragment thereof (e.g. a Fab or an scFv) . In a fourth aspect, the present invention provides a composition comprising a Checkpoint 1 (Chkl) inhibitor for use in a method of treatment of a cancer in a mammalian subject, wherein the cancer comprises one or more cells that carry mutations in or are deficient in DNA polymerase alpha and/or DNA polymerase epsilon.

In some embodiments the method of treatment comprises determining whether or not the subject has a cancer with a mutation in or which is deficient in DNA polymerase alpha and/or DNA polymerase epsilon. This may be done actively by such as by testing a sample obtained from the subject (e.g. containing one or more tumour cells) or may be confirmatory in nature (e.g. by consulting a data record, prior test result or interrogating a database of patient records) .

Likewise, comparison of a test sample reading with a control sample reading may be active (wherein the control sample is directly tested) or confirmatory, e.g., wherein a test sample reading is compared with a predetermined control value, reference range or other reading. The control reading may, for example, be a reading derived from a non-cancerous tissue of the same individual as the test sample (preferably, a reading taken from the same individual at an earlier time point (e.g. pre-disease) or a reading taken from another individual (e.g. a subject not suffering from a cancer, who may optionally be matched, such as age-matched and/or gender matched) ) . The control reading may in some cases be derived from a plurality of subjects (e.g. a population reference range) . Where an active test is carried out, this may be by any suitable method such as those known in the art for detecting DNA mutations and/or down- regulated proteins and/or inactive DNA polymerase. In particular embodiments of this and other aspects of the present invention, the method may comprise:

(a) determining in a test sample obtained from the subject whether a population of cells of said cancer has:

(i) a loss-of-function mutation in the POLA1 gene, the POLE gene and/or the P0LE2 gene;

(ii) a decreased level of POLA1 protein, POLE protein and/or POLE2 protein relative to the level of the respective protein in a population of cells from a control sample obtained from a cancer-free tissue from the same subject as the test sample or obtained from a cancer-free control subject; and/or

(iii) a decreased activity of DNA polymerase alpha and/or DNA polymerase epsilon relative to the activity of the respective polymerase in a population of cells from a control sample obtained from a cancer-free tissue from the same subject as the test sample or obtained from a cancer-free control subject; and

(b) having determined that test sample in (a) has said loss- of-function mutation, said decreased protein level and/or said decreased polymerase activity, administering the Chkl inhibitor to the subject.

In particular embodiments of this and other aspects of the present invention, the cancer may comprise cells:

(i) that carry mutations in the POLE gene and/or the P0LE2 gene,

(ii) that have decreased level of POLE protein and/or POLE2 protein, and/or

(iii) that are deficient in DNA polymerase epsilon activity.

In particular embodiments of this and other aspects of the present invention, the method may comprise:

(i) a loss-of-function mutation in the POLE gene and/or the P0LE2 gene;

(ii) a decreased level of POLE protein and/or POLE2 protein relative to the level of the respective protein in a population of cells from a control sample obtained from a cancer-free tissue from the same subject as the test sample or obtained from a cancer-free control subject; and/or

(iii) a decreased activity of DNA polymerase epsilon relative to the activity of DNA polymerase epsilon in a population of cells from a control sample obtained from a cancer-free tissue from the same subject as the test sample or obtained from a cancer-free control subject; and

(b) having determined that test sample in (a) has said loss- of-function mutation, said decreased protein level and/or said decreased DNA polymerase epsilon activity, administering the Chkl inhibitor to the subject.

As with the first to third aspects of the invention, the Chkl inhibitor may inhibit Chkl with an IC50 of ≤ 1 μΜ, for example ≤ 100 nM, < 10 nM, or < 1 nM. Additionally or alternatively, the Chkl inhibitor may exhibit at least 10-fold (e.g. at least 100-fold or at least 1000-fold) selectivity for Chkl relative to Chk2.

In some embodiments, the Chkl inhibitor may be selected from the group consisting of: CCT245737, LY-2606368, MK-8776, GDC-0575, V158411, LY2603618, GDC-0425, AZD7762, PF-477736, XL844, CHIR-124 and CEP-3891.

In some embodiments, the method of treatment further comprises administering an inhibitor of DNA polymerase epsilon.

In some embodiments in accordance with any aspect of the present invention, the cancer may be selected from the group consisting o a lung cancer, a colorectal cancer and an endometrial cancer.

In particular, the lung cancer may be Non-small-cell lung carcinoma and/or the colorectal cancer may be a colorectal adenocarcinoma.

In some embodiments in accordance with any aspect of the present invention, the cancer may comprise a mutation in the POLE gene and/or the P0LE2 gene. As described further in the Examples herein, antagonism and/or downregulation of Pol ε was found to sensitise cancer cells to the toxic effects of Chkl inhibition. Without wishing to be bound by any particular theory, the present inventors believe that cancers that are deficient in Pol ε, such as those having one or more POLE gene mutations and/or one or more P0LE2 gene mutations will be more susceptible to the anti-cancer effects of Chkl inhibition therapy. A number of clinically relevant POLE gene mutations have been described (see, for example, Hansen et al . , Familial Cancer, 2015, Vol. 14, pp. 437-448; Stenzinger et al . , Cancer Medicine, 2014, Vol. 3(6), pp. 1527-1538; Ahn et al . ,

Oncotarget, 2016, Advance Publication, pp. 1-12; and Lange et al., Nature Reviews Cancer, 2011, Vol. 11, pp. 96-110 - all of which are expressly incorporated herein by reference) . In certain cases, the POLE gene mutation may be selected from the Exon 9, Exon 11, Exon 13 and Exon 14 mutations set forth in Table 2, on page 1532 of

Stenzinger et al . , Cancer Medicine, 2014, Vol. 3(6), pp. 1527-1538, which table is expressly incorporated herein by reference. For example, mutations such as C.1033OT (p.Q345*) that are described in said table as "Truncating" may result in partial or total loss of Pol ε activity. In certain cases, the mutation in the POLE gene may be selected from the group consisting of: c.l373A>T (p . Tyr458Phe) , and a mutation encoding the amino acid substitution Pro286Arg.

In a fifth aspect, the present invention provides a method of treatment of a cancer in a mammalian subject, the method comprising administering a therapeutically effective amount of a Checkpoint 1 (Chkl) inhibitor to the subject in need thereof, wherein the Chkl inhibitor is administered simultaneously, separately or sequentially with an inhibitor of DNA polymerase epsilon.

