Aberrant regulation of programmed cell death (PCD) has been tied to an array of human pathologies ranging from cancers to autoimmune disorders to diverse forms of neurodegeneration. Pharmacologic modulation of PCD signalling is therefore of central interest to a number of clinical and biomedical applications. A key component of PCD signalling involves the modulation of pro- and anti-apoptotic Bcl-2 family members. Among these, Bax translocation represents a critical regulatory phase in PCD. In the present study, we have employed a high-content high-throughput screen to identify small molecules which inhibit the cellular process of Bax re-distribution to the mitochondria following commitment of the cell to die. Screening of 6246 Generally Recognized As Safe compounds from four chemical libraries post-induction of cisplatin-mediated PCD resulted in the identification of 18 compounds which significantly reduced levels of Bax translocation. Further examination revealed protective effects via reduction of executioner caspase activity and enhanced mitochondrial function. Consistent with their effects on Bax translocation, these compounds exhibited significant rescue against in vitro and in vivo cisplatin-induced apoptosis. Altogether, our findings identify a new set of clinically useful small molecules PCD inhibitors and highlight the role which cAMP plays in regulating Bax-mediated PCD.

Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair pathway that is of critical importance during the S and G2 phases of the mammalian cell cycle. The limiting step in the HR pathway is the generation of single-stranded DNA (ssDNA) by resection of DNA ends at the DSB site. We have established a high-throughput immunofluorescence-based assay that monitors DNA end resection by plate-based laser scanning cytometry. Employing this assay we screened a genome-scale siRNA library that targeted approximately 18,500 genes. Remarkably, the top hits in our screen were known regulators of resection including CtIP and all three subunits of the MRN complex. I will present ongoing work on one of the hits from the screen, the zinc finger protein ZNF335. The ZNF335 gene was recently found to be mutated in a syndrome that causes some of the worst cases of neonatal microcephaly ever observed. Numerous genome instability syndromes have been documented to display microcephaly as a clinical feature, including Seckel syndrome, which can be caused by mutations in the known resection genes CtIP and DNA2. We first validated the results of the screen by demonstrating that ZNF335 promotes resection and repair by HR. We determined that the four C-terminal zinc finger domains of ZNF335 are necessary and sufficient for its function in resection. Depletion of ZNF335 inhibits phosphorylation of CHK1 at a characterized ATR consensus site, suggesting that ZNF335 is needed for activation of ATR following a DSB. We examined the recruitment of known resection factors to DSB sites and determined that the localization of CtIP and BLM were impaired in cells depleted of ZNF335. Our data suggests that ZNF335 plays an important role in promoting resection and ATR activation in response to DSBs. Citation Format: Jordan Young, Mikhail Bashkurov, Andrea McEwan, Thomas Sun, Alessandro Datti, Daniel Durocher. A genome-scale screen identifies the microcephaly gene, ZNF335, as a regulator of DNA end resection [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B45.

Triple negative breast cancer (TNBC) includes basal-like and claudin-low subtypes for which only chemotherapy and radiation therapy are currently available. The retinoblastoma (RB1) tumor suppressor is frequently lost in human TNBC. Knockdown of RB1 in luminal BC cells was shown to affect response to endocrine, radiation and several antineoplastic drugs. However, the effect of RB1 status on radiation and chemo-sensitivity in TNBC cells and whether RB1 status affects response to divergent or specific treatment are unknown. Using multiple basal-like and claudin-low cell lines, we hereby demonstrate that RB-negative TNBC cell lines are highly sensitive to gamma-irradiation, and moderately more sensitive to doxorubicin and methotrexate compared to RB-positive TNBC cell lines. In contrast, RB1 status did not affect sensitivity of TNBC cells to multiple other drugs including cisplatin (CDDP), 5-fluorouracil, idarubicin, epirubicin, PRIMA-1met, fludarabine and PD-0332991, some of which are used to treat TNBC patients. Moreover, a non-biased screen of ∼3400 compounds, including FDA-approved drugs, revealed similar sensitivity of RB-proficient and -deficient TNBC cells. Finally, ESA+/CD24−/low/CD44+ cancer stem cells from RB-negative TNBC lines were consistently more sensitive to gamma-irradiation than RB-positive lines, whereas the effect of chemotherapy on the cancer stem cell fraction varied irrespective of RB1 expression. Our results suggest that patients carrying RB-deficient TNBCs would benefit from gamma-irradiation as well as doxorubicin and methotrexate therapy, but not necessarily from many other anti-neoplastic drugs.

