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AZD1208 and quizartinib co-treatment was well tolerated in the orthotopic model.

Purpose: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML), and these patients have short disease-free survival. FLT3 inhibitors have limited and transient clinical activity, and concurrent treatment with inhibitors of parallel or downstream signaling may improve responses. The oncogenic serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD and also promotes its signaling in a positive feedback loop, suggesting benefit of combined Pim and FLT3 inhibition.Experimental Design: Combinations of clinically active Pim and FLT3 inhibitors were studied in vitro and in vivo.Results: Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated in vitro growth of FLT3-ITD, but not wild-type FLT3 (FLT3-WT), cell lines. AZD1208 cotreatment increased FLT3 inhibitor–induced apoptosis of FLT3-ITD, but not FLT3-WT, cells measured by sub-G1 fraction, annexin V labeling, mitochondrial membrane potential, and PARP and caspase-3 cleavage. Concurrent treatment with AZD1208 and the FLT3 inhibitor quizartinib decreased growth of MV4-11 cells, with FLT3-ITD, in mouse xenografts, and prolonged survival, enhanced apoptosis of FLT3-ITD primary AML blasts, but not FLT3-WT blasts or remission marrow cells, and decreased FLT3-ITD AML blast colony formation. Mechanistically, AZD1208 and quizartinib cotreatment decreased expression of the antiapoptotic protein Mcl-1. Decrease in Mcl-1 protein expression was abrogated by treatment with the proteasome inhibitor MG132, and was preceded by downregulation of the Mcl-1 deubiquitinase USP9X, a novel mechanism of Mcl-1 regulation in AML.Conclusions: The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. Clin Cancer Res; 24(1); 234–47. ©2017 AACR.

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BackgroundLong-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive given the relatively intact immune system of patients with LGG and the slow tumor growth rate. However, accumulation of the oncometabolite D-2-hydroxyglutarate (D-2HG) in IDH-mutated gliomas leads to suppression of inflammatory pathways in the tumor microenvironment, thereby contributing to the ‘cold’ tumor phenotype. Inhibiting D-2HG production presents an opportunity to generate a robust antitumor response following tumor antigen vaccination and immune checkpoint blockade.MethodsAn IDH1R132H glioma model was created in syngeneic HLA-A2/HLA-DR1-transgenic mice, allowing us to evaluate the vaccination with the human leukocyte antigens (HLA)-DR1-restricted, IDH1R132H mutation-derived neoepitope. The effects of an orally available inhibitor of mutant IDH1 and IDH2, AG-881, were evaluated as monotherapy and in combination with the IDH1R132H peptide vaccination or anti-PD-1 immune checkpoint blockade.ResultsThe HLA-A2/HLA-DR1-syngeneic IDH1R132H cell line expressed the IDH1 mutant protein and formed D-2HG producing orthotopic gliomas in vivo. Treatment of tumor-bearing mice with AG-881 resulted in a reduction of D-2HG levels in IDH1R132H glioma cells (10 fold) and tumor-associated myeloid cells, which demonstrated high levels of intracellular D-2HG in the IDH1R132H gliomas. AG-881 monotherapy suppressed the progression of IDH1R132H gliomas in a CD4+ and CD8+ cell-dependent manner, enhanced proinflammatory IFNγ-related gene expression, and increased the number of CD4+ tumor-infiltrating T-cells. Prophylactic vaccination with the HLA-DR1-restricted IDH1R132H peptide or tumor-associated HLA-A2-restricted peptides did not enhance survival of tumor-bearing animals; however, vaccination with both HLA-A2-IDH1R132H and DR1-IDH1R132H peptides in combination with the IDH inhibitor significantly prolonged survival. Finally, tumor-bearing mice treated with both AG-881 and a PD-1 blocking antibody demonstrated improved survival when compared with either treatment alone.ConclusionThe development of effective IDH1R132H-targeting vaccine may be enhanced by integration with HLA class I-restricted cytotoxic T cell epitopes and AG-881. Our HLA-A2/HLA-DR1-syngeneic IDH1R132H glioma model should allow us to evaluate key translational questions related to the development of novel strategies for patients with IDH-mutant glioma.

Acute myeloid leukemias (AML) are comprised of multiple cell types with distinct capabilities to propagate the disease and resist therapy. Approximately 20% of AML patients carry gain- of-function mutations in IDH1 or IDH2 that result in over-production of the onco-metabolite 2-HG. Although IDH inhibitors can induce complete morphological remission, almost all patients eventually relapse. Analysis of clinical samples suggests that a population of IDH mutant cells is able to persist during treatment eventually acquiring 2-HG independence and drug resistance. Herein we characterized the molecular and cellular responses to the clinical IDH1 inhibitor AG-120 at high resolution using a novel multi-allelic mouse model of IDH1 mutant AML. We demonstrate that AG-120 exerts cell type-dependent effects on leukemic cells promoting delayed disease regression. Although IDH1 inhibition alone was not able to fully eradicate the disease, we uncovered that it increases cycling of rare leukemic stem cells and triggers transcriptional upregulation of the pyrimidine salvage pathway. Accordingly, AG-120 sensitized IDH1 mutant AML to azacitidine with the combination of AG-120 and azacitidine showing vastly improved efficacy in vivo. Our data highlight the impact of non- genetic heterogeneity on treatment response and provide mechanistic rationale for a drug combination that is being tested in clinical trials.STATEMENT OF SIGNIFICANCEInhibition of mutant IDH1 in AML is insufficient to eliminate the disease but promotes proliferation of quiescent leukemic stem cells. Our data provide a mechanistic explanation for the observed synergy between IDH inhibitors and azacitidine and suggest that IDH inhibitors may also synergize with other drugs that preferentially target actively dividing cells.

Anticancer efficacy is driven not only by dose but also by frequency and duration of treatment. We describe a multiscale model combining cell cycle, cellular heterogeneity of B-cell lymphoma 2 family proteins, and pharmacology of AZD5991, a potent small-molecule inhibitor of myeloid cell leukemia 1 (Mcl-1). The model was calibrated using in vitro viability data for the MV-4-11 acute myeloid leukemia cell line under continuous incubation for 72 hours at concentrations of 0.03-30 μM. Using a virtual screen, we identified two schedules as having significantly different predicted efficacy and showed experimentally that a "short" schedule (treating cells for 6 of 24 hours) is significantly better able to maintain the rate of cell kill during treatment than a "long" schedule (18 of 24 hours). This work suggests that resistance can be driven by heterogeneity in protein expression of Mcl-1 alone without requiring mutation or resistant subclones and demonstrates the utility of mathematical models in efficiently identifying regimens for experimental exploration.

Targeting the menin-MLL protein-protein interaction is being pursued as a new therapeutic strategy for the treatment of acute leukemia carrying MLL-rearrangements (MLLr leukemia). Herein, we report M-1121, a covalent and orally active inhibitor of the menin-MLL interaction capable of achieving complete and persistent tumor regression. M-1121 establishes covalent interactions with Cysteine 329 located in the MLL binding pocket of menin and potently inhibits growth of acute leukemia cell lines carrying MLL translocations with no activity in cell lines with wild-type MLL. Consistent with the mechanism of action, M-1121 drives dose-dependent down-regulation of HOXA9 and MEIS1 gene expression in the MLL-rearranged MV4;11 leukemia cell line. M-1121 is orally bioavailable and showed potent antitumor activity in vivo with tumor regressions observed at tolerated doses in the MV4;11 subcutaneous and disseminated models of MLL-rearranged leukemia. Together, our findings support development of an orally active covalent menin inhibitor as a new therapy for MLLr leukemia.