In a sixth aspect, the present invention provides a method of treatment of a cancer in a mammalian subject, the method comprising administering a therapeutically effective amount of a DNA polymerase epsilon inhibitor to the subject in need thereof, wherein the DNA polymerase epsilon inhibitor is administered simultaneously, separately or sequentially with an inhibitor of Checkpoint 1 (Chkl) . In a seventh aspect, the present invention provides a method of treatment of a cancer in a mammalian subject, the method comprising administering a therapeutically effective amount of a composition (e.g. a pharmaceutical composition) comprising a Checkpoint 1 (Chkl) inhibitor and a DNA polymerase epsilon inhibitor to the subject in need thereof. The composition may further comprise one or more pharmaceutically acceptable carriers in addition to the Chkl inhibitor and the DNA polymerase epsilon inhibitor. While it is envisaged that the Chkl inhibitor and the DNA polymerase epsilon inhibitor may be combined in a mixed composition (i.e. two actives in a single carrier) , it is also specifically contemplated herein that the Chkl inhibitor and the DNA polymerase epsilon inhibitor may be provided as a co-formulation in which the two actives are kept physically separate, such as in a dual chamber vial or syringe.

In accordance with the fifth, sixth or seventh aspect of the present invention, the Chkl inhibitor and/or the DNA polymerase epsilon inhibitor may be as defined in connection with the first, second, third or fourth aspects of the invention.

In an eight aspect the present invention provides a method of treatment of a cancer in a mammalian subject, the method comprising administering a therapeutically effective amount of a Checkpoint 1 (Chkl) inhibitor to the subject, wherein the cancer comprises one or more cells that carry mutations in or are deficient in DNA

polymerase alpha and/or DNA polymerase epsilon.

In some embodiments the method of treatment comprises determining whether or not the subject has a cancer with a mutation in or which is deficient in DNA polymerase alpha and/or DNA polymerase epsilon. This may be done actively by such as by testing a sample obtained from the subject (e.g. containing one or more tumour cells) or may be conformational in nature (e.g. by consulting a data record, prior test result or interrogating a database of patient records) . Where an active test is carried out, this may be by any suitable method such as those known in the art for detecting DNA mutations and/or down-regulated proteins and/or inactive DNA polymerase. In

particular embodiments of this and other aspects of the present invention, the method may comprise:

(ii) a decreased level of P0LA1 protein, POLE protein and/or POLE2 protein relative to the level of the respective protein in a population of cells from a control sample obtained from a cancer-free tissue from the same subject as the test sample or obtained from a cancer-free control subject; and/or

(i) that carry mutations in the POLE gene and/or the P0LE2 gene,

(ii) that have decreased level of POLE protein and/or POLE2 protein, and/or

(iii) that are deficient in DNA polymerase epsilon activity.

(i) a loss-of-function mutation in the POLE gene and/or the P0LE2 gene;

(b) having determined that test sample in (a) has said loss- of function mutation, said decreased protein level and/or said decreased DNA polymerase epsilon activity, administering the Chkl inhibitor to the subject.

In some embodiments, the method of treatment further comprises administering an inhibitor of DNA polymerase epsilon. In accordance with this aspect of the present invention, the cancer, including any mutations carried therein, may be as defined in connection with the fourth aspect of the invention.

In a ninth aspect, the present invention provides use of a

Checkpoint 1 (Chkl) inhibitor in the preparation of a medicament for treatment of a cancer in a mammalian subject, wherein the Chkl inhibitor is administered simultaneously, separately or sequentially with an inhibitor of DNA polymerase epsilon.

In a tenth aspect, the present invention provides use of a DNA polymerase epsilon inhibitor in the preparation of a medicament for treatment of a cancer in a mammalian subject, wherein the DNA polymerase epsilon inhibitor is administered simultaneously, separately or sequentially with an inhibitor of Checkpoint 1 (Chkl) .

In an eleventh aspect, the present invention provides use of a composition (e.g. a pharmaceutical composition) comprising a

Checkpoint 1 (Chkl) inhibitor and a DNA polymerase epsilon inhibitor in the preparation of a medicament for treatment of a cancer in a mammalian subject.

In accordance with the ninth, tenth or eleventh aspect of the present invention, the Checkpoint 1 (Chkl) inhibitor, the DNA polymerase epsilon inhibitor, or a composition ( s ) thereof, may be as defined in connection with the first, second or third aspects of the invention. The medicament ( s ) may be for use in any method as defined in connection with the fifth, sixth, seventh or eighth aspects of the invention.

In accordance with any aspect of the present invention, the subject may be a human, a companion animal (e.g. a dog or cat) , a laboratory animal (e.g. a mouse, rat, rabbit, pig or non-human primate), a domestic or farm animal (e.g. a pig, cow, horse or sheep) .

Preferably, the subject is a human.

Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying figures. However various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

The present invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or is stated to be expressly avoided. These and further aspects and embodiments of the invention are described in further detail below and with reference to the accompanying examples and figures.

Brief Description of the figures

Figure 1 shows a summary of siRNA screen hits. The left-hand panel shows results for the A549 cancer cell line; the right-hand panel shows results for the SW620 cancer cell line. Data shown as the average difference in robust Z-score between CCT245737 treated and untreated conditions; the greater difference in robust Z-score the greater the difference in cell viability. Top hits common to both cell lines highlighted as closed coloured circles. Black open circles (0) represent individual Dharmacon ON-TARGET pooled siRNA' s targeting -6500 genes. n≥3.

Figure 2 shows validation of hits in A549 and SW620. (A+C) Cells were transfected with either 25 nM Allstars negative control, WEE1 siRNA, death siRNA or individual Qiagen POLA1 , POLE or P0LE2 siRNA at a range of concentrations for 48 h. Mock cells treated with 0.3 % HiPerFect alone. Cells were then treated with screening

concentration of CCT245737 for 82 h, n>3. (B+C) Western blot analysis of cell lysates harvested 48 h after transfection with individual Qiagen POLA1 , POLE and P0LE2 siRNAs at a range of concentrations (n≥l) . GAPDH used as loading control. Statistical analysis used unpaired Student's t-test *p<0.05, **p<0.01,

***p<0.001, ****p<0.0001.

Figure 3 shows CCT245737 GI50 determinations in combination with non- lethal concentrations of POLA1 /POLE/P0LE2 siRNA. (A-B) Cells were transfected for 48 h; Allstars negative siRNA at 25 nM and POLA1 (#3), POLE (#2), and P0LE2 (#4) siRNA at 0.1 nM, 0.3 nM and 0.3 nM in A549 cells, respectively, or at 1 nM in SW620 cells (n≥2) . Cells were then treated with a range of CCT245737 concentrations for 82 h. CCT245737 GI50S were determined using GraphPad Prism 6.0. n≥3. (A, mean ± SD. B, average and range) . (C-D) Boxplot displaying GI50S calculated from SRB data. Statistical analysis used unpaired students t-test *p<0.05, ***p<0.001, ****p<0.0001, NS= not

significant . Figure 4 shows hit validation in additional NSCLC and colon cancer cell lines. (A-D) Cells were transfected with either 25 nM Allstars negative control, WEE1 siRNA, death siRNA or individual Qiagen POLA1, POLE or P0LE2 siRNA at a range of concentrations for 48 h. Mock cells treated with 0.2 % HiPerFect alone. Cells were then treated with optimum non-lethal concentration of CCT245737 for 82 h, n≥3. Statistical analysis used unpaired students t-test *p<0.05, ***p<0.001.

