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The transcription factor IRF4 is essential for the survival of both normal plasma cells and the plasma cell malignancy multiple myeloma (MM). However the basis for this dependency remains uncertain, with the prevailing view that IRF4 loss in MM induces "death by a thousand cuts." Here we have explored the genetic basis for IRF4 addiction through CRISPR-Cas9 mediated deletion of IRF4 in three lenalidomide sensitive MM cells lines (MM1S, OPM2 and H929) and one resistant cell line (RPMI-8226). Loss of IRF4 resulted in marked loss of viability, irrespective of lenalidomide sensitivity, which was driven by two distinct mechanisms: 1) induction of apoptosis, which was rescued through prior deletion of apoptotic mediators BAX and BAK, and 2) proliferative arrest, which persisted despite rescue from apoptosis. RNA-sequencing following IRF4 inactivation identified a core group of 86 genes whose dysregulation was shared across all 4 MM cell lines. We identified 31 common down-regulated genes (IRF4 activated), including several with previously described roles in survival (such as KLF2, TNFRSF17/BCMA and MYB). Furthermore, MYC, a previously identified target of IRF4 known to play an important role in MM pathogenesis and survival, was down-regulated in 3 of the 4 cell lines. Most surprisingly, IRF4 inactivation resulted in 55 common up-regulated genes that included two BH3-only pro-apoptotic proteins, BMF and BCL2L11 (BIM). Up-regulation of BMF appeared more marked than BIM with an average fold change of 6.5x (range 2.7 - 10.3) vs 1.9x (range: 1.8 - 2.2) respectively. Remarkably, genetic ablation of BMF in the OPM2 cell line and BMF/BIM in the MM1S largely protected from the cell death observed following IRF4 inactivation, suggesting that IRF4 maintains MM survival through the transcriptional repression of BMF and BIM. Our results confirm the vital role of IRF4 for MM proliferation and survival, and shed further light into its critical downstream transcriptional targets. Most interestingly, we have demonstrated that the 'killer blow' appears to be the concerted effect of the BH3-only pro-apoptotic proteins BMF and BIM. Disclosures Huang: Genentech: Patents & Royalties: DCSH is an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax.

Multiple myeloma is a malignancy of plasma cells. The abnormal clonal accumulation of plasma cells interferes with the blood cell production, thereby causing cytopenias and aberrant, excessive antibody production. Patients can also experience bone pain, neurological symptoms and renal failure. With the exception of bone marrow transplantation that is associated with significant morbidity and mortality, there is currently no cure for patients with this disease. Thus, novel therapies are desperately needed for treating patients especially those harboring disease with adverse prognostic markers or when they become refractory to current treatment regimens. Recently, we have screened a panel of well-established myeloma cell lines against multiple anti-myeloma agents, including standard-of-care agents such as bortezomib, lenalidomide, dexamethasone or melphalan, as well as novel targeted agents such as the BH3 mimetic compounds (e.g. venetoclax/ABT-199 to target BCL2) or HDAC inhibitors (e.g. panobinostat). Specifically, we sought to determine whether their cytotoxic action depends on the induction of BAX/BAK-mediated apoptosis. When activated, these two proteins drive permeabilization of the outer mitochondrial membrane. In their absence, apoptosis cannot proceed and we could readily establish if BAX/BAK-mediated apoptosis is critical by comparing the sensitivity of isogenic cell lines lacking these two cell death mediators to their wild-type parental counterparts. As anticipated, the action of the BH3 mimetic compounds such as venetoclax/ABT-199 to inhibit BCL2 or small molecule inhibitors to selectively target relatives of BCL2, such as BCLxL and MCL1, rely exclusively on BAX and BAK. In the their absence, the treated cells remained viable. While the action of some drugs such as dexamethasone is already known to be mediated in part by BAX/BAK-driven apoptosis, we were very surprised that lenalidomide, an immunomodulatory drug (IMiD), can also kill some myeloma cell lines through BAX/BAK mediated apoptosis. A more potent relative of lenalidomide, pomalidamide, does likewise. While the cytotoxic action of these IMiDs, most notably the induction of cell cycle arrest upon activation of the E3 ligase cereblon (CRBN) is well known, our data is the first to definitively demonstrate that the IMiDs can actively induce BAX/BAK-mediated apoptosis. Next we explored the mechanisms by which the IMiDs can trigger apoptosis. Most stress signals and many cytotoxic drugs activate the BH3-only proteins such as BIM, BAD, BID or PUMA to initiate apoptosis. We checked whether killing by lenalidomide might also depend on specific BH3-only proteins and found that the genetic deletion of BIM conferred almost complete resistance to treatment with lenalidomide in vitro. We are currently investigating the precise mechanism by which the IMiDs can activate BIM to drive apoptosis. While lenalidomide can trigger BAX/BAK-mediated apoptosis in some myeloma cell lines, many others were spared. We expect that by deciphering the mechanisms by which anti-myeloma agents can induce BAX/BAK-mediated apoptosis of myeloma cell lines, we can probably improve the utility of drugs such as the IMiDs and understand why they might fail in some patients. Disclosures Gong: The Walter and Eliza Hall Institute of Medical Research: Employment. Segal:The Walter and Eliza Hall Institute of Medical Research: Employment. Riffkin:The Walter and Eliza Hall Institute of Medical Research: Employment. van Delft:The Walter and Eliza Hall Institute of Medical Research: Employment. Roberts:Walter and Eliza Hall Institute of Medical Research: Employment. Huang:The Walter and Eliza Hall Institute of Medical Research: Employment.

