defective chronic lymphocytic leukemia cells

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Regular Article LYMPHOID NEOPLASIA

ATR inhibition induces synthetic lethality and overcomes chemoresistance in TP53- or ATM-defective chronic lymphocytic leukemia cells Marwan Kwok,1,2,* Nicholas Davies,1,* Angelo Agathanggelou,1 Edward Smith,1 Ceri Oldreive,1 Eva Petermann,1 Grant Stewart,1 Jeff Brown,3 Alan Lau,4 Guy Pratt,1,5 Helen Parry,1,2 Malcolm Taylor,1 Paul Moss,1,2 Peter Hillmen,6 and Tatjana Stankovic1,2 1

School of Cancer Sciences, University of Birmingham, Birmingham, United Kingdom; 2Centre for Clinical Haematology, Queen Elizabeth Hospital Birmingham, Birmingham, United Kingdom; 3Oncology iMed, AstraZeneca Pharmaceuticals, Waltham, MA; 4R&D Oncology iMed, AstraZeneca Pharmaceuticals, Alderley Park, United Kingdom; 5Birmingham Heartlands Hospital, Birmingham, United Kingdom; and 6Section of Experimental Haematology, Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom

TP53 and ataxia telangiectasia mutated (ATM) defects are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukemia • ATR inhibition is synthetically (CLL). Currently, therapies capable of providing durable remissions in relapsed/ refractory TP53- or ATM-defective CLL are lacking. Ataxia telangiectasia and Rad3lethal to TP53- or ATMrelated (ATR) mediates response to replication stress, the absence of which leads to defective CLL cells. collapse of stalled replication forks into chromatid fragments that require resolution • ATR targeting induces through the ATM/p53 pathway. Here, using AZD6738, a novel ATR kinase inhibitor, we selective cytotoxicity and investigated ATR inhibition as a synthetically lethal strategy to target CLL cells with chemosensitization in TP53TP53 or ATM defects. Irrespective of TP53 or ATM status, induction of CLL cell or ATM-defective CLL cells proliferation upregulated ATR protein, which then became activated in response to in vitro and in vivo. replication stress. In TP53- or ATM-defective CLL cells, inhibition of ATR signaling by AZD6738 led to an accumulation of unrepaired DNA damage, which was carried through into mitosis because of defective cell cycle checkpoints, resulting in cell death by mitotic catastrophe. Consequently, AZD6738 was selectively cytotoxic to both TP53- and ATM-defective CLL cell lines and primary cells. This was confirmed in vivo using primary xenograft models of TP53- or ATM-defective CLL, where treatment with AZD6738 resulted in decreased tumor load and reduction in the proportion of CLL cells with such defects. Moreover, AZD6738 sensitized TP53- or ATM-defective primary CLL cells to chemotherapy and ibrutinib. Our findings suggest that ATR is a promising therapeutic target for TP53- or ATMdefective CLL that warrants clinical investigation. (Blood. 2016;127(5):582-595)

Key Points

Introduction Chronic lymphocytic leukemia (CLL) is characterized by biological and clinical heterogeneity.1-4 Two important genes associated with adverse prognosis, TP53 and ataxia telangiectasia mutated (ATM), govern the cellular response to DNA damage, whereby CLL cells, having accumulated sufficient genotoxic damage, are directed to undergo cell cycle arrest or apoptosis.5,6 Disruption of these genes, through gene deletion (ie, 17p for TP53 or 11q for ATM) and/or mutation, confers genomic instability and chemoresistance, leading to an adverse clinical outcome.3,7-16 There is increasing recognition that therapeutic failure and relapse associated with chemotherapy or chemoimmunotherapy can be attributed to selection of DNA damage response (DDR)– defective subclones.17,18 As a result, a paradigm shift has emerged toward the use of DDR-independent therapies, such as B-cell

receptor (BCR) signaling inhibitors, in CLL with TP53 defects.19,20 However, although treatment-na¨ıve del(17p) patients exhibited durable response to the BCR signaling inhibitor ibrutinib,21 previously treated individuals with del(17p) or del(11q) continued to demonstrate inferior progression-free and overall survival compared with individuals without these aberrations.19,22 Furthermore, Bruton tyrosine kinase or PLCg2 mutations,23 clonal evolution, 24 early relapses, and Richter transformation25 following ibrutinib treatment have been reported predominantly in patients with del(17p) or del(11q). Many of these patients also acquired complex cytogenetics, 23-25 thus underscoring the relevance of genomic instability to therapeutic resistance even for p53independent treatments. Patients relapsing from BCR signaling inhibitors have limited salvage options and poor clinical outcome,

Submitted May 8, 2015; accepted October 31, 2015. Prepublished online as Blood First Edition paper, November 12, 2015; DOI 10.1182/blood-2015-05644872.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

*M.K. and N.D. contributed equally to this study. The online version of this article contains a data supplement.

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© 2016 by The American Society of Hematology

BLOOD, 4 FEBRUARY 2016 x VOLUME 127, NUMBER 5

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with a median survival of 3.1 months following discontinuation of ibrutinib as reported recently by Jain et al.25 Therefore, therapeutic strategies specifically targeting genomically unstable DDR-defective subclones may be of value for these CLL patients. Synthetically lethal approaches, in which collaborating pathways that DDR-defective cells depend on for survival are exploited for therapeutic targeting, offer a potential avenue to eradicate CLL cells with DDR defects.26,27 In this respect, we previously demonstrated the utility of poly (ADP-ribose) polymerase inhibition as a targeted therapy for ATM-defective CLL.28 However, synthetically lethal approaches targeting the TP53-defective CLL phenotype have not hitherto been investigated. Replication stress can arise during physiological cell cycles and is frequently elevated in malignant cells. It occurs when replication fork progression is disrupted and cells continue to cycle despite the presence of unreplicated DNA. 29 Ataxia telangiectasia and Rad3-related (ATR) is a serine/threonine protein kinase that regulates replication initiation, preventing aberrant and excessive replication origin firing, which depletes the cellular pool of nucleotides and replication proteins. Inhibition of ATR therefore induces replication stress, manifested by the accumulation of slowed and stalled replication forks with unprotected single-stranded DNA that inevitably breaks. This results in fragmented, partially replicated sister chromatids with free DNA double-stranded ends (DSEs). ATR also mediates the response to replication stress, delaying cell cycle progression, and stabilizing and repairing DNA replication forks.29-31 If ATR is inhibited, maintenance of genome integrity becomes dependent on functional ATM and p53, with ATM being essential for homologous recombination repair (HRR) to restore replication fork topology, and both ATM and p53 for arresting cell cycle progression to permit repair.5,6 ATR, therefore, represents an attractive synthetically lethal target for p53 or ATM deficiency.32,33 Evidence for this synthetically lethal interaction has been previously provided by deletion of ATR in p53-deficient mice34 and by inhibition of ATR in tumor cell lines, which resulted in selective killing of cells harboring p53 or ATM defects.35-37 No study to date has addressed the impact of ATR inhibition on primary tumor samples or xenotransplantation models of hematologic malignancies with DDR defects. Here, we used AZD6738 (AstraZeneca, Alderley Park, United Kingdom), a novel, highly specific, and orally bioavailable ATR kinase inhibitor to examine the effect of ATR inhibition in primary CLL cells, cell lines, and primary CLL xenografts. We present evidence for synthetic lethality and selective cytotoxicity in CLL cells with TP53 or ATM defects, as well as sensitization of these cells to chemotherapeutic agents and ibrutinib.

Materials and methods Cells and reagents Details of cell lines and their short hairpin RNA transfections are provided in the supplemental Materials and Methods (available on the Blood Web site). Primary CLL samples (supplemental Table 1; supplemental Figure 1) were obtained from local hospitals with research ethics committee approval. Methods for induction of CLL cell proliferation69 and drugs used are detailed in the supplemental Materials and Methods and supplemental Figure 2. Western blotting and cytotoxicity assays Western blotting was carried out as previously described.38 Cytotoxicty was assessed by measurement of propidium iodide uptake and/or CellTiter-Glo

ATR TARGETING IN CLL WITH TP53 OR ATM DEFECTS

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luminescence cell viability assay (Promega, Southampton, United Kingdom) as described in the supplemental Materials and Methods. Primary CLL xenograft models Animal experiments were conducted in accordance with United Kingdom Home Office regulations. Protocol for the generation of CLL xenografts and treatment regimens are provided in the supplemental Materials and Methods and supplemental Figure 3. Cell cycle analysis and immunofluorescence microscopy Cell cycle profiles were obtained using the Accuri C6 flow cytometer (BD Biosciences, Oxford, United Kingdom). Foci and DNA replication tracts were examined using a Nikon E600 Eclipse microscope (Kingston upon Thames, United Kingdom) as described in the supplemental Materials and Methods. Statistical analysis Statistical significance was determined using GraphPad Prism (GraphPad Software, San Diego, CA). P # .05 was considered significant.