Figure 5 shows replication stress markers increase with combination. Cells were treated transfected with 0.1 nM (A549) or 1 nM (SW620) POLA1 #3, POLE #2 or P0LE2 #4 siRNA for 48 h. Allstars negative control at 25 nM, mock treated with lipid only. Cells were then treated with 0.4 or 0.8 μΜ CCT245737, A549 and SW620 respectively, for 24 h. GAPDH used as loading control. N=2. ^Λ υ ' =untransfected cells with no drug, ^Λ+' = CCT245737, ^,-^Λ = no drug.

Figure 6 shows γΗ2ΑΧ intensity in cancer cells treated with

CCT245737, aphidicolin or combination. Cells plated in 96 well plates and incubated for 48 h, cell were then treated for 24 h.

Cells were fixed and permeabilised and incubated with yH2AX primary antibody (1:500) O/N at 4C. Cells were then incubated with goat- anti-mouse secondary antibody Alexafluor 488 (1:1000) for 1 h at RT, then incubated with DAPI (1:1000) for 5 min at RT . Cells were imaged on IN Cell and foci were analysed on IN Cell analyser workstation 3.7.2. Results shown as γΗ2ΑΧ intensity per well (mean ± SD, n≥3) . The panels A-H correspond to the cell lines as follows: A) A549; B) Calu-6; C) NCI-H1975; D) SW620; E) HCT116; F) RKO; G) NCI-H460; and H) H23. Figure 7 shows a schematic depiction of a potential mechanism behind the synthetic lethality observed for combination inhibition of B- family DNA polymerase (via Aphidicolin treatment) and inhibition of CHK1 (via CCT245737 treatment) . The left-hand panel depicts inhibition with Aphidicolin only. The right-hand panel depicts combination inhibition with Aphidicolin and CHK1 inhibition (e.g. via CCT245737 treatment) .

Detailed description of the invention

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

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

CHK1 Inhibitors

As used herein Checkpoint 1 (Chkl) is a Serine/threonine-protein kinase that coordinates the DNA damage response (DDR) and cell cycle checkpoint response. Activation of Chkl results in the initiation of cell cycle checkpoints, cell cycle arrest, DNA repair and cell death to prevent damaged cells from progressing through the cell cycle. The human Chkl (Serine/threonine-protein kinase Chkl) protein has the UniProt Accession No. 014757 (version 2; sequence last modified 11 January 2011; Checksum: 0ABD0FAB67E60F67 ) .

A "Chkl inhibitor" is an agent (e.g. a small molecule, a nucleic acid, such as an siRNA, an antibody, or binding fragment thereof) that antagonises, blocks, reduces the expression, activity or function of Chkl. A number of Chkl inhibitors are known and many are or have been the subject of clinical trials, including for the treatment of various cancers. Exemplary Chkl inhibitors are shown in Table 1 below.

Table 1 : Chkl Inhibitors

DNA polymerase epsilon inhibitors DNA polymerase epsilon is a member of the DNA polymerase family of enzymes found in eukaryotes . It is composed of the following four subunits: POLE (central catalytic unit), POLE2 (subunit 2), POLE3 (subunit 3) , and POLE4 (subunit 4) . It is thought to play a major role in leading strand DNA synthesis and nucleotide and base excision repair.

The human POLE protein (DNA polymerase epsilon catalytic subunit A) has the UniProt Accession No. Q07864 (version 5; sequence last modified 17 October 2006; Checksum: A213AE1EA8437DEC) .

The human POLE2 protein (DNA polymerase epsilon subunit 2) has the UniProt Accession No. P56282 (version 2; sequence last modified 15 December 1998; Checksum: AFA7AF7C2C0BFF15 ) . As used herein, "DNA polymerase epsilon inhibitor" is an agent (e.g. a small molecule, a nucleic acid, such as an siRNA, an antibody, or binding fragment thereof) that antagonises, blocks, reduces the expression, activity or function of the DNA polymerase epsilon complex. In some cases, the DNA polymerase epsilon inhibitor may target POLE and/or POLE2. A number of small molecule and peptide DNA polymerase epsilon inhibitors have been described. Exemplary DNA polymerase epsilon inhibitors are shown in Table 2 below.

Table 2 : DNA polymerase epsilon inhibitors Compound Name Reference

HO-CH2-CH2-CH (NH₂) -CO-NH-CH2-CH2-

JP 2005002102

OH

Kuriyama et al., Bioorganic &

L-homoserylaminoethanol Medicinal Chemistry, 2004, Vol.

12 (5) , pp. 957-962

Mizushina et al., Biochemical sulphoquinovosyl diacylglycerol Journal, 2003, Vol. 370(1), pp.

299-305

Riman, FEBS Letters, 2001, Vol.

Carbonyldiphosphonate (COMDP)

505(1), pp. 141-146

Determining DNA polymerase mutation and/or deficiency

As described in detail in the Examples herein, the present inventors have found that antagonism and/or downregulation of Pol ε and/or Pol a sensitises cancer cells to the toxic effects of Chkl inhibition. Without wishing to be bound by any particular theory, the present inventors believe that cancers that are deficient in Pol ε, such as those having one or more POLE gene mutations, one or more P0LE2 gene mutations, and/or decreased levels of POLE protein, POLE2 protein and/or DNA polymerase epsilon activity will be more susceptible to the anti-cancer effects of Chkl inhibition therapy. Likewise, cancers that are deficient in Pol a, such as those having one or more POLA1 gene mutations, decreased levels of POLA1 protein, and/or decreased DNA polymerase alpha activity will be more susceptible to the anti-cancer effects of Chkl inhibition therapy

As used herein, the term "loss-of-function mutation" refers to any mutation (including natural variations such as single nucleotide polymorphism minor alleles) that results in decreased expression of the gene product or which results in the gene product having less function or no function relative to the non-mutated gene. In particular, a loss-of-function mutation (e.g. in a POLA1, POLE and/or P0LE2 gene) may be a gene deletion, a lower copy number of the gene, a substitution, deletion or insertion of one or more nucleotides in the gene sequence or a regulatory sequence (e.g. promoter) associated with said gene, which substitution, deletion or insertion results in reduced expression, increased degradation or which results in a gene product having reduced activity or no activity. For example, a truncating mutation which results in an abnormally short polypeptide, a frameshift mutation or a missense mutation that results in an altered protein may give rise to a loss of function.

The skilled person will be aware of numerous techniques for

determining whether a sample (such as a tumour sample) carries a mutation, has a decreased level of a protein and/or has lowered enzyme activity.

In particular embodiments, determining protein expression and/or level (e.g. POLE, POLE2 or POLA1 protein) comprises one or more of: determining protein expression in a tumour sample using

immunohistochemistry, measuring protein levels in a cell lysate by ELISA or Western blotting, and/or using a binding agent capable of specifically binding to the protein (e.g. POLE, POLE2 or POLA1 protein), or a fragment thereof.

In certain embodiments, determining whether the individual has a mutated or deficient cancer is performed on genomic nucleic acid extracted from a sample of cells obtained from the cancer, from a sample of cancer cells circulating in blood and/or from circulating tumour DNA (ctDNA) in blood or plasma.