PurposeMantle-cell lymphoma (MCL) has a variable natural history but is incurable with current therapies. MicroRNAs (miRs) are useful in prognostic assessment of cancer. We determined an miR signature defining aggressiveness in B-cell non-Hodgkin lymphomas (NHL) and assessed whether this signature aids in MCL prognosis.MethodsWe assessed miR expression in a training set of 43 NHL cases. The miR signature was validated in 44 additional cases and examined on a training set of 119 MCL cases from four institutions in Canada. miRs significantly associated with overall survival were examined in an independent cohort of 114 MCL cases to determine association with patient outcome. miR expression was combined with current clinical prognostic factors to develop an enhanced prognostic model in patients with MCL.ResultsFourteen miRs were differentially expressed between aggressive and indolent NHL; 11 of 14 were validated in an independent set of NHL (excluding MCL). miR-127-3p and miR-615-3p were significantly associated with overall survival in the MCL training set. Their expression was validated in an independent MCL patient set. In comparison with Ki-67, expression of these miRs was more significantly associated with overall survival among patients with MCL. miR-127-3p was combined with Ki-67 to create a new prognostic model for MCL. A similar model was created with miR-615-3p and Mantle Cell Lymphoma International Prognostic Index scores.ConclusionEleven miRs are differentially expressed between aggressive and indolent NHL. Two novel miRs were associated with overall survival in MCL and were combined with clinical prognostic models to generate novel prognostic data for patients with MCL.

Triple-negative breast cancer (TNBC) represents an aggressive subtype, for which radiation and chemotherapy are the only options. Here we describe the identification of disulfiram, an FDA-approved drug used to treat alcoholism, as well as the related compound thiram, as the most potent growth inhibitors following high-throughput screens of 3185 compounds against multiple TNBC cell lines. The average IC50 for disulfiram was ~300 nM. Drug affinity responsive target stability (DARTS) analysis identified IQ motif-containing factors IQGAP1 and MYH9 as direct binding targets of disulfiram. Indeed, knockdown of these factors reduced, though did not completely abolish, cell growth. Combination treatment with 4 different drugs commonly used to treat TNBC revealed that disulfiram synergizes most effectively with doxorubicin to inhibit cell growth of TNBC cells. Disulfiram and doxorubicin cooperated to induce cell death as well as cellular senescence, and targeted the ESA+/CD24-/low/CD44+ cancer stem cell population. Our results suggest that disulfiram may be repurposed to treat TNBC in combination with doxorubicin.

