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Supplementary Table S1. Overall activity and predictors of treatment failure (PD or less than partial response by IWG criteria). Supplementary Figure S1. BCL-2 family protein index at screening. Supplementary Figure S2. Mean (+ SD) venetoclax plasma concentration-time profile at week 6 day 1 (800 mg). Supplementary Figure S3. Study design.

Supplementary Data from Acquired Resistance to Combination Treatment with Temozolomide and ABT-888 Is Mediated by Both Base Excision Repair and Homologous Recombination DNA Repair Pathways

The covalent inhibitor of Bruton’s tyrosine kinase ibrutinib and the specific Bcl-2 inhibitor venetoclax are both highly efficacious single-agent drugs in the treatment of chronic lymphocytic leukemia (CLL). Based on their complementary modes of action, ibrutinib and venetoclax are hypothesized to act in a synergistic fashion. Currently, it is unclear whether combined treatment is indeed superior to continuous single-agent treatment and what mechanisms underlie the resistance to combination treatment. In addition, the effects of such treatment on the skewed T-cell compartment characteristic of CLL are as yet unknown. In the murine Eµ-TCL1 adoptive transfer model resembling aggressive CLL, we found that combined treatment resulted in the deepest responses, with the longest duration related to a combination of decreased proliferation and increased induction of apoptosis. In addition, alterations in T-cell subsets were most prominent after combination treatment, with increased naive cells and reduced effector memory cells. Remarkably, effects of single agents but also combination treatment were eventually interrupted by relapse, and we found downregulation of BIM expression as a plausible cause of acquired drug resistance. Nevertheless, in this murine model, the combination of venetoclax and ibrutinib has increased efficacy over single agents, accompanied by a restoration of the T-cell compartment.

One of the most recent advances in the treatment of HER2+ breast cancer is the development of the antibody-drug conjugate, T-DM1, composed of trastuzumab (T) linked to the cytotoxic maytansinoid (DM1). T-DM1 has proven clinical benefits for patients with advanced and/or metastatic breast cancer who have progressed on prior HER2-targeted therapies. However, T-DM1 resistance ultimately occurs and represents a major obstacle in the effective treatment of this disease. We previously identified BCL2 upregulation as a critical component and biomarker of the adaptive response to inhibition of PI3K/mTOR or HER2, and thus examined whether BCL2/XL combinatorial strategies could improve the initial efficacy of T-DM1. Here, we demonstrate that combined inhibition of BCL2/XL plus T-DM1 significantly enhances the cytotoxicity of T-DM1 in vivo. The effectiveness of T-DM1 plus BCL2/XL inhibition was evaluated in two patient-derived xenograft (PDX) models of advanced HER2+ER- resistant disease (PDX8;PDX12). Animals were randomized into one of four groups: T-DM1, ABT-737, T-DM1 + ABT-737 or vehicles. Our initial results after a 14d treatment period indicate that combined treatment with T-DM1 and ABT-737, the dual BCL2/XL inhibitor, confers an exceptional tumor cell cytotoxic advantage characterized by widespread elimination of the tumor cells. To evaluate whether ABT-263, the clinically relevant BCL2/XL inhibitor, mimics ABT-737, we randomized animals into one of four groups: T-DM1, ABT-263, T-DM1 + ABT-263 or vehicles. To minimize thrombocytopenia that is induced by ABT-263, we included a fifth group that received pulsed treatment of ABT-263 + T-DM1. Notably, unlike continuous treatment, pulsed administration of ABT-263 reduced weight loss to vehicle levels and allowed recovery of platelets. Evaluation of pathological responses by H&E staining indicated that T-DM1 + ABT-263 mimics T-DM1 + ABT-737. To better distinguish tumor cells from stromal elements, we used epithelial membrane antigen IHC to specifically visualize tumor cells and Trichrome stain to visualize stromal content and scored the tissue sections blindly. ABT-263 had no observable effect. T-DM1 induced a 38.75% and 20% average reduction in tumor cell content in the two PDX models, whereas the combined treatment caused a 74% and 54% average reduction after the 14d treatment period. The loss of tumor cell content was associated with an increased stromal reaction at the tumor bed. T-DM1 treated tumors contained 27.5% and 47.5% average stromal content, whereas combination treated tumors contained 86% and 85.6% average stromal content. Importantly, T-DM1 + pulsed ABT-263 elicited a similar response as continuous treatment in the PDX8 model, but was not as effective in PDX12. The dramatic improvement in tumor regression observed in these preclinical studies, together with the safety benefits of modified dosing of ABT-263, provides substantial rationale for the clinical investigation of this drug combination. Citation Format: Jason J. Zoeller, Roderick T. Bronson, Deepak Sampath, Joel Leverson, Joan S. Brugge. Neutralization of BCL2/XL enhances the cytotoxicity of T-DM1 in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4358.