In certain cases, determining the expression of the gene of interest (e.g. the POLE, P0LE2 or POLA1 gene) comprises extracting RNA from a sample of cancer cells and measuring expression by real time PCR and/or by using a probe capable of hybridising to RNA encoding the the protein (e.g. POLE, POLE2 or POLA1 protein), or a fragment thereof .

Several methods have been developed for the detection of mutations in a sample. The sample may be of normal cells from the individual where the individual has a mutation in the gene or the sample may be of cancer cells, e.g. where the cells forming a tumour contain one or more mutations . Alternatively, the sample may be a DNA, RNA or protein sample directly obtained from the individual. When cells are used as the sample, the first step is generally to extract DNA or RNA from the sample. In the case of RNA, mutations can be detected by first carrying out reverse transcription- polymerase chain reaction (RT-PCR) to amplify the cDNA sequence of the target gene. RT-PCR methods have previously been used to determine mutations in the BCR/ABL fusion gene that are associated with resistance to imatinib.

Methods for detecting the presence of a mutation in a DNA sample preferably include amplifying at least a portion of the DNA obtained from a sample by PCR using a pair of primers. Primer pairs include a first primer that binds upstream of the target DNA sequence

(forward (F) primer) and a second primer that binds downstream of the DNA sequence (reverse (R) primer) , such that a portion of the target DNA sequence comprising the mutation is amplified.

Preferably, the presence of the mutation can be detected in the amplified DNA or cDNA by direct Sanger sequencing or Next-generation Sequencing (NGS) . Additional methods to detect the mutation include matrix-assisted laser desorption/ionization time-of-flight (MALDI- TOF) spectrometry, restriction fragment length polymorphism (RFLP) , high-resolution melting (HRM) curve analysis, and denaturing high performance liquid Chromatography (DHPLC) . Other PCR-based methods for detecting mutations include allele specific oligonucleotide polymerase chain reaction (ASO-PCR) and sequence-specific primer (SSP)-PCR. Alternatively, the DNA sample can be directly sequenced without an amplification step.

NGS offers the speed and accuracy required to detect somatic mutations in cancer, either through whole-genome sequencing (WGS) or by focusing on specific regions or genes using whole-exome

sequencing (WES) or targeted gene sequencing. Examples of NGS techniques include methods employing sequencing by synthesis, sequencing by hybridisation, sequencing by ligation, pyrosequencing, nanopore sequencing, or electrochemical sequencing.

Fluorescent in situ hybridisation (FISH) is a technique used to detect and localise the presence of specific DNA and RNA sequences. FISH uses fluorescent probes to bind to sequences that show a high degree of complementarity. FISH can be used to identify specific genetic aberrations and to detect the presence or absence of specific cancer biomarkers.

Alternatively or additionally, the present invention the

determination of whether a patient has a Pol ε and/or Pol a mutated cancer can be carried out by determining whether the POLE, POLE2 or POLA1 protein contains one or more mutations. The presence or amount of mutated protein may be determined directly using a binding agent, such as an antibody, capable of specifically binding to the mutant protein, or fragments thereof. The binding agent may be labelled to enable it to be detected or capable of detection following reaction with one or more further species, for example using a secondary antibody that is labelled or capable of producing a detectable result, e.g. in an ELISA type assay. As an alternative a labelled binding agent may be employed in a Western blot to detect mutant protein.

Additionally, the activity or level of the Pol ε and/or Pol a may be determined by using techniques well known in the art such as Western blot analysis, immunohistology, chromosomal abnormalities, enzymatic or DNA binding assays and plasmid-based assays. Activity may be determined relative to a control, for example in the case of defects in cancer cells, relative to non-cancerous cells, preferably from the same tissue. DNA polymerase epsilon activity may be measured using techniques known in the art, such as an in vitro assay described in Mizushina et al . , Biochem. J., 2003, Vol. 370, pp. 299- 305 (and references 25-27 cited therein) - the contents of which are expressly incorporated herein by reference.

Samples A "test sample" as used herein may be a cell or tissue sample (e.g. a biopsy), a biological fluid, an extract (e.g. a protein or DNA extract obtained from the subject) . In particular, the sample may be a tumour sample, a blood sample (including plasma or serum sample) , a cerebrospinal fluid sample, or a non-tumour tissue sample. The sample may be one which has been freshly obtained from the subject or may be one which has been processed and/or stored prior to making a determination (e.g. frozen, fixed or subjected to one or more purification, enrichment or extractions steps) . When the mutation (e.g. a POLE, P0LE2 or POLA1 mutation) is a germline mutation it may be convenient to use a non-tumour sample (e.g. a cheek swab, blood sample, hair sample or similar DNA-containing sample) to determine the presence or absence of a mutation. When the mutation (e.g. a POLE, P0LE2 or POLA1 mutation) is a somatic mutation, for example a mutation that has triggered and/or developed with the cancer, the sample will generally be obtained directly from the tumour, obtained from circulating cancer cells and/or

circulating tumour DNA. Techniques for enriching a blood or plasma sample for circulating tumour DNA (e.g. based on fragment size) have been described. Moreover, sequencing techniques for identifying cancer-associated mutations in ctDNA have been described (e.g. based on digital PCR, targeted deep sequencing, nested real-time PCR, and the like) .

A "control sample" may be employed as a reference to determine relative levels of a protein or activity of interest (e.g. POLE, POLE2 or POLA1 protein and/or Pol ε or Pol a activity) . Generally, the control sample will be chosen for its similarity to the test sample, save for its level of the protein or activity of interest. In some cases, the control sample may be a sample obtained from a cancer that has been verified as not being deficient in POLE, POLE2 or POLA1 protein and/or Pol ε or Pol a activity. In some cases, the control sample may be a sample obtained from the same subject as the test sample, but from a non-cancerous tissue (e.g. from another location in the same organ or region as the cancer or from the same organ or region at an earlier (pre-cancer) point in time) . In some cases, the control sample may be a sample obtained from a control subject that is healthy or otherwise cancer-free. The control subject may be, for example, age-matched and/or gender matched with the test subject. In certain cases, the control sample may be a pooled sample obtained from a plurality of individuals (e.g. a population of cancer-free individuals) . The sample may comprise cells of the corresponding tissue or cell type affected by the cancer (e.g. a non-cancerous colorectal, lung or endometrial tissue or cell sample) .

As used herein, the term "tissue" (for example in the context of "a control sample obtained from a cancer-free tissue") may be given a broad interpretation, in particular, specifically including any one or more cells, a biopsy of a solid tissue, or a biological liquid (e.g. blood, plasma, cerebrospinal fluid, urine or faeces) that contains the protein or nucleic acid of interest. Nevertheless, in certain cases, "tissue" may be taken to mean an ensemble of similar cells from the same origin that together carry out a specific function .

