WO2020104777A1

WO2020104777A1 - Compounds for telomere length-related treatment of cancer

Info

Publication number: WO2020104777A1
Application number: PCT/GB2019/053261
Authority: WO
Inventors: Christopher PEPPER; Duncan Martin Baird; Christopher Fegan; Joseph Thomas BIRKETT
Original assignee: Telonostix Ltd
Priority date: 2018-11-19
Filing date: 2019-11-18
Publication date: 2020-05-28
Also published as: GB201818792D0

Abstract

The invention relates to a DNA damage response inhibitor (DDRi) for use, optionally in combination with a DNA damaging agent and/or a DNA damaging procedure, in treating cancer in a patient, characterized in that the patient's cancerous cells have a mean telomere length, determined using high- resolution telomere length analysis, that is less than or equal to a threshold value; and to methods for treating cancer in said patient by administering a DDRi and, optionally in combination with a DNA damaging agent and/or a DNA damaging procedure.

Description

Compounds for Telomere Length-Related Treatment of Cancer

The invention relates to a DNA damage response inhibitor (DDRi) for use, optionally in combination with a DNA damaging agent and/or a DNA damaging procedure, in treating cancer in a patient, characterized in that the patient’s cancerous cells have a mean telomere length, determined using high- resolution telomere length analysis, that is less than or equal to a threshold value; and to methods for treating cancer in said patient by administering a DDRi, optionally in combination with a DNA damaging agent and/or a DNA damaging procedure.

Background of Invention

Telomeres are nucleoprotein structures composed of repetitive DNA sequences that cap the ends of linear eukaryotic chromosomes, protecting them from deterioration or fusion with adjacent chromosomes. For humans, the repeat sequence of nucleotides in telomeres is TTAGGG, the complementary DNA strand being CCCTAA, with a single-stranded CCCTAA overhang. This sequence of CCCTAA is repeated approximately 2,500 times in humans. In humans, the average telomere length declines from about 1 1 kilobases at birth to less than 4 kilobases in old age, with the average rate of decline being greater in men than in women.

During replication of DNA, the ends of chromosomes are vulnerable to degradation; telomeres however prevent this happening by themselves being degraded during each stage of cell division, essentially ‘capping’ the chromosome. Telomere ends are, however, maintained in certain cell types such as germ cells, stem cells and certain white blood cells, by the reverse transcriptase telomerase that catalyses the RNA templated addition of telomere repeats. Telomere length is a key determinant of telomeric function and it has been shown that short dysfunctional telomeres can drive genomic instability and tumourigenesis in mouse models. Furthermore, deregulation of telomerase has been shown to drive oncogenesis. Additionally, the loss of telomeres in somatic cells has been linked to replicative senescence preventing genomic instability and cancer. Conversely, it has also been shown that malignant cells can by-pass this senescence and become immortalised via telomere extension using aberrant activation of telomerase.

Consistent with the role of telomere biology in tumour progression, there is now a substantial body of evidence indicating that telomere length can provide prognostic information in many human malignancies including Chronic lymphocytic leukaemia (CLL)^1-8 CLL is the most common adult leukaemia, characterised by an accumulation of immuno-incompetent, monoclonal CD5⁺ B-lymphocytes. CLL has a heterogeneous clinical course with survival ranging from a few months to many decades. Treatment strategies vary with staging or disease progression and include chemotherapy, radiotherapy, monoclonal antibody therapy or bone marrow transplantation, although, early stage patients often receive no treatment. Early clinical intervention is required for patients with an aggressive form of the disease, whereas patients with a more benign form simply need monitoring for disease progression until a point is reached where appropriate treatment is administered. In this latter respect, it has been shown that early stage CLL intervention does not improve survival rate. It is therefore inappropriate to expose someone presenting with a disease that is unlikely to be life-threatening for up to 30 years with highly dangerous chemotherapeutic drugs.

In previous studies, we have developed single-molecule technologies that allow us to accurately detect the presence of critically shortened telomeres^{9 10} and to characterise telomere end-end fusions^{11 12} Single telomere length analysis (STELA) allows complete resolution of telomere lengths at specific chromosome ends, including telomeres in a length range, termed the fusogenic range, at which telomere end-end fusions occur^{9 11}. STELA therefore permits detection of shortened telomeres that are potentially dysfunctional and capable of fusion. In particular, our previous investigations have used telomere length and fusion analysis to identify the longest mean telomere length at which telomere end-end fusion events can be detected for a number of chromosomes (examples are shown in Table 1 ). Further we have shown that CLL patients with telomere lengths within this fusogenic range (i.e. less than or equal to the to the longest mean telomere length at which telomere end-end fusion events can be detected), have inferior clinical outcomes when compared to patients with telomere lengths above the longest mean telomere length at which telomere end-end fusion events can be detected¹³. Indeed, these investigations demonstrated that telomere lengths can accurately predict response to chemotherapy¹⁴ and chemo-immunotherapy¹⁵, with short telomere patients having significantly worse progression-free survival and overall survival.

In the present investigations, we show that patients with short telomeres, i.e. telomere length profiles within the fusogenic range, could particularly benefit from the administration of a DDRi, optionally when used in combination with a DNA damaging agent (e.g. chemotherapy agent) and/or a DNA damaging procedure (e.g. radiotherapy). In particular, cells with short telomeres are shown to be inherently more sensitive to DDRi and also the combination of a DDRi and a DNA damaging agent than cells with telomeres above the fusogenic range.

Statements of Invention

According to a first aspect of the invention, there is therefore provided a DNA damage response inhibitor (DDRi) for use in treating cancer in a patient whose cancer cells have a mean telomere length, determined using high-resolution telomere length analysis, that is less than or equal to a threshold value selected according to the chromosome to be tested from the group consisting of: i) 3.81 kb, preferably 2.26 kb, for XpYp;

ii) 4.81 kb, preferably 2.57 kb, for 17p;

iii) 5.01 kb, preferably 3.01 kb, for 2p;

iv) 4.49 kb, preferably 2.94 kb, for 16p;

v) 4.47 kb, preferably 2.66 kb, for 18q;

vi) 4.27 kb, preferably 3.05 kb, for 7q; or

vii) a mean telomere length threshold determined using any two or more of the above threshold values identified in i) to vi) depending upon the combination of different chromosomes that are tested.

In particular, it has been found that patients with cancerous cells having a mean telomer length below a threshold value show a preferential response to treatment with said DDRi, optionally in combination with said DNA damaging agent and/or a DNA damaging procedure.

The threshold values indicated in i) to vi) above each represents an established chromosome-specific upper limit of the threshold value. Therefore, if the patient’s mean telomere length is determined using a mean average value derived from two or more of the above chromosomes, then the mean telomere length threshold is calculated as the mean of the relevant chromosome- specific upper limits identified in i) to v) above. For example:

• if the patient’s mean telomere length threshold is calculated for XpYp and 17p, the mean telomere length threshold is (3.81 +4.81 ) 1 2 = 4.31 kb;

• if the patient’s mean telomere length threshold is calculated for XpYp, 17p and 2p, the mean telomere length threshold is (3.81 +4.81 +5.01 ) / 3 = 4.54 kb;

• if the patient’s mean telomere length threshold is calculated for XpYp, 17p, 2p and 16p, the mean telomere length threshold is (3.81 +4.81 +5.01 +4.49) / 4 = 4.53 kb; and • if the patient’s mean telomere length threshold is calculated for XpYp, 17p, 2p, 16p and 18q, the mean telomere length threshold is (3.81 +4.81 +5.01 +4.49+4.47) / 5 = 4.52 kb.

These thresholds represent the longest mean telomere length at which telomere end-end fusion events can be detected for a specific chromosome. Below each threshold is a range, termed herein a fusogenic range, in which shortened telomeres become dysfunctional.

Additionally, or alternatively, the above said threshold value is determined for any other chromosome (such as one or more of 1 1 q, 9p, 5p and 12q) and once this value is established the above invention is worked by using a DNA damage response inhibitor (DDRi) for use in treating cancer in a patient whose cancer cells have a mean telomere length, determined using high-resolution telomere length analysis, that is less than or equal to the said threshold value established for said any other chromosome or a mean telomere length threshold determined using any two or more of the above threshold values identified in i) to vii) depending upon the combination of different chromosomes that are tested.

High-resolution telomere length analysis (STELA) is known to be suitable for determining mean telomere length for most, if not all, chromosomes, in particular XpYp, 17p, 2p, 16p, 18q, 7q, 1 1 q, 9p, 5p and 12q. Therefore, the invention may be practiced by those skilled in the art by analyzing any other chromosome to find the relevant threshold or combination of chromosomes (optionally together with one or more of XpYp, 17p, 2p, 16p, 18q and/or 7q) to find the mean threshold: the threshold value being the longest mean telomere length at which telomere end-end fusions can be detected in samples of tissue from a plurality of individuals presenting with the same cancer condition.

Whilst when working the invention the form of cancer to be treated is not limited to any particular cancer type, but in a preferred embodiment, the cancer is a B-cell malignant condition, such a condition includes, but is not limited to, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B- cell lymphoma (MZL), mucosa-associated lymphatic tissue lymphoma (MALT), chronic lymphocytic leukaemia, (CLL), Mantle cell lymphoma (MCL), B cell Acute lymphoblastic leukaemia (B-ALL), Non- Hodgkin’s Lymphoma (NHL), Hodgkin’s Lymphoma (HL) and Multiple myeloma (MM).

In a particularly preferred embodiment, the B-cell malignant condition is CLL, the cancerous cells of which may or may not possess large scale chromosomal abnormalities / deletions. Chromosomal abnormalities that are often associated with CLL include, but are not limited to, deletions of 1 1 q and 17p chromosomes.