Results ATR signaling is active in proliferating CLL cells and is inhibited by AZD6738

The ATR pathway has been shown to be suppressed in quiescent lymphocytes.39 Consistent with this, we observed that ATR protein expression was low in resting primary CLL cells but induced when cells were stimulated to proliferate by coculturing with CD40 ligand (CD40L)–expressing murine embryonic fibroblasts and interleukin (IL) 21 (CD40L/IL-21). This was irrespective of p53 or ATM status (Figure 1A). We used phosphorylation of checkpoint kinases Chk1 and Chk2, which are downstream targets of ATR and ATM, respectively, as surrogate markers for ATR and ATM pathway activation. Upon treatment with hydroxyurea (HU), which induces replication stress, ATR-dependent Chk1 phosphorylation was observed in proliferating (Figure 1B, lanes 2, 3, and 10) but not quiescent (lanes 6 and 8) CLL cells. The background Chk2 phosphorylation in quiescent cells (lanes 5 and 6) likely reflected cellular stress when cryopreserved samples were preincubated in culture media prior to treatment. This was absent when fresh samples were used without preincubation (lanes 7 and 8). As 24-hour HU treatments (lane 4) appeared to be associated with S-phase checkpoint adaptation, a process in which Chk1 phosphorylation is lost despite persistent replication stress,40 we adopted 5-hour HU treatments for subsequent experiments. The specificity of Chk1 as a marker for ATR activation was evident by minimal Chk1 phosphorylation following ionizing radiation (IR), which generates DNA double-strand breaks and activates the ATM/Chk2 pathway (Figure 1C, lanes 8-11). To investigate the effect of AZD6738 on ATR and ATM pathways, we pretreated cycling primary CLL cells and cell lines with 1 mM AZD6738 and/or 10 mM ATM inhibitor KU-55933 (ATMi) for 2 hours prior to HU or IR exposure. AZD6738 treatment led to suppression of ATR signaling as indicated by a reduction in HU-induced Chk1 phosphorylation, independent of ATM status (Figure 1C, lane 4 vs 6; Figure 1D, lanes 3 vs 4, 9 vs 10). Complete abolition of HU-induced Chk1 phosphorylation was evident at AZD6738 doses $3 mM (supplemental Figure 4A). The specificity of AZD6738 for ATR inhibition was demonstrated by its lack of effect on IR-induced ATM/Chk2 phosphorylation

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KWOK et al


Figure 1. ATR signaling is activated in response to replication stress in proliferating primary CLL cells and is inhibited by AZD6738. (A) Stimulation of primary CLL cell proliferation by coculture with CD40L-expressing murine embryonic fibroblasts in the presence of IL-21 (CD40L/IL-21) for 4 days resulted in induction of ATR expression in primary CLL cells irrespective of ATM or TP53 status. Cyclin A expression is a marker of proliferating cells. Actin is the loading control. (B) Cryopreserved or fresh primary CLL cells cultured with or without CD40L/IL-21 (lanes 1-4 and 9 and 10, and lanes 5-8, respectively) were treated with HU. Cryopreserved samples not cocultured with CD40L/ IL-21 were resuspended and preincubated in culture media for 24 hours prior to treatment (lanes 5 and 6), whereas fresh cells were treated immediately upon isolation from peripheral blood without preincubation (lanes 7 and 8). Exposure to HU (1 mM), which induces replication stress, led to Chk1 phosphorylation in primary CLL cells cocultured with CD40L/IL-21 (lanes 2, 3, and 10). (C) TP53/ATM wild-type (TP53/ATM-wt) primary CLL cells cocultured with CD40L/IL-21 (C) and CII cells (D), both CII-GFPsh and CII-ATMsh, were treated with AZD6738 (1 mM) and/or the ATM inhibitor KU-55933 (ATMi; 10 mM) for 2 hours, or left untreated, prior to exposure to HU (1 mM) or IR (6 Gy) for a further 5 hours. AZD6738 treatment inhibited ATR signaling as indicated by a reduction in HU-induced Chk1 phosphorylation (panel C, lane 4 vs 6; panel D, lanes 3 vs 4 and 9 vs 10). In ATM-proficient CLL cells, this also led to ATM activation as evidenced by ATM phosphorylation and Chk2 phosphorylation (panel C, lane 4 vs 6; panel D, lane 3 vs 4). In panels B-C, representative blots from experiments on 3 CLL samples are shown.

(Figure 1C, lane 8 vs 10; Figure 1D, lane 5 vs 6). Importantly, in ATM-proficient primary CLL cells and cell line, AZD6738 treatment resulted in ATM activation as evidenced by ATM phosphorylation and HU-induced Chk2 phosphorylation (Figure 1C, lane 4 vs 6; Figure 1D, lane 3 vs 4). This indicated dependence on the ATM pathway in the absence of functional ATR. Because DNA protein kinase (DNA-PK) was reported to have some redundant activity with ATR,41 we assessed the impact of DNA-PK inhibition in primary CLL cells. We detected no change in HU-induced Chk1 phosphorylation upon treatment with DNA-PK inhibitor (supplemental Figure 4B), suggesting that such redundancy is not present in CLL cells. ATR inhibition is selectively cytotoxic to TP53- or ATM-defective CLL cells in vitro and in vivo

To assess the therapeutic potential of ATR inhibition for DDRdefective CLL, we investigated the cytotoxic effects of AZD6738 on CLL cells. ATM-deficient CII-ATMsh and p53-defective Mec1 cells displayed significantly greater AZD6738 sensitivity (50% effective concentration [EC50], 1.6 mM and 1.1 mM, respectively) following 96-hour treatment compared with ATM/p53-proficient CII-GFPsh cells (EC50, 8.4 mM; Figure 2A). Reintroduction of wild-type TP53 significantly reduced the sensitivity of Mec1 cells to AZD6738 (EC50,

3.2 mM vs 1.0 mM; Figure 2B). The effect was, however, limited because of a moderate level of wild-type p53 expression achieved by transfection relative to expression of the intrinsic mutant TP53 allele (supplemental Figure 5). Following 96-hour AZD6738 treatment, a panel of 29 CD40L/ IL-21 cocultured primary CLL samples provided additional evidence for selective cytotoxicity toward CLLs with DDR defects (Figure 2C-D; supplemental Table 1), with a significantly lower EC50 in ATMdefective (8.7 mM; 95% confidence interval [CI], 3.4-13.9 mM; n 5 6) and TP53-defective (8.2 mM; 95% CI, 3.5-12.9 mM; n 5 6) CLL samples compared with either ATM/TP53 wild-type CLLs (38.3 mM; 95% CI, 28.8-47.8 mM; n 5 17) or healthy donor peripheral blood mononuclear cells (PBMCs; 87.6 mM; 95% CI, 50.0-125.3 mM; n 5 4). No significant difference in AZD6738 sensitivity was found when these samples were analyzed according to clinical stage, prior treatment, or IGHV mutational status (Figure 2E). As expected, AZD6738 had little cytotoxic effect on cells cultured without CD40L/IL-21, regardless of TP53 or ATM status (supplemental Figure 6). This suggests that AZD6738 specifically targets the proliferating CLL population. To assess the effectiveness of ATR inhibition in vivo, we used primary CLL xenografts. We engrafted 4 representative primary CLL samples carrying either del(17p) and a TP53 mutation (p.I251S or p.M237I) or del(11q) and an ATM mutation (p.I1407T or p.L3013Q)



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Figure 2. ATR inhibition is selectively cytotoxic to both ATM-defective and TP53-defective CLL cells in vitro and in vivo. (A) CII-GFPsh, CII-ATMsh (ATM-deficient), and Mec1 (p53-defective) cells were treated with AZD6738 for 4 days, and viability was measured using the CellTiter-Glo assay. Surviving fraction is expressed relative to untreated controls. AZD6738 induced significantly greater dose-dependent cytotoxicity with significantly lower AZD6738 EC50 in CII-ATMsh and Mec1 cells compared with CII-GFPsh cells. (B) Mec1 cells transfected with either wild-type TP53 (Mec1-p53-pcDNA3.1) or GFP (Mec1-GFP-pcDNA3.1, as control) were treated with AZD6738 for 4 days, and viability was measured using the CellTiter-Glo assay. Surviving fraction is expressed relative to untreated controls. AZD6738-induced cytotoxicity was reduced with significantly higher AZD6738 EC50 in Mec1-p53-pcDNA3.1 cells compared with Mec1-GFP-pcDNA3.1 cells. (C) Carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled primary CLL cells with or without ATM/TP53 defects and healthy donor peripheral blood mononuclear cells (PBMCs) cocultured with CD40L/IL-21 were treated with AZD6738 for 4 days. Viability was measured by propidium iodide exclusion of the proliferating cell population that was identified by reduction in CFSE


KWOK et al

into NOD/Shi-scid/IL-2Rgnull mice. Following treatment with AZD6738 (n 5 10) or vehicle (n 5 11) for 2 weeks, tumor load was significantly reduced in AZD6738-treated animals compared with vehicle-treated controls (P # .01; Figure 2F). When analyzed separately according to genotype, both TP53- and ATM-defective AZD6738-treated xenografts showed significant reduction in tumor load (P # .05; Figure 2G). Furthermore, in the CLL26 xenograft where tumor cell recovery allowed monitoring of del(11q) cells, we observed a significant reduction in the percentage of cells with del(11q) (37% vs 100%, P # .05) in AZD6738-treated animals compared with vehicle-treated controls (Figure 2H). Taken together, our in vitro and in vivo data demonstrate efficacy and specificity of AZD6738 for TP53- or ATM-defective CLL. ATR inhibition induces DNA damage and mitotic catastrophe in TP53- or ATM-defective CLL cells

