ALBERT

Description: Background: BCR-ABL+ acute lymphoblastic leukemia (ALL) in adults has a poor prognosis with allogeneic stem cell transplantation (SCT) considered the best curative option for suitable patients. BH3 mimetics induce mitochondrial outer membrane permeabilization (MOMP) linked to apoptosis induction by releasing BH3-only proteins BIM and/or BID from the anti-apoptotic factors BCL2, BCL-XL, MCL1, BCLW and BFL1. The BCL2-specific BH3 mimetic venetoclax (ABT-199) may provide an opportunity to improve pharmacotherapy of BCR-ABL+ ALL in particular for elderly patients not suitable for SCT. Aim: We aimed to rationally design optimized combination therapies for BCR-ABL+ ALL based on the molecular mechanisms of apoptosis induction by BH3 mimetics. Methods: We first biochemically characterized binding of BIM, a BH3 only activator of mitochondrial apoptosis, to BCL2, BCLXL and MCL1 and its release by BH3 mimetics in two BCR-ABL+ ALL cell lines. We next visualized and quantitated MOMP-induction by BH3 mimetics in viable cells. We then characterized the effects of dexamethasone and tyrosine kinase inhibitors (TKI) imatinib and dasatinib on BIM expression and calculated dose-effect combination indices (CI) for combination therapies in cell lines and two BCR-ABL+ ALL primograft models co-cultured with mesenchymal stem cells ex vivo. We finally used in vivo bioluminescence and survival analyses in murine xenotransplantation models to evaluate therapeutic efficacy in vivo. Results: In BCR-ABL+ BV173 and SUPB15 cells BIM but not BID binds to BCL2. BIM is rapidly released from BCL2 by venetoclax in a time and dose dependent manner. Release of BIM induces both MOMP (as defined by a decrease in mitochondrial membrane potential) and apoptosis (as defined by PARP cleavage and propidium iodide staining). Furthermore, BIM is strongly required for cytotoxicity of venetoclax, dasatinib and dexamethasone. Primary BCR-ABL+ ALL cells are more resistant against MOMP induction by venetoclax than BCR-ABL-negative ones, and BIM expression is reduced in these cells. Both, TKIs and dexamethasone augment BIM expression in BV173 and SUPB15 cells and act synergistically with venetoclax in cell lines and two BCR-ABL+ primografts (CI for the triple combination therapy of venetoclax, dexamethasone and dasatinib between 0.1 and 0.15, CI〈 1.0 considered synergistic). Triple combinations with venetoclax, dexamethasone and TKIs efficiently attenuate leukemia progression in xenotransplantation models and, notably, the dasatinib- but not the imatinib-containing combination led to treatment- and leukemia-free long-term survival in a BCR-ABL+ mouse model. Conclusions: These data demonstrate efficacy of venetoclax in ALL. Although BCR-ABL inhibits venetoclax cytotoxicity, this inhibition can be overcome by triple combination therapy with venetoclax, dexamethasone and dasatinib. Since the triple combination therapy can be curative in preclinical xenotranplatation models clinical studies with oral chemotherapy-free regimens may be considered in particular for elderly patients not suitable for allogeneic SCT. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees.