The DDRi may be any compound or complex that has an inhibitory effect on one or more cellular DNA damage response pathways (e.g. signaling or repair). In a preferred embodiment, the DDRi is selected from the group consisting of inhibitors of one or more of the following proteins: ATM serine/threonine kinase (ATM), ATR serine/threonine-protein kinase (ATR), Checkpoint kinase 1 (Chk1 ), Checkpoint kinase 2 (CHk2), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), DNA polymerase theta (POLQ), Wee1 -like protein kinase (WEE1A, also known as WEE1 ), Wee1 -like protein kinase 2 (WEE1 B, also known as WEE2), and poly ADP ribose polymerase (PARP). More preferably, the DDRi is selected from the group consisting of inhibitors of one or more of Chk1 , WEE1A and PARP. In a particularly preferred embodiment, the DDRi is a PARP inhibitor.

Preferably, the Chk1 inhibitor is GDC-0575 (i.e. (R)-N-(4-(3-aminopiperidin-1 - yl)-5-bromo-1 H-pyrrolo[2,3-b]pyridin-3-yl)cyclopropanecarboxamide (CAS No. 1 196541 -47-5)). The chemical structure of GDC-0575 is shown below:

Preferably the WEE1A inhibitor is MKK-1775 (i.e. 1 -[6-(2-Hydroxypropan-2- yl)pyridin-2-yl]-6-[4-(4-methylpiperazin-1 -yl)anilino]-2-prop-2-enylpyrazolo [3,4-d]pyrimidin-3-one (CAS No. 955385-80-7)), which is commonly known as

Adavosertib. The chemical structure of MKK-1775 is shown below.

Preferred PARP inhibitors are selected from the group consisting of: olaparib, rucaparib, niraparib, talazoparib, veliparib, 3-aminobenzamide, and iniparib. In a particularly preferred embodiment, the PARP inhibitor is olaparib. The chemical structures of each of the preferred PARP inhibitors are shown below:

Olaparib

aazoparb

Veliparib

3-aminobenzamide

Iniparib

In a further preferred embodiment of the invention, the DDRi is used in combination with a DNA damaging agent and/or a DNA damaging procedure. A typically, non-limiting, example of a DNA damaging procedure is radiotherapy.

In a particularly preferred embodiment, the DDRi is used in combination with a DNA damaging agent.

Preferred DNA damaging agents are selected from the group consisting of nucleoside analogues and alkylating agents. A particularly preferred nucleoside analogue is fludarabine. Particularly preferred alkylating agents are selected from the group consisting of chlorambucil, bendamustine, mafosfamide, cyclophosphamide and 4-hydroxy-cyclophosphamide (i.e. the active metabolite of both mafosfamide and, following hepatic activation, cyclophosphamide).

The chemical structures of each of these preferred DNA damaging agents are shown below:

Fludarabine

cyclophosphamide

4-hydroxy-cyclophospham ide

High resolution telomere length analysis is typically measured using a single telomere length analysis (STELA) method, which allows complete resolution of telomere lengths at specific chromosome ends^{9 11}. However, any other method that can measure the full range of telomere length from one telomere repeat to several kb of telomere length may be utilised. In some embodiments, telomere length is detected for a single chromosome. Alternatively, a mean telomere length may be detected for a plurality of different chromosomes and, in such embodiments, an average mean telomere length is calculated. In preferred embodiments, mean telomere length is determined for the XpYp chromosome, either alone or in combination with one or more further chromosomes. In preferred embodiments, the mean telomere length is determined using at least one, any two, any three, any four, any five, any six, any seven, any eight, any nine or all ten of the following chromosomes: XpYp, 17p, 2p, 16p, 18q, 7q, 1 1 q, 9p, 5p and 12q. More preferably, the mean telomere length is determined using at least any one, any two, any three, any four, five or all six of the following chromosomes: XpYp, 17p, 2p, 16p, 18q and 7q chromosomes.

Whilst in principle telomere length can be assessed using any type of cell, the mean telomere length will typically be determined from at least a sample of the cancerous cell type. Therefore, in preferred embodiments, the mean telomere length is determined using at least a sample of B-cells isolated from the patient to be treated.

According to a second aspect of the invention, there is provided a method for treating cancer in a patient, the method comprising: a. using high-resolution telomere length analysis to determine the mean telomere length (in kb) of at one chromosome selected from XpYp, 17p, 2p, 16p, 18q and 7q in at least a sample of cancer cells from said patient; and

b. where said mean telomere length is less than or equal to a threshold value selected according to the chromosome that was tested from the group consisting of:

i. 3.81 kb, preferably 2.26 kb, for chromosome XpYp;

ii. 4.81 kb, preferably 2.57 kb, for chromosome 17p;

iii. 5.01 kb, preferably 3.01 kb, for chromosome 2p;

iv. 4.49 kb, preferably 2.94 kb, for chromosome 16p;

v. 4.47 kb, preferably 2.66 kb, for chromosome 18q;

vi. 4.27 kb, preferably 3.05 kb, for 7q; or

vii. a mean telomere length threshold determined using any two or more of the above threshold values identified in i) to vi) depending upon the combination of different chromosomes that are tested,

c. administering a DDRi to said patient.