Abstract 1586 Background Mantle cell lymphoma (MCL) is a subtype of B-cell non-Hodgkin lymphoma (NHL) characterized by the t(11;14) translocation and concomitant over-expression of cyclin D1. MCL has a variable natural history; while some patients have prolonged survival similar to other indolent B-cell lymphomas, most follow an aggressive course with short survival. While the t(11;14) is pathognomonic of MCL, it is not necessary for disease pathogenesis, as a subset of MCL cases lack the translocation. Furthermore, in vivo models demonstrate that cyclin D1 over-expression alone is unable to bring about the disease, and that deregulation of additional cellular pathways is required for its pathogenesis. Assessment of microRNA (miR) expression in MCL may help determine mechanisms of gene deregulation and reveal pathways involved in disease pathogenesis. In this study we examined MCL in relation to both aggressive and indolent B-cell NHL to determine a miR signature that characterizes MCL. Design and Methods Total RNA from a training set of 36 B-cell NHL cases (19 indolent and 17 aggressive) and 32 MCL was applied to a high-throughput quantitative real-time PCR platform assessing the expression of 365 miRs [TaqMan Human MicroRNA Array v1.0 (Early Access) or TLDA]. miRs that were differentially expressed between MCL and aggressive NHL, and between MCL and indolent NHL were then validated using RNA from a second, independent, set of B-cell NHL cases (28 indolent and 28 aggressive) and 50 MCL cases. Validated miRs were determined and potential targets for each miR were examined. A map of targets common to the MCL miR signature was created, revealing important proteins involved in MCL pathogenesis. Results 66 miRs (11 over-expressed, 55 under-expressed) were differentially expressed between MCL and aggressive B-cell NHL and 8 miRs (7 over-expressed, 1 under-expressed) were differentially expressed between MCL and indolent B-cell NHL (false discovery rate = 0.2). 6 miRs from each group were chosen for validation in an independent set of MCL and NHL cases. Of these 12 miRs, 7 miRs validated (2 were under-expressed in MCL relative to aggressive B-cell NHL, and 5 were over-expressed in MCL relative to indolent B-cell NHL). Genes and pathways involved in disease pathogenesis are most likely targeted by multiple miRs. We thus determined a set of 123 genes predicted to be targets of this MCL miR signature, based on five miR target prediction databases from the mirDIP (microRNA data integration) portal. These genes were significantly enriched for focal adhesion and integrin signalling, proteasome-mediated degradation, and the PI3K signalling pathway. Conclusions Using the largest set of MCL cases evaluated to date, a 7-miR signature characteristic of MCL was discovered. The gene targets of these miRs are enriched for roles in proteasome-mediated protein degradation, consistent with the reported sensitivity of MCL to proteasome inhibitors. In addition, these miRs are predicted to be involved in regulation of PI3K/AKT signalling, confirming reports of the importance of this pathway in MCL pathogenesis. Enrichment of target genes involved in focal adhesion and integrin signalling indicate the importance of MCL-stromal interactions and motivates further study into the role of the tumor microenvironment in MCL pathogenesis. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 61 To identify new therapeutic strategies for AML, we compiled and screened an in-house library of on-patent and off-patent drugs to identify agents cytotoxic to leukemia cells. From this screen, we identified mefloquine, an off-patent drug indicated for the treatment and prophylaxis of malaria. In secondary assays, mefloquine decreased the viability of 9/10 human and murine leukemia cell lines (EC50 3.25–8.0 μM). Moreover, it reduced the viability of 4/5 primary AML samples, but was not cytotoxic to normal hematopoietic cells (EC50>31 μM). Importantly, mefloquine reduced the clonogenic growth of primary AML samples, but not normal hematopoietic cells, and completely inhibited engraftment of primary AML cells into immune deficient mice. Finally, systemic treatment with oral mefloquine (50 mg/kg/day) decreased leukemic burden without evidence of toxicity in 4 mouse models of leukemia, including mice engrafted with primary AML cells. Thus, mefloquine effectively targets leukemic cells, including leukemia stem cells, at concentrations that appear pharmacologically achievable and are not toxic to normal hematopoietic cells. To identify the mechanisms of mefloquine-mediated cell death in AML cells, we performed a binary drug combination screen, hypothesizing that drugs that synergized with mefloquine may share overlapping mechanism of action. From this combination screen of 550 drugs, we identified 18 that reproducibly synergized with mefloquine as measured by the Excess over Bliss additivism score, including 3 members of the artemisinin class of anti-malarials: artemisinin, artesunate and artenimol. Strikingly, 10/18 synergistic compounds, including the artemisinins, were known generators of reactive oxygen species (ROS). Therefore we tested mefloquine's ability to increase ROS in leukemic cells. Mefloquine increased ROS production in leukemia cells in a dose- and time-dependent manner. Co-treatment with ROS scavengers α-tocopherol and N-acetyl-cysteine abrogated mefloquine-induced ROS production and cell death, indicating that ROS production was functionally important for mefloquine-mediated cell death. Moreover, the artemisinins induced ROS as single agents, and synergistically increased ROS when combined with mefloquine. To identify cellular target(s) of mefloquine's anti-leukemic effects, we performed a yeast genome-wide functional screen to identify heterozygous gene deletions that rendered yeast more sensitive to mefloquine. 21/37 genes whose depletion conferred >4-fold sensitivity to mefloquine were associated with function of the yeast vacuole, equivalent to the mammalian lysosome. Consistent with these data, fluorescent confocal microscopy demonstrated that mefloquine and artesunate disrupted lysosomes. Cell death after mefloquine and artesunate treatment was caspase-independent and associated with increased incorporation of monodancylcadaverin in autophagosomes, consistent with the effect of these drugs on the lysosomes. To further explore the anti-leukemic activity of lysosomal disruption, we evaluated the anti-leukemic effects of the known lysosomal disrupter L-leucine-leucine methyl ether (LeuLeuOMe). Similar to mefloquine and artesunate, LeuLeuOMe induced cell death in leukemia cells, increased ROS production, and disrupted the lysosomes. Highlighting the potential clinical utility of lysosomal disrupters for the treatment of leukemia, a patient with relapsed/refractory juvenile myelomonocytic leukemia self-administered artemisinin. The artemisinin cleared the circulating blasts from the circulating blasts and the patient proceeded to allotransplant. Finally, to investigate the basis of leukemic cell hypersensitivity to lysosomal disruption, we assessed lysosomal characteristics of primary AML and normal hematopoietic cells. By gene expression analysis, AML patient samples had higher mRNA levels of the lysosomal cathepsins A, B, C, D, H, L, S and Z, compared to CD34+ normal hematopoietic cells, and cathepsins C, D and Z were significantly over-expressed in the LSC compartment, compared to normal HSCs. In summary, our data demonstrate that lysosomal disruption preferentially targets AML cells and AML stem cells through a mechanism related to increased ROS production. Thus, this work highlights lysosomal disruption as a novel therapeutic strategy for AML. Disclosures: Off Label Use: This study includes a case report of off-label use of the anti-malarial artemisinin in the treatment of a case of juvenile myelomonocytic leukemia.

Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin, a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks.