ALBERT

Description: In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Kandela et al., 2015) that described how we intended to replicate selected experiments from the paper "BET bromodomain inhibition as a therapeutic strategy to target c-Myc" (Delmore et al., 2011). Here we report the results of those experiments. We found that treatment of human multiple myeloma (MM) cells with the small-molecular inhibitor of BET bromodomains, (+)-JQ1, selectively downregulated MYC transcription, which is similar to what was reported in the original study (Figure 3B; Delmore et al., 2011). Efficacy of (+)-JQ1 was evaluated in an orthotopically xenografted model of MM. Overall survival was increased in (+)-JQ1 treated mice compared to vehicle control, similar to the original study (Figure 7E; Delmore et al., 2011). Tumor burden, as determined by bioluminescence, was decreased in (+)-JQ1 treated mice compared to vehicle control; however, while the effect was in the same direction as the original study (Figure 7C-D; Delmore et al., 2011), it was not statistically significant. The opportunity to detect a statistically significant difference was limited though, due to the higher rate of early death in the control group, and increased overall survival in (+)-JQ1 treated mice before the pre-specified tumor burden analysis endpoint. Additionally, we evaluated the (−)-JQ1 enantiomer that is structurally incapable of inhibiting BET bromodomains, which resulted in a minimal impact on MYC transcription, but did not result in a statistically significant difference in tumor burden or survival distributions compared to treatment with (+)-JQ1. Finally, we report meta-analyses for each result.

Description: In 2015, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Kandela et al., 2015) that described how we intended to replicate selected experiments from the paper “Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs“ (Sugahara et al., 2010). Here we report the results of those experiments. We found that coadministration with iRGD peptide did not have an impact on permeability of the chemotherapeutic agent doxorubicin (DOX) in a xenograft model of prostate cancer, whereas the original study reported that it increased the penetrance of this cancer drug (Figure 2B; Sugahara et al., 2010). Further, in mice bearing orthotopic 22Rv1 human prostate tumors, we did not find a statistically significant difference in tumor weight for mice treated with DOX and iRGD compared to DOX alone, whereas the original study reported a decrease in tumor weight when DOX was coadministered with iRGD (Figure 2C; Sugahara et al., 2010). In addition, we did not find a statistically significant difference in TUNEL staining in tumor tissue between mice treated with DOX and iRGD compared to DOX alone, while the original study reported an increase in TUNEL positive staining with iRGD coadministration (Figure 2D; Sugahara et al., 2010). Similar to the original study (Supplemental Figure 9A; Sugahara et al., 2010), we did not observe an impact on mouse body weight with DOX and iRGD treatment. Finally, we report meta-analyses for each result.

Description: Background: The BCL-2 family of anti-apoptotic proteins (e.g., BCL-2, BCL-XL, MCL-1) are important regulators of lymphocyte development and are therapeutic targets for hematologic malignancies, including multiple myeloma (MM). Venetoclax (Ven) is a highly selective, potent, oral BCL-2 inhibitor that induces apoptosis in MM cells and has shown synergistic activity with bortezomib (Velcade; V) and dexamethasone (Dex or d). Combination of the CD38 monoclonal antibody (mAb) daratumumab (D) with Ven is hypothesized to further increase anti-myeloma activity based upon dual mechanisms of pro-apoptotic effects on tumor cells as well as enhanced immune stimulation. Pre-clinically and in healthy human subjects, Ven treatment leads to enrichment of CD8+ T effector memory cells, while resulting in loss of CD4+ and CD8+ naïve T-cells, however the potential immunomodulatory effect of Ven in MM is unknown. Results presented herein describe the pharmacodynamic changes observed in immune cell subsets in relapsed/refractory (R/R) MM patients (pts) treated with VenDex, VenDd, and VenDVd. Methods: Peripheral blood samples were collected at day 1 of cycles 1-5 to characterize pharmacodynamic changes in B- and T-cell sub-populations by multicolor flow cytometry in Ven MM clinical trials M13-367 (NCT01794520) and M15-654 (NCT03314181). M13-367 is a phase 1/2 study of VenDex in t(11;14) R/R MM, and M15-654 is a phase 1/2 study of VenDd in t(11;14) R/R MM (Part 1) and VenDVd in R/R MM (Part 2). As of 12 June 2019, 15 out of 31 pts treated with VenDex in phase 2 of M13-367, 19 out of 24 pts treated in Part 1 (VenDd), and 19 out of 24 pts treated in Part 2 (VenDVd) of M15-654 had baseline and post-treatment specimens available for analysis. Results: Consistent with previous findings that B-cells are highly dependent upon BCL-2 for cell survival, each Ven-containing regimen (VenDex, VenDd, and VenDVd) resulted in rapid and sustained reduction (~90% decrease from baseline by end of cycle 1) in peripheral B-cell (CD19+/CD5-) counts (Figure). In subgroup analyses, naïve B-cells (CD27-/IgM+) were significantly reduced in pts treated with each regimen, however regulatory B-cells (CD27+/CD24+) remained largely unaffected. In addition, a decrease in plasmablasts (CD27+CD38+CD20-) were only observed in pts treated with D-containing regimens (VenDd and VenDVd), which is consistent with expression of CD38 in this subgroup. In contrast to B-cells, CD3+ T-cells were minimally reduced (~20-30% reduction from baseline through end of cycle 4) with VenDex and VenDd treatment. A more prominent reduction was observed in pts treated with VenDVd (~50-60% reduction from baseline through end of cycle 4). CD4+ T-cells were more sensitive than CD8+ T-cells to VenDex and VenDd, while both CD4+ and CD8+ T-cells were reduced in VenDVd treated pts (Figure). T-cell subgroup analyses were also performed, including naïve T-cells (CD45RA+/CD197+), effector (CD45RA+/CD197-), effector memory (CD45RA-/CD197-), and central memory (CD45RA-/CD197+). Overall, there were no significant changes in the composition of the T-cell pool (Tnaive, TCM, TEM, TEMRA) or in Th1 (CD4+/CD183+/CD196-), Th2 (CD4+/CD183-/CD196-), and Th17 (CD4+/CD183-/CD196+) cells in pts treated with VenDex, VenDd, or VenDVd. Finally, a decrease in Tregs (CD4+/CD25+/CD127low/CD194+) was observed in VenDd and VenDVd treated pts, but not in VenDex treated pts (Figure). While no reduction in overall Tregs was observed with VenDex, a decrease in the relative proportion of activated Tregs (HLA-DR+/CD45RO+) to naïve (HLA-DR-/CD45RO-) and memory (HLA-DR-/CD45RO+) Tregs was observed. Conclusions: Comprehensive immunophenotyping studies were conducted to characterize the potential effects of Ven in B- and T-cell subsets when used in combination with standard of care anti-myeloma agents of various mechanisms of action (i.e. glucocorticoid, CD38 mAb and/or proteasome inhibitor). While each Ven combination regimen resulted in significant reductions in circulating B-cells, T-cell effects were largely dependent upon the combination regimen. Minimal reduction in total T-cell counts were observed with VenDex and VenDd, which were primarily due to reduction in CD4+ T-cells. However, the extent of T-cell depletion (CD4+ and CD8+ T-cells) was greatest in pts treated with the bortezomib-containing regimen VenDVd. Correlative studies between immune profiles and pt outcomes are ongoing. Disclosures Vishwamitra: AbbVie: Employment, Other: stock or other options. Mantis:AbbVie Inc: Employment, Other: Stock/stock options. Haribhai:AbbVie: Employment, Other: stock or other options. Uziel:AbbVie: Employment, Other: stock or other options. Leverson:AbbVie Inc: Employment, Other: Stock or options. Pauff:AbbVie Inc: Employment, Other: may own stock or stock options. Bueno:AbbVie: Employment, Other: Stock/stock options. Maciag:AbbVie: Employment, Other: Stock/stock options. Ross:AbbVie: Employment, Other: Stock/stock options.

Description: Background Acute myeloid leukemia (AML) is a highly heterogeneous disease with multiple prognostic indicators of outcomes to conventional treatments; including age, cytogenetic risk, and presence of genomic abnormalities (NPM1, TP53, FLT3 and IDH1/2). Additionally, overexpression of BCL-2 is a predictor for poor response to chemotherapy and can lead to therapeutic resistance in AML (Campos et. al. Blood, 1993). Venetoclax (Ven), an oral selective BCL-2 inhibitor, in combination with hypomethylating agents (HMA) or low dose cytarabine (LDAC), was recently approved for treatment naive patients (pts) with AML who were not fit for standard induction therapy on the basis of two phase 1b/2 studies (DiNardo et.al, Lancet Onclogy, 2018; Wei et.al., J Clin. Oncol., 2019). Here, we present clinical outcome results from the phase 1b/2 study populations in subgroups defined by molecular markers and correlations with BCL-2 family expression. Methods: The data cut-off dates were 31-Aug-2018 (VEN+HMA study M14-358, NCT02203773) and 22-Aug-2018 (VEN+LDAC study M14-387 NCT02287233). Pt responses were assessed according to the IWG criteria for AML (Cheson et.al., J Clin. Oncol.,2003). The presence of AML associated mutations (NPM1, IDH1, IDH2, TP53 and FLT3) were determined centrally in bone marrow aspirates (BMA) at baseline. BCL-2 (BCL2) gene expression was defined centrally by quantitative polymerase chain reaction (qPCR) using the deltaCt (ΔCt) method. BCL2 expression normalized to a reference gene (2-ΔCt) was evaluated for pts with 〉 50% AML blasts in baseline BMA (median 75%; range 50-99%). The composite complete remission (CR) and CR with incomplete marrow recovery (CRi) rate, overall survival (OS), time to first response (TTR), and duration of response (DOR), and BCL-2 gene expression were correlated with presence of baseline molecular markers. Results: Data from 209 pts who received Ven 400 mg or 600 mg in combination with either HMA or LDAC are reported here. The median age was 74 years (range: 61-90 years) and 61% (n=127) were male (Table 1). Of the 167 pts with centrally assessed molecular markers, IDH1 or IDH2 mutations were detected in 25.7%, NPM1 mutations in 15.6%, TP53 mutations in 22.2%, and FLT3 mutations in 18.0% pts. Pts may have more than one of these mutations (i.e co-mutations of NPM1 and IDH1or IDH2, NPM1 with FLT3, or NPM1 with TP53) identified in baseline BMA. The CR/CRi rates were 83.7% for pts with IDH1/IDH2 mutations, 84.6% for pts with NPM1 mutations, 59.5% for pts with TP53 mutations, and 53.3% for pts with FLT3 mutations (Table 2). The median OS was not reached (NR) for pts with IDH1, IDH2 or NPM1 mutations, while the median OS for pts with detectable TP53 mutations or FLT3 mutations was 8.9 mos and 12.4 mos respectively. The remissions (CR or CRi) were rapid (median TTR between 1.1 mos to 1.8 mos) for each group (Table 2) and durable responses were observed for pts with IDH1, IDH2, NPM1, or FLT3 mutations (median DOR was either NR for IDH and NPM1 and 19.9 mos for FLT3). Pts with TP53 mutations are known to have poor prognosis and the DOR in this cohort was 5.6 mos. The median time on study was 11.6 mos (range: 0.3-44 mos). The range of BCL2 mRNA expression (2-ΔCt) in bone marrow blasts was 0.03 to 2.93, with a median of 0.86. The Cox hazard ratio for OS was 1.06, p=0.8565 based on BCL2 above the median (〉 0.86 2-ΔCt). The multivariate analysis included age, cytogenetic risk, AML type (primary vs secondary) and trial (M14-387 vs M14-358). Albeit not statistically significant and small sample sets, pts with either IDH1 or IDH2 mutations (N = 21) tended to have higher BCL2 expression (median 2-ΔCt 1.1 ; range 0.19 -2.93) while pts with TP53 mutations (N = 7) had lower levels of BCL2 expression (median 2-ΔCt 0.55; range 0.03 - 