ALBERT

Description: Toxicity and drug resistance has impeded clinical improvements in acute myeloid leukaemia (AML). We hypothesized that directly targeting pro-survival proteins with BH3-mimetics may have therapeutic rationale in AML and potentially sensitize chemoresistant cases to chemotherapy. This work will therefore assess which Bcl-2 family members mediate pro-survival activity in AML and the potential benefit of combining BH3-mimetics with standard AML drugs. Immunoblotting of fresh primary AML samples revealed strong Bcl-2 expression in 12/13 (92%) cases, Bcl-xL expression in 3/13 (23%) cases and Mcl-1 expression in 8/13 cases (62%). Sensitivity to ABT-199 (targets Bcl-2) or ABT-737 (targets Bcl-2, -xL, -w) was assessed by incubation of freshly harvested AML samples in RPMI + 20% FBS for 48 hrs and viable cells enumerated by exclusion of Sytox Blue using flow cytometry. There were 2 discrete populations identified; one that was sensitive to ABT-199 with an LC50 (concentration of drug to kill 50% of AML blasts) 〈 10nM (5/13 cases; 39%) and a more resistant population (LC50 〉 10μM in 6/13 cases, 46%). A strong correlation in the sensitivity of AML cells to ABT-199 and ABT-737 was observed (r=0.9628, p 〈 0.0001), suggesting Bcl-xL was not a dominant survival factor in AML. To identify relevant Bcl-2 pro-survival targets for elimination of AML in vivo, MV4;11 cells were engineered to express under doxycycline control 1) BimSwt targeting all pro-survival proteins, 2) BimS2A targeting Mcl-1 only, 3) BimSBad targeting Bcl-2, -x and –w; or 4) an inert BimS4E, which served as a negative control. 1x 105 MV4;11 cells per mouse were xenografted into cohorts of 6 NSG mice and doxycycline-rich water introduced on day 5 after transplantation. Mice transplanted with MV4;11 cells expressing the inert BimS4E succumbed to leukemia by ~ day 40. Expression of BimSBad or BimS2A delayed, but did not prevent leukemia-related death, whereas mice engrafted with MV4;11 cells enforced to express BimSwt were alive and leukemia-free after 100 days (Figure 1A). Similar outcomes were demonstrated using an OCI-AML xenograft model (not shown). As on-target toxicity to platelets is an undesirable consequence of targeting Bcl-xL (Mason, Cell 2006), the effectiveness of targeting only Bcl-2 and Mcl-1 in vivo was next investigated. Mice engrafted with MV4;11 cells expressing BimS2A (to target Mcl-1) were fed doxycycline water from day 5 onwards. In addition, the selective Bcl-2 inhibitor ABT-199 75mg/kg was administered daily by oral gavage between days 5-12 post engraftment. Strikingly, this combination strategy led to eradication of AML by day 100 in all mice, in contrast to delayed, but inevitable leukemic death in most mice receiving treatment directed at either Bcl-2 (ABT-199) or Mcl-1 (Bims2A) alone (Figure 1B). These results suggest that AML eradication in vivo is best achieved through combined targeting of Bcl-2 and Mcl-1. In the absence of direct inhibitors of Mcl-1, standard AML drugs were examined for their capacity to suppress Mcl-1. In the MV4;11 cell line, anthracyclines such as idarubicin rapidly (〈 3 hrs) suppressed Mcl-1 at concentrations between 300nM and 1μM. In contrast, 100μM cytarabine did not suppress Mcl-1 after exposure for 6 hrs. Further studies in primary AML cells demonstrated synergy between ABT-199 and idarubicin in ABT-199 resistant AML cells. We conclude that 1) a subset of primary AML cells is highly sensitive to Bcl-2 inhibition, 2) that eradication of AML in vivo is best through combined Bcl-2/Mcl-1 targeting and 3) that anthracyclines are effective at suppressing Mcl-1 and could be combined with ABT-199 to treat patients with AML resistant to Bcl-2 targeted therapy. A Figure 1 A) Cohorts of 6 NSG mice were xenografted with MV4;11 cells expressing BimS ligand variants under doxycycline control. Doxycycline water was fed to mice from day 5+ and survival assessed. B. Cohorts of 6 NSG mice were xenografted with MV4;11 cells expressing BimS2A under doxycycline control. Mice were allocated to treatment with either vehicle or ABT-199 on days 5-12 +/- doxycycline water from day 5+ and survival assessed. All mice in the arm combining Doxycycline (to suppress Mcl-1) and ABT-199 (to target Bcl-2) were alive and leukemia-free on day 100. Figure 1. A) Cohorts of 6 NSG mice were xenografted with MV4;11 cells expressing BimS ligand variants under doxycycline control. Doxycycline water was fed to mice from day 5+ and survival assessed. B. Cohorts of 6 NSG mice were xenografted with MV4;11 cells expressing BimS2A under doxycycline control. Mice were allocated to treatment with either vehicle or ABT-199 on days 5-12 +/- doxycycline water from day 5+ and survival assessed. All mice in the arm combining Doxycycline (to suppress Mcl-1) and ABT-199 (to target Bcl-2) were alive and leukemia-free on day 100. B Figure 2 Figure 2. Disclosures Huang: AbbVie: Consultancy, Honoraria. Wei:Abbvie: Consultancy, Honoraria.

