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TP53 and ataxia telangiectasia mutated (ATM) defects are associated with genomic instability, clonal evolution, and chemoresistance in chronic lymphocytic leukemia (CLL). Currently, therapies capable of providing durable remissions in relapsed/refractory TP53- or ATM-defective CLL are lacking. Ataxia telangiectasia and Rad3-related (ATR) mediates response to replication stress, the absence of which leads to collapse of stalled replication forks into chromatid fragments that require resolution through the ATM/p53 pathway. Here, using AZD6738, a novel ATR kinase inhibitor, we investigated ATR inhibition as a synthetically lethal strategy to target CLL cells with TP53 or ATM defects. Irrespective of TP53 or ATM status, induction of CLL cell proliferation upregulated ATR protein, which then became activated in response to 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 ATM-defective CLL that warrants clinical investigation.

Overexpression of P-glycoprotein (P-gp), the protein product of the multidrug resistance gene (MDR1), confers a drug resistant phenotype on cells. This phenotype is reminiscent of human T-cell leukemia virus (HTLV)-transformed leukemic cells, for which no consistently effective chemotherapeutic regime has been found. The presence of an active multiple drug resistance (MDR) phenotype in freshly isolated peripheral blood mononuclear cells (PBMC) from HTLV-I–infected subjects was investigated. Significant P-gp–mediated efflux activity and enhanced MDR1 mRNA expression was observed in nine of 10 HTLV-infected subjects. The development of MDR phenotypes was found to be independent of disease type or status with significant MDR activities being observed in adult T-cell leukemia (ATL), HTLV-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), and asymptomatic HTLV-infected individuals. P-gp–mediated drug efflux was also found to be restricted to CD3+ T-cell populations. Furthermore, we show the novel finding that theMDR1 gene promoter is transcriptionally activated by the HTLV-I tax protein, suggesting a molecular basis for the development of drug resistance in HTLV-I infections. These observations open up the possibility of new chemotherapeutic approaches to HTLV-associated diseases through the use of P-gp inhibitors.

DNA damage response (DDR) defects, particularly TP53 or biallelic ATM aberrations, are associated with chemoresistance in chronic lymphocytic leukemia (CLL). Chemoimmunotherapy or B-cell receptor signaling inhibitors alone may not be sufficient to overcome adverse prognosis or provide durable response in TP53 or biallelic ATM inactivated CLL. In particular, genomic instability resulting from impaired DDR facilitates rapid clonal evolution leading to treatment refractoriness or disease relapse. Development of therapeutic approaches specifically targeting DDR defects is therefore necessary for effective long-term control of DDR-defective CLL. We previously demonstrated selective cytotoxicity of the ATR inhibitor AZD6738 towards TP53 or ATM null CLL cells, and validated this in CLL xenograft models for biallelic TP53 or ATM loss. Here, we provide mechanistic insight into the synthetically lethal interactions between ATR pathway inhibition and TP53 or ATM loss in CLL, and offer experimental evidence supporting the use of ATR inhibition in combination with conventional chemotherapies and other targeted therapies in CLL. To determine the mechanism of ATR inhibition in CLL, we first investigated its effect on DDR-intact cycling primary cells. We observed compensatory activation of the ATM/p53 pathway in AZD6738-treated cells in response to hydroxyurea, providing evidence for crosstalk between ATR and ATM/p53 pathways. Next, we explored the cellular consequence of ATR inhibition in CLL cells with DDR loss. To determine the impact of ATR inhibition on DNA replication, we performed DNA fiber analysis, which revealed significantly increased replication fork stalling and firing of replication origins upon AZD6738 treatment in ATM/p53-defective CLL. To investigate the effect of ATR inhibition on DNA damage, we measured γH2AX and 53BP1 foci formation, markers of DNA double-strand breaks. Significant induction of γH2AX and 53BP1 foci was seen in ATM/p53-defective CLL cells upon AZD6738 treatment, suggesting that ATR inhibition exacerbates replication stress in ATM/p53 defective cells by imposing requirement for DDR through the ATM/p53 pathway. On the contrary, 53BP1 bodies, a marker of unreplicated DNA, appeared in ATM/p53 proficient but not in ATM/p53 deficient cells following AZD6738 exposure, indicating that cell cycle arrest in response to replication stress is ATM/p53 dependent. This was corroborated by cell cycle profiling and co-labeling experiments of γH2AX with phosphohistone H3 ser10, a marker of mitosis, showing that ATM/p53-defective CLL cells carrying unrepaired DNA damage continued to cycle. Thus, ATR inhibition results in accumulation of intolerable levels of DNA damage in DDR-defective CLL, leading to cell death by mitotic catastrophe, which we have confirmed both in vitro and in AZD6738-treated murine xenograft models. The benefit of ATR inhibition as a therapeutic strategy for DDR-defective CLL lies in its ability to alter the subclonal landscape in favor of less unstable DDR-proficient subclones, which are less susceptible to clonal evolution, thus reducing the likelihood of disease relapse. We reasoned that this provides a strong rationale for addition of AZD6738 to existing therapeutic agents for the treatment of CLL with DDR defects. We demonstrated synergistic and additive effects of low-dose AZD6738 in ATM/p53-defective CLL cells with DNA damaging agents such as chlorambucil, fludarabine, bendamustine and cyclophosphamide, BCR-signaling inhibitors and the PARP inhibitor olaparib. We validated the AZD6738 plus chlorambucil combination in biallelic TP53 or ATM inactivated primary CLL xenografts, where combined AZD6738/chlorambucil treatment was superior to chlorambucil alone, as evidenced by significantly greater reduction in tumor load and percentage of CLL subclones with del(17p) or del(11q) in animals treated with combination regimen compared to single-agent chlorambucil. Similar investigations evaluating AZD6738 plus ibrutinib versus ibrutinib monotherapy in primary CLL xenografts are currently underway. We conclude that ATR inhibition is a suitable approach for targeting the loss of p53 function in aggressive CLL subclones and should be considered as a valuable addition to DNA damaging agents and current targeted treatments. Disclosures Off Label Use: ATR inhibitor AZD6738 targets TP53-null or ATM-null phenotype inducing synthetic lethality. Brown:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Lau:AstraZeneca Pharmaceuticals: Employment.