Suppression of aberrant origin firing is an important mechanism whereby ATR protects DNA replication forks from collapse.29-31 We therefore hypothesized that ATR inhibition in CLL cells would result in increased origin firing. To verify this, we first determined the impact of ATR inhibition on DNA replication in CII-ATMsh and Mec1 cells by DNA fiber analysis that visualizes origins of replication and replication fork progression (Figure 3A-C). One hour after treatment with AZD6738, we observed increased replication initiation as indicated by decreased interorigin distance (Figure 3B). We also observed decreased replication fork progression rates (Figure 3C), consistent with fork slowing or stalling and therefore increased replication stress upon ATR inhibition.30,31,42 We next assessed the impact of ATR inhibition on cell cycle progression. In response to AZD6738, CII-GFPsh cells accumulated in G0/G1 consistent with G1/S cell cycle arrest (Figure 3D; supplemental Figure 7). In contrast, neither G1/S nor G2/M arrest was evident in CII-ATMsh and Mec1 cells despite replication stress (Figure 3E-F; supplemental Figure 7), indicating dependence on ATM and p53 for cell cycle regulation in the absence of ATR. These cell cycle effects were recapitulated in primary CLL cells upon treatment with AZD6738, with G1/S arrest occurring in TP53/ATM wild-type but not TP53-defective samples (Figure 3G-H). Because ATR inhibition leads to S-phase chromatid fragments,31,32 for which effective resolution requires ATM-mediated HRR and ATM/ p53-dependent cell cycle checkpoint activation,5,6 we reasoned that ATR inhibition should produce more fragmented chromatids in ATM/ p53-defective CLL cells. Consistent with this, we observed accumulation of markers of DNA damage, gH2AX and 53BP1 foci, in Mec1 and ATMi pretreated CII cells but not in CII cells without ATMi pretreatment, 24 and 48 hours, respectively, following exposure to 1 mM AZD6738 (Figure 4A-E; supplemental Figure 8). Moreover, consistent with loss of both G1/S and G2/M checkpoints in the absence


of functional ATR and ATM/p53, Mec1 and ATMi pretreated CII cells carrying unrepaired DNA damage progressed into mitosis, as indicated by dual positivity for gH2AX and the mitotic marker pH3 following 24-hour incubation with 1 mM AZD6738 (Figure 4A-B). Replication stress leads to an increase in the amount of underreplicated regions within the genome.29 As the sister chromatids try to separate during anaphase, these catenated regions of DNA result in ultrafine DNA bridges, which often break and are transmitted to daughter cells at the following G1. This can be visualized as 53BP1 nuclear bodies, which serve to protect DNA from endonuclease degradation and facilitate repair.43,44 We observed an accumulation of 53BP1 bodies in ATM/p53-proficient CII cells following 48 hours of AZD6738 treatment (Figure 4F). This was not seen in ATM/p53defective CLL cells, suggesting that formation of 53BP1 bodies was dependent on the ATM pathway as previously demonstrated.44 Failure to initiate this protective mechanism could precipitate further DNA damage in ATM/p53-defective cells. We postulated that abolition of cell cycle checkpoints because of the combined loss of functional ATR and ATM/p53 would permit entry into mitosis despite the accumulation of partially replicated chromatid fragments, resulting in mitotic catastrophe. To confirm this, we probed CLL cells for pH3 and lamin B. Cells undergoing normal mitosis stain positive for pH3 but lose lamin B staining. In contrast, cells undergoing mitotic catastrophe do not stain for pH3 but retain lamin B staining, whereas expression of both markers is lost when cells undergo apoptosis (Figure 4G). We found significant levels of mitotic catastrophe 72 hours following 1 mM AZD6738 in Mec1 and ATMi pretreated CII cells, but not in CII cells without ATMi pretreatment (Figure 4H). Because of incomplete DNA replication in the presence of AZD6738, we did not observe accumulation of tetraploid cells (G2/M population) that can accompany mitotic catastrophe (Figure 3E-F,H).45 In ATM/p53-proficient CII cells, we detected low-level apoptotic activity with AZD6738, which is likely a result of p53 activation in response to replication stress (supplemental Figure 9A-B). Mec1 cells also exhibited low levels of apoptotic markers poly (ADP-ribose) polymerase and caspase cleavage (supplemental Figure 9C), but such levels were unlikely to account for their differential sensitivity to ATR inhibition compared with DDR-proficient cells. Indeed, the negligible impact of pan-caspase inhibitor Z-VAD-FMK on the viability of AZD6738-treated Mec1 cells supports this notion (supplemental Figure 9D). ATR inhibition sensitizes TP53- or ATM-defective CLL cells to chemotherapy and ibrutinib

We reasoned that the synthetically lethal effects of ATR inhibition on ATM- or TP53-defective CLL cells could sensitize these chemoresistant cells to chemotherapeutic agents through potentiation of DNA damage. We therefore assessed the impact of combining AZD6738 with chemotherapy in CII-ATMsh and Mec1 cells. Although resistance to

Figure 2 (continued) fluorescence intensity as shown in supplemental Figure 2. Surviving fraction is expressed relative to untreated controls. AZD6738 induced significantly greater dose-dependent cytotoxicity in ATM/TP53-defective CLL cells than either ATM/TP53 wild-type CLL cells or healthy donor PBMCs. (D) The EC50 of AZD6738 was significantly lower for ATM/TP53-defective primary CLL samples than both ATM/TP53 wild-type samples and healthy donor PBMCs. A list of CLL samples assessed and their respective EC50 values are provided in supplemental Table 1. (E) There was no significant difference in the EC50 of AZD6738 between subgroups of CLL samples stratified according to Binet stage, prior treatment, and IGHV mutational status. (F) Primary CLL samples (CLL29, CLL31, CLL25, CLL26) with biallelic TP53 or ATM defects were engrafted into NOD/Shi-scid/IL-2Rgnull mice and treated with vehicle (n 5 11) or AZD6738 (n 5 10). Collectively, AZD6738 treatment significantly reduced tumor load compared with vehicle treatment in TP53/ATM-defective xenografts. (G) When analyzed separately, both TP53 and ATM defective xenografts showed significant reduction in tumor load following AZD6738 treatment compared with vehicle-treated controls. (H) Fluorescence in situ hybridization probes for 11q were applied on human CD451 CD191 cells collected from the spleens of CLL26 xenografts (harboring del(11q) and L3013Q ATM mutation) treated with AZD6738 (n 5 3) or vehicle (n 5 3). Two hundred cells were analyzed per mouse. The proportion of CLL cells with del(11q) was significantly reduced following AZD6738 treatment compared with vehicle-treated controls. All data are displayed as mean 6 standard error of the mean (SEM). Statistical significance was determined using 2-way analysis of variance (ANOVA) with Bonferroni post hoc analysis (A,B,C,G), 1-way ANOVA (A,D), or Student t test (B,F,H). Statistical significance vs CII-GFPsh (A), Mec1-GFP-pcDNA3.1 (B), ATM/TP53 wild-type samples (*) or healthy donor PBMCs (†) (C,D), or vehicle (F-H) is indicated by *,†P , .05, **,††P , .01, and ***,†††P , .001. Nonsignificant results are denoted by n.s.



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Figure 3. ATR inhibition leads to increased replication stress and ATM/p53 dependent G1/S cell cycle arrest in CLL cells. (A) AZD6738 (1 mM) treatment of 1 hour led to increased replication stress in Mec1 (p53-defective) and CII-ATMsh (ATM-deficient) cells as demonstrated by DNA fiber analysis. Replicating DNA in cycling cells was sequentially labeled with CldU and IdU for 20 min each, after which DNA fibers were analyzed by immunofluorescence microscopy. Representative images are displayed. (B-C) AZD6738 (1 mM) treatment of 1 hour significantly reduced interorigin distance (B) and fork progression rate (C) in Mec1 and CII-ATMsh cells. (D-F) Cell cycle analyses on CII-GFPsh (D), CII-ATMsh (E), and Mec1 (F) cells were carried out following 24-hour treatment with either AZD6738 (1 mM or 3 mM) or RPMI media. AZD6738 treatment induced G1/S cell cycle arrest in CII-GFPsh cells but not CII-ATMsh or Mec1 cells. (G-H) Cell cycle analyses on TP53/ATM-wt CLL samples (CLL03, CLL06, CLL07) (G) and TP53-defective samples (CLL32, CLL35, CLL36) (H) were carried out following 48-hour treatment with AZD6738 (3 mM or 10 mM) or RPMI media. Analyses in panels G-H were performed on the viable cell population by expressing the G0/G1, S, and G2/M populations as a percentage of the total viable cells. Data are displayed as mean 6 SEM of triplicate experiments. Statistical significance was determined using Student t test (B), MannWhitney U test (C), or 2-way ANOVA with Bonferroni post hoc analysis (D-H). Statistical significance vs dimethylsulfoxide (DMSO)–treated controls (B) or untreated controls (D-H) is indicated by *P , .05 and **P , .01.

chlorambucil, fludarabine, 4-hydroperoxycyclophosphamide, or bendamustine monotherapy was evident even at relatively high doses, we discovered significant sensitization to a range of doses of these agents upon addition of 1 mM AZD6738 (Figure 5; Table 1). Of note, CII-GFPsh cells were sensitive to chemotherapy. However, addition

of AZD6738 to chemotherapeutic agents led to further sensitization (supplemental Figure 10; supplemental Table 2). We then proceeded to evaluate these findings in DDR-defective primary CLL cells cocultured with CD40L/IL-21. AZD6738 (1 or 3 mM) enhanced sensitivity to (Figure 6A-B) and synergized


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Figure 4. ATR inhibition results in accumulation of DNA damage and mitotic catastrophe in CLL cells with ATM or p53 deficiency. CII cells pretreated for 2 hours with ATMi (10 mM), CII cells without ATMi pretreatment, and Mec1 (p53-defective) cells were exposed to AZD6738. (A-B) Cells treated with AZD6738 (1 mM) for 24 hours were colabeled with anti-gH2AX and anti-pH3 antibodies and analyzed by immunofluorescence microscopy using a 360 lens. A cell was considered gH2AX positive if .5 gH2AX foci were present. At least 200 mitotic (phosphohistone H3 ser-10 [pH3]–positive) cells were analyzed in each sample. AZD6738 induced gH2AX foci in Mec1 and ATMi pretreated CII cells, but not in CII cells without ATMi pretreatment. (C) The pattern of 53BP1 labeling distinguishes 53BP1 bodies from 53BP1 foci. 53BP1 bodies are characterized by robust blocklike staining and indicate underreplicated DNA. 53BP1 foci are characterized by discrete punctate staining and indicate DNA damage. A cell was considered 53BP1 foci positive if .5 53BP1 foci were present. (D-F) Cells treated with AZD6738 (1 mM) for 48 hours were labeled with anti-53BP1 antibodies, and at least 200 cells were then analyzed in each sample using a 360 lens. AZD6738 treatment led to an accumulation of 53BP1 foci in Mec1 and ATMi pretreated CII cells and an