Multiple myeloma is a clonal B-cell malignancy characterized by complex genetic aberrations. Despite recent advances in our understanding of genetic basis of this disease and the introduction of novel therapies, the outcome for many patients remains poor. For example, 20% of newly diagnosed patients with stage 3 myeloma have disease marked by high lactate dehydrogenase levels or unfavorable cytogenetic abnormalities (e.g. 17p deletion, t(4;14)). Half of these patients die within 2 years of diagnosis in spite of advanced therapies. A promising class of novel agents to treat patients with other B-cell malignancies is the BH3 mimetic compounds, which targets the pro-survival BCL2 proteins. For example, the majority of patients with CLL (chronic lymphocytic leukaemia) respond to venetoclax (ABT-199), a BCL2-selective inhibitor. Myeloma cell lines with t(11;14) translocations are also reported to be sensitive to BCL2 inhibition and venetoclax is in clinic trails for patients with relapsed or refractory multiple myeloma. We are eager to know whether the other pro-survival BCL2 proteins, namely BCLXL, BCLW, MCL1 and BCL2A1 (BFL1) play a role in maintaining the survival of myeloma cells. To determine this, we screened a large panel of immortalized (25) and low-passage (7) myeloma cell lines to killing by BH3 mimetics with varying specificities. We found that a fraction of the myeloma cell lines were rapidly killed when BCL2 (25%, 8/32) or BCLXL (25%, 8/32) were targeted. Interestingly, we also identified a distinct t(4;14) subgroup, as well as the previous recognized t(11;14) subgroup, to be highly sensitive to BCL2 inhibition. Unlike CLL, in which the majority patients respond to venetoclax, BCL2 inhibition is only likely to account for some cases. We then asked which other pro-survival BCL2 protein keeps the other myeloma cell lines alive. A prime candidate is MCL1 since normal plasma cells rely on it and previous studies had implicated MCL1 in myeloma. By targeting the expression of the pro-survival BCL2 proteins using CRISPR/Cas9 genome editing technology, we found that a significant fraction (74%, 14/19) of our cell line panel died rapidly in the absence of MCL1. The importance of MCL1 was confirmed using an orthogonal approach: we found that expression of a MCL1-selective peptidyl ligand BIM2A rapidly killed a sizeable fraction of both immortalized (17/25) and low-passage (5/7) myeloma cell lines. This activity strongly correlated with the genetic targeting of MCL1 by CRISPR/Cas9. Importantly, even cell lines that harbor the poor prognostic t(4;14) chromosomal translocation were readily killed by BIM2A and the cell lines were killed regardless of their TP53 status. Moreover, targeting MCL1 also constrained the growth of myeloma in vivo. Since the inhibition of MCL1, BCLXL or BCL2 can on their own can kill almost all the myeloma cell lines screened, we next identified the molecular mechanisms by which these BH3 mimetics act. Firstly, we tested likely candidates for their role in driving the killing induced by a specific BH3 mimetic, for example whether deleting the BH3-only protein BIM impact upon killing by venetoclax. Secondly, we undertook genome-wide recessive CRISPR/Cas9 screens for genes that are critical for the action of the BH3 mimetics. We have identified a number of hits from such screens and are in the process of validating these. By identifying the unique susceptibility of the myeloma cell lines to the BH3 mimetics and elucidating how they act to kill, our studies are critical for the clinical testing of BH3 mimetics that target BCL2 or its close pro-survival relatives in patients with multiple myeloma. Disclosures Gong: The Walter and Eliza Hall Institute of Medical Research: Employment. Segal:The Walter and Eliza Hall Institute of Medical Research: Employment. van Delft:The Walter and Eliza Hall Institute of Medical Research: Employment. Roberts:The Walter and Eliza Hall Institute of Medical Research: Employment. Huang:The Walter and Eliza Hall Institute of Medical Research: Employment.