Most targeted cancer therapies fail to achieve complete tumor regressions or attain durable remissions. To understand why these treatments fail to induce robust cytotoxic responses despite appropriately targeting oncogenic drivers, here we systematically interrogated the dependence of cancer cells on the BCL-2 family of apoptotic proteins after drug treatment. We observe that multiple targeted therapies, including BRAF or EGFR inhibitors, rapidly deplete the pro-apoptotic factor NOXA, thus creating a dependence on the anti-apoptotic protein MCL-1. This adaptation requires a pathway leading to destabilization of the NOXA mRNA transcript. We find that interruption of this mechanism of anti-apoptotic adaptive resistance dramatically increases cytotoxic responses in cell lines and a murine melanoma model. These results identify NOXA mRNA destabilization/MCL-1 adaptation as a non-genomic mechanism that limits apoptotic responses, suggesting that sequencing of MCL-1 inhibitors with targeted therapies could overcome such widespread and clinically important resistance., MAPK-targeted therapies fail to achieve complete remission. Here, the authors show that anti-apoptosis resistance is acquired in these targeted therapies through the mRNA destabilization of NOXA which leads to dependence on MCL-1, and that sequential combination of MCL-1 inhibition with targeted therapies overcomes this resistance.

Mcl-1 is a member of the Bcl-2 family of proteins that promotes cell survival by preventing induction of apoptosis in many cancers. High expression of Mcl-1 causes tumorigenesis and resistance to anticancer therapies highlighting the potential of Mcl-1 inhibitors as anticancer drugs. Here, we describe AZD5991, a rationally designed macrocyclic molecule with high selectivity and affinity for Mcl-1 currently in clinical development. Our studies demonstrate that AZD5991 binds directly to Mcl-1 and induces rapid apoptosis in cancer cells, most notably myeloma and acute myeloid leukemia, by activating the Bak-dependent mitochondrial apoptotic pathway. AZD5991 shows potent antitumor activity in vivo with complete tumor regression in several models of multiple myeloma and acute myeloid leukemia after a single tolerated dose as monotherapy or in combination with bortezomib or venetoclax. Based on these promising data, a Phase I clinical trial has been launched for evaluation of AZD5991 in patients with hematological malignancies (NCT03218683)., High expression of Mcl-1 promotes tumorigenesis and resistance to anticancer therapies. Here they report a macrocyclic molecule with high selectivity and affinity for Mcl-1 that exhibits potent anti-tumor effects as single agent and in combination with bortezomib or venetoclax in preclinical models of multiple myeloma and acute myeloid leukemia.

Most targeted cancer therapies fail to achieve complete tumor regressions or attain durable remissions. To understand why these treatments fail to induce robust cytotoxic responses despite appropriately targeting oncogenic drivers, here we systematically interrogated the dependence of cancer cells on the BCL-2 family of apoptotic proteins after drug treatment. We observe that multiple targeted therapies, including BRAF or EGFR inhibitors, rapidly deplete the pro-apoptotic factor NOXA, thus creating a dependence on the anti-apoptotic protein MCL-1. This adaptation requires a pathway leading to the destabilization of the NOXA mRNA transcript. We find that interruption of this mechanism of anti-apoptotic adaptive resistance dramatically increases cytotoxic responses in cell lines and a murine melanoma model. Durable control of disease in vivo can be achieved with combinations of targeted therapy with an MCL-1 inhibitor. However, consistent with an adaptive resistance mechanism we have identified, the timing of drug treatment strongly influences the efficacy of the combination therapy. Collectively, our results identify NOXA mRNA destabilization/MCL-1 adaptation as a non-genomic mechanism that limits apoptotic responses. Our data indicate that optimal sequencing of MCL-1 inhibitors with targeted therapies could overcome widespread and clinically important therapeutic resistance, and achieve prolonged tumor responses in solid tumors. Citation Format: Joan Montero, Cecile Gstalder, Daniel J. Kim, Dorota Sadowicz, Wayne Miles, Michael Manos, Justin R. Cidado, J. Paul Secrist, Adriana E. Tron, Keith Flaherty, F. Stephen Hodi, Charles H. Yoon, Anthony Letai, David E. Fisher, Rizwan Haq. Destabilization of NOXA mRNA as a common resistance mechanism to targeted therapies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 62.

Dysregulation of the mitochondrial apoptotic pathway controlled by members of the Bcl-2 protein family plays a central role in cancer development and resistance to conventional cytotoxic as well as targeted therapies. Hence, selective inhibition of pro-survival Bcl-2 family of proteins to activate apoptosis in malignant cells represents an exciting anti-cancer strategy. The remarkable clinical performance of the selective Bcl-2 antagonist venetoclax has highlighted the potential for selective inhibitors of the other pro-survival members of the Bcl-2 family, particularly Mcl-1. Here we review the latest progress on the discovery and development of selective inhibitors of Mcl-1 that are undergoing clinical evaluation for cancer therapy.

Leukemia and lymphoma cell lines have been pivotal in the cytogenetic and molecular analysis of recurring chromosomal translocations, elucidating the pathogenesis of several hematological malignanc...

Purpose: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML), and these patients have short disease-free survival. FLT3 inhibitors have limited and transient clinical activity, and concurrent treatment with inhibitors of parallel or downstream signaling may improve responses. The oncogenic serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD and also promotes its signaling in a positive feedback loop, suggesting benefit of combined Pim and FLT3 inhibition. Experimental Design: Combinations of clinically active Pim and FLT3 inhibitors were studied in vitro and in vivo. Results: Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated in vitro growth of FLT3-ITD, but not wild-type FLT3 (FLT3-WT), cell lines. AZD1208 cotreatment increased FLT3 inhibitor–induced apoptosis of FLT3-ITD, but not FLT3-WT, cells measured by sub-G1 fraction, annexin V labeling, mitochondrial membrane potential, and PARP and caspase-3 cleavage. Concurrent treatment with AZD1208 and the FLT3 inhibitor quizartinib decreased growth of MV4-11 cells, with FLT3-ITD, in mouse xenografts, and prolonged survival, enhanced apoptosis of FLT3-ITD primary AML blasts, but not FLT3-WT blasts or remission marrow cells, and decreased FLT3-ITD AML blast colony formation. Mechanistically, AZD1208 and quizartinib cotreatment decreased expression of the antiapoptotic protein Mcl-1. Decrease in Mcl-1 protein expression was abrogated by treatment with the proteasome inhibitor MG132, and was preceded by downregulation of the Mcl-1 deubiquitinase USP9X, a novel mechanism of Mcl-1 regulation in AML. Conclusions: The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. Clin Cancer Res; 24(1); 234–47. ©2017 AACR.

Acute myeloid leukemia (AML) and Diffuse Large B-cell Lymphoma (DLBCL) are challenging hematological malignancies due to complex mutational patterns within and across patients and have unmet medical needs for therapies tailored for different disease subtypes. Translating AML and DLBCL preclinical findings into meaningful clinical outcomes can be inefficient and costly, as evidenced by failure of 85% of oncology drugs entering clinical testing to gain FDA approval and costs of bringing drugs to market exceeding $2 billion dollars. There is a pressing need for disease focused comprehensive knowledgebases to provide insight into whether preclinical models represent patient disease, assist in selecting accurate models to define proof of mechanism and advance therapeutic strategies. We provided Hemebase as the first comprehensive functional-genomic map for a one-stop-shop solution to enable real-time assessment of the relationships between thousands of patients and preclinical models within DLBCL or AML. Hemebase is an interactive, web-based tool to browse, compare, and visualize models and patients from the largest functional-genomic knowledgebase to date, including over 600 AML patients, over 1000 DLBCL patients, over 60 AML/DLBCL cell lines and sources of PDX mouse models (Crown/Champion). The multimodal data incorporated into Hemebase include somatic mutations, copy number alterations, expression values, pathway activity scores, drug screening data, and clinical features (i.e. diagnosis, previous treatment, and clinical outcomes). Importantly, several unbiased methods of clustering cross preclinical models and patients are incorporated, including a non-negative matrix factorization (NMF) that enables interactive assessment of model-to-patient similarity on molecular basis. NMF integration allowed us to recreate the methods used in the Schipp lab’s recent Nature Medicine paper to determine where cell lines lie within the the relevant 5 clusters of published patient data and perform real time analysis of how the clusters change based on varying the model inputs. To enable predictive biomarker discovery, clinically relevant filters can be simultaneously applied across models to analyze drug sensitivity - this was used to reveal the specific patient subpopulations that carry mutations that conferred drug sensitivity. These methods enabled the identification of an AML patient population that closely resembles previously used PDX models in terms of genomics and drug response, allowing for broader understanding of pharmacological response between these two models and a path for future investigation. Taken together, Hemebase provides unique opportunities to reveal the full landscapes of AML and DLBCL disease biology and therapeutics from enabling a comprehensive model-to-patient genomic map for AML and DLBCL. Citation Format: Kathleen A. Burke, Tyler Faits, Adriana E. Tron, Jay Mettetal, Andrew Bloecher, Zhongwu Lai, Jonathan Dry, Bolan Linghu. HemeBase: a comprehensive model-to-patient genomic map for AML and DLBCL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2462.