Pharmaceutical compositions and therapy

The active agents disclosed herein for the treatment of cancer may be administered alone, but it is generally preferable to provide them in pharmaceutical compositions that additionally comprise with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents .

Examples of components of pharmaceutical compositions are provided in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

The compounds listed in Tables 1 and 2 above or derivatives of them may be used in the present invention for the treatment of cancer. As used herein "derivatives" of the therapeutic agents includes salts, coordination complexes, esters such as in vivo hydrolysable esters, free acids or bases, hydrates, prodrugs or lipids, coupling partners . Salts of the compounds of the invention are preferably

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

Derivatives which as prodrugs of the compounds are convertible in vivo or in vitro into one of the parent compounds. Typically, at least one of the biological activities of compound will be reduced in the prodrug form of the compound, and can be activated by conversion of the prodrug to release the compound or a metabolite of it .

Other derivatives include coupling partners of the compounds in which the compounds is linked to a coupling partner, e.g. by being chemically coupled to the compound or physically associated with it. Examples of coupling partners include a label or reporter molecule, a supporting substrate, a carrier or transport molecule, an

effector, a drug, an antibody or an inhibitor. Coupling partners can be covalently linked to compounds of the invention via an appropriate functional group on the compound such as a hydroxyl group, a carboxyl group or an amino group. Other derivatives include formulating the compounds with liposomes.

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

The active agents disclosed herein for the treatment of cancer according to the present invention are preferably for administration to an individual in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the

individual patient, the site of delivery, the method of

administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, Lippincott, Williams & Wilkins. A composition may be administered alone or in combination with other treatments, either simultaneously or

sequentially, dependent upon the condition to be treated.

The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of

pharmacy. Such methods include the step of bringing the active compound into association with a carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

The agents disclosed herein for the treatment of cancer may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal;

vaginal; parenteral, for example, by injection, including

subcutaneous, intradermal, intramuscular, intravenous,

intraarterial, intracardiac, intrathecal, intraspinal,

intracapsular, subcapsular, intraorbital, intraperitoneal,

intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal ; by implant of a depot, for example, subcutaneously or intramuscularly . Formulations suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

Formulations suitable for parenteral administration (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants , buffers, preservatives, stabilisers,

bacteriostats , and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non- aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's

Solution, or Lactated Ringer's Injection. Typically, the

concentration of the active compound in the solution is from about 1 ng/ml to about 10 mg/ml, for example from about 10 ng/ml to about 1 mg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs.

Compositions comprising agents disclosed herein for the treatment of cancer may be used in the methods described herein in combination with standard chemotherapeutic regimes or in conjunction with radiotherapy. Examples of other chemotherapeutic agents include Amsacrine (Amsidine) , Bleomycin, Busulfan, Capecitabine (Xeloda) , Carboplatin, Carmustine (BCNU) , Chlorambucil (Leukeran) , Cisplatin, Cladribine (Leustat) , Clofarabine (Evoltra) , Crisantaspase

(Erwinase) , Cyclophosphamide, Cytarabine (ARA-C) , Dacarbazine

(DTIC) , Dactinomycin (Actinomycin D) , Daunorubicin, Docetaxel

(Taxotere) , Doxorubicin, Epirubicin, Etoposide (Vepesid, VP-16) , Fludarabine (Fludara) , Fluorouracil (5-FU) , Gemcitabine (Gemzar) , Hydroxyurea (Hydroxycarbamide, Hydrea) , Idarubicin (Zavedos) .

Ifosfamide (Mitoxana) , Irinotecan (CPT-11, Campto) , Leucovorin (folinic acid), Liposomal doxorubicin (Caelyx, Myocet) , Liposomal daunorubicin (DaunoXome®) Lomustine, Melphalan, Mercaptopurine, Mesna, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin

(Eloxatin) , Paclitaxel (Taxol), Pemetrexed (Alimta) , Pentostatin (Nipent) , Procarbazine, Raltitrexed (Tomudex®) , Streptozocin

(Zanosar®), Tegafur-uracil (Uftoral), Temozolomide (Temodal),

Teniposide (Vumon) , Thiotepa, Tioguanine (6-TG) (Lanvis), Topotecan (Hycamtin) , Treosulfan, Vinblastine (Velbe) , Vincristine (Oncovin) , Vindesine (Eldisine) and Vinorelbine (Navelbine) .

Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. In general, a suitable dose of the active compound is in the range of about 100 mg to about 250 mg per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent compound, and so the actual weight to be used is increased proportionately.

In combination therapy envisaged by the present invention (i.e. a Chkl inhibitor combined with a DNA polymerase epsilon inhibitor) the two active agents (or compositions comprising them) may be

administered at the same time or spaced apart in time and/or site of administration. In some cases, the time between administration of the first of the two active agents and administration of the second of the two active agents may be between 1 minute and 1 week, e.g., between 5 minutes and 1 day, or between 5-60 minutes. It is specifically contemplated herein that the Chkl inhibitor may be administered first, followed by the DNA polymerase epsilon

inhibitor. Alternatively, the DNA polymerase epsilon inhibitor may be administered first followed by the Chkl inhibitor. Repeat doses of the respective inhibitors may be the same or different.

Generally, the dosing pattern of each of the two active agents will be dictated by the pharmacokinetics and pharmacodynamics of the respective agents in the subject to be treated. Thus, where one agent is metabolised or cleared more quickly than the other, it may require more frequent dosing in order to maintain effective

combination therapy.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific

disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the

invention in diverse forms thereof.

While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described .

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise" and "include", and variations such as "comprises", "comprising", and "including" will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent "about," it will be understood that the particular value forms another embodiment. The term "about" in relation to a numerical value is optional and means for example +/-

The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.

Examples

Materials and Methods

Cell lines were obtained from the ATCC, with the exception of SKLU1 which was obtained from the Health Protection Agency. NSCLC cell lines were cultured in RPMI 1640 supplemented with 10% foetal bovine serum (FBS) and colon cancer cell lines were cultured in DMEM with 5mM L-glutamine, 1% NEAA and 10% FBS. Cells were incubated at 37 ^°C in a humidified atmosphere of 5% CO2. A PCR based screen was performed every 6 months to ensure cell lines were free from

Mycoplasma contamination.

Drugs

The CHK1 inhibitor CCT245737 (PNT737) (MW 379.34) was synthesised in-house and aphidicolin was purchased from Sigma. Stock solutions of PNT737 and aphidicolin were prepared in DMSO. All stored at - 80^°C.

Cell lysis and Western blotting

Total cell lysates were prepared from cultured cells using 0.3% NP40 lysis buffer containing protease and phosphatase inhibitors (Roche, Lewes, UK) . Protein concentration was determined using a

bicinchoninic acid (BCA) assay. Cell lysates (10-50 g) were mixed in Laemmli sample buffer and heated for 3min at 95 °C. Protein was separated by SDS-PAGE on pre-cast 3-8% tris-acetate or 10% tris- glycine gels (Invitrogen) , at 150V for 60-90min. Proteins were then transferred onto PVDF membranes (Merck Millipore, MA, USA) at 100V for 90 min. Membranes were then incubated in blocking 5% milk or 5%BSA in TNT for 1 hour at room temperature (RT) and then incubated overnight at 4°C with primary antibodies; POLA1 (ab31777), POLE

(GTX132100), POLE2 (ab57298), CHK1 pS345 (CST-2348), CHK1 (SC-8408), RPA32 (abl25681), Cleaved PARP (CST-9541) and GAPDH (ab8245) .