As is the case for the first aspect of the invention, the cancer may be any type of cancer but according to a preferred embodiment, the form of cancer is a 13- cell malignant condition. Similarly, in a particularly preferred embodiment, the B-cell malignant condition is CLL, the cancerous cells of which may or may not possess large scale chromosomal abnormalities / deletions, such abnormalities / deletions usually being found in, but not limited to, chromosomes 1 1 q and 17p.

In a preferred embodiment of the second aspect, the DDRi is selected from the group consisting of inhibitors of one or more proteins selected from the group consisting of an ATM, ATR, Chk1 , CHk2, DNA-PKcs, POLQ, WEE1A, WEE1 B, and PARP. More preferably the DDRi is selected from the group consisting of inhibitors of one or more of Chk1 , WEE1A and PARP. In a particularly preferred embodiment, the DDRi is a PARP inhibitor. A preferred Chk1 inhibitor is GDC-0575. A preferred WEE1A inhibitor is MKK- 1775. Preferred PARP inhibitors are selected from the group consisting olaparib, rucaparib, niraparib, talazoparib, veliparib, 3-aminobenzamide, and iniparib. In a particularly preferred embodiment, the PARP inhibitor is olaparib.

In a further preferred embodiment of the invention, the DDRi is administered in combination with a DNA damaging agent and/or before, during or after a DNA damaging procedure. As noted above, a typical, non-limiting, example of a DNA damaging procedure is radiotherapy. In particularly preferred embodiments, the DDRi is administered in combination with a DNA damaging agent.

As noted above, preferred DNA damaging agents are selected from the group consisting of nucleoside analogues and alkylating agents. A particularly preferred nucleoside analogue is fludarabine. Particularly preferred alkylating agents are selected from the group consisting of chlorambucil, bendamustine, mafosfamide, cyclophosphamide and 4-hydroxy-cyclophosphamide.

Again, high resolution telomere length analysis is typically measured using a single telomere length analysis (STELA) method. However, any other method that can measure the full range of telomere length from one telomere repeat to several kb of telomere length may be utilised in the second aspect of the invention. In some embodiments, telomere length is detected for a single chromosome. Alternatively, a mean telomere length may be detected for a plurality of different chromosomes and, in such embodiments, an average mean telomere length is calculated. In a preferred embodiment, mean telomere length is determined for the XpYp chromosome, either alone or in combination with one or more of said further chromosomes. In preferred embodiments, the mean telomere length is determined using at least one, two, three, four, five, six, seven, eight, nine or all ten of the following chromosomes: XpYp, 17p, 2p, 16p, 18q, 7q, 1 1 q, 9p, 5p and 12q. In more preferred embodiments, the mean telomere length is determined using at least one, two, three, four, five or all six of the following chromosomes: XpYp, 17p, 2p, 16p, 18q and 17q chromosomes. Mean telomere length is typically determined from at least a sample of cancer cell type. Therefore, in preferred embodiments, the mean telomere length is determined using at least a sample of B-cells isolated from the patient to be treated.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word“comprises”, or variations such as“comprises” or“comprising” is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties 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.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

The invention will now be described by way of example only with reference to the following tables and figures:

Table 1. Longest mean telomere length at which telomere end-end fusion events can be detected for a range of chromosomes, including the mean thereof and (derived therefrom) the prognostic mean telomere length for each one of said chromosomes, including the mean thereof. Data adapted from WO 2013/024264.

Table 2. LD50 values for tested individual DNA Damaging agents and DDRis in monoculture conditions.

Figure 1. Comparative cytotoxicity of (A) fludarabine (B) chlorambucil (C) bendamustine (D) mafosfamide (E) rituximab and (F) olaparib in monoculture and co-culture for 48h. The figures show composite dose-response curves for 10 individual patients; all drugs were tested in the same patient samples (n = 10).

Figure 2. Comparison of the cytotoxic effect in primary CLL cell monocultures treated with (A) fludarabine (B) chlorambucil (C) bendamustine (D) mafosfamide (E) olaparib in patient samples grouped according to telomere length.

Figure 3. Comparison of the cytotoxic effect in primary CLL cells co-cultured on CD40L-expressing mouse fibroblasts and treated with (A) fludarabine (B) chlorambucil (C) bendamustine (D) mafosfamide (E) olaparib in patient samples grouped according to telomere length.