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Figure 5. ATR inhibition sensitizes CII-ATMsh and Mec1 CLL cells to cytotoxic chemotherapy. CII-shATM (A) and Mec1 (B) cells were treated with chlorambucil, fludarabine, 4-hydroperoxycyclophosphamide (4HC), or bendamustine with or without coadministration of AZD6738 (1 mM). Viability was assessed 96 hours later by the CellTiter-Glo assay. Surviving fraction is expressed relative to untreated controls for chemotherapy treatment alone (no AZD6738) and relative to 1 mM AZD6738 monotherapy for the cotreated samples. Addition of AZD6738 significantly enhanced sensitivity of CII-shATM and Mec1 cells to cytotoxic chemotherapeutic agents. Data are displayed as mean 6 SEM of triplicate experiments. Statistical significance was determined using 2-way ANOVA with Bonferroni post hoc analysis. Statistical significance vs no AZD6738 is indicated by *P , .05, **P , .01, and ***P , .001. (C-D) AZD6738 is synergistic with chlorambucil, fludarabine, 4HC, and bendamustine in CII-shATM (C) and Mec1 (D) cells across a range of effective drug doses. Combination indices (CI) were calculated using the median-effect method. Each point represents the mean CI value obtained from 3 independent experiments plotted against the corresponding affected fraction that is expressed relative to untreated controls. CI ,0.9 represents synergism, CI 5 0.9-1.1 represents additive effect, and CI .1.1 represents antagonism. The actual values are presented in Table 1.

with (Figure 6C-D; Table 2) chlorambucil, fludarabine, and 4-hydroperoxycyclophosphamide in primary CLL samples with defective ATM or TP53 (n 5 6). However, because of prosurvival properties of

the coculture system, the sensitization effect (Figure 6A-B) was less profound than in CLL cell lines (Figure 5A-B). Interestingly, in these primary CLL samples, we also found additive to synergistic interaction

Figure 4 (continued) accumulation of 53BP1 bodies in CII cells without ATMi pretreatment. (G) Colabeling with anti-lamin B and anti-pH3 antibodies allows apoptotic CLL cells to be distinguished from cells undergoing mitotic catastrophe. At least 200 cells were analyzed in each sample. (H) AZD6738 exposure for 72 hours resulted in significantly elevated levels of mitotic catastrophe in Mec1 and ATMi pretreated CII cells. Data are displayed as mean 6 SEM of triplicate results from a representative experiment. Statistical significance was determined using Student t test. Statistical significance vs untreated controls is indicated by *P , .05, ** P , .01, and ***P , .001.



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Table 1. Combination indices of AZD6738 (1 mM) with cytotoxic chemotherapy in CII-ATMsh and Mec1 cells CII-ATMsh Drug combined with AZD6738 and dose (mM)

Mec1

Fraction affected

Combination index (mean 6 SEM)

Synergism*

Fraction affected


1

0.43

0.45 6 0.16

111

0.51

0.75 6 0.16

11

2.5

0.54

0.48 6 0.09

111

0.61

0.49 6 0.11

111

5

0.73

0.27 6 0.14

1111

0.71

0.29 6 0.03

1111

10

0.89

0.16 6 0.08

1111

0.76

0.26 6 0.03

1111

1

0.35

0.89 6 0.29

1

0.44

1.06 6 0.14

6

2.5

0.37

0.93 6 0.28

6

0.55

0.69 6 0.14

111

5

0.51

0.69 6 0.17

111

0.61

0.56 6 0.18

111

10

0.71

0.55 6 0.13

111

0.72

0.34 6 0.07

111

0.25

0.40

0.75 6 0.33

11

0.48

0.97 6 0.30

6

0.5

0.56

0.53 6 0.26

111

0.57

0.69 6 0.28

111

Synergism*

Chlorambucil

Fludarabine

4-Hydroperoxycyclophosphamide

1

0.73

0.36 6 0.15

111

0.68

0.42 6 0.14

111

2

0.89

0.30 6 0.07

1111

0.78

0.26 6 0.10

1111

10

0.24

1.08 6 0.15

6

0.39

1.10 6 0.08

6

25

0.31

0.86 6 0.15

1

0.49

0.89 6 0.29

1

50

0.40

0.66 6 0.15

111

0.52

0.76 6 0.18

11

100

0.65

0.34 6 0.11

111

0.66

0.41 6 0.01

111

Bendamustine

*1111, strong synergism; 111, synergism; 11, moderate synergism; 1, slight synergism; 6, additive.

between AZD6738 and cytotoxic doses of ibrutinib46 (Figure 6C-D; Table 2). We addressed mechanisms behind the AZD6738-ibrutinib interaction. We discovered modest dose-dependent reduction in Erk/Syk phosphorylation with AZD6738 and dose-dependent reduction in HU-induced Chk1 phosphorylation with ibrutinib (supplemental Figure 11), suggesting that off-target effects of AZD6738 and ibrutinib may account for the potentiating effect at higher dose combinations involving 4 mM or 16 mM ibrutinib. We also observed that whereas AZD6738 primarily targeted cycling CLL cells for killing, ibrutinib affected both the cycling and noncycling populations (supplemental Figure 12). This could potentially account for the sensitization seen with 1 mM ibrutinib. Finally, we assessed the AZD6738-chlorambucil combination in a primary xenograft model with del(11q) and ATM mutation p.I407T. Twenty engrafted mice were treated with AZD6738, chlorambucil, the AZD6738-chlorambucil combination, or vehicle (n 5 5 in each arm). AZD6738 monotherapy resulted in significant reduction in tumor load. Moreover, combination treatment yielded significantly greater reductions in tumor load than chlorambucil monotherapy (Figure 6E-F), although synergism could not be appropriately assessed from this experiment. Collectively, our results support the combined use of ATR inhibitor with a range of existing therapeutic agents for CLL.

Discussion In this study, we presented in vitro and in vivo data demonstrating efficacy of ATR inhibition in CLL with TP53 or ATM defects. This report is, to our knowledge, the first demonstration of a therapeutic approach that provides specific targeting of TP53-defective CLL cells. Our findings offer insight into the cytotoxic mechanism underpinning ATR inhibition in CLL, which is consistent with our current understanding of the role of ATR in resolving replication stress (Figure 7A).29-33,47-50 When ATR function is abolished, stalled replication forks with persistent single-stranded DNA are susceptible to nicking and collapse resulting in a partially replicated sister

chromatid with a DSE that has become disengaged from the replication template (Figure 7B). Reengagement of the DSE with the replication template requires the HRR stimulatory activity of ATM. Similarly, functional p53 becomes required in the context of ATR inhibition for cell cycle arrest to permit repair of collapsed forks and HRR-directed reengagement of DSEs to restore replication fork geometry (Figure 7C). In the absence of HRR, nonhomologous end joining can ligate 2 independently generated partially replicated sister chromatid DSEs, at the expense of potential sequence deletions and aberrant chromosomal translocations. CLL cells with defective p53 or ATM accumulate collapsed forks and DSEs, whereupon lack of effective cell cycle regulation permits subsequent unrestricted entry into mitosis, resulting in mitotic catastrophe (Figure 7D). This mechanistic model accounts for the selective cytotoxicity of ATR inhibition for CLL cells with TP53 or ATM defects. Importantly, ATR inhibition is capable of circumventing the protective effect of the microenvironment, which often hinders effective clearance of genomically unstable, proliferating CLL populations. This is evidenced by the ability of AZD6738 to overcome the prosurvival signals provided by the CD40L/IL-21 coculture system that mimics interaction of CLL cells with T cells in proliferation centers and, in xenograft experiments, by the loss of tumor burden in murine spleens upon treatment with AZD6738. Interestingly, whereas TP53- or ATM-defective primary CLL samples were uniformly and substantially more sensitive to ATR inhibition than healthy donor PBMCs, TP53/ATM wild-type primary CLL cells displayed variable sensitivity toward ATR inhibition. There are several potential explanations for this finding. Firstly the acquisition of additional, as yet uncharacterized DDR defects could render some CLL patients sensitive to ATR inhibition even in the absence of defective TP53 or ATM. Secondly, such variability in sensitivity could be because of different amounts of endogenous DNA damage in each sample, reflecting varying degrees of genomic instability. ATR inhibition may therefore potentially be most useful in heavily pretreated relapsed/refractory patients, where higher levels of genomic instability could be attributable, in part, to enhanced replication stress. Finally, alternative lengthening of telomeres has been reported in CLL cells51