1.27). Determination of baseline expression of other family members (BCL2L1, MCL1, BCL2A1, BCLw, NOXA, etc) is ongoing and additional univariate and multivariate analyses will be presented at the meeting. Conclusions: These analyses demonstrate that VEN + HMA or LDAC has efficacy across multiple molecular markers in AML. This activity is rapid and durable, and is observed across different levels of BCL2 expression in AML blasts. Disclosures Chyla: Abbvie, Inc: Employment, Other: Stock or options. Harb:AbbVie Inc: Employment, Other: Stock/stock options. Mantis:AbbVie Inc: Employment, Other: Stock/stock options. Riehm:AbbVie Inc.: Employment, Other: Stock/stock options. Ross:AbbVie: Employment, Other: Stock/stock options. Sun:AbbVie: Employment, Other: Stock/stock options. Huang:AbbVie Inc: Employment, Other: Stock/stock options. Jiang:AbbVie Inc.: Employment, Other: Stock/stock options. Dail:Genentech: Employment, Equity Ownership. Peale:Genentech Inc.: Employment, Other: Stock/stock options. Potluri:AbbVie, Inc.: Employment, Other: Stock/stock options. Hayslip:AbbVie Inc: Employment, Other: Stock/stock options. OffLabel Disclosure: Venetoclax is a BCL-2 inhibitor that is FDA-approved in some indications. This presentation will focus on venetoclax for treatment in acute myeloid leukemia, which is not an approved indication.

Description: Background: Multiple myeloma (MM) is a clonal plasma cell malignancy characterized by a complex genomic landscape with molecular and clinical heterogeneity. Venetoclax (Ven) is a selective, potent, oral BCL-2 inhibitor currently under investigation as a potential targeted therapy for the treatment of relapsed/refractory (RR) MM. Using a next-generation sequencing (NGS)-based approach, the mutational landscape and gene expression profile (GEP) of patients (pts) with RRMM in BELLINI was evaluated for potential impact on observed clinical outcomes in key biomarker subgroups. Methods: BELLINI (NCT02755597) is a randomized, double-blind, multicenter Phase 3 study of bortezomib + dexamethasone (Bd) + either Ven or placebo (Pbo) in pts with RRMM who received 1-3 prior therapies and were sensitive or naïve to proteasome inhibitors. Pts were randomized 2:1 to receive Ven 800 mg/d or Pbo + Bd. The following biomarker analyses were performed by central laboratory assessments of CD138-enriched bone marrow mononuclear cells collected at baseline: BCL2 gene expression by quantitative PCR (qPCR), cytogenetic abnormalities by interphase fluorescence in situ hybridization (FISH), identification of somatic mutations by whole-exome sequencing (WES), and transcriptomic analysis by RNA sequencing (RNAseq). Correlation between mutational status, BCL2high status (Harrison et al. Blood. 2019:134 suppl:142), cytogenetics, GEP, and survival outcomes were examined by pairwise t-test and by hazard ratio (HR) from Cox proportional hazard model. Results: As of the data cutoff (13 Sep 2019), 291 pts were randomized (194 to Ven and 97 to Pbo); 262 pts (90.0%) were evaluable by FISH, 257 pts (88.3%) by qPCR, 197 pts (67.7%) by WES, and 184 pts (63.2%) by RNAseq. The mutational landscape of evaluable pts was highly heterogeneous (Figure 1A). MAPK pathway mutations were the most prevalent (62/197 pts; 31.5%); however, recurrent mutations in the NF-kB pathway (TRAF3 or CYLD) were also observed (12/197 pts; 6.1%). Within the MAPK pathway, the most frequently mutated genes were KRAS (39/197 pts; 19.8%), NRAS (8/197 pts; 4.1%), BRAF (8/197 pts; 4.1%), and NF1 (12/197 pts; 6.1%). Although a higher rate of pts with a MAPK mutation was observed in the t(11;14) and BCL2high subgroup compared to pts without these biomarkers (39/101 pts [38.6%] vs 23/95 pts [24.2%]; P=.04), improvement in progression-free survival (PFS) with Ven+Bd was observed in pts with (HR=0.08; 95% CI, 0.02-0.31; P