Figure 4. Comparison of the synergistic cytotoxic effect in primary CLL cell monocultures treated with a combination of olaparib and (A) fludarabine, (B) chlorambucil, (C) bendamustine, and (D) mafosfamide. The short telomere subset showed significantly enhanced synergy with all four drug combinations in comparison with that observed for the long telomere subset.

Figure 5. Comparison of the synergistic cytotoxic effect in primary CLL cells co-cultured on CD40L-expressing mouse fibroblasts that were treated with a combination of olaparib and (A) fludarabine, (B) chlorambucil, (C) bendamustine, and (D) mafosfamide. The short telomere subset showed strong synergy with all four drug combinations whereas the long telomere subset demonstrated additive effects or weak synergy. Figure 6. Comparison of the synergistic cytotoxic effect in primary CLL cell monocultures treated with a combination of MK-1775 and (A) bendamustine or (B) mafosfamide. The short telomere subset showed significantly enhanced synergy with both drug combinations in comparison with that observed for the long telomere subset.

Figure 7. Comparison of the synergistic cytotoxic effect in primary CLL cell monocultures treated with a combination of GDC-0575 and (A) bendamustine or (B) mafosfamide. The short telomere subset showed significantly enhanced synergy with both drug combinations in comparison with that observed for the long telomere subset.

METHODS

CLL Patients

Peripheral blood samples from CLL consenting patients, in accordance with the Declaration of Helsinki and as approved by the South East Wales local research ethics committee (LREC# 02/4806). CLL was defined by clinical criteria as well as cellular morphology, and also the co-expression of CD19 and CD5 in lymphocytes simultaneously displaying restriction of light-chain rearrangement. All of the samples were collected at, or close to, the time of diagnosis and staging was based on the Binet classification system¹⁶.

Isolation of peripheral blood mononuclear cells from CLL patients

Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA venous blood of CLL patients by density centrifugation using Ficoll-Hypaque (Invitrogen).

Purification of CLL B-cells

CD19⁺ B-cells were positively selected by using CD19⁺ dynabeads from Life Technologies. 10pL of the desired bead was used for the isolation of 500,000 target cells form isolated PBMCs. The cell preparation containing the cells of interest and the washed dynabeads was incubated for 20 minutes at 4°C. Cells were isolated by placing the microtube back into the magnetic particle concentrator for 2 minutes. After this time, the supernatant was aspirated and discarded and the microtube was removed from the magnetic particle concentrator.

Telomere length analysis using STELA

DNA was extracted using the Maxwell® 16 Blood DNA Purification Kit on the Maxwell® 16 Instrument (Promega, Madison, Wl, USA). For telomere length analysis at the XpYp telomere we used an adaptation of the chromosome- specific single telomere length analysis (STELA) assay, as previously described^{9 11}, to allow for high-throughput analysis (HT-STELA). In particular, the STELA protocol was adapted to use telomere-adjacent primers specific for the XpYp telomere (XpYpC: 5' -C AG G G AC C G G GAC AAAT AG AC-3' ) , in triplicate 30pl PCR reactions each containing 30 ng of genomic DNA. Thermal cycling conditions were: 23 cycles of 94°C for 20 s, 65°C for 30 s and 68°C for 5 mins. Amplified fragments were resolved using capillary gel electrophoresis and mean telomere length determined using PROSize software (AATI, Ankeny, Iowa, USA).

CLL PBMC Cultures

Primary CLL cells have the propensity to die very quickly once removed from the patient. However, it has previously been shown that such‘spontaneous apoptosis’ can be inhibited by the addition of interleukin 4 (IL-4) into the culture media¹⁷. Therefore, all culture data generated in this report was obtained using Roswell Park Memorial Institute (RPMI) 1640 (Sigma) culture media supplemented with 5ng/mL interleukin 4 (IL-4).

The cytotoxicity of all the agents were first tested in the following monoculture conditions: 1x10⁶ CLL cells were resuspended in 1 mL of cell culture medium - RPMI (1640 Sigma), which was supplemented with: 2mM L-Glutamine (Life Technologies); 100units/ml Penicillin and 100g/mL streptomycin (Life Technologies); and 10% foetal Calf Serum (FCS) (Life Technologies) supplemented with 5ng/pL IL-4 (Biosource).

In addition to monoculture experiments, we also tested the cytotoxicity of all the agents in CD40L co-culture: CD40L-expressing mouse fibroblast cells were suspended in 5m L of fibroblast media and irradiated (8000RADs). 1x10⁵ irradiated cells were then seeded into a 24-well plate. The plate was incubated overnight at 37°C with 5% CO2 to allow the fibroblast cells to adhere to the well surface. Subsequently, duplicate aliquots of 1x10⁶ CLL patient’s PBMCs were taken and re-suspended in cell culture medium (3ml) - RPMI as above, which was supplemented with: 2mM L-Glutamine (Life Technologies); 100units/ml Penicillin and 100g/mL streptomycin (Life Technologies); and 10% FCS (Life Technologies) supplemented with 5ng/pL IL-4 (Biosource). These samples were left to incubate at 37°C in a 5% CO2 moist chamber for 1 hour. The 1x10⁶ CLL patient’s PBMCs were then added to the surface of the irradiated cells and incubated at 37°C in a 5% CO2 humidified chamber.