591

Figure 6. ATR inhibition synergizes with existing therapeutic agents both in ATM-defective and TP53-defective primary CLL cells. CFSE-labeled primary CLL cells with ATM defect (CLL22, CLL23, CLL24) (A) or TP53 defect (CLL29, CLL31, CLL32) (B) cocultured with CD40L/IL-21 were treated with chlorambucil, fludarabine, 4HC, or ibrutinib with or without coadministration of AZD6738 (1 mM). Viability was assessed after 96 hours by propidium iodide exclusion of the proliferating cell population as identified by reduction in CFSE fluorescence intensity. Surviving fraction is expressed relative to untreated controls for chemotherapy treatment alone (no AZD6738) and relative to 1 mM AZD6738 monotherapy for the cotreated samples. Addition of AZD6738 significantly enhanced sensitivity of ATM-defective primary CLL samples to chlorambucil and 4HC, and TP53-defective primary CLL samples to these therapies and also to fludarabine and ibrutinib at $1 dose combination. Data are displayed as mean 6 SEM. Statistical significance was determined using 2-way ANOVA with Bonferroni post hoc analysis. Statistical significance vs no AZD6738 is indicated by *P , .05, **P , .01, and ***P , .001. (C-D) AZD6738 is synergistic with chlorambucil, fludarabine, 4HC, and ibrutinib in primary CLL samples with ATM (C) or TP53 (D) defect across a



KWOK et al

Table 2. Combination indices of AZD6738 (1 or 3 mM) with cytotoxic chemotherapy or BCR signaling inhibitor in ATM- or TP53-defective primary CLL cells cocultured with CD40L/IL-21 ATM-defective CLL (n 5 3) Drug combined with AZD6738 and dose (mM)

AZD6738 dose (mM)

Fraction affected


4

1

0.37

16

1

0.45

16

3

1 4 16

TP53-defective CLL (n 5 3)

Synergism*

Fraction affected


Synergism*

0.30 6 0.05

1111

0.27

0.47 6 0.20

111

0.34 6 0.04

111

0.35

0.38 6 0.17

111

0.50

0.78 6 0.29

11

0.59

0.34 6 0.08

111

1

0.22

0.83 6 0.29

11

0.18

0.81 6 0.24

11

1

0.29

0.77 6 0.27

11

0.27

0.56 6 0.16

111

1

0.44

0.71 6 0.21

11

0.49

0.37 6 0.05

111

1

1

0.39

0.32 6 0.11

111

0.29

0.36 6 0.11

111

4

1

0.49

0.33 6 0.10

111

0.40

0.34 6 0.13

111

4

3

0.60

0.12 6 0.02

1111

0.71

0.22 6 0.07

1111

1

1

0.37

0.36 6 0.00

111

0.20

0.84 6 0.27

11

4

1

0.47

0.46 6 0.04

111

0.27

0.83 6 0.20

11

16

1

0.60

1.00 6 0.13

6

0.44

1.05 6 0.17

6

Chlorambucil

Fludarabine

4-Hydroperoxycyclophosphamide

Ibrutinib

*1111, strong synergism; 111, synergism; 11, moderate synergism; 1, slight synergism; 6, additive.

and could provide an additional mechanism accounting for their sensitivity to ATR inhibition.52 Further work is required to explore these hypotheses and identify markers of sensitivity to ATR inhibition in CLL other than TP53 or ATM defects. With respect to TP53 or ATM defects, the specificity of ATR inhibition for these lesions could allow alteration of the subclonal landscape in favor of less genomically unstable DDR-proficient subclones, which are less susceptible to clonal evolution, thus reducing the likelihood of therapeutic resistance or disease relapse. The specificity of ATR inhibition for TP53-defective CLL cells may possibly also reduce the likelihood of Richter transformation because TP53 defects have been implicated in this process.53,54 Inhibitors of ATR such as AZD6738 could therefore potentially augment current therapies for TP53- or ATM-defective CLL. This is likely to be attributable to potentiation by chemotherapeutic agents of AZD6738-induced replication stress to which TP53- or ATM-defective cells are distinctively susceptible. In CLL, deep remissions attained with chemotherapy or chemoimmunotherapy are translatable into longterm treatment-free survival.55,56 Hence, combination of ATR inhibitor with cytotoxic chemotherapy could provide a realistic salvage option for TP53- or ATM-defective patients relapsing from signaling inhibitors. The synergism of AZD6738 with chlorambucil and bendamustine is particularly attractive given their milder toxicity profiles, making these combinations potentially suitable for older or frailer CLL patients. Although the in vivo toxicity profile and long-term effects of AZD6738 remain to be established in a clinical setting, it is important to note the lack of detrimental effect on normal tissues reported in mice subjected to ATR suppression to 10% of its normal levels.57 Corroborating this report, the AZD6738 dose used in our study to achieve effective tumor load reduction in TP53/ATM-defective xenografts was well tolerated. In addition, the discrepancy between sensitivity of TP53/ATM-defective CLL cells and healthy donor

PBMCs to AZD6738 argues for the existence of a substantial therapeutic window making it suitable for clinical use. A potential caveat is that the prolonged use of the ATR inhibitor could allow accumulation of postreplicative damage in nontumor cells as well as ATM/p53 wild-type CLL cells.58 Hence, our current data support the use of ATR inhibition specifically in CLL patients with TP53 or ATM defects, where there is distinct reliance on ATR and sensitivity to ATR inhibition. Unexpectedly, we found, depending on the dose, additivity or synergism between AZD6738 and ibrutinib in DDR-defective primary CLL cells. The underlying mechanism of the potentiating interaction at higher dose combinations may involve off-target effects of AZD6738 and ibrutinib. At clinically relevant doses (with #1 mM ibrutinib), however, this is likely to be accounted for by the limited overlap in the cellular populations that are targeted by these 2 compounds. Should AZD6738 be used in combination with BCR signaling inhibitors, consideration needs to be given to the sequence of treatment because signaling inhibitors, which evict CLL cells from proliferation centers, may render them quiescent and no longer sensitive to ATR inhibition.19 In patients with TP53/ATM-defective CLL, we envisage daily administration of AZD6738 until the attainment of maximum response. Given the relatively high CLL proliferation rate associated with clinically aggressive, relapsed/refractory disease,59 and the potent cytotoxic effect of AZD6738 against proliferating TP53/ATM-defective CLL cells, we anticipate that this could be profound and achievable over weeks to months. Combination with chemotherapy or other targeted therapies would allow simultaneous targeting of both the proliferating and nonproliferating populations, and both DDR proficient and deficient subclones. When combined with ibrutinib, AZD6738 should be initiated first, with ibrutinib added subsequently and dual therapy continued until maximum response is attained.

Figure 6 (continued) range of effective drug doses. CI values were calculated using the median-effect method. Each point represents the mean CI value of 3 samples plotted against the corresponding mean affected fraction that is expressed relative to untreated controls. CI ,0.9 represents synergism, CI 5 0.9-1.1 represents additive effect, and CI .1.1 represents antagonism. The actual values are presented in Table 2. (E-F) A primary CLL xenograft (CLL25) with a biallelic ATM defect (del(11q) and 1407I.T ATM mutation) was randomized into 4 treatment arms (n 5 5 each): AZD6738, chlorambucil, AZD6738-chlorambucil cotreatment, and vehicle. AZD6738 treatment alone or in combination with chlorambucil significantly reduced tumor load relative to vehicle, and the addition of AZD6738 to chlorambucil led to a significantly greater reduction in tumor load relative to chlorambucil monotherapy. The relative number of CLL cells in panel F was normalized to vehicle-treated controls. Data are displayed as mean 6 SEM. Statistical significance was determined using 2-way ANOVA with Bonferroni post hoc analysis and is indicated by *P , .05, **P , .01, and ***P , .001.



593

Figure 7. A model for synthetic lethality in CLL cells with ATM or p53 deficiency by inhibition of ATR. ATM and ATR are master regulators of DDR, with ATM being activated in response to DNA double-strand breaks, and ATR in response to replication stress. (A) Activation of the ATR pathway leads to cell cycle arrest mediated primarily through the G2/M checkpoint and repair of stalled replication forks. This leads to the resolution of replication stress. (B-C) Inhibition of ATR by AZD6738 directly induces replication stress by slowing and stalling replication forks. The inability of CLL cells to resolve stalled forks as a result of suppressed ATR signaling leads to collapse of stalled replication forks into fragmented, partially replicated sister chromatids with free DNA DSEs that necessitate repair through the ATM/p53 pathway. This involves cell cycle arrest mediated primarily through the G1/S checkpoint and HRR. (D) In cells with defective ATM or p53, inhibition of ATR by AZD6738 results in an intolerable accumulation of unrepaired DNA damage. This arises from impaired HRR because of defective ATM and/or impaired cell cycle regulation resulting from combined loss of functional ATR and ATM/p53. PCNA, proliferating cell nuclear antigen; Pole, DNA polymerase e.

Finally, TP53 and ATM defects are poor prognostic markers in other hematologic malignancies including mantle cell lymphoma,60 T-prolymphocytic leukemia,61,62 acute myeloid leukemia,63,64 myelodysplastic syndrome,65 multiple myeloma,66,67 and diffuse large B-cell lymphoma.68 Our work on CLL provides a model of how ATR

inhibition could selectively target TP53- or ATM-defective cells, and its use in these malignancies could be explored in future studies. In conclusion, ATR inhibition allows selective targeting of genomically unstable DDR-defective CLL cells, therefore potentially helping to avert clonal evolution, a major cause of treatment



KWOK et al

refractoriness and disease relapse. The ATR kinase inhibitor AZD6738 is currently being trialed for refractory solid tumors (registered at www. clinicaltrials.gov as #NCT02223923). Our preclinical data provide a basis for similar investigations for refractory TP53/ATM-defective CLL.

Acknowledgments The authors thank Drs Elaine Willmore, Andy Turnell, and Roger Grand for helpful discussions. This work was supported by a Leukaemia & Lymphoma Research Programme Grant (11045) (T.S.) and Clinical Research Training Fellowship (13059) (M.K.).