Internal tandem duplication of fms-like tyrosine kinase-3 (FLT3-ITD) is frequent (30 percent) in acute myeloid leukemia (AML), and is associated with short disease-free survival following chemotherapy. The serine threonine kinase Pim-1 is a pro-survival oncogene transcriptionally upregulated by FLT3-ITD that also promotes its signaling in a positive feedback loop. Thus inhibiting Pim-1 represents an attractive approach in targeting FLT3-ITD cells. Indeed, co-treatment with the pan-Pim kinase inhibitor AZD1208 or expression of a kinase-dead Pim-1 mutant sensitized FLT3-ITD cell lines to apoptosis triggered by chemotherapy drugs including the topoisomerase 2 inhibitors daunorubicin, etoposide and mitoxantrone, but not the nucleoside analog cytarabine. AZD1208 sensitized primary AML cells with FLT3-ITD to topoisomerase 2 inhibitors, but did not sensitize AML cells with wild-type FLT3 or remission bone marrow cells, supporting a favorable therapeutic index. Mechanistically, the enhanced apoptosis observed with AZD1208 and topoisomerase 2 inhibitor combination treatment was associated with increased DNA double-strand breaks and increased levels of reactive oxygen species (ROS), and co-treatment with the ROS scavenger N-acetyl cysteine rescued FLT3-ITD cells from AZD1208 sensitization to topoisomerase 2 inhibitors. Our data support testing of Pim kinase inhibitors with topoisomerase 2 inhibitors, but not with cytarabine, to improve treatment outcomes in AML with FLT3-ITD.

Summary The approximately 300 human cullin-RING ligases (CRLs) are multisubunit E3s in which a RING protein, either RBX1 or RBX2, recruits an E2 to catalyze ubiquitination. RBX1-containing CRLs also can bind Glomulin (GLMN), which binds RBX1's RING domain, regulates the RBX1-CUL1-containing SCF FBW7 complex, and is disrupted in the disease Glomuvenous Malformation. Here we report the crystal structure of a complex between GLMN, RBX1, and a fragment of CUL1. Structural and biochemical analyses reveal that GLMN adopts a HEAT-like repeat fold that tightly binds the E2-interacting surface of RBX1, inhibiting CRL-mediated chain formation by the E2 CDC34. The structure explains the basis for GLMN's selectivity toward RBX1 over RBX2, and how disease-associated mutations disrupt GLMN-RBX1 interactions. Our study reveals a mechanism for RING E3 ligase regulation, whereby an inhibitor blocks E2 access, and raises the possibility that other E3s are likewise controlled by cellular proteins that mask E2-binding surfaces to mediate inhibition.

Fbw7, a substrate receptor for Cul1-RING-ligase (CRL1), facilitates the ubiquitination and degradation of several proteins, including Cyclin E and c-Myc. In spite of much effort, the mechanisms underlying Fbw7 regulation are mostly unknown. Here, we show that Glomulin (Glmn), a protein found mutated in the vascular disorder glomuvenous malformation (GVM), binds directly to the RING domain of Rbx1 and inhibits its E3 ubiquitin ligase activity. Loss of Glmn in a variety of cells, tissues, and GVM lesions results in decreased levels of Fbw7 and increased levels of Cyclin E and c-Myc. The increased turnover of Fbw7 is dependent on CRL and proteasome activity, indicating that Glmn modulates the E3 activity of CRL1(Fbw7). These data reveal an unexpected functional connection between Glmn and Rbx1 and demonstrate that defective regulation of Fbw7 levels contributes to GVM.

Apoptosis is controlled through the dynamic interactions of the Bcl2 protein family, and cancers have evolved mechanisms to hijack this pathway to evade apoptosis, often by upregulating anti-apoptotic proteins (e.g. Mcl1, Bcl2). This survival adaptation creates a dependency that could be exploited therapeutically, which is why considerable effort has been made to develop small molecule inhibitors of the anti-apoptotic Bcl2 family proteins. This class of drug was clinically validated with the approval of venetoclax, a selective Bcl2 inhibitor, for the treatment of CLL. Venetoclax is undergoing evaluation in numerous other clinical trials for predominantly hematologic malignancies. Despite impressive responses observed with venetoclax in CLL (ORR 79%), acquired resistance is beginning to emerge. Likewise, other hematologic indications are more intrinsically resistant to venetoclax, exhibiting much lower response rates in the respective Phase I clinical trials (AML = 38%, NHL = 44%, MM = 12%). Preclinical studies with venetoclax have reported increased levels of other anti-apoptotic proteins as a likely mechanism contributing to both de novo and acquired resistance. Therefore, combining cell death inducing agents that inhibit Mcl1 or BclxL could be a means of combating resistance. Inhibition of cyclin-dependent kinase 9 (CDK9), which regulates transcription elongation, has been reported to reduce protein levels of genes with short-lived transcripts and proteins, such as MCL1. AZD4573 is a novel and selective CDK9 inhibitor that shows potent single agent activity, inducing cell death in vitro and tumor regressions in vivo in a diverse set of hematologic cancers (Cidado et. al., AACR Annual Meeting 2018). AZD4573 is currently being evaluated in a Phase I clinical trial for patients with hematological malignancies (NCT03263637). This study evaluates whether combinations of AZD4573 with other cell death inducing agents could overcome de novo venetoclax monotherapy resistance. A panel of 12 AML and 6 DLBCL cell lines were treated for 6 h with venetoclax, AZD4573, and a selective BclxL probe compound (AZ'3202) either as single agents or in combinations and assayed for caspase activation. The Loewe model was used to calculate synergy scores to assess benefit over monotherapy, and combinations with scores >5 were deemed beneficial. Treatment with AZD4573+venetoclax and AZD4573+AZ'3202 resulted in beneficial combinations for 13/18 and 10/18 cell lines, respectively. On the other hand, venetoclax+AZ'3202 showed significant combination benefit in only two cell lines (NB4, SUDHL4), suggesting a primary dependency upon Mcl1 for most of these hematologic cancer cell lines. Interestingly, cell lines sensitive to single agent AZ'3202 (4 AML, 0 DLBCL) did not show any combination benefit when treated with AZD4573+venetoclax, highlighting the exquisite dependency of those four models upon BclxL and mutual exclusivity with Mcl1. Cell lines benefitting from the AZD4573+venetoclax combination tended to fall into one of two categories: having single agent activity to either agent that is enhanced by the combination or having no single agent activity but the combination shifts the cell line into a responder. SUDHL4 cells were sensitive to AZD4573 (caspase activation EC50 = 18 nM) but not venetoclax (EC50 = 476 nM) while OCI-AML3 was insensitive to both (EC50 > 30 µM). In vitro biomarker kinetic analysis revealed an increase in Mcl1 levels (~2-fold) after 3 h of venetoclax treatment that was abrogated upon combination treatment, providing a mechanistic rationale for the combination benefit. Furthermore, when tested in an OCI-AML3 tumor xenograft study in mice, AZD4573 or venetoclax monotherapy exhibited minimal tumor growth inhibition (44% and 16%, respectively) while the combination led to tumor regressions (64%) with minimal effect on body weight. In a SUDHL4 tumor xenograft study, venetoclax monotherapy displayed minimal tumor growth inhibition (25%), but intermittent dosing of AZD4573 exhibited 94% tumor growth inhibition. Still, combination therapy demonstrated a clear benefit as it led to complete tumor regressions with 6/8 mice remaining tumor-free until the end of study (150 days). Together, this work presents supporting evidence that combining cell death inducing agents would be effective at overcoming de novo or acquired resistance associated with monotherapy treatments. Disclosures Cidado: AstraZeneca: Employment, Equity Ownership. Boiko:AstraZeneca: Employment. Proia:AstraZeneca: Employment. Woods:AstraZeneca: Employment. Tavana:AstraZeneca: Employment. San Martin:AstraZeneca: Employment. Tron:AstraZeneca: Employment. Shao:AstraZeneca: Employment. Drew:AstraZeneca: Employment.