On day 3 or 4, 1 mL of fresh culture medium was added to each sample well. Every 7 days the cells were transferred to new CD40L feeder wells. We have previously shown that co-culturing CLL cells in this way causes CLL cell activation and proliferation¹⁷.

RESULTS

The in vitro cytotoxicity of a range of DNA damaging agents was assessed in monoculture (i.e. CLL cells alone) and co-culture (i.e. CLL cells on CD40L- expressing mouse fibroblasts) to determine whether sensitivity to these agents was related to the telomere length profiles of the tumour cells. Subsequently, these agents were combined with various DDRi compounds, in particular olaparib (a PARP inhibitor), GDC-0575 (a Chk1 inhibitor) and MKK-1775 (a WEE1A inhibitor), in the same in vitro models. Analysis of the efficacy of chemotherapy agents, rituximab and the PARP inhibitor, olaparib, to kill primary CLL cells in monoculture and CD40L- expressing co-culture

We have analysed the comparative cytotoxicity of fludarabine, chlorambucil, bendamustine, mafosfamide, rituximab and olaparib as single agents in both a monoculture system and a co-culture system (Figure 1 ).

Our results demonstrate that the purine nucleoside analogue (fludarabine) and the three alkylating agents (chlorambucil, bendamustine and mafosfamide) all show reduced cytotoxicity under co-culture conditions, and rituximab showed little cytotoxicity under either monoculture or co-culture conditions.

In contrast, olaparib was shown to enhance the cytotoxic effect when applied to CLL cells in co-culture. Although not bound to any particular theory (at least because the mechanism for this increased potency was not explored in this study), one may speculate that this increased cellular activation and tumour cell division induced by the co-culture conditions may make cells more prone to DNA damage and hence more reliant on DNA repair mechanisms. Therefore, DDRis such as PARP inhibitors may, even as monotherapies, be more effective in proliferating cells within a tumour.

The impact of telomere length on the ability of chemotherapy agents, rituximab and olaparib to kill primary CLL cells in monoculture and coculture

To assess if there was any relationship between telomere length and the cytotoxic response to drugs, the telomere length distribution in our CLL patient samples was analysed using single telomere length analysis (STELA) at the XpYp telomere, and the patients were divided into two groups based on telomere length (i.e. telomere length below 3.81 kb or a telomere length at/above 3.81 kb, 3.81 kb representing the upper threshold at which telomere end-end fusion events can be detected) before the response to drugs in our in vitro models was compared between said groups (Figure 2 and Figure 3).

These results demonstrated that the short telomere group was significantly resistant to killing with the purine nucleoside analogue (fludarabine) and the three alkylating agents (chlorambucil, bendamustine and mafosfamide) in both monoculture (Figure 2A-2D) and co-culture (Figure 3A-3D). In contrast, the same cells showed increased sensitivity to olaparib under both sets of in vitro conditions (Figure 2E and Figure 3E respectively).

It was also noted that CLL cells derived from patients harbouring an 1 1 q deletion or a 17p deletion showed no significant difference in response to the drugs when the samples were grouped according to telomere length. This implies that CLL cells with short telomeres are inherently more resistant to the cytotoxic effect of DNA damaging agents, and more sensitive to the cytotoxic effects of DDRis such as PARP inhibitors, regardless of their cytogenetic background.

Analysis of the cytotoxic efficacy of chemotherapy agents and rituximab, when used in combination with olaparib, against primary CLL cells

To assess if there was any antagonistic, additive or synergistic effect upon the cytotoxic efficacy of DNA damaging agents, the experimentally determined LD50 values of each of the individual agents (Table 2) from the monoculture studies detailed above were used to determine the molar ratios for the combination studies. Specifically, potential synergistic enhancement of cytotoxic efficacy was evaluated in both monoculture (Figure 4) and co-culture (Figure 5) experiments using the following DNA Damaging AgentDDRi molar ratios:

• 1 : 1 for chlorambucil:olaparib combinations;

• 1 :2 for bendamustine:olaparib combinations;

• 1 :40 for fludarabine:olaparib combinations; and

• 1 : 100 for mafosfamide combinations. These results not only demonstrate that the PARP inhibitor olaparib was synergistic with fludarabine, chlorambucil, bendamustine and mafosfamide (particularly in DNA Damaging Agent: DDRi molar ratios ranging from 1000: 1 to 1 :1000, preferably 500: 1 to 1 : 1000, more preferably 10: 1 to 1 :1000, and still more preferably from 5:1 to 1 :500), but also that a significantly stronger synergy was observed for the short telomere patient group (Figures 4 and 5).