Abstract 1289 Hematopoietic stem cells due to their life-long function should protect genome integrity to avoid accumulation of genetic aberrations leading to malignant transformation or bone marrow failure. We reported recently that human HSC of cord blood origin are exquisitely sensitive to DNA damage-induced apoptosis. Thus, following 3Gy of ionizing radiation HSC show evidence of persistent DNA damage response and greater p53-dependent apoptosis in comparison with commited myeloid progenitors. To elucidate the molecular basis of these observations we carried out a genome-wide loss-of-function genetic screen using a library of 80 000 shRNA vectors targeting more than 16 000 human genes. A screen was performed on immortalized (by TLS-ERG infection) cord blood cells (TEX), which radio-sensitivity is similar to early hematopoietic cells. To find out the radio-protective hits we exposed infected cells to four rounds of 4Gy irradiation in three independent experiments. TEX cells exhibited steady increase of their proliferative potential after each irradiation exposure. Cells were gathered after each irradiation round and their DNA was subjected to sequencing to determine protective hits. Upon the analysis we recognized known regulators of DNA damage response (e.g. p53) and identified many genes that previously were not connected to genotoxic stress response. Thus, the knockdown of these genes was at least as effective as p53 knockdown in protection of TEX cells against gamma-irradiation. The validation of the chosen hits on TEX cells showed that about half of them indeed mediate the protection against irradiation. Further validation included real-time PCR of infected cells to ensure the absence of off-target effect along with Western blot analysis of affected protein. We followed up the investigation of chosen hits on primary human lineage-negative cord blood cells and observed that only few candidates were mediating the same effect on HSC/progenitors cells as on TEX cells. To further validate the effect of the most prominently effective hits we employed several shRNA vectors for the same target gene. Following this step of validation, we choose to investigate the knockdown of CHEK2, the gene with reported role in DNA damage response in several murine tissues. Our preliminary studies in long-term cytokine-supplemented cultures demonstrated that CHEK2 is involved in DNA damage response of human HSC and progenitors. The further investigation of the effect of CHEK2 knockdown on repopulating human HSC is currently underway. An integrated analysis of our observations revealed several putative CHEK2-centered molecular networks, which connect DNA damage response to HSC function. Disclosures: No relevant conflicts of interest to declare.

Lymphoid tumors often respond poorly to conventional cytotoxics, a common cause being their impaired sensitivity to apoptosis, such as that caused by Bcl-2 overexpression. A strategy to overcoming this is to use mimics of the natural antagonists of pro-survival Bcl-2, the BH3 only proteins. A promising BH3 mimetic is ABT-737, which targets Bcl-2 and closely related pro-survival proteins. We evaluated its potential utility by testing it on cell lines, clinical samples and on a relevant mouse lymphoma model. We assessed the sensitivity of B cell lymphoma cell lines and primary CLL samples to ABT-737, either alone or in combination. To ascertain its efficacy in vivo, we utilized a mouse model based on the Eμ-myc tumor that is readily transplantable and amenable to genetic manipulation. When syngeneic recipient mice were inoculated with tumors, they develop widespread lymphoma, fatal unless treated by agents such as cyclophosphamide. We found that ABT-737, on its own, was cytotoxic only to a subset of cell lines and primary CLL samples. However, it can synergize potently with agents such as dexamethasone, suggesting that this agent might be useful in combination with currently used chemotherapeutics. In the Eμ myc mouse lymphoma model, treatment with ABT-737 alone did not control the disease as multiple independently derived tumors proved refractory to treatment with this agent. However, ABT-737 was partially effective as a single agent for treating bitransgenic tumors derived from crosses of the Eμmyc and Eμ-bcl-2 transgenic mice. ABT-737 therapy prolonged the survival of recipient mice transplanted with tumors from 30 to 60 days. When combined with a low dose of cyclophosphamide (50mg/kg), long term