Multiple myeloma is a malignancy of long-lived plasma cells of the bone marrow that is rarely curable. Thus, despite recent advances in the development of new therapies, additional approaches are required. We investigated potential molecular vulnerabilities in the BCL2 family. Using the MMRF CoMMpass (NCT0145429) study (IA13), we determined the frequency of nonsynonymous coding mutations in the BCL2 family. Analysis of baseline samples from 982 patients revealed that mutations in the BCL2 family are relatively rare events. No mutations were observed in the 3 pro-apoptotic effector genes BAX, BAK1 and BOK. Similarly, in the BH3-only genes, mutations were rare with no mutations in BCL2L11 (BIM), BAD, BID, HRK and BMF and only single mutations in BBC3 (PUMA) and PMAIP1 (NOXA). In the anti-apoptotic BCL2 genes, mutations were also rare with no mutations in BCL2, BCL2L1 (BCLX) and BCL2L10 (BCLB). A single sample had a mutation in BCL2A1 (A1) while 2 samples had mutations in BCL2L2 (BCLW). Interestingly MCL1 was mutated in 10 baseline samples (1.02%) and the frequency of the mutations in these samples was high (median 0.391, range 0.066-0.531). Therefore, we further investigated the functional consequences of the MCL1 mutant alleles. Of the 10 mutations detected, 1 was in the N-terminal region (G32R) and 4 were in the PEST domain in the N-terminal half of MCL1 that is associated with regulating protein stability (V140I, P142S, E149Q and E173K). An additional mutation was found in an uncharacterized region between the PEST and BH1 domains (L186F). We focused on the 4 mutations that lie within or near the functional BH1 (V249L and L267V) and within the BH3 (N223S, and R214Q) domains. Wild type (WT) MCL1 and the four mutant MCL1 constructs were introduced into a murine B-ALL cell line that has endogenous murine MCL1 flanked with LoxP sites and confirmed expression by western blot analysis. Human MCL1 can replace murine MCL1 in this cell model, therefore we are determining if the myeloma-derived mutants of MCL1 can complement loss of mouse Mcl1 and will report on these findings. However, we began to functionally characterize these mutations by taking advantage of an anomaly in the development of inhibitors of human MCL1. To date, no inhibitor developed against human MCL1 is as effective against murine Mcl1. Thus, differences in the activity of these inhibitors reflects changes in dependence on the introduced human MCL1. We treated cells with increasing concentrations of the MCL1 inhibitor S63845 and measured cell death (Annexin V/PI) at 24 hours. As expected, cells where the empty vector was introduced were highly resistant to S63845-induced cell death (less than 20% at 1000 nM) while cells expressing the human WT MCL1 were significantly more susceptible to the MCL1 inhibitor across a concentration range of 100 to 1000 nM. The V249L, N223S and R214Q mutations mimicked the sensitivity of the WT MCL1 suggesting they did not alter MCL1 function in the cells. In contrast the L267V mutation resulted in a dose curve that was more similar to the empty vector control suggesting this mutation either resulted in loss of function or in an MCL1 molecule that could not be inhibited by the drug. Since drug binding can stabilize MCL1 by competing for E3 ligase binding, we determined the effect of S63845 on MCL1 protein levels. We found that with all the mutants, S63845 dramatically increased human MCL1 protein expression ruling out lack of drug binding. We next performed MCL1 co-immunoprecipitation assays, and found that BIM release correlated with S63845 sensitivity. In addition to not releasing BIM, the L267V did not effectively release NOXA and BAK after S63845 treatment. Taken together, the L267V mutation does not prevent the binding of S63845 to free MCL1, rather it blocks the ability of drug to displace pro-apoptotic proteins required to induce cell death. We then tested another MCL-1 inhibitor in clinical trials, AZD-5991. The L267V mutation was completely resistant to AZD-5991-induced apoptosis despite evidence of drug binding. Interestingly the other 3 mutations also resulted in diminished killing activity when compared to cells expressing the WT MCL1, suggesting that these mutations may also influence drug function. Together these data suggest that myeloma-derived mutations in MCL1 may not necessarily influence MCL1 function, however they could alter responses to an emerging class of inhibitors where 3 drugs are currently in clinical trials. Disclosures Secrist: AstraZeneca: Employment. Cidado:AstraZeneca: Employment, Equity Ownership. Tron:AstraZeneca: Employment. Lonial:Amgen: Research Funding. Boise:Abbvie: Consultancy; AstraZeneca: Honoraria.