Analysis of the cytotoxic efficacy of chemotherapy agents when used in combination with WEE1A inhibitors or Chk1 inhibitors, against primary CLL cells

To assess if the synergistic effect upon the cytotoxic efficacy of DNA damaging agents for combinations of DNA damaging agents and DDRis was specific to combinations comprising PARP inhibitor DDRis, cytotoxicity efficacy was also evaluated in monoculture experiments using combinations comprising the WEE1A inhibitor MK-1775 (Figure 6) or the Chk1 inhibitor GDC-0575 (Figure 7) using the following DNA Damaging Agent: DDRi molar ratios:

• 500: 1 for bendamustine:MKK-1775 and bendamustine:GDC-0575 combinations; and

• 50: 1 for mafosfamide:MKK-1775 and bendamustine combinations.

Consistent with the results obtained using combinations comprising the PARP inhibitor olaparib, these results not only demonstrate that the WEE1A inhibitor MKK-1775 and Chk1 inhibitor GDC-0575 were each synergistic with the DNA damaging agents bendamustine and mafosfamide, but also that a significantly stronger synergy was observed for the short telomere patient group. In particular, these synergistic effects were shown for combinations comprising DNA Damaging Agent: DDRi molar ratios ranging from 1000: 1 to 1 :1000, more preferably from 750: 1 to 25: 1 and still more preferably from 500: 1 to 50: 1 . Summary

The main findings of this study can be summarised as follows:

The work presented here provides a rationale for using DDRis such as PARP inhibitors, WEE1A inhibitors or Chk1 inhibitors, alone or in combination with DNA damaging agents/procedures, for the treatment of cancer.

The enhanced synergistic efficacy observed in the short telomere group suggests that tumour cells with short telomeres are inherently more sensitive to inhibition of DNA damage response pathways.

It is also noted that short telomeres are associated with genomic complexity in a number of human cancers. Therefore, one can speculate that patients with these adverse clinical characteristics may preferentially benefit from the combination of DNA damage response pathway inhibitors and DNA damaging agents.

Consequently, tumour cell telomere length potentially could be used to risk- stratify patients (routinely or in clinical trials) and assign them to drug treatment regimens specifically designed to exploit their telomere length-associated weakness.

References

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Table 1. Upper limit and mean telomere length at which telomere end-end fusion events can be detected for five different chromosome ends

value calculated based on XpYp, 17p, 2p, 16p and 18q determinations only

Table 2. LD50 values for tested individual DNA Damaging agents and DDRis in monoculture conditions

Claims

1 . A DNA damage response inhibitor (DDRi) for use in treating cancer in a patient whose cancer cells have a mean telomere length, determined using high-resolution telomere length analysis, that is less than or equal to a threshold value selected according to the chromosome to be tested from the group consisting of: i) 3.81 kb for XpYp;

ii) 4.81 kb for 17p;

iii) 5.01 kb for 2p;

iv) 4.49 kb for 16p;

v) 4.47 kb for 18q;

vi) 4.27 kb for 7q; or

2. The DDRi for use according to claim 1 , wherein the mean telomere length of said cells is determined as the average of the mean telomere lengths determined for each of XpYp, 17p, 2p, 16p and 18q, and wherein said threshold value is 4.52 kb.

3. The DDRi for use according to claim 1 or claim 2, wherein said cancer is a B-cell malignant condition.

4. The DDRi for use according to claim 3, wherein said B-cell malignant condition is chronic lymphocytic leukaemia (CLL).

5. The DDRi for use according to claim 4, wherein said cancer cells are free of chromosomal abnormalities (cytogenetically normal).

6. The DDRi for use according to 4, wherein said cancer cells possess one or more chromosomal abnormalities.

7. The DDRi for use according to claim 6, wherein said chromosomal abnormalities are selected from deletions in one or more chromosomes selected from the group consisting of 1 1 q and 17p.

8. The DDRi for use according to any one of claims 1 -7, wherein said DDRi is an inhibitor of one or more proteins selected from the group consisting of: an ATM serine/threonine kinase (ATM), ATR serine/threonine- protein kinase (ATR), Checkpoint kinase 1 (Chk1 ), Checkpoint kinase 2 (CHk2), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), DNA polymerase theta (POLQ), Wee1 -like protein kinase (WEE1A), Wee1 -like protein kinase 2 (WEE1 B), and poly ADP ribose polymerase (PARP).

9. The DDRi for use according to claim 8, wherein said DDRi is an inhibitor of one or more proteins selected from the group consisting of WEE1 A, Chk1 and PARP.

10. The DDRi for use according to claim 9, wherein:

a. said DDRi is a WEE1A inhibitor, optionally MKK-1775; or b. said DDRi is a Chk1 inhibitor, optionally GDC-0575.

1 1 . The DDRi for use according to claim 9, wherein said DDRi is a PARP inhibitor optionally selected from the group consisting of: olaparib, rucaparib, niraparib, talazoparib, veliparib, 3-aminobenzamide, and iniparib.