Authorship Contribution: M.K., N.D., and T.S. designed the study; M.K., N.D., A.A., E.S., and E.P. performed experiments; J.B. and A.L. provided AZD6738; G.P., H.P., and P.M. contributed clinical samples; M.K. wrote the manuscript; and M.K., T.S., P.H., G.S., E.P., C.O., and M.T. revised the manuscript. Conflict-of-interest disclosure: J.B. and A.L. are employees of AstraZeneca Pharmaceuticals. The remaining authors declare no competing financial interests. Correspondence: Tatjana Stankovic, School of Cancer Sciences, University of Birmingham, Institute for Biomedical Research, Vincent Drive, Edgbaston, Birmingham B15 2TT, United Kingdom; e-mail: [email protected].

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2016 127: 582-595 doi:10.1182/blood-2015-05-644872 originally published online November 12, 2015

ATR inhibition induces synthetic lethality and overcomes chemoresistance in TP53- or ATM-defective chronic lymphocytic leukemia cells Marwan Kwok, Nicholas Davies, Angelo Agathanggelou, Edward Smith, Ceri Oldreive, Eva Petermann, Grant Stewart, Jeff Brown, Alan Lau, Guy Pratt, Helen Parry, Malcolm Taylor, Paul Moss, Peter Hillmen and Tatjana Stankovic

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ATR inhibition induces synthetic lethality and overcomes chemoresistance in TP53 or ATM defective chronic lymphocytic leukemia cells

Data Supplement

1

Supplemental Materials & Methods

CII and Mec1 CLL cell lines

CII and Mec1 are CLL cell lines obtained from Prof. A. Rosen, Linkoping University, Sweden.

The CII cell line was derived from a 47 year old female with CLL who presented with lymphadenopathy, hepatosplenomegaly, a white cell count of 152000/mm3 with 88% lymphocytes, and increased lymphocytes in the bone marrow. This cell line was established by infecting mononuclear cells with the B95-8 strain of Epstein-Barr virus. The CII cell line shows expression of mature B cell markers (CD5+, CD19+, CD20+ and CD23+) with immunoglobulin production restricted to the IgM lambda subtype, similar to the patient CLL clone. Moreover, the cell line is characterized by unmutated IGHV-69/IGHD3-10/IGHJ6 and IGLV1-4/IGLJ3 gene rearrangements showing HCDR3 sequence homology with stereotyped CLL subset-5. The CII cell line shows low CD38 but high ZAP-70 and TCL1 expression levels, and exhibits trisomy 12, which was also present in the in vivo CLL clone.1-3

The Mec1 cell line was derived from a 58 year old male with CLL in prolymphocytoid transformation, clinically characterized by constitutional symptoms, lymphadenopathy and splenomegaly. The Mec1 cell line was established by spontaneous outgrowth from peripheral blood lymphocytes, taken prior to treatment at a time when the white cell count was 39 x 103/ml. The Mec1 cell line displays mature B-cell markers (CD19+, CD20+ and CD23+) with mutated IGHV4-59/IGHD2-21/IGHJ4 rearrangement identical to that of the patient’s CLL clone. Mec1 cells lack CD5 and express low levels of CD38 and TCL1, with absent ZAP70 expression. Mec1 cells possess a deleterious TP53 mutation, which results in a truncation at the N-terminus, producing a lower molecular weight protein (approx. 47 kDa compared to wild-type p53 which is 53 kDa) that is non-functional and cannot be induced by DNA damaging agents such as fludarabine.3-5 We have independently confirmed 2

by TP53 sequencing the presence of c.950insC mutation with absent wild type allele in Mec1 cells.

shRNA transfections in CII cells

shRNA transfections in CII cells were carried out using RNA oligonucleotide (Dharmacon, Lafayette, CO) targeting either green fluorescent protein (GFP) as a negative control (CIIGFPsh), or ATM (CII-ATMsh) as described previously.6,7 In brief, stable knockdown of ATM was achieved using two siRNA sequences corresponding to positions 912 and 8538 respectively of the ATM transcript. The sequences were cloned into a retroviral vector, and transfected CII cells were selected using 1 μg/ml puromycin.

shRNA transfections in Mec1 cells

Mec1 cells were transfected by electroporation (Amaxa system, Lonza Biologics, Berkshire, UK) with the vector pcDNA3.1 (kind gift from Dr. A. Turnell, University of Birmingham, UK) containing either wild type TP53, or GFP as control. Forty-eight hours after transfection, the culture media was supplemented with Geneticin (Life Technologies, Paisley, UK) to select for the transfected cells.

Primary CLL samples

A list of primary CLL samples used and their characteristics is provided in Table S1. The methodology for determination of DNA damage response (DDR) in each sample is described in Figure S1. CLL and healthy donor PBMCs were isolated from peripheral blood using Lymphoprep (Stemcell Technologies, Cambridge, UK) gradient centrifugation.

3

Cell culture and induction of CLL cell proliferation

Cells were cultured in vitro in RPMI-1640 medium (Sigma, Poole, UK) supplemented with 10% fetal bovine serum (FBS; Sigma).

To induce proliferation, primary CLL cells (Table S1) labeled with 0.5 μM carboxyflourescein succinindyl ester (CFSE; Life Technologies) were co-cultured for 4 days with irradiated CD40 ligand-expressing murine embryonic fibroblasts (CD40L) in the presence of IL-21 (25 ng/ml) as previously described.8 Proliferation was confirmed using flow cytometry which demonstrates successive reduction in CFSE fluorescence intensity with each cell division (Figure S2).9

Drugs and inhibitors

AZD6738, hydroxyurea (HU; Sigma), chlorambucil (Sigma), fludarabine (Teva, Castleford, UK), bendamustine (Sigma) and ibrutinib (Seleckchem, Suffolk, UK) were dissolved in DMSO and used at the specified concentration. In vitro studies involving cyclophosphamide were conducted using its metabolized form, 4-hydroperoxycyclophosphamide (4HC; Niomech, Bielefeld, Germany). Pharmacological inhibition of ATM, DNA protein kinase (DNA-PK) and caspases was carried out respectively using the ATM inhibitor KU-55933 (ATMi; Calbiochem, Watford, UK), the DNA-PK inhibitor NU7441 (Seleckchem) and Z-VADFMK (Enzo, Exeter, UK).

The ATR inhibitor AZD6738

AZ20 is a sulfoximine morpholinopyrimidine with potent and selective activity against the ATR kinase. It has been generated through an AstraZeneca lead compound discovery and optimization program, which involved screening a compound library with structural similarity 4

to phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol 3-kinase-related kinase (PIKK) inhibitors, and modification of the identified lead compound. Detailed information regarding the synthesis of AZ20 is provided by Foote and colleagues.10 AZD6738 is a newly synthesized analog of AZ20, with improved pharmacodynamic and pharmakokinetic properties, and is suitable for oral administration. Its discovery is referenced in Jones et al and in recent review articles.11-13

The ATM inhibitor KU-55933

The structure and chemistry of KU-55933 is detailed in Hickson et al.14 We tested the effect of 10 μM KU-55933 over time in both primary CLL samples co-cultured with CD40L/IL-21 (n=3) and CII CLL cells, measuring viability after 24 and 48 hours. In primary CLL cells, the surviving fraction was 95.4% (±1.6%) relative to untreated cells following exposure to KU55933 for 24 hours, and 88.9% (±4.1%) at 48 hours. In CII CLL cells, this was 99.8% at 24 hours and 93.2% at 48 hours. Therefore, when used at the conditions specified in Figures 1C and 4, the ATMi KU-55933 is unlikely to have substantial impact on the viability of CLL cells.

Antibodies used for Western blotting

Western blotting was carried out with primary antibodies against ATR (Santa Cruz, Heidelberg, Germany), ATM (Sigma), phospo-ATM (R&D Systems, Abingdon, UK), Chk1 (Santa Cruz), Chk2 (Santa Cruz), phospho-Chk1 (Cell Signaling, Hitchin, UK), phosphoChk2 (Cell Signaling), phospho-SMC1 (Bethyl, Cambridge, UK), phospho-p53 (Cell Signaling), p53 (Dr. R. Grand, University of Birmingham, UK), p21 (Santa Cruz), PARP (Cell Signaling), caspase 7 (Cell Signaling), cyclin A (Thermo Scientific, Loughborough, UK), DNA-PK (Cell Signaling), phospho-DNA-PK (Cell Signaling), Erk (Cell Signaling), phospho-

5

Erk (Cell Signaling), Syk (Cell Signaling) or phospho-Syk (Cell Signaling). Anti-actin (Sigma) or anti-SMC1 antibodies (Bethyl) were used as the loading control.

Analysis of cytotoxicity and calculation of drug combination indices

CFSE-labeled primary CLL cells were plated onto 96-well plates at 105 cells per well, cocultured with CD40L/IL-21 to induce proliferation and treated with escalating drug doses in triplicates. Viability was ascertained by flow cytometric measurement of propidium iodide (PI; Sigma) uptake using Accuri C6 flow cytometer (BD Biosciences, Oxford, UK) on the proliferating cell population. Cytotoxicity was assessed on cell lines (plated at 104 cells per well) using CellTiter-Glo luminescence cell viability assay (Promega, Southampton, UK) according to the manufacturer’s instructions. Drug doses which would provide 50% cell killing (EC50) and drug combination indices (CI) were calculated by the median effect method using Calcusyn (Biosoft, Cambridge, UK).

Generation of primary CLL xenografts

Primary CLL xenografts were generated by injecting 2-5×107 CLL cells intravenously into NOD/Shi-scid/IL-2Rγnull (NOG) mice, alongside 105 autologous T lymphocytes which were stimulated for 3-7 days prior to inoculation into mice with CD3/CD28 Dynal beads (Life Technologies) in the presence of human IL-2 (30 units/ml; Peprotec, London, UK).15 Engraftment was confirmed by the presence of ≥1% human CD45+ CD19+ cells in peripheral blood. Animals were treated with AZD6738 (50 mg/kg orally 5 days/week for 2 weeks) and/or chlorambucil (16 mg/kg on day 1, and 5 mg/kg on day 3, intraperitoneally), or vehicle, and culled 3 weeks following the initiation of treatment (Figure S3A).