Membranes were then washed and incubated with horseradish peroxidase conjugated secondary antibodies (mouse/rabbit) for 1 hour at RT . Proteins were visualised using enhanced chemiluminescence (ECL, Pierce) and hyperfilm (GE Healthcare) .

Small Interfering RNA screening

siRNA library master plates containing lyophilised siRNA were re- suspended in RNAse free water to 5 nM. 0.5 μΐ of siRNA was then transferred from 384 well master plate to 96 well daughter plates using the Echo dispenser to make a final assay concentration of 25 nM. The screen was carried out using reverse transfection; 50 μΐ of HiPerFect in Opt-MEM (Invitrogen, USA) was added to each well at a final assay concentration of 0.3% and incubated for 20 minutes at room temperature. 50 μΐ of cells, at previously determined optimum seeding density, were added to each well and incubated for 48 h at 37°C. Then plates were treated with optimum screening concentration of PNT737 (100 μΐ/ well) for 84 h at 37 °C. Plates were fixed with 10% trichloroacetic acid and stained with 0.4% SRB in 1% acetic acid. SRB was solubilised with 10 mM TRIS base and plates were read at 490 nm using a Wallac Victor 1420 multilabel counter (Perkin- Elmer) .

Statistical tests: Robust z-score and Z' factor

A Z' -factor was calculated for the entire screen to ensure a wide assay window for hit identification was maintained. A Z' -factor score ≥ 0.5 is excellent and 0 is borderline for a cell-based assay (Birmingham et al . , Nature methods., 2009, Vol. 6(8), pp. 569-575) . Robust Z-scores were calculated to identify significant hits from the screen. The difference in robust Z-score, between ± PNT737, was then calculated for each siRNA for each biological repeat and averaged. The most significant hits had the largest difference in robust Z-score. Hit validation: siRNA and protein knock-down

Hits were validated with both newly purchased Dharmacon pooled siRNA and Qiagen individual siRNAs . HiPerFect was added at 0.3% for A549 and SW620 and 0.2% for Calu-6, NCI-H1975, RKO and HCT116. 40 ul HiPerFect in opti-MEM was added to 10 ul siRNA in RNAse free water per well and incubated for 20 min at room temperature. 50 ul cells were then added at optimal seeding density and incubated for 48 h at 37 °C. Plates were treated with optimum concentration of PNT737 (100 μΐ/ well) for 84 h at 37 °C. Plates were fixed and stained with SRB and read and analysed as described previously.

Knock-down levels of protein were determined using western blot. Knock-down experiments were carried out in 6 well plates using reverse transfection . siRNA and HiPerFect were added at 3x the final assay concentrations (500 ul/well) and incubated for 20 min at room temperature. 500 ul of cells were then added to each well and incubated for 6 h at 37. 2 ml of media was then added to each well to make lx siRNA and HiPerFect concentration and cells were

incubated for 48 h at 37 °C.

GI₅₀ determinations with POLAl, POLE and POLE2 knock-down

Using non-lethal concentrations of siRNA cells were transfected as previously outlined. Plates were treated with a range of PNT737 concentrations (0.01-1 μΜ) (100 μΐ/ well) for 84 h at 37 °C. Plates were fixed and stained with SRB and read and analysed as described previously. Data was analysed using GraphPad Prism 6.0.

Combination studies

Cells were plated in 96 well plates at optimum seeding densities and incubated for 36 h at 37°C. Plates were then treated with single agent PNT737 or aphidicolin or in combination at a 1:1 ratio of the GI50' s (previously determined) and incubated for 4 doubling times at 37°C. Plates were fixed and stained with SRB and read and analysed as described previously. Data was analysed using the Chau and

Talalay method to generate a combination index (CI) (Chou, T. and P. Talalay, Advances in enzyme regulation, 1984, 22; Chou, T.C., Cancer Res._r 2010, Vol. 70(2), pp. 440-446) . IN Cell analysis

Cells were plated in 96 well clear bottom opaque sided plates and incubated for 36 h at 37 °C. Cells were then treated with GI50 concentration of aphidicolin or PNT737 or combination and incubated for 24 h at 37 °C. Gemcitabine at 200 nM was added as control. Cells were then fixed in 4% formaldehyde for 20 min at RT and

permeabilised with 0.25% TritonXlOO for 10 min at RT . Cells were blocked with 3% FBS in TBST for 1 h at RT and γΗ2ΑΧ antibody

(Upstate #05636) was added at 1:500 O/N at 4 °C. Cells were washed with PBS and secondary antibody (Alexa Fluor 488 goat anti-mouse IgG, A11001 Invitrogen) was added at 1:1000 for 1 h at RT . Cells were washed and DAPI was added at 1:1000 for 5 min at RT . Cells were washed and 200 ul/well PBS added and imaged on IN Cell analyser and analysed on IN Cell analyser workstation 3.7.2.

Example 1 - Identification of gene products the inhibition of which is synthetically lethal in combination with CHKl inhibition

In order to identify genes products whose loss is synthetically lethal in combination with CHKl inhibition, an siRNA screen of the druggable genome was performed in combination with a non-lethal concentration of CHKl inhibitor CCT245737 in A549 and SW620 cancer cell lines. CCT245737 is a highly selective, potent and orally bioavailable and is currently in phase I clinical trial for advanced solid cancers

(https : //clinicaltrials . gov/ct2/show/NCT02797977?term=CCT245737&rank =1 and

https : //clinicaltrials . gov/ct2/show/NCT02797964?term=CCT245737&rank= 2) . The Dharmacon druggable genome siRNA library consists of four pooled siRNAs per gene and targets genes from a number of different families; this screen included the kinase, ubiquitin conjugates and 'drug targets' families which sum -6300 genes. The screen was performed in two cell lines, A549 (NSCLC) and SW620 (colon cancer) which have low sensitivityto CCT245737. Transfection conditions were optimised for both cell lines including lipid concentration, seeding density and CCT245737 concentration. Each repeat of the screen was performed both with and without CCT245737 (n=3) . Robust Z-scores were calculated for each siRNA with and without CCT245737 to identify significant hits; the difference in the robust Z-score between treated and untreated conditions was then calculated for each siRNA. The differences in robust Z-scores were then ranked, the largest difference indicating the largest difference in cell viability between the treated and untreated conditions. Graphical representations of the results are shown in figure 1, with

prioritised hits highlighted (coloured circles) . The most

significant hits common to both cancer cell lines were POLAl, POLE and POLE2, all members of the B-family DNA polymerases, highlighted in figure 1.