stable remissions were achieved, which were sustained even longer than control mice treated with much higher doses of cyclophosphamide (300mg/kg). We found that ABT-737 was well tolerated as a single agent and when combined with low doses of cytotoxics such as cyclophosphamide. Thus, ABT-737 may prove to be efficacious for those tumors highly dependent on Bcl-2 for their survival. We found that despite its high affinity for Bcl-2, Bcl-xL and Bcl-w, many cell types proved refractory to ABT-737 as a single agent. We show that this resistance reflects its inability to target another pro-survival relative Mcl-1. Down-regulation of Mcl-1 by several strategies conferred sensitivity to ABT-737. Furthermore, enforced Mcl-1 expression in the Eμmyc/bcl-2 bitransgenic mouse lymphoma model conferred marked resistance as mice transplanted with such tumors died as rapidly as the untreated counterparts. However, enhanced Bcl-2 overexpression on these tumors had little impact on the in vivo response, suggesting that ABT-737 can be utilized even when Bcl-2 is markedly overexpressed. ABT-737 appears to be a promising agent for the clinic. It potently sensitizes certain lymphoid tumors to conventional cytotxics in vitro. The synergy observed between dexamethasone and ABT-737 on some lymphoid lines in culture suggests that it is attractive for clinical testing. Encouragingly, ABT-737 appeared efficacious in vivo against Bcl-2 overexpressing tumors when combined with a reduced dose of cyclophosphamide, suggesting that it will be useful for treating even those Bcl-2-overexpressing tumors that are normally highly chemoresistant.

As overactivity of pro-survival Bcl-2 proteins promotes neoplasia and enhances resistance of malignant cells to cytotoxic therapies, a promising approach for incurable lymphoid tumors is to directly target the pro-survival proteins. One approach is to mimic their physiological antagonists, the BH3-only proteins. We initially determined which of the diverse BH3-only proteins would be optimal to mimic. The interactions of the BH3 domains of this family with a groove on the Bcl-2-like proteins have been considered promiscuous. However, we found that the interactions between eight BH3 peptides and five Bcl-2-like proteins varied over 10,000 fold in affinity, and that only certain protein pairs associate inside cells (Chen et al Mol Cell;17:393–403, 2005). Bim and Puma potently engaged all the pro-survival proteins comparably. Bad, however, bound preferentially to Bcl-2, Bcl-xL and Bcl-w. Strikingly, Noxa bound only Mcl-1 and A1. In accord with their complementary binding, Bad and Noxa cooperated to induce potent killing. We next determined the mechanism of action of putative BH3 mimetic compounds. Because killing by the BH3-only proteins require the action of the essential cell death mediators, Bax and Bak, we initially screened putative BH3 mimetic compounds on cells genetically engineered to lack both Bax and Bak. Only those compounds that mimicked the BH3-only proteins (i.e, inert on BaxBak double null cells, but active in Bax or Bak expressing cells) were evaluated further. Surprisingly, among seven putative BH3 mimetics tested, we found that only the recently described ABT-737 (Abbott Laboratories; Oltersdorf et al, Nature435:677–81, 2005), required the pro-apoptotic protein Bax or Bak to induce apoptosis. The cytotoxicity of ABT-737 alone was modest in a range of hematopoietic and non-hematopoietic cell types. Further investigations revealed that, like Bad, ABT-737 only targeted Bcl-2, Bcl-xL and Bcl-w. As hematopoietic cells typically express Mcl-1, we attempted to augment the activity of ABT-737 by concomitantly neutralizing Mcl-1. Targeting of Mcl-1 by overexpressing the BH3-only protein Noxa, or Mcl-1 down-regulation by RNAi, cytokine deprivation or cytotoxic agents, allowed ABT-737 to efficiently kill diverse cell types, even when Bcl-2 was over-expressed. We conclude that ABT-737 is a highly selective and specific BH3 mimetic compound that should prove highly efficacious in tumors where Mcl-1 is low, or when combined with agents that down-regulate Mcl-1. This hypothesis is currently being tested in vitro in primary human lymphoid malignancies, and in vivo using immunocompetent murine models of lymphoma. ABT-737 also provides strong proof-of-principle that targeting pro-survival Bcl-2 proteins is feasible, but the optimal utility of such BH3 mimetics depends on a thorough understanding of the pathways to apoptosis.