Assessment of tumor load and cytogenetics

6

Tumor load was assessed by flow cytometric quantitation of human CD45+ CD19+ cells in infiltrated spleens (Figure S3B-C). Fluorescence in situ hybridization (FISH) was carried out on human CD45+ CD19+ cells using 11q FISH probes (Abbott Molecular, Maidenhead, UK) according to a standard protocol.

Cell cycle analysis

Cell cycle analysis was performed using flow cytometry on cells fixed in 100% ethanol, treated with RNAse A (Life Technologies, Paisley, UK) and stained with PI (Sigma) as previously described.16

Foci labeling

Cells were fixed with 4% paraformaldehyde (Sigma) onto poly-L lysine-coated slides, and stained with anti-γH2AX (Millipore), anti-53BP1 (Santa Cruz), anti-phosphohistone H3 ser-10 (pH3; Cell Signaling) and/or anti-lamin B (Santa Cruz) antibody prior to examination using immunofluorescence microscopy.

DNA fiber analysis

DNA fiber analysis was carried out by pulse-labeling cells with thymidine analogs 25 μM 5chloro-2’-deoxyuridine (CidU; Sigma) and 250 μM 5-iodo-2’-deoxyuridine (IdU; Sigma) as previously described.17,18 Foci and DNA replication tracts were examined using a Nikon E600 Eclipse microscope (Kingston upon Thames, UK) and analyzed using the Volocity software (Perkin Elmer, Coventry, UK). Replication rates in kb/min were obtained by measuring DNA fiber length in microscopy images. Fiber length in μm was converted into kb using the factor 2.59 kb/μm as determined by spreading and staining viral genomes of a defined length.17 7

Supplemental References

1. Fialkow PJ, Najfeld V, Reddy AL, Singer J, Steinmann L. Chronic lymphocytic leukaemia: Clonal origin in a committed B-lymphocyte progenitor. Lancet. 1978;2(8087):444446. 2. Karande A, Fialkow PJ, Nilsson K, et al. Establishment of a lymphoid cell line from leukemic cells of a patient with chronic lymphocytic leukemia. Int J Cancer. 1980;26(5):551556. 3. Lanemo Myhrinder A, Hellqvist E, Bergh AC, et al. Molecular characterization of neoplastic and normal "sister" lymphoblastoid B-cell lines from chronic lymphocytic leukemia. Leuk Lymphoma. 2013;54(8):1769-1779. 4. Stacchini A, Aragno M, Vallario A, et al. MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation. Leuk Res. 1999;23(2):127-136. 5. Almazi JG, Mactier S, Best OG, Crossett B, Mulligan SP, Christopherson RI. Fludarabine nucleoside induces accumulations of p53, p63 and p73 in the nuclei of human B-lymphoid cell lines, with cytosolic and mitochondrial increases in p53. Proteomics Clin Appl. 2012;6(5-6):279-290. 6. Weston VJ, Oldreive CE, Skowronska A, et al. The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo. Blood. 2010;116(22):4578-4587. 7. Biton S, Dar I, Mittelman L, Pereg Y, Barzilai A, Shiloh Y. Nuclear ataxiatelangiectasia mutated (ATM) mediates the cellular response to DNA double strand breaks in human neuron-like cells. J Biol Chem. 2006;281(25):17482-17491. 8. Pascutti MF, Jak M, Tromp JM, et al. IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells. Blood. 2013;122(17):30103019. 9. Bagnara D, Kaufman MS, Calissano C, et al. A novel adoptive transfer model of chronic lymphocytic leukemia suggests a key role for T lymphocytes in the disease. Blood. 2011;117(20):5463-5472. 10. Foote KM, Blades K, Cronin A, et al. Discovery of 4-{4-[(3R)-3-Methylmorpholin-4-yl]6-[1-(methylsulfonyl)cyclopropyl]pyrimidin-2-y l}-1H-indole (AZ20): a potent and selective inhibitor of ATR protein kinase with monotherapy in vivo antitumor activity. J Med Chem. 2013;56(5):2125-2138. 11. Jones CD, Blades K, Foote KM, et al. Discovery of AZD6738, a potent and selective inhibitor with the potential to test the clinical efficacy of ATR kinase inhibition in cancer patients. Cancer Res. 2013;73:Abstract 2348. 12. Weber AM, Ryan AJ. ATM and ATR as therapeutic targets in cancer. Pharmacol Ther. 2015;149:124-138. 13. Foote KM, Lau A, Nissink JW. Drugging ATR: progress in the development of specific inhibitors for the treatment of cancer. Future Med Chem. 2015;7(7):873-891. 8

14. Hickson I, Zhao Y, Richardson CJ, et al. Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM. Cancer Res. 2004;64(24):9152-9159. 15. Patten PE, Chen S-S, Bagnara D, et al. Engraftment of CLL-derived T cells in NSG mice is feasible, can support CLL cell proliferation, and eliminates the need for third party antigen presenting cells [abstract]. Blood. 2011;118(21):Abstract 975. 16. Pozarowski P, Darzynkiewicz Z. Analysis of cell cycle by flow cytometry. Methods Mol Biol. 2004;281:301-311. 17. Jackson DA, Pombo A. Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. J Cell Biol. 1998;140(6):1285-1295. 18. Petermann E, Woodcock M, Helleday T. Chk1 promotes replication fork progression by controlling replication initiation. Proc Natl Acad Sci U S A. 2010;107(37):16090-16095.

9

Table S1. Clinical and biological characteristics of primary CLL samples

Number Flud of prior refractory therapies

DNA % cells with Other known AZD6738 ATM/TP53 damage mutations EC50 defect response*

IGHV mut status

Cytogenetics

ATM mutation

TP53 mutation

No

nk

Normal

No

No

-

nk

No

66.8

Figure 1, 2C

2

Yes

U

Normal

No

No

-

Normal

NOTCH1

38.9

Figure 1, 2C, S6

B

1

Yes

M

Normal

No

No

-

Normal

No

-

Figure 1, 3

F

A

0

No

M

Normal

No

No

-

Normal

No

9.3

Figure 1, 2C

70

M

A

0

No

nk

Normal

No

No

-

nk

No

19.6

Figure 2C

CLL06

67

M

A

2

No

U

Normal

No

No

-

Normal

No

21.2

Figure 1, 2C, 3, S6

CLL07

72

F

C

1

No

M

del13q, trisomy12

No

No

-

Normal

No

25.6

Figure 1, 2C, 3, S6

CLL08

80

M

C

1

No

nk

del13q

No

No

-

Normal

No

67.4

Figure 2C

CLL09

57

F

A

0

No

M

Normal

No

No

-

Normal

No

40.2

Figure 2C

CLL10

66

F

A

0

No

M

del13q

No

No

-

Normal

No

26.8

Figure 2C

CLL11

70

M

B

2

No

M

Normal

No

No

-

Normal

No

45.0

Figure 2C

CLL12

61

M

A

2

No

nk

Normal

No

No

-

nk

No

29.4

Figure 2C

CLL13

51

F

A

0

No

M

Normal

No

No

-

Normal

No

33.2

Figure 2C

CLL14

65

M

A

0

No

U

del13q

No

No

-

Normal

No

76.0

Figure 2C

CLL15

69

F

C

1

No

M

Normal

No

No

-

Normal

No

44.1

Figure 2C

CLL16

35

F

A

1

No

nk

trisomy12

No

No

-

nk

No

22.9

Figure 2C

CLL17

77

F

A

4

No

M

Normal

No

No

-

nk

No

52.2

Figure 2C

CLL18

55

F

A

1

No

M

Normal

No

No

-

Normal

No

32.9

Figure 2C

CLL19

70

M

A

1

No

U

del11q, trisomy12

c.5224G>C, p.A1724P

No

nk

Defective

No

7.1

Figure 2C

CLL20

70

M

A

1

No

nk

del11q

c.2282delCT, p.761fs

No

nk

Defective

No

9.2

Figure 2C

CLL21

79

M

A

0

No

U

del11q

c.2466+2T>G, splicing site

No

nk

Defective

No

18.5

Figure 1, 2C

CLL22

88

M

A

0

No

M

del11q

c.222T>A, p.C74X

No

41.7

Defective

No

5.8

Figure 1, 2C, 6A

CLL23

61

F

A

1

No

M

del13q

c.1229T>C, p.V410A

No

48.0

Defective

No

5.1

Figure 1, 2C, 6A, S6

ID

Age

Sex

Binet stage

CLL01

66

M

A

0

CLL02

73

M

C

CLL03

74

F

CLL04

58

CLL05

Sample used in

10

ID

Age

Sex

Binet stage

CLL24

61

M

A

Number Flud of prior refractory therapies 1

No

IGHV mut status

Cytogenetics

ATM mutation

M

Normal

c.6067G>A, p.G2023R c.4220T>C, p.I1407T

No

c.9038T>A, p.L3013Q

CLL25

78

F

C

2

No

nk

del11q, del6q, trisomy12

CLL26

74

M

B

1

No

U

del11q

TP53 mutation No

c.4220T>C, p.I1407T c.6966C>G, p.S2322R

DNA % cells with Other known AZD6738 ATM/TP53 damage mutations EC50 defect response* 56.4