Description: Despite advances in both targeted therapies with ABL-specific tyrosine kinase inhibitors and in allogeneic stem cell transplantation, BCR-ABL positive acute lymphoblastic leukemia (ALL) remains a very high-risk disease, necessitating the development of novel treatment strategies. miRNAs are small non-coding RNAs which regulate gene expression posttranscriptionally in a sequence-specific manner. miRNAs usually repress the expression of many target genes. We hypothesized that miRNAs may help to identify potential therapeutic targets if (i) they are expressed in a disease-specific manner and if (ii) modulating their expression induces a desired phenotype, such as apoptosis of tumour cells, in appropriate experimental models. Based on our observation that miR∼17-92-encoded miRNAs are significantly less abundant in primary BCR-ABL-positive as compared to negative ALL-cells, we studied the expression and function of miRNAs encoded by the miR∼17-92 derivative miR∼17-19b in a murine pro-B-cell line with inducible BCR-ABL-expression (TonB). Induction of BCR-ABL expression in TonB cells reduced endogenous miR-17, miR-18a, and miR-19 by 2 to 3.5-fold, confirming that expression of the miR∼17-92 cluster is controlled by BCR-ABL. Interestingly, over-expression of miR∼17-19b by lentiviral gene transfer led to a substantial induction of apoptosis in TonB cells in a BCR-ABL-dependent manner. To identify potential miRNA targets, we used a proteomic approach based on stable isotope labeling of amino acids in cell culture (SILAC) followed by liquid chromatography and mass spectroscopy (LC-MS) in miR∼17-19b transgenic TonB cells. Several apoptosis-related proteins were differentially expressed including Bcl2, an established inhibitor of mitochondrial pro-apoptotic pathways. The miRNA target prediction program RNA22 predicted several miR∼17-19b miRNA-binding sites within both murine and human Bcl2 mRNA, and we demonstrated direct miRNA binding to Bcl2 mRNA by luciferase reporter and anti-AGO2 RIP chip analyses. As with miR∼17-19b over-expression, Bcl2 specific RNAi strongly induced apoptosis in murine TonB and the human BCR-ABL-positive cell lines BV-173, Tom1 and SupB15. BCR-ABL positive human ALL-cell lines were also more sensitive than negative ones to pharmacological BCL2 inhibition with the BH3 mimetic ABT 737. In addition, inhibition of BCL2 by ABT 737 and BCR-ABL kinase activity by Imatinib exert different anti-leukemic effects with differential impact on miR∼17-92 miRNA-expression. To assess the therapeutic potential of BCL2 inhibition we used a xenotransplantation assay with real time in vivo monitoring of drug therapies by bioluminescent imaging. ABT-737 treatment substantially inhibited expansion of luciferase-expressing human primary BCR-ABL-positive ALL xenografts in NOD/LtSz-scid IL-2Rγ null (NSG) mice and significantly lengthened their median survival. Taken together, our data identify BCL2 as a therapeutic target of particular relevance in BCR-ABL-positive ALL and indicate involvement of miR∼17-92-encoded miRNAs in regulation of apoptosis in these cells. The validity of this miRNA-based approach to identify potential drug targets is demonstrated by the efficacy of the BCL2 inhibitor ABT-737 in an in vivo model of human BCR-ABL positive ALL, suggesting that BCL2 inhibition should be considered for early phase clinical testing as a strategy to improve disease outcomes. Disclosures: No relevant conflicts of interest to declare.

Description: Cardiac levels of the signal transducer and activator of transcription factor-3 (STAT3) decline with age, and male but not female mice with a cardiomyocyte-specific STAT3 deficiency conditional knockout (CKO) display premature age-related heart failure associated with reduced cardiac capillary density. In the present study, isolated male and female CKO-cardiomyocytes exhibit increased prostaglandin (PG)-generating cyclooxygenase-2 (COX-2) expression. The PG-degrading hydroxyprostaglandin-dehydrogenase-15 (HPGD) expression is only reduced in male cardiomyocytes, which is associated with increased prostaglandin D2 (PGD2) secretion from isolated male but not female CKO-cardiomyocytes. Reduced HPGD expression in male cardiomyocytes derive from impaired androgen receptor (AR)–signaling due to loss of its cofactor STAT3. Elevated PGD2 secretion in males is associated with increased white adipocyte accumulation in aged male but not female hearts. Adipocyte differentiation is enhanced in isolated stem cell antigen-1 (SCA-1)+ cardiac progenitor cells (CPC) from young male CKO-mice compared with the adipocyte differentiation of male wild-type (WT)-CPC and CPC isolated from female mice. Epigenetic analysis in freshly isolated male CKO-CPC display hypermethylation in pro-angiogenic genes (Fgfr2, Epas1) and hypomethylation in the white adipocyte differentiation gene Zfp423 associated with up-regulated ZFP423 expression and a shift from endothelial to white adipocyte differentiation compared with WT-CPC. The expression of the histone-methyltransferase EZH2 is reduced in male CKO-CPC compared with male WT-CPC, whereas no differences in the EZH2 expression in female CPC were observed. Clonally expanded CPC can differentiate into endothelial cells or into adipocytes depending on the differentiation conditions. ZFP423 overexpression is sufficient to induce white adipocyte differentiation of clonal CPC. In isolated WT-CPC, PGD2 stimulation reduces the expression of EZH2, thereby up-regulating ZFP423 expression and promoting white adipocyte differentiation. The treatment of young male CKO mice with the COX inhibitor Ibuprofen or the PGD2 receptor (DP)2 receptor antagonist BAY-u 3405 in vivo increased EZH2 expression and reduced ZFP423 expression and adipocyte differentiation in CKO-CPC. Thus, cardiomyocyte STAT3 deficiency leads to age-related and sex-specific cardiac remodeling and failure in part due to sex-specific alterations in PGD2 secretion and subsequent epigenetic impairment of the differentiation potential of CPC. Causally involved is the impaired AR signaling in absence of STAT3, which reduces the expression of the PG-degrading enzyme HPGD.