12. The DDRi for use according to claim 1 1 , wherein said PARP inhibitor is olaparib.

13. The DDRi for use according to any one of claims 1 -12, wherein said DDRi is used in combination with a DNA damaging agent and/or a DNA damaging procedure.

14. The DDRi for use according to claim 13, wherein said DNA damaging agent is selected from the group consisting of nucleoside analogues and alkylating agents.

15. The DDRi for use according to claim 14, wherein said nucleoside analogue is fludarabine, and/or wherein said alkylating agent is selected from the group consisting of: chlorambucil, bendamustine, mafosfamide, cyclophosphamide and 4-hydroxy-cyclophosphamide.

16. The DDRi for use according to any one of claims 13-15, wherein said DNA damaging procedure is radiotherapy.

17. The DDRi for use according to any one of claims 1 -16, wherein said mean telomere length is determined in at least a sample of B-cells isolated from said patient.

18. The DDRi for use according to any one of claims 1 -17, wherein said mean telomere length is determined for a single chromosome.

19. The DDRi for use according to any one of claims 1 -17, wherein said mean telomere length is determined for a plurality of different chromosomes.

20. The DDRi for use according to any one of claims 1 -19, wherein said mean telomere length is determined for chromosome XpYp.

21.A method for treating cancer in a patient, the method comprising: a. using high-resolution telomere length analysis to determine the mean telomere length (in kb) of at one chromosome selected from XpYp, 17p, 2p, 16p, 18q and 7q in at least a sample of cancer cells from said patient; and

i. 3.81 kb for chromosome XpYp;

ii. 4.81 kb for chromosome 17p;

iii. 5.01 kb for chromosome 2p;

iv. 4.49 kb for chromosome 16p;

v. 4.47 kb for chromosome 18q;

vi. 4.27 kb for 7q; or

c. administering a DDRi to said patient.

22. The method according to claim 21 , wherein the mean telomere length of said cells is determined as the average of the mean telomere lengths determined for each of XpYp, 17p, 2p, 16p and 18q, and wherein said threshold value is 4.52 kb.

23. The method according to claim 21 or claim 22, wherein said cancer is a B-cell malignant condition.

24. The method according to claim 23, wherein said B-cell malignant condition is chronic lymphocytic leukemia (CLL).

25. The method according to claim 24, wherein said cancerous cells are free of chromosomal abnormalities (cytogenetically normal).

26. The method according to 24, wherein said cancerous cells possesses one or more chromosomal abnormalities.

27. The method according to claim 26, wherein said chromosomal abnormalities are selected from deletions in one or more chromosomes selected from the group consisting of 11 q and 17p.

28. The method according to any one of claims 21 -27, wherein said DDRi is an inhibitor of one or more proteins selected from the group consisting of: an ATM serine/threonine kinase (ATM), ATR serine/threonine- protein kinase (ATR), Checkpoint kinase 1 (Chk1 ), Checkpoint kinase 2 (CHk2), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), DNA polymerase theta (POLQ), Wee1 -like protein kinase (WEE1A), Wee1 -like protein kinase 2 (WEE1 B), and poly ADP ribose polymerase (PARP).

29. The method according to claim 28, wherein said DDRi is an inhibitor of one or more proteins selected from the group consisting of WEE1A, Chk1 and PARP.

30. The method according to claim 29, wherein:

a. Said DDRi is a WEE1 inhibitor, optionally MKK-1775; or b. said DDRi is a Chk1 inhibitor, optionally GDC-0575.

31. The method according to claim 29, wherein said DDRi is a PARP inhibitor optionally selected from the group consisting of: olaparib, rucaparib, niraparib, talazoparib, veliparib, 3-aminobenzamide, and iniparib.

32. The method according to claim 31 , wherein said PARP inhibitor is olaparib.

33. The method according to any one of claims 21 -33, wherein said DDRi is administered in combination with a DNA damaging agent and/or a DNA damaging procedure.

34. The method according to claim 33, wherein said DNA damaging agent is selected from the group consisting of nucleoside analogues and alkylating agents.

35. The method according to claim 34, wherein said nucleoside analogue is fludarabine, and/or wherein said alkylating agent is selected from the group consisting of: chlorambucil, bendamustine, mafosfamide, cyclophosphamide and 4-hydroxy-cyclophosphamide.

36. The method according to any one of claims 33-35, wherein said DNA damaging procedure is radiotherapy.

37. The method according to any one of claims 21 -36, wherein said mean telomere length is determined in at least a sample of B-cells isolated from said patient.

38. The method according to any one of claims 21 -37, wherein said mean telomere length is determined for a single chromosome.

39. The method according to any one of claims 21 -37, wherein said mean telomere length is detected for a plurality of different chromosomes.

40. The method according to any one of claims 21 -39, wherein said mean telomere length is detected for chromosome XpYp.