Example 2 - POLAl, POLE and POLE2 validate as hits In A549 and SW620 cancer cell lines

POLAl, POLE and P0LE2 were validated as hits with newly purchased pooled Dharmacon siRNA and individual Qiagen siRNAs at a range of siRNA. All three hits validated with Dharmacon and Qiagen siRNAs in both cell lines. The largest window between treated and untreated was seen at the lower concentrations of siRNA tested (Figure 2A and C, A549 and SW620 respectively) . Protein knock-down levels of all three hits were confirmed at the lower concentrations of siRNA using western blot (Figure 2B and D, A549 and SW620 respectively) .

Encouragingly the siRNAs that produce the largest window also seem to produce the best protein knock-down.

Example 3 - POLAl, POLE and POLE2 siRNA sensitise to A549 and SW620 cancer cell lines to CHKll

We have shown that knock-down of POLAl, POLE and P0LE2 in A549 and SW620 cell lines increases sensitivity to CCT245737, but we were interested to determine the degree to which the cell lines had been sensitised. Therefore CCT245737 GI50 determinations were carried out in A549 and SW620 cell lines transfected with non-lethal

concentrations of POLAl, POLE and P0LE2 siRNA (Figure 3A and B) . Knock-down of POLAl, POLE or P0LE2 significantly increased the sensitivity of both cell lines to CCT245737, when compared to mock and Allstars negative control, showing between 6 and 14-fold sensitisation (Figure 3C and D) . Example 4 - POLAl, POLE and POLE2 siRNA synthetically lethal in combination with CHKll in other NSCLC and colon cancer cell lines We then determined whether the validated hits in A549 and SW620 can be extended into other NSCLC and colon cancer cell lines. The most effective Qiagen siRNA for each of POLA1 , POLE and P0LE2 was tested at a range of concentrations in NSCLC cell lines NCI-H1975 and Calu- 6 and colon cancer cell lines HCT116 and RKO. POLE and P0LE2 siRNA significantly enhanced sensitivity to CCT245737 in all four cell lines (Figure 4), POLA1 siRNA only significantly enhanced

sensitivity in NCI-H1975 and Calu-6 (Figure 4A and B, respectively) . POLA1 siRNA in the HCT116 and RKO reduced cell viability to < 10% regardless of drug (Figure 4C and D, respectively) .

Example 5 - Chou and Talalay combination studies with CCT245737 and aphidicolin

Aphidicolin has been reported to specifically inhibit the B-family DNA polymerases and therefore was considered to be a useful chemical tool to use in combination studies with CCT245737. Chou and Talalay analysis was conducted on the combination data to determine the combination index (CI) which indicates if the combination is synergistic, additive or antagonistic. Table 3 below summarises the combination data for panel of NSCLC and colon cancer cell lines, 8 out of the 9 cell lines showed synergism between aphidicolin and CCT245737.

Table 3: Summary of CI's in panel of NSCLC and colon cancer cell lines .

Cells were plated in 96 well plates at optimum seeding densities and incubated for 36 h at 37°C. Plates were then treated with single agent CCT245737 or aphidicolin or in combination at a 1:1 ratio of the GI50' s (previously determined) and incubated for 4 doubling times at 37 °C. Data was analysed using the Chau and Talalay method to generate a CI. Data mean + SD n≥3.

Example 6 - POLAl, POLE and POLE2 knock-down in combination with CHKli increases markers of replication stress

It has been reported that high levels of replication stress (RS) increase the sensitivity to CHK1 inhibitors, therefore we wanted to test the hypothesis that knock-down of the polymerases increases levels of RS which can then be exploited by addition of a CHK1 inhibitors. Also, whether the combination of knock-down and CHK1 inhibitors further increases RS . Markers of replication stress include PhosphoRPA32 , as RPA32 becomes phosphorylated by different kinases on a number of sites in response to RS, and pS345 on CHK1 which is phosphorylated by ATR in response to RS .

Figure 5 shows that POLAl knock-down alone increases levels of pRPA32 and pS345 in both cell lines suggesting increased RS . pRPA32 is further increased when POLAl, POLE and P0LE2 knock-down is combined with CCT245737 in both cell lines, suggesting increased levels of RS . In the SW620 cell line, pS345 is further increased when POLA1, POLE and P0LE2 knock-down is combined with CCT245737, suggesting more activation of CHKl by ATR is response to RS . However this is not the case in the A549 cell line where the pS345 signal is comparable to CCT245737 alone (Mock +/Allstars +) . Lastly, C-PARP a marker of apoptosis, increases with POLA1 and P0LE2 knock-down in combination with CCT245737 in the A549 cell line and with all three knock-down combinations in the SW620 cell line. This suggests that combination of knock-down and CCT245737 is increasing cell death. Example 7 - Combination of CCT737 and aphidicolin increase DNA damage compared to single agent

We have shown that POL knock-down in combination with CHKli

increases RS, however if replication stress is unresolved it can lead to DSBs and DNA damage. CHKl inhibition reduces DNA repair capabilities, cell cycle arrest and replication fork stabilisation effects, therefore we hypothesised that DNA damage levels would increase with inhibition of both CHKl and the DNA polymerases compared to inhibition of either alone. A panel of cell lines were treated with GI50 concentrations of CCT245737, aphidicolin or a combination of CCT245737 and aphidicolin, at 1:1 of the GI50, and levels of γΗ2ΑΧ intensity (DNA damage marker) were determined using immunofluorescence. 6 out of 7 cell lines, which showed synergy in combination studies (Figure 6 and Table 3), also show significantly higher levels of γΗ2ΑΧ intensity when treated with the combination in comparison to either agent alone.

All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was

specifically and individually indicated to be incorporated by reference in its entirety.

The specific embodiments described herein are offered by way example, not by way of limitation. Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.

Claims

1. A composition comprising a Checkpoint 1 (Chkl) inhibitor for use in a method of treatment of a cancer in a mammalian subject, wherein the cancer comprises one or more cells that carry mutations in or are deficient in DNA polymerase epsilon and/or DNA polymerase alpha .

2. The composition for use according to claim 1, wherein the method of treatment comprises:

3. The composition for use according to claim 1 or claim 2, wherein the cancer comprises cells: that carry mutations in the POLE gene and/or the P0LE2 gene, that have decreased level of POLE protein and/or POLE2 protein, and/or that are deficient in DNA polymerase epsilon activity.

4. The composition for use according to claim 3, wherein the method of treatment comprises:

(i) a loss-of-function mutation in the POLE gene and/or the P0LE2 gene;

5. The composition for use according to any one of the preceding claims ,

wherein the Chkl inhibitor inhibits Chkl with an IC50 of ≤ 1 μΜ and/or has at least 10-fold selectivity for Chkl relative to Chk2.

6. The composition for use according to any one of the preceding claims, wherein the Chkl inhibitor is selected from the group consisting of: CCT245737, LY-2606368, MK-8776, GDC-0575, V158411, LY2603618, GDC-0425, AZD7762, PF-477736, XL844, CHIR-124 and CEP- 3891.