We and others have previously demonstrated that MM is often dependent on MCL1 or co-dependent on MCL1 and BCLXL or BCL2 for survival. Therefore, drug development targeting MCL1 has been a top priority. Here we report on AZD5991, a specific small molecule inhibitor of MCL1. We treated 17 MM cell lines with increasing concentrations of AZD5991 for 24 h and measured Annexin V staining to determine the IC50s. Nine of the cell lines tested were highly sensitive to AZD5991 with IC50 values below 100 nM, 6 lines exhibited an intermediate sensitivity (IC50 100-1000 nM), and only 2 cell lines tested were resistant (IC50 >1000 nM). Six of the highly sensitive lines are t(11;14) and sensitive to venetoclax suggesting co-dependence on BCL2 and MCL1 for survival. We also determined the effect of the bone marrow microenvironment on the response of MM cell lines to AZD5991. We reported that IL-6 protects MM cell lines and patient samples from apoptosis by making the cells more MCL1 dependent. Based on this, we predicted IL-6 would have little to no effect on AZD5991-induced cell death. We treated 12 cell lines with AZD5991 in the presence of 1 ng/mL IL-6 or 10% Hs5 conditioned medium (CM) for 24 h and found that only 3/12 and 2/12 lines were protected from apoptosis in the presence of IL-6 and CM, respectively. Interestingly, when co-cultured with the stromal cell line Hs5, 7/11 lines tested were protected from AZD5991-induced cell death, suggesting cell-cell contact is influencing the response. This is in contrast to ABT-737 and venetoclax where cell-cell contact provided no additional protection than CM. Mechanistically apoptosis induced via MCL1 inhibition is not dependent on BIM expression as is the case with BCLXL and BCL2 inhibition. KMS26 and LP1 MM cell lines contain a bi-allelic deletion of BIM and we have reported their resistance to ABT-737. However, both cell lines respond to AZD5991 with IC50 values in an intermediate sensitivity range. Co-immunoprecipitation (CoIP) studies were employed to determine the protein bound to MCL1 that could be promoting apoptosis upon release. We found NOXA and BAK bound in KMS26 and LP1 and both were released from MCL1 in response to AZD5991. Additionally, CoIPs performed on cell lines expressing BIM showed NOXA, BIM, and BAK bound to MCL1 and released following treatment. To further investigate we used CRISPR-cas9 to generate MM cell lines lacking expression of NOXA, BAK, BAX, or BIM. In KMS26 and LP1, deletion of NOXA and BAX had little effect on AZD5991-induced cell death while the BAK deletion significantly inhibited apoptosis in both cell lines. Similar results were observed in the BIM expressing cell line OCI-My5, with no protection from AZD5991-induced apoptosis in the NOXA and BAX edited lines, significant protection in the BAK-deleted line, and an intermediate degree of protection in the BIM knockout line. In KMS18, BIM deficiency had a minimal effect on apoptosis following MCL1 inhibition, however both BAX and BAK were required for AZD-induced cell death. Additionally, we have tested 41 samples from 37 patients for sensitivity to AZD5991. Samples were treated with increasing concentrations to determine IC50 values in the same manner as the MM cell lines. The samples segregated into 4 groups based on IC50. The most sensitive group (N=3) had an IC50 below 10 nM. The largest group had an IC50 range of 50-114 nM (N=26). The last two cohorts were more resistant with a range of 500-916 nM (N=10) and 2 samples with an IC50 over 1300 nM. Since MCL1 is on 1q21, a frequently amplified region in MM, we determined if 1q21 gain was associated with sensitivity. For the 35 samples where FISH data were available, 18 had 1q21 gains by FISH while 17 were negative. There is a trend for the 1q21 gain cohort to be more sensitive (P=0.0573), with only 2/18 having an IC50 above 109 nM. In contrast for the 1q21 negative 7/17 were in the resistant groups. Thus 1q21 may be a marker of sensitivity to MCL1 inhibitors. The data reported here demonstrate that AZD5991 is effective at inducing apoptosis in MM and can overcome soluble microenvironment resistance factors that influence the response to venetoclax. This appears to be due to differential requirements for pro-apoptotic factors for BCL2 and MCL1 inhibition and suggests an underappreciated complexity in the role of BCL2 and MCL1 in cell survival. Finally these findings also suggest that 1q21 gain may be a marker for AZD5991 sensitivity. A clinical trial is currently ongoing in myeloma. Disclosures Secrist: AstraZeneca: Employment. Cidado:AstraZeneca: Employment, Equity Ownership. Tron:AstraZeneca: Employment. Neri:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria. Bahlis:Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Kaufman:Roche: Consultancy; Abbvie: Consultancy; Karyopharm: Other: data monitoring committee; Janssen: Consultancy; BMS: Consultancy. Heffner:Pharmacyclics: Research Funding; Genentech: Research Funding; ADC Therapeutics: Research Funding; Kite Pharma: Research Funding. Lonial:Amgen: Research Funding. Nooka:Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive technologies: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Spectrum Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Boise:AstraZeneca: Honoraria; Abbvie: Consultancy.

Mcl-1 is a member of the Bcl/Mcl family of proteins that promotes cell survival by preventing induction of apoptosis in a broad range of cancers. High expression of Mcl-1 has been linked to tumor development and resistance to anticancer therapies, underscoring the potential of Mcl-1 inhibitors as anticancer drugs. We have previously shown that AZD5991, a rationally designed macrocycle with sub-nanomolar affinity for Mcl-1 and high selectivity, induces rapid and irreversible commitment to apoptosis in Mcl-1-dependent cancer cells in a manner dependent on proapoptotic BAK. Here, we demonstrate that AZD5991 exhibits cytotoxic activity (GI50 Citation Format: Adriana E. Tron, Matthew A. Belmonte, Steven Criscione, Edwin A. Clark, Eric Gangl, Francis D. Gibbons, Jeffrey W. Tyner, Stephen E. Kurtz, Qing Ye, Alexander W. Hird, Alwin Schuller, J. Paul Secrist. Selective Mcl-1 inhibition by AZD5991 induces on-target cell death and achieves antitumor activity in multiple myeloma and acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 302.

Purpose: Internal tandem duplication of fms-like tyrosine kinase 3 (FLT3-ITD) is a common (30%) molecular abnormality in acute myeloid leukemia (AML), associated with a high relapse rate and short disease-free survival. The oncogenic kinase Pim-1 is upregulated downstream of FLT3-ITD and contributes to its proliferative and anti-apoptotic effects. We recently showed that Pim kinase inhibition sensitizes AML cells with FLT3-ITD to apoptosis induction by topoisomerase 2 (TOP-2) inhibitors by increasing DNA double-strand breaks (DSBs) and oxidative stress. Here, we studied whether altered DSB repair activity contributes to the increase in DNA damage. Methods: Ba/F3 cells expressing FLT3-ITD (Ba/F3-ITD) or wild-type FLT3 (Ba/F3-WT) were studied. The pan-Pim kinase inhibitor AZD1208 was provided by AstraZeneca. TOP-2 inhibitors included daunorubicin (DNR), mitoxantrone (MXR) and etoposide (VP-16). Nuclear expression of repair proteins was measured by immunoblotting. Functional changes in DSB repair activities were measured in cell lines with stable chromosomal integration of the green fluorescent protein (GFP)-based DSB repair reporters DR-GFP, EJ5-GFP and EJ2-GFP, measuring homologous recombination (HR), classical non-homologous end-joining (C-NHEJ) and alternative (Alt-) NHEJ, respectively. Results: Nuclear expression of the HR component Rad51 decreased progressively (to 40%) in Ba/F3-ITD cells after 12 hours of co-treatment with AZD1208 and DNR, compared to DNR alone. Progressive decrease (to 60%) in nuclear expression of the Alt-NHEJ component DNA ligase 3 and the C-NHEJ component Ku-70 was also seen with combination treatment. Functionally, co-treatment with AZD1208 and DNR led to a 50% decrease in Alt-NHEJ repair activity, compared to DNR alone, but no significant change in C-NHEJ or HR repair activity. Ba/F3-WT cells did not show a significant change in Alt-NHEJ, HR or C-NHEJ activity with combination treatment. Decreased Alt-NHEJ activity was also seen in Ba/F3-ITD, but not Ba/F3-WT, cells co-treated with AZD1208 and MXR or VP-16. Conclusions: While nuclear expression of multiple DNA DSB repair proteins decreased, only Alt-NHEJ repair activity decreased in FLT3-ITD cells co-treated with Pim kinase and TOP-2 inhibitors. Alt-NHEJ, a highly error-prone DNA DSB repair pathway, is upregulated in cells with FLT3-ITD and contributes to genomic instability in these cells. Decreasing Alt-NHEJ repair activity in cells treated with TOP-2 inhibitors has the potential to decrease the genomic instability that may contribute to disease progression in AML patients with FLT3-ITD. Citation Format: Kshama A. Doshi, Pratik K. Nagaria, Adriana E. Tron, Feyruz V. Rassool, Maria R. Baer. Concurrent treatment with Pim kinase inhibitor decreases alternative non-homologous end-joining repair of DNA damage induced by topoisomerase 2 inhibitors in cells with FLT3-ITD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4145. doi:10.1158/1538-7445.AM2017-4145

The FBXL subfamily is composed of 22 members including the well-characterized FBXL1 (also known as Skp2) and FBXL2 to FBXL21, each containing an F-box motif and a C-terminal Leu-rich repeat (LRR) domain (Fig. 3.1). Intensive studies have revealed an oncogenic role for Skp2, but the potential roles of other FBXL subfamily members in tumorigenesis have just begun to be appreciated. In this chapter, we primarily focus on summarizing the recent genetic, pathological as well as the biochemical evidence pinpointing a possible tumor suppressor or oncogenic role for each of the FBXL subfamily member proteins. In the following paragraphs, we discuss current advances in three major categories, including the physiological (mouse modeling), pathological (human clinical cancer relevance), and biochemical evidence (updated ubiquitin substrates). These three experimental evidence categories will provide insights to facilitate our understanding for their roles in tumorigenesis (Table 3.4). As stated in previous chapters, given the fact that physiological evidence (mouse modeling results) is considered as the strongest supportive data to implicate any given F-box protein in tumorigenesis (Tables 3.2 and 3.3), we choose to summarize FBXL members with available mouse genetic models.