Ataxia Telangiectasia Mutated (ATM) protein coordinates responses to DNA double strand breaks (DSBs) and the ATM-null status caused by biallelic ATM gene inactivation in chronic lymphocytic leukemia (CLL) results in resistance to p53-dependent apoptosis. Accordingly, alternative strategies to target ATM-null CLL are needed. ATM is a serine/threonine protein kinase that synchronises rapid DNA damage response (DDR) to DNA double strand breaks (DSBs) with activation of cell cycle checkpoints, DNA repair and apoptosis via p53 activation. ATM-null cells are defective in a type of DSB repair that involves homologous recombination and rely on co-operating and compensatory DNA repair pathways for their survival. Therefore, inhibition of DNA repair pathways to which CLL cells with loss of ATM signalling become addicted could provide ‘synthetic lethality’ and induce tumour specific killing. Indeed, we have recently shown that inhibition of a single strand break protein PARP induces differential killing of ATM-null CLL tumours. Here we expand the concept of synthetic lethality in ATM-null CLL and address the question of whether ATM-null deficient CLL cells can be targeted by inhibition of the ATR protein that governs responses to post-replicative damage and co-operates with ATM. First, we addressed the status of the ATR pathway in primary CLL cells and consistent with previous findings we observed that initiation of cell cycling is required for both ATR upregulation and activation of ATR target Chk1 in response to replicating stress inducing agent hydroxyurea. We then proceeded with testing viability of the isogenic CLL cell line CII, with and without stable ATM knock down, in the presence or absence of increasing doses of ATR inhibitor AZD6738. We observed a uniform loss of cellular viability in the presence of 1 or 3 μM of inhibitor in ATM-null cells but not in the ATM-wt counterpart. Similar observation was made in primary CLL cells initiated to cycle in the presence of stimulatory oligonucleotide-ODN2006/IL2 support. To confirm the cytotoxic effect of AZD6738 in vivo we used an ATM null primary CLL xenograft model. Representative primary CLL tumour cells with 15% bialleic ATM inactivation, as assessed by percentage of 11q deletion and allelic frequency of ATM mutation 4220T>C, was engrafted in the presence of activated autologous T lymphocytes into 10 NOG mice. Upon detection of engraftment in peripheral blood, animals were treated by oral administration of either AZD6738 (50mg/kg) or vehicle alone over a 2 week period, and tumour load measured by FACS analysis of CD45+ CD19+ human cells in infiltrated spleens. We observed a reduction in tumour cell numbers in AZD6738-treated compared to vehicle-treated spleens and current investigations are underway to determine whether this difference can be attributed to the selective disappearance of CLL population with biallelic ATM loss. We suggest that targeting ATR pathway provides an attractive approach for selective killing of ATM-null CLL cells and that this approach should be considered as a future therapeutic strategy for this CLL subtype. Disclosures: Off Label Use: ATR inhibitor AZD6738 targets ATM-null phenotype inducing synthetic lethality. Jeff:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Lau:AstraZeneca Pharmaceuticals: Employment.