7. The composition for use according to any one of the preceding claims, wherein the method of treatment further comprises

administering an inhibitor of DNA polymerase epsilon.

8. A composition comprising a Checkpoint 1 (Chkl) inhibitor for use in a method of treatment of a cancer in a mammalian subject, wherein the Chkl inhibitor is administered simultaneously, separately or sequentially with an inhibitor of DNA polymerase epsilon.

9. A composition comprising a DNA polymerase epsilon inhibitor for use in a method of treatment of a cancer in a mammalian subject, wherein the DNA polymerase epsilon inhibitor is administered simultaneously, separately or sequentially with an inhibitor of Checkpoint 1 (Chkl) .

10. A composition comprising a Checkpoint 1 (Chkl) inhibitor and a DNA polymerase epsilon inhibitor for use in a method of treatment of a cancer in a mammalian subject.

11. The composition for use according to any one of claims 8 to 10, wherein the Chkl inhibitor inhibits Chkl with an IC50 of ≤ 1 μΜ and/or has at least 10-fold selectivity for Chkl relative to Chk2.

12. The composition for use according to any one of claims 8 to 11, wherein the Chkl inhibitor is selected from the group consisting of: CCT245737, LY-2606368, MK-8776, GDC-0575, V158411, LY2603618, GDC- 0425, AZD7762, PF-477736, XL844, CHIR-124 and CEP-3891.

13. The composition for use according to any one of claims 8 to 12, wherein the DNA polymerase epsilon inhibitor inhibits DNA polymerase epsilon with an IC50 of ≤ 1 μΜ and has at least 10-fold selectivity for DNA polymerase epsilon relative to DNA polymerase alpha and/or DNA polymerase delta.

14. The composition for use according to claim 13, wherein the DNA polymerase epsilon inhibitor inhibits the POLE subunit and/or the POLE2 subunit of DNA polymerase epsilon.

15. The composition for use according to any one of claims 8 to 14, wherein the DNA polymerase epsilon inhibitor is selected from the group consisting of: HO-CH₂-CH₂-CH (NH₂) -CO-NH-CH₂-CH₂-OH, L- homoserylaminoethanol , sulphoquinovosyl diacylglycerol and

carbonyldiphosphonate .

16. The composition for use according to any one of the preceding claims, wherein the cancer is selected from the group consisting of: a lung cancer, a colorectal cancer and an endometrial cancer.

17. The composition for use according to claim 16, wherein the lung cancer is Non-small-cell lung carcinoma and/or the colorectal cancer is a colorectal adenocarcinoma.

18. The composition for use according to any one of the preceding claims, wherein the cancer comprises a mutation in the POLE gene.

19. The composition for use according to claim 18, wherein the mutation in the POLE gene is selected from the group consisting of: c.l373A>T (p.Tyr458Phe) , and Pro286Arg.

20. A method of treatment of a cancer in a mammalian subject, the method comprising administering a therapeutically effective amount of a Checkpoint 1 (Chkl) inhibitor to the subject in need thereof, wherein the Chkl inhibitor is administered simultaneously,

separately or sequentially with an inhibitor of DNA polymerase epsilon .

21. A method of treatment of a cancer in a mammalian subject, the method comprising administering a therapeutically effective amount of a DNA polymerase epsilon inhibitor to the subject in need thereof, wherein the DNA polymerase epsilon inhibitor is

administered simultaneously, separately or sequentially with an inhibitor of Checkpoint 1 (Chkl) .

22. A method of treatment of a cancer in a mammalian subject, the method comprising administering a therapeutically effective amount of a composition comprising a Checkpoint 1 (Chkl) inhibitor and a DNA polymerase epsilon inhibitor to the subject in need thereof.

23. The method according to any one of claims 20 to 22, wherein the Chkl inhibitor inhibits Chkl with an IC50 of ≤ 1 μΜ and/or has at least 10-fold selectivity for Chkl relative to Chk2.

24. The method according to any one of claims 20 to 23, wherein the Chkl inhibitor is selected from the group consisting of: CCT245737, LY-2606368, MK-8776, GDC-0575, V158411, LY2603618, GDC-0425,

AZD7762, PF-477736, XL844, CHIR-124 and CEP-3891.

25. The method according to any one of claims 20 to 24, wherein the DNA polymerase epsilon inhibitor inhibits DNA polymerase epsilon with an IC50 of ≤ 1 μΜ and has at least 10-fold selectivity for DNA polymerase epsilon relative to DNA polymerase alpha and/or DNA polymerase delta.

26. The method according to claim 25, wherein the DNA polymerase epsilon inhibitor inhibits the POLE subunit and/or the POLE2 subunit of DNA polymerase epsilon.

27. The method according to any one of claims 20 to 26, wherein the DNA polymerase epsilon inhibitor is selected from the group

consisting of: HO-CH₂-CH₂-CH (NH₂) -CO-NH-CH₂-CH₂-OH, L- homoserylaminoethanol , sulphoquinovosyl diacylglycerol and

carbonyldiphosphonate .

28. A method of treatment of a cancer in a mammalian subject, the method comprising administering a therapeutically effective amount of a Checkpoint 1 (Chkl) inhibitor to the subject, wherein the cancer comprises one or more cells that carry mutations in or are deficient in DNA polymerase alpha and/or DNA polymerase epsilon.

29. The method according to claim 28, wherein the method of treatment comp

30. The method according to claim 28 or claim 29, wherein the cancer comprises cells: that carry mutations in the POLE gene and/or the P0LE2 gene, that have decreased level of POLE protein and/or POLE2 protein, and/or that are deficient in DNA polymerase epsilon activity .

31. The method according to claim 30, wherein the method of treatment comprises:

(i) a loss-of-function mutation in the POLE gene and/or the P0LE2 gene; (ii) a decreased level of POLE protein and/or POLE2 protein relative to the level of the respective protein in a population of cells from a control sample obtained from a cancer-free tissue from the same subject as the test sample or obtained from a cancer-free control subject; and/or

32. The method according to any one of claims 28 to 31,

33. The method according to any one of claims 28 to 32, wherein the Chkl inhibitor is selected from the group consisting of: CCT245737, LY-2606368, MK-8776, GDC-0575, V158411, LY2603618, GDC-0425,

AZD7762, PF-477736, XL844, CHIR-124 and CEP-3891.

34. The method according to any one of claims 28 to 33, wherein the method of treatment further comprises administering an inhibitor of DNA polymerase epsilon.

35. The method according to any one of claims 20 to 34, wherein the cancer is selected from the group consisting of: a lung cancer, a colorectal cancer and an endometrial cancer.

36. The method according to claim 35, wherein the lung cancer is Non-small-cell lung carcinoma and/or the colorectal cancer is a colorectal adenocarcinoma.

37. The method according to any one of claims 20 to 36, wherein the cancer comprises a mutation in the POLE gene.

38. The method according to claim 37, wherein the mutation in the POLE gene is selected from the group consisting of: c.l373A>T

(p. Tyr458Phe) , and Pro286Arg.