Premature senescence functions as a tumor suppressor mechanism in response to oncogenic stimuli. It is characterized by irreversible cell cycle arrest mediated by tumor suppressors such as p53, Rb and the Promyelocytic Leukemia (PML) protein. PML mainly localizes in sub-nuclear structures known as PML nuclear bodies. These nuclear bodies accumulate in senescent cells largely due to increased PML gene transcription driven by p53 and/or the interferon pathways. PML exerts its pro-senescence activity by modulating both the p53 and Rb pathways, the major regulators of the cellular senescence program. Mechanistically, PML binds and promotes p53 modifications to generate a positive feedback loop, thereby triggering the senescence program. In addition, PML associates with Rb and may function in Rb/E2F-mediated gene silencing in senescent cells. Moreover, PML bodies recruit DNA damage sensing and repair proteins, thereby linking PML to the activation of the DNA damage response pathway, a pathway frequently activated in senescence. Therefore, elucidation of key factors controlling PML protein abundance will help to better understand how cells become transformed by avoiding senescence and allowing continued cellular proliferation in the presence of oncogenic signals. These findings will also be crucial in aiding scientists and physicians in the development of novel anti-cancer therapies by restoring PML to trigger senescence.

Plant homeodomain-leucine zipper proteins, unlike most animal homeodomains, bind DNA efficiently only as dimers. In the present work, we report that the deletion of the homeodomain N-terminal arm (first nine residues) of the homeodomain-leucine zipper protein Hahb-4 dramatically affects its DNA-binding affinity, causing a 70-fold increase in dissociation constant. The addition of the N-terminal arm of Drosophila Antennapedia to the truncated form restores the DNA-binding affinity of dimers to values similar to those of the native form. However, the Antennapedia N-terminal arm is not able to confer increased binding affinity to monomers of Hahb-4 lacking the leucine zipper motif, indicating that the inefficient binding of monomers must be due to structural differences in other parts of the molecule. The construction of proteins with modifications at residues 5 to 7 of the homeodomain suggests strongly that positively charged amino acids at these positions play essential roles in determining the DNA-binding affinity. However, the effect of mutations at positions 6 and 7 can be counteracted by introducing a stretch of positively charged residues at positions 1 to 3 of the homeodomain. Sequence comparisons indicate that all homeodomain-leucine zipper proteins might use contacts of the N-terminal arm with DNA for efficient binding. The occurrence of a homeodomain with a DNA-interacting N-terminal arm must then be an ancient acquisition in evolution, earlier than the separation of lines leading to metazoa, fungi and plants.

// Jiateng Zhong 1,2,* , Shavali Shaik 1,* , Lixin Wan 1 , Adriana E. Tron 1 , Zhiwei Wang 1 , Liankun Sun 2 , Hiroyuki Inuzuka 1 and Wenyi Wei 1 1 Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 2 Department of Pathophysiology, Norman Bethune College of Medicine, Jilin University, Changchun, P. R. China * These two authors contributed equally to this work Correspondence: Hiroyuki Inuzuka, email: // Correspondence: Wenyi Wei, email: // Keywords : tumor suppressor, MTSS1, ubiquitination, phosphorylation, migration Received : September 25, 2013 Accepted : November 4, 2013 Published : November 6, 2013 Abstract Metastasis suppressor 1 (MTSS1) is an important tumor suppressor protein, and loss of MTSS1 expression has been observed in several types of human cancers. Importantly, decreased MTSS1 expression is associated with more aggressive forms of breast and prostate cancers, and with poor survival rate. Currently, it remains unclear how MTSS1 is regulated in cancer cells, and whether reduced MTSS1 expression contributes to elevated cancer cell proliferation and migration. Here we report that the SCF β-TRCP regulates MTSS1 protein stability by targeting it for ubiquitination and subsequent destruction via the 26S proteasome. Notably, depletion of either Cullin 1 or β-TRCP1 led to increased levels of MTSS1. We further demonstrated a crucial role for Ser322 in the DSGXXS degron of MTSS1 in governing SCF β-TRCP -mediated MTSS1 degradation. Mechanistically, we defined that Casein Kinase Iδ (CKIδ) phosphorylates Ser322 to trigger MTSS1’s interaction with β-TRCP for subsequent ubiquitination and degradation. Importantly, introducing wild-type MTSS1 or a non-degradable MTSS1 (S322A) into breast or prostate cancer cells with low MTSS1 expression significantly inhibited cellular proliferation and migration. Moreover, S322A-MTSS1 exhibited stronger effects in inhibiting cell proliferation and migration when compared to ectopic expression of wild-type MTSS1. Therefore, our study provides a novel molecular mechanism for the negative regulation of MTSS1 by β-TRCP in cancer cells. It further suggests that preventing MTSS1 degradation could be a possible novel strategy for clinical treatment of more aggressive breast and prostate cancers.

Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) are present in acute myeloid leukemia (AML) cells in 30% of cases and are associated with high relapse rate and short disease-free survival following both chemotherapy and allogeneic hematopoietic stem cell transplantation. Inhibitors of FLT3 signaling have shown activity in clinical trials in FLT3-ITD AML, but efficacy has generally been limited and transient. Concurrent inhibition of other targets in FLT3-ITD signaling pathways is being explored as an approach to increasing the depth and duration of responses to FLT3 inhibitors. The oncogenic serine/threonine kinase Pim-1 is transcriptionally upregulated downstream of FLT3-ITD and phosphorylates and stabilizes FLT3, thereby promoting FLT3 signaling in a positive feedback loop in cells with FLT3-ITD. Pim kinase inhibitors are in clinical trials. We previously showed that combinations of clinically active Pim kinase and FLT3 inhibitors at pharmacologically relevant concentrations enhance apoptosis and decrease clonogenic growth of FLT3-ITD AML cell lines and primary patient cells in vitro and suppress growth of FLT3-ITD AML cells in vivo, in relation to treatment with FLT3 or Pim inhibitors alone. Here we studied the mechanistic effects of concurrent Pim kinase and FLT3 inhibition, demonstrating a novel mechanism of Mcl-1 downregulation in FLT3-ITD AML cells. Ba/F3-ITD cells, transfected with FLT3-ITD, were cultured with the pan-Pim kinase inhibitor AZD1208 at 1 μM, a concentration chosen based on in vitro and phase I clinical trial data, and/or the FLT3 inhibitor quizartinib at 1 nM, its IC50 concentration, and expression of the anti-apoptotic proteins Mcl-1, Bcl2 and Bcl-xL and the pro-apoptotic proteins BAD/S112 p-BAD, BAK, BAX and Bim was measured by western blot analysis. Mcl-1 expression decreased in a time-dependent manner with AZD1208 and quizartinib co-treatment, but not with treatment with either inhibitor alone, while levels of the other proteins did not change. Mcl-1 downregulation with Pim kinase and FLT3 inhibitor combination treatment was then confirmed in the human FLT3-ITD AML cell lines MV4-11 and MOLM-14. Mcl-1 expression is regulated at multiple levels, and we next sought to determine the mechanism(s) by which it is downregulated by concurrent Pim and FLT3 inhibition. While Mcl-1 protein levels decreased, Mcl-1 mRNA levels did not change, indicating post-transcriptional regulation. Additionally, levels of miR-29b, a negative regulator of Mcl-1 translation,decreased similarly in Ba/F3-ITD cells treated with AZD1208 and quizartinib, compared to quizartinib alone. Polysome profiling showed decreased total mRNA translation, but no selective reduction in Mcl-1 translation. In contrast, the progressive decrease in Mcl-1 protein expression with AZD1208 and quizartinib co-treatment was abrogated by addition of the proteasome inhibitor MG-132, demonstrating that Mcl-1 protein is downregulated by enhanced Mcl-1 proteasomal degradation. This mechanism was further confirmed by demonstration of an increase in ubiquitinated Mcl-1 prior to Mcl-1 downregulation in cells co-treated with AZD1208 and quizartinib, but not with each inhibitor alone or with DMSO control. The deubiquitinase USP9X decreases Mcl-1 ubiquitination and consequent proteasomal degradation, and we found that USP9X expression is downregulated prior to the increase in ubiquitinated Mcl-1 and the subsequent decrease in Mcl-1 protein levels during AZD1208 and quizartinib co-treatment, but was not altered by treatment with either inhibitor alone. In contrast, expression of the ubiquitin E3 ligases Mule/ARF-BP1, SCFβ-TrCP and Trim17, which mediate Mcl ubiquitination, did not change prior to Mcl-1 downregulation. Preclinical studies in our laboratory and others have shown in vitro and in vivo efficacy of combination treatment with Pim kinase and FLT3 inhibitors in FLT3-ITD AML, suggesting clinical promise of this approach. Here we show that, mechanistically, concurrent Pim kinase and FLT3 inhibition causes a post-translational decrease in expression of the anti-apoptotic protein Mcl-1 via enhanced proteasomal degradation, preceded by downregulation of the Mcl-1 deubiquitinase USP9X and an increase in ubiquitinated Mcl-1, a novel mechanism of Mcl-1 downregulation in FLT3-ITD AML cells. Disclosures Tron: AstraZeneca: Employment; AstraZeneca: Employment. Huszar:AstraZeneca: Employment.