Description: 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.

Description: 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.

Description: Abstract 550 It is widely held that megakaryocytes undergo a specialized form of apoptosis in order to shed platelets. Conversely, it is also believed that a range of insults including chemotherapeutic agents, autoantibodies and viruses, cause thrombocytopenia by inducing the apoptotic death of megakaryocytes and their progenitors. However, the apoptotic pathways that megakaryocytes possess, and the role they play in survival and platelet production are ill-defined. We recently demonstrated that platelets contain a classical intrinsic mitochondrial apoptosis pathway that regulates their life span in vivo. The key components of this pathway are the Bcl-2 family pro-survival protein Bcl-xL, and pro-death Bak and Bax. Deletion of Bak and Bax—the gatekeepers of the intrinsic pathway—blocks platelet apoptosis in response to genetic mutation or pharmacological insult, and significantly extends circulating platelet life span. To elucidate the role of the intrinsic apoptosis pathway in megakaryocytes, we generated both hematopoietic- and megakaryocyte lineage-specific deletions of Bak and Bax in mice. Surprisingly, we found that the ability of Bak−/−Bax−/− animals to produce platelets, both at steady state and under conditions of thrombopoietic stress, was unperturbed. Megakaryocyte numbers, morphology and ploidy were normal. Bak−/−Bax−/− megakaryocytes cultured in vitro showed no impairment of pro-platelet formation. Thus, classical intrinsic apoptosis is not required by megakaryocytes for the process of platelet shedding. Given that in platelets, Bak and Bax must be kept in check to maintain survival, we reasoned that the same might be true of megakaryocytes. If so, then it would be expected that one or more members of the Bcl-2 family of pro-survival proteins restrain Bak and Bax. Since Bcl-xL fulfills this role in platelets, we generated mice lacking Bcl-xL in the megakaryocyte lineage. Platelet counts in Bcl-xPf4CΔ/Pf4CΔ animals were approximately 2% of those observed in Bcl-xfl/fl littermates. Platelet life span was reduced to 5 hours, versus 5 days in controls, underscoring the requirement for Bcl-xL in mediating platelet survival. In addition, reticulated platelet analyses combined with mathematical modeling suggested that Bcl-xPf4CΔ/Pf4CΔ mice had an underlying platelet production defect. Further examination revealed that megakaryocyte numbers were significantly increased in both the bone marrow and spleen of Bcl-xPf4CΔ/Pf4CΔ animals relative to Bcl-xfl/fl controls. Megakaryocyte progenitor numbers were doubled, and serum TPO levels were dramatically reduced, indicating a megakaryocyte compartment under considerable thrombopoietic stress. In vitro cultures confirmed that Bcl-xPf4CΔ/Pf4CΔ megakaryocytes were able to develop and mature. Strikingly, however, at the point of pro-platelet formation, they underwent an abortive attempt to generate extensions and died. Death was accompanied by a dramatic increase in apoptotic effector caspase activity. This suggested that, like platelets, megakaryocytes possess a functional intrinsic apoptosis pathway that must be restrained in order to survive, and that Bcl-xL is the factor that does so during pro-platelet formation and platelet shedding. To establish whether Bak and Bax can mediate megakaryocyte death, we examined the effect on mature wild type megakaryocytes of three pharmacological agents that activate the intrinsic apoptosis pathway in other cell types: etoposide, staurosporine, and a BH3 mimetic that inhibits Bcl-xL, Bcl-2 and Bcl-w. All three triggered mitochondrial damage, caspase activation and cell death. Remarkably, genetic deletion of Bak and Bax rendered megakaryocytes resistant to etoposide and the BH3 mimetic, but not staurosporine. Our results demonstrate that megakaryocytes can undergo classical Bak- and Bax-mediated apoptotic death. They do not activate the intrinsic pathway to facilitate platelet shedding, rather, the opposite is true: they must restrain it in order to survive and generate platelets. These findings offer a potential mechanism for the death of megakaryocytes in response to insults such as cancer chemotherapy. They also suggest that additional megakaryocyte cell death pathways remain to be elucidated. Disclosures: Roberts: Abbott: Research Funding.