Sample used in

Defective

No

6.3

Figure 2C, 6A

nk

Defective

BIRC3, NOTCH1

-

Figure 2F, 6E

No

65.0

Defective

No

-

Figure 2F

No

76.0

Defective

BIRC3

-

Figure S6

No

46.6

Defective

No

-

Figure S6

CLL27

65

F

A

3

No

U

del11q, trisomy12

CLL28

80

M

A

0

No

M

del11q, trisomy12

CLL29

56

M

C

2

Yes

U

del17p, trisomy12

No

c.752T>G, p.I251S

27.2

Defective

BIRC3

16.0

Figure 2C, 2F, 6B, S6

CLL30

55

F

C

4

Yes

U

del17p

c.968T>A, p.I323K

No

30.1

Defective

No

15.7

Figure 2E only

CLL31

78

F

C

2

Yes

M

del17p, del13q, trisomy12

No

c.711G>A, p.M237I

98.2

Defective

No

2.8

Figure 2C, 2F, 6B

CLL32

65

M

A

1

Yes

M

del17p, del13q

No

c.377A>G, p.Y126C

75.3

Defective

No

2.9

Figure 1, 2C, 3, 6B

CLL33

77

M

A

1

No

M

del17p

No

No

50.8

nk

SF3B1

-

Figure 1

54.7

Defective

No

5.6

Figure 2C, S6

90.3

Defective

No

15.7

Figure 2C, 3, S6

53.5

Defective

SF3B1

7.0

Figure 2C, 3, S6

CLL34

74

F

C

2

No

U

Normal

No

CLL35

86

F

B

2

No

nk

Normal

No

CLL36

60

M

B

3

No

M

del17p, del13q

No

c.849_850insC, p.T284fs c.626_627del, p.209_209del c.743G>A, p.R248Q

†

ID, sample identifier; Flud refractory, fludarabine refractory; IGHV mut status, IGHV mutational status; U, unmutated; M, mutated; nk, not known * DNA damage response (ATM and p53 function) was assessed using methodology specified in Figure S1. †

This sample was originally used in the experiment presented in Figure 2C-D but subsequently was excluded from analysis, as it contained both ATM and TP53 defects.

11

Table S2. Combination indices of AZD6738 (1 µM) with cytotoxic chemotherapy in CIIGFPsh cells

Drug combined with AZD6738

Chlorambucil

Fludarabine

4-hydroperoxycyclophosphamide

Bendamustine

Dose, µM

Fraction Affected

Combination Index (mean ± SEM)

Synergism

1 2.5 5 10 1 2.5 5 10 0.25 0.5 1 2 10 25 50 100

0.33 0.53 0.70 0.85 0.20 0.28 0.39 0.64 0.29 0.37 0.53 0.83 0.26 0.32 0.42 0.60

0.74 ± 0.42 0.46 ± 0.14 0.40 ± 0.03 0.34 ± 0.03 0.98 ± 0.24 1.10 ± 0.27 1.29 ± 0.36 1.09 ± 0.33 0.67 ± 0.10 0.78 ± 0.10 0.92 ± 0.22 0.79 ± 0.04 0.41 ± 0.09 0.45 ± 0.10 0.45 ± 0.10 0.36 ± 0.04

++ +++ +++ +++ ± ± −− ± +++ ++ ± ++ +++ +++ +++ +++

−−, moderate antagonism. All other abbreviations are explained in Table 1.

12

Figure S1

A

p53 p21 Actin Lanes:

1

2

3

4

5

6

7

8

9

10

11

12

B

Determination of DNA damage response (DDR) in primary CLL samples. Two strategies were used to assess DDR in primary CLL samples and the impact of the identified mutations in the ATM or TP53 gene. (A) Cellular lysates were obtained from CLL cells before and 8 hours following in vitro treatment with 5 Gy IR. Subsequent assessment of the levels of p53 and its target p21 allowed identification of defective p53 function in CLL29 and defective ATM function in CLL26. Both samples are characterized by the absence of IR-induced p53 and p21 levels, but in the p53 mutant CLL29 this is coupled with a high p53 basal level. (B) Analysis of IR-induced ATM targets (phosphorylated ATM/p53 and SMC1) 45 minutes after 5 Gy IR allows detection of normal ATM function in CLL07, CLL11, CLL15 and CLL09 and defective p53 function in CLL34 where p53 cannot be phosphorylated, despite normal SMC1 phosphorylation that is suggestive of functional ATM.

13

Figure S2

>7 7 6 5 4 3 2

1 0

>6 6 5 4 3 2 1

0

CD19 +ve

CD19 -ve

Induction of CLL cell proliferation by the CD40L/IL-21 co-culture system. CFSE (0.5 M) labeled CLL cells were co-cultured for 4 days with irradiated CD40 ligand-expressing murine embryonic fibroblasts (CD40L) at a 10:1 ratio in RPMI media containing 10% FBS and IL-21 (25 ng/ml). Thereafter, CLL cells were dislodged from the adherent fibroblasts and subjected to flow cytometric analysis. (A) Induction of proliferation is evidenced from the pattern of CFSE labeling. The rightmost peak represents non-cycling CLL cells. Each cell division is accompanied by reduction in CFSE fluorescence intensity indicated by a shift of the peaks to its left. Each peak represents cells that have undergone a particular number of cell divisions, indicated on the top of the histogram (in red). (B) Co-labeling with anti-CD19 antibody demonstrates induction of CLL cell proliferation. The numbers on the top of the flow cytometric plot indicate the number of cell divisions undergone.

14

Figure S3

Primary CLL xenografts enable assessment of the effect of AZD6738 in vivo. (A) Schematic diagram summarizing the methodology. Tumor load was measured by flow cytometric analysis of splenic cells obtained from xenografts at the end of treatment. The strategy for identifying CLL cells is shown in panels B-C. (B) Infiltrated human cells (P2) in murine spleens were identified by positive staining for human CD45 and negative staining for murine CD45. (C) Human CLL cells were selected by further analysis of the human CD45positive, murine CD45-negative gated population for cells positive for CD19 and negative for CD3 (Q4-1).

15

Figure S4

A

Chk1 pSer345 Chk1 Actin Lanes:

1

2

3

4

5

6

7

8

B

DNAPK pSer2056 DNAPK Chk1 Chk1 Actin Lanes:

1

2

3

4

5

6

Complete abolition of Chk1 activity can be achieved with higher doses of AZD6738 and is not dependent on DNA-PK activity. (A) Primary CLL cells were treated with escalating doses of AZD6738 for 1 hour prior to exposure to HU. Complete elimination of HU-induced Chk1 phosphorylation was observed at 3 μM AZD6738 or above (lanes 7-8). (B) Cells were pre-treated with the DNA-PK inhibitor NU7441 and/or AZD6738, or neither, for 1 hour, exposed to HU or IR, and incubated for a further 5 hours before analysis. Treatment with 10 μM NU7441 resulted in >60% reduction of IR-induced DNA-PK phosphorylation (lane 5 vs 6). However, treatment with NU7441, either alone (lane 1 vs 2) or in combination with 1 μM AZD6738 (lane 3 vs 4) did not produce a detectable impact on HU-induced Chk1 phosphorylation in primary CLL cells. 16

Figure S5

A

B

Reintroduction of wild-type TP53 in Mec1 cells. Mec1 cells were transfected with p53pcDNA3.1 and GFP-pcDNA3.1. (A) Fluorescence microscopy of GFP-transfected cells shows that 15-20% transfection efficiency was achieved. (B) Western blotting shows expression of wild type p53 (wt-p53) in p53-pcDNA3.1 transfected Mec1 cells. Irradiated CII cells were loaded as control for wt-p53 and the position of the mutant p53 (mut-p53) expressed by Mec1 cells is indicated. Actin was used for loading control.

17

Figure S6

A

No CD40L/IL-21 (24 hours)

B

No CD40L/IL-21 (96 hours)

The cytotoxic effect of AZD6738 monotherapy is dependent on CLL cell proliferation. Primary CLL samples [n=10, including 3 TP53/ATM-wild type (CLL02, CLL06 and CLL07), 3 ATM-defective (CLL23, CLL27 and CLL28), and 4 TP53-defective samples (CLL29, CLL34, CLL35 and CLL36)] were treated with escalating doses of AZD6738 without co-culture with CD40L/IL-21. Cytotoxicity was assessed using flow cytometric measurement of PI uptake. Surviving fraction was expressed as a percentage of untreated controls. In the absence of cycling induced by CD40L/IL-21, AZD6738 had little impact on the viability of CLL cells, regardless of TP53 or ATM status. 18

Figure S7

The effect of ATR inhibition on the cell cycle profiles of CLL cells with defective ATM or p53. CII-GFPsh, CII-ATMsh and Mec1 cells were analyzed 24 (A) or 48 (B) hours after treatment with either AZD6738 (1 μM or 3 μM) or RPMI media. AZD6738 treatment induced G1/S cell cycle arrest in CII-GFPsh cells but not in CII-ATMsh or Mec1 cells. Representative cell cycle profiles from 3 independent experiments are displayed. The percentages shown represent the proportion of cells within each phase of the cell cycle. 19

Figure S8

A pH3

B

(10 µM)

ATR inhibition results in accumulation of DNA damage in both ATM-deficient and p53defective CLL cells. (A) Co-labeling of CLL cells with γH2AX and pH3 allows identification of both mitotic (pH3-positive) and non-mitotic (pH3-negative) cells that have accumulated DNA damage (γH2AX). Images were captured using a 60x lens. (B) The effect of AZD6738 (1 µM) treatment on the proportion of γH2AX-positive CLL cells, both mitotic and non-mitotic. Statistical significance compared to untreated controls, assessed by two-way ANOVA with Bonferroni post-hoc analysis, is indicated by *P