Proteins in the BCL-2 family are key regulators of apoptosis, or programmed cell death. We have conducted studies to evaluate a highly potent and orally available BCL-2 selective inhibitor, venetoclax (ABT-199), for efficacy and mechanism of action in the lupus prone NZB/W F1 mouse model and in human cells in vitro. Venetoclax treatment dose-dependently reduced the incidence of severe proteinuria and prolonged survival compared to vehicle controls and attenuated glomerulonephritis, tubular dilatation, immune cell infiltrates and IgG deposition in the kidney. Venetoclax mediated a significant reduction in the numbers of splenic T cells but conferred a preferential reduction in select B cell subsets. Interestingly, venetoclax did not impair the number of CD138+ long-lived plasma cells in the bone marrow, which was consistent with unaltered circulating anti-dsDNA titers in these animals. Venetoclax efficacy also correlated with a dose -dependent reduction of lymphocytes in peripheral blood of NZB/WF1 mice. Consistent with these findings, human lymphocytes from both healthy donors and systemic lupus erythematosus (SLE) patients treated with venetoclax in vitro have reduced lifespan. Taken together, these data underscore the essential role of BCL-2 in the pathogenesis of lupus and support further exploration of selective BCL-2 inhibition in autoimmune diseases such as SLE/Lupus Nephritis. The design, study conduct, and financial support for this research were provided by AbbVie.

The expression of Bcl-2 family proteins is perturbed in multiple types of cancers, including leukemias, and is associated with disease progression and resistance to chemotherapy. ABT-199 (GDC-0199) is a new BH3 mimetic that was developed to specifically target Bcl-2 while sparing Bcl-XL, hence avoiding thrombocytopenia intrinsic to 1st generation BH3 mimetics like ABT-737 (Souers et al., Nat Med, 2013). In this study, we report proteomic profiling of Bcl-2 family members in a large series of ALL patients (pts) and pre-clinical activity of ABT-199. Expression of 20 pro- and anti-apoptotic proteins was studied in 186 newly diagnosed ALL using reverse phase protein arrays (RPPA). Supervised clustering demonstrated distinct differences in 11 proteins in ALL with different cytogenetic and FAB characteristics (Fig. 1, p Figure 1 RPPA profiling of apoptosis regulators in ALL. Heatmap of differentially expressed proteins based on cytogenetics and immunophenotype. Black box, Burkitt's leukemia; red box, t(4;11). Figure 1. RPPA profiling of apoptosis regulators in ALL. Heatmap of differentially expressed proteins based on cytogenetics and immunophenotype. Black box, Burkitt's leukemia; red box, t(4;11). The potential of ABT-199 to disrupt interactions between Bcl-2 and different pro-apoptotic proteins was studied using Bimolecular Fluorescence Complementation (BiFC, J Biol Chem 288:4935, 2013). The coding sequences for human Bcl-2, Bim, Bak, Bax and Noxa were subcloned into BiFC plasmids containing Venus fragments and transfected into HeLa cells. Approximately 60-70% of transfected cells were positive for Venus fluorescence due to association between Bcl-2 and Bim, Noxa, Bax or Bak. ABT-199 (2.5 µM, 24 hrs) significantly reduced Venus signal, indicating an inhibition of the interactions of Bcl-2 with these proteins, most potently with the multidomain proteins Bax and Bak (95%±18% and 85%±15% inhibition, respectively). ABT-199 rapidly induced apoptotic cell death in ALL cell lines and in primary ALL samples. Pre-B ALL cells (Nalm-6, REH, SEMK2 and RS4;11) were sensitive to ABT-199 and ABT-737 (IC50 0.007-1.4µM (199) and 0.035-0.7µM (737)). Notably, ABT-199 was more cytotoxic than ABT-737 against MLL-rearranged SEMK2 and RS4;11 cells, consistent with the notion of the greater Bcl-2 dependency of these cells. Lentiviral silencing of Bcl-XL sensitized REH cells to apoptosis by ABT-199 and ABT-737. T-ALL cells (PF-382, MOLT-4, P-12) expressed lower levels of Bcl-2 and were uniformly less sensitive to ABT-199 compared to ABT-737 (IC50 3.7±1.1µM vs 0.7±0.3µM, p=0.01). Burkitt's lymphoma cells Ramos and Raji had low Bcl-2 and high Mcl-1 expression, and were resistant to both agents (IC50>4µM). Next, the cytotoxic activity of ABT-199 was tested against a panel of 12 genetically diverse primary ALL samples, including 6 from pts with relapsed or refractory disease. Ten out of twelve samples (83%) were exquisitely sensitive to both agents, with IC50 values of 0.0001-0.14µM for ABT-199 and 0.0004-0.3µM for ABT-737. One of the four Ph+ samples was resistant to both agents, and one of the two T-ALL was less sensitive to ABT-199 compared to ABT-737. Two samples with t(4;11) were highly sensitive to ABT-199. All primary ALL samples tested (n=7) expressed high levels of Bcl-2, and no significant correlation between sensitivity and expression of Bcl-2 family members was found. Importantly, three human-derived xenografts from pediatric pre-B-ALL samples (1345, 1809, 0398) were very sensitive to ABT-199 (IC50 3nM, 0.1nM and 2.3nM, respectively). Finally, anti-leukemia activity of ABT-199 was tested in MLL-rearranged patient-derived xenograft NSG mice. Treatment with ABT-199 at 100mg/kg/d by oral gavage days 13-23 significantly reduced leukemia tumor burden as determined by bioluminescence imaging (average 70% reduction in BLI signal in 4 ABT-treated mice compared to 4 control animals at 9 weeks, p=0.03). In summary, proteomic profiling and patterns of sensitivity to Bcl-2 inhibition indicate that ALL, with exception of Burkitt's lymphoma, represents a Bcl-2 dependent disease. These results provide strong rationale for introducing ABT-199, which recently showed impressive efficacy in CLL trials, into the clinical armamentarium of ALL therapy. Disclosures: Konopleva: AbbVie, Inc: Research Funding. Leverson:AbbVie, Inc.: Employment, Equity Ownership.