Fbw7 is the substrate recognition component of the SCF (Skp1-Cullin-F-box)-type E3 ligase complex and a well-characterized tumor suppressor that targets numerous oncoproteins for destruction. Genomic deletion or mutation of FBW7 has been frequently found in various types of human cancers, however, little is known about the upstream signaling pathway(s) governing Fbw7 stability and cellular functions. Here we report that Fbw7 protein destruction and tumor suppressor function are negatively regulated by the prolyl isomerase Pin1. Pin1 interacts with Fbw7 in a phoshorylation-dependent manner and promotes Fbw7 self-ubiquitination and protein degradation by disrupting Fbw7 dimerization. Consequently, over-expressing Pin1 reduces Fbw7 abundance and suppresses Fbw7’s ability to inhibit proliferation and transformation. By contrast, depletion of Pin1 in cancer cells leads to elevated Fbw7 expression, which subsequently reduces Mcl-1 abundance, sensitizing cancer cells to Taxol. Thus, Pin1-mediated inhibition of Fbw7 contributes to oncogenesis and Pin1 may be a promising drug target for anti-cancer therapy.

CUL7 and the p53-associated, PARkin-like cytoplasmic protein (PARC) were previously reported to form homodimers and heterodimers, the first demonstration of cullin dimerization. Although a CUL7-based SKP1/CUL1/F-box (SCF)–like complex has been observed, little is known about the existence of a PARC-based SCF-like complex and how PARC interacts with CUL7-based complexes. To further characterize PARC-containing complexes, we examined the ability of PARC to form an SCF-like complex. PARC binds RBX1 and is covalently modified by NEDD8, defining PARC as a true cullin. However, PARC fails to bind SKP1 or F-box proteins, including the CUL7-associated FBXW8. To examine the assembly of PARC- and CUL7-containing complexes, tandem affinity purification followed by multidimensional protein identification technology were used. Multidimensional protein identification technology analysis revealed that the CUL7 interaction with FBXW8 was mutually exclusive of CUL7 binding to PARC or p53. Notably, although heterodimers of CUL7 and PARC bind p53, p53 is not required for the dimerization of CUL7 and PARC. The observed physical separation of FBXW8 and PARC is supported functionally by the generation of Parc−/−, Fbxw8−/− mice, which do not show exacerbation of the Fbxw8−/− phenotype. Finally, all of the PARC and CUL7 subcomplexes examined exhibit E3 ubiquitin ligase activity in vitro. Together, these findings indicate that the intricate assembly of PARC- and CUL7-containing complexes is highly regulated, and multiple subcomplexes may exhibit ubiquitin ligase activity. [Cancer Res 2007;67(5):2006–14]

Homeodomain-leucine zipper (HD-Zip) proteins, unlike most homeodomain proteins, bind a pseudopalindromic DNA sequence as dimers. We have investigated the structure of the DNA complexes formed by two HD-Zip proteins with different nucleotide preferences at the central position of the binding site using footprinting and interference methods. The results indicate that the respective complexes are not symmetric, with the strand bearing a central purine (top strand) showing higher protection around the central region and the bottom strand protected toward the 3′ end. Binding to a sequence with a nonpreferred central base pair produces a decrease in protection in either the top or the bottom strand, depending upon the protein. Modeling studies derived from the complex formed by the monomeric Antennapedia homeodomain with DNA indicate that in the HD-Zip/DNA complex the recognition helix of one of the monomers is displaced within the major groove respective to the other one. This monomer seems to lose contacts with a part of the recognition sequence upon binding to the nonpreferred site. The results show that the structure of the complex formed by HD-Zip proteins with DNA is dependent upon both protein intrinsic characteristics and the nucleotides present at the central position of the recognition sequence. Fil: Tron, Adriana E.. Universidad Nacional del Litoral; Argentina Fil: Comelli, Raul Nicolas. Universidad Nacional del Litoral; Argentina Fil: Gonzalez, Daniel Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina

Plant homeodomain-leucine zipper (HD-Zip) proteins, unlike many animal homeodomains (HDs), are unable to bind DNA as monomers. To investigate the molecular basis of their different behavior, we have constructed chimeras between the HD of the sunflower HD-Zip protein Hahb-4 and that of Drosophila engrailed (EN). Analysis of the interaction of these proteins with the pseudopalindromic Hahb-4 binding site and the monomeric EN binding site suggests that the loop located between helix I and helix II (amino acids 21-28) of EN is enough to confer efficient DNA binding activity to the Hahb-4 HD. Accordingly, the combined mutation of residues 24 and 25 of Hahb-4 to those present in EN (S24R/ R25Y) originated an HD able to interact with the EN binding site, while single mutations were ineffective. We have also determined that a protein with the leucine zipper and helix III of Hahb-4 fused to the rest of the EN HD binds to the Hahb-4 pseudopalindomic binding site with increased affinity and shows extended contacts with DNA respective to Hahb-4. We conclude that the loop located between helix I and helix II of the HD must be regarded as one of the segments that contribute to the present-day diversity in the properties of different HDs. Fil: Tron, Adriana E.. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina Fil: Welchen, Elina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Gonzalez, Daniel Hector. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina

Pim kinases have been shown to play a key role as downstream effectors of growth factor signaling in hematological malignancies. We have previously described AZD1208, a novel, orally bioavailable, highly selective pan Pim kinase inhibitor, undergoing clinical testing in AML. Specifically, we had demonstrated the anti-proliferative activity of AZD1208 in models of AML and had shown that AZD1208 treatment of AML cell lines results in the dose dependent reduction of pBAD at serine 112 and p4EBP1 at serine 65 as well as pp70S6K at threonine 389 and pS6 at serine 235/236, associated with global effects on protein translation (Keeton et al. Blood 2014). Since AZD1208 is about 10-15 fold more potent against Pim-1 than Pim-2 in both enzymatic and cellular assays, we sought to assess the dependency of each of these markers on Pim-1 and/or Pim-2 for phosphorylation using a Pim-1/3 selective inhibitor (Pim-1/3) that lacks Pim-2 inhibitory activity. In vitro studies demonstrate that while the modulation of pBAD does not require inhibition of Pim-2, the regulation of 4EBP1 as well as p70S6K and S6 are dependent, at least in part, on Pim-2 activity. As these proteins are canonically regulated by mTORC1, these data are consistent with Pim-2 acting upstream of this complex; perhaps through phosphorylation of TSC2 as reported for multiple myeloma cell lines (Lu et al. Blood 2013). These observations are also supported by in vivo data. Analysis of pBAD and p4EBP1 levels in Molm16 xenografts show that maximal inhibition of pBAD (70-80% inhibition) is achieved at AZD1208 plasma concentration of about 1000 ng/ml, consistent with the estimated cellular IC90 of pBAD inhibition in vitro. However, maximal inhibition of p4EBP1 is achieved at concentrations 3-5 fold higher. This observation is consistent with the differential potency of AZD1208 against Pim-1 and Pim-2, and with a requirement for Pim-2 inhibition for maximal p4EBP1 inhibition in this AML model. pBAD and p4EBP1 were selected as the pharmacodynamic endpoints for evaluating clinical target inhibition in patient samples. Bone marrow and peripheral blood samples were collected pre- and post-dose from relapsed, refractory AML patients enrolled in the AZD1208 Phase I dose escalation. Analysis of pBAD inhibition on day 1 of dosing shows at least 60-70% inhibition across all of the dosing cohorts. With clinical exposures on day 1 approaching 1000 ng/ml at the first dose level and exceeding 1000 ng/ml as the dose increases, the extent of pBAD inhibition seen in patients appears to be consistent with Pim-1 inhibition, as seen in preclinical models. Furthermore, and also similar to our preclinical observations, maximal inhibition of p4EBP1 in patients is achieved only at higher exposures. These data strengthen the hypothesis that BAD phosphorylation is primarily dependent on Pim-1, whereas suppression of Pim-2 activity is required for maximal inhibition of p4EBP1 in AML cells. In summary, Pim inhibition in AML cell line models and in patients treated with AZD1208 results in the inhibition of the downstream targets of Pim signaling, pBAD and p4EBP1. Invitro and in vivo, the inhibition of pBAD is consistent with inhibition of Pim-1 while inhibition of p4EBP1 indicates a requirement for Pim-2 inhibition as well. These observations are validated in patients and provide further evidence for the relevance of these biomarkers as a measure of Pim signaling in AML. Disclosures McEachern: AstraZeneca: Employment, Equity Ownership. O'Connor:AstraZeneca: Employment, Equity Ownership. DuPont:AstraZeneca: Employment, Equity Ownership. Gibbons:AstraZeneca: Employment, Equity Ownership. Pablo:AstraZeneca: Employment, Equity Ownership. Vishwanathan:AstraZeneca: Employment, Equity Ownership. McCoon:AstraZeneca: Employment, Equity Ownership. Cortes:AstraZeneca: Research Funding. Neumann:AstraZeneca: Employment, Equity Ownership. Keating:AstraZeneca: Employment, Equity Ownership. Pease:AstraZeneca: Employment, Equity Ownership. Brown:AstraZeneca Pharmaceuticals: Employment, Patents & Royalties. Barrett:AstraZeneca: Employment, Equity Ownership.

Internal tandem duplication (ITD) mutations of the receptor tyrosine kinase fms-like tyrosine kinase 3 (FLT3) are present in acute myeloid leukemia (AML) cells in 30% of cases and are associated with high relapse rate and short disease-free survival. FLT3 inhibitors have clinical activity, but their activity is limited and transient. New therapeutic approaches combining FLT3 inhibitors and inhibitors of downstream or parallel signaling pathways may increase depth and duration of responses. The Pim-1 serine/threonine kinase is transcriptionally upregulated by FLT3-ITD. We previously demonstrated that Pim-1 phosphorylates and stabilizes FLT3 and thereby promotes its signaling in a positive feedback loop. Pim kinase inhibitors are in clinical trials. Here we studied the effect of combinations of the Pim kinase inhibitor AZD1208 and clinically active FLT3 inhibitors on AML with FLT3-ITD in vitro and in vivo. Ba/F3-ITD cells, with FLT3-ITD, were grown in medium with the Pim kinase inhibitor AZD1208 at 1 μM and/or the FLT3 inhibitors quizartinib (Q), sorafenib (S) or crenolanib (C) at their IC50values of 1, 2.5 and 20 nM, respectively, and viable cells were measured at serial time points. While Q, S, C or AZD1208 treatments reduced cell numbers, compared to DMSO control, combined AZD1208 and Q, S or C treatments abrogated proliferation. Because FLT3-ITD cells remain responsive to FLT3 ligand (FLT3L) despite constitutive FLT3 activation and increased FLT3L levels following chemotherapy have been hypothesized to contribute to relapse, we repeated the proliferation experiments in the presence of 0, 1, 3 and 10 ng/ml FLT3L. FLT3L produced a concentration-dependent increase in proliferation and, while Q, S, C or AZD1208 treatments individually reduced cell numbers, combined AZD1208 and Q, S or C abrogated proliferation at all FLT3L concentrations tested, suggesting that these combinations overcome growth stimulation by FLT3L. To understand the anti-proliferative effect of combined Pim-1 and FLT3 inhibitors, we first studied cell cycle effects of AZD1208 and Q, S or C in Ba/F3-ITD cells and of AZD1208 and Q in the additional FLT3-ITD cell lines 32D-ITD, MV4-11 and MOLM14. We found a progressive increase in sub-G1 phase cells at 24, 48 and 72 hours, consistent with induction of apoptosis. Synergistic induction of apoptosis was confirmed by Annexin V/propidium iodide labeling of Ba/F3-ITD and 32D-ITD cells treated for 48 hours with AZD1208 combined with Q (p Mechanistically, combined AZD1208 and Q treatment in vitro did not increase reactive oxygen species, compared to each drug alone, but increased both cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP) levels, and caspase 3 cleavage was reduced by co-incubation with the pan-caspase inhibitor Z-VAD. Moreover, combined AZD1208 and Q treatment caused a synergistic decrease in expression of the anti-apoptotic Mcl-1 and of Bcl-xL proteins, but did not significantly alter Bim-1, p-Bad, Bad, Bax, Bak or Bcl-2, pro- and anti-apoptotic protein levels. Bcl-xL mRNA expression decreased along with protein levels, but Mcl-1 mRNA levels remain unchanged, indicating post-transcriptional down-regulation of Mcl-1 by the combination treatment. In summary, synergistic cytotoxicity of AZD1208 and clinically active FLT3 inhibitors was demonstrated in FLT3-ITD cell lines and patient samples in vitro and in cell lines in vivo, via caspase-mediated apoptosis, associated with a synergistic decrease in Mcl-1 and Bcl-xL expression. Our data suggest clinical promise for combination therapy with Pim kinase and FLT3 inhibitors in patients with AML with FLT3-ITD. Disclosures No relevant conflicts of interest to declare.

Four groups of plant homeodomain proteins contain a dimerization motif closely linked to the homeodomain. We here show that two sunflower homeodomain proteins, Hahb-4 and HAHR1, which belong to the Hd-Zip I and GL2/Hd-Zip IV groups, respectively, show different binding preferences at a defined position of a pseudopalindromic DNA-binding site used as a target. HAHR1 shows a preference for the sequence 5′-CATT(A/T)AATG-3′, rather than 5′-CAAT(A/T)ATTG-3′, recognized by Hahb-4. To analyze the molecular basis of this behavior, we have constructed a set of mutants with exchanged residues (Phe→Ile and Ile→Phe) at position 47 of the homeodomain, together with chimeric proteins between HAHR1 and Hahb-4. The results obtained indicate that Phe47, but not Ile47, allows binding to 5′-CATT(A/T)AATG-3′. However, the preference for this sequence is determined, in addition, by amino acids located C-terminal to residue 53 of the HAHR1 homeodomain. A double mutant of Hahb-4 (Ile47→Phe/Ala54→Thr) shows the same binding behavior as HAHR1, suggesting that combinatorial interactions of amino acid residues at positions 47 and 54 of the homeodomain are involved in establishing the affinity and selectivity of plant dimeric homeodomain proteins with different DNA target sequences. Fil: Tron, Adriana. Universidad Nacional del Litoral; Argentina Fil: Bertoncini, Carlos Walter. Universidad Nacional del Litoral; Argentina Fil: Palena, Claudia. Universidad Nacional del Litoral; Argentina Fil: Chan, Raquel Lia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina Fil: Gonzalez, Daniel Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina