Erlichman, Charles

Abstract Abstract 3928 Background: Accumulating data support the critical role of PI3K/Akt in CLL B cell receptor (BCR) mediated signal transduction, cell proliferation and survival. In addition recent preclinical and clinical studies indicate that specific PI3K blockade results in robust preclinical and clinical efficacy in CLL. In our model system of CLL B cell-stromal cultures which feature their interaction, platelet derived growth factor (PDGF) present in CLL culture medium drives VEGF production through PI3K/Akt activation in stromal cells (Blood. 2010. 116:2984). Indeed Akt was found to be activated in leukemic cells during the CLL-stroma interaction (Leuk Res. 2008. 32:1565). Therefore, we hypothesized that Akt inhibition should promote CLL B cell apoptosis and abrogate BCR mediated cytokine production. MK2206 is an orally bioavailable highly specific allosteric Akt inhibitor. It has been tested in patients with refractory solid tumors and was demonstrated to be safely administered in a phase I trial. Therefore the goal of this study was to test the preclinical efficacy of MK2206 on both the survival and the BCR mediated cytokine production of CLL leukemic B cells. Methods: Peripheral blood mononuclear cells isolated from CLL patients (n=37) were treated with escalating concentrations of MK2206 (1–16 μM) for 24 hours, 48 hours or 72 hours. The levels of leukemic B cell viability were tested using an (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. The potential impact (antagonistic/additive/synergistic) of Bendamustine in combination with MK2206 was also tested by using the MTT assay. We used the Calcusyn system to calculate the effect of drug interactions. The combination index (CI) as calculated by the program usually indicates synergy when ≤ 0.8 and indicates additive outcomes when between 0.8–1.2. A CI >1.2 indicates antagonism. Downstream signals of Akt activation in CLL B cells were evaluated by testing their expression of Mcl-1, 4EBP1 and p70S6K using immunoblot. The impact of Akt inhibition by MK2206 on cytokine production in response to B-cell receptor ligation with anti-IgM was also tested using a multiplex cytokine analysis (Invitrogen) in a time-course experiment. Results: MK2206 treatment induced concentration- and time-dependent apoptosis in CLL leukemic cells. At 72 hours, the IC50 of MK2206 in the experiments using CLL leukemic cells in vitro is ∼8 mM. MK2206 incubation at 1 or 5 mM cultured with CLL B cells over a 48-hour period abolished of Akt and p70S6K phosphorylation while native PARP was cleaved into the 85 kD polypeptide fragment. However, the expression level of the upstream signal molecule, PI3K, was not changed. Among the CLL patients tested (n = 37), we did not find any difference in sensitivity to MK2206 induced apoptosis based on critical prognostic factors of CD38, ZAP-70, IGHV and del(17p) status. Importantly, we detected synergistic or additive activity between MK2206 and Bendamustine in 11 tested CLL samples when these combinations were used to treat CLL cells in vitro for 72hrs. Thus the median CI value for this group of patients was 0.8 (0.1 – 1.1). Six were found to have CI ≤ 0.8 and five fell within the additive CI values (0.8 – 1.2). Production of immune or chemotactic cytokines (e.g. CCL3, CCL4, MCP-1, IL-1Ra, IL-8 and IL-2R) at 24 hour incubation increased significantly above baseline when CLL cells were stimulated anti-IgM. Akt inhibition with MK2206 selectively abrogated upregulation of CCL3, CCL4, MCP-1 and IL-2R production, but not for IL-8 or IL-1Ra secretion. MK2206 also abolished BCR mediated Akt activation and decreased Erk activation. Conclusion: MK2206, a robust and selective Akt inhibitor, induced significant in vitro apoptosis of CLL B-cells in vitro. Preclinical evidence of a synergistic effect between MK2206 and Bendamustine was also observed independent of prognostic risk. MK2206 abolished BCR mediated Akt activation and selectively abrogates BCR mediated production of cytokines that may promote apoptotic resistance. These findings support the use of MK2206 in treating CLL and indeed we have initiated a phase I/II trial of MK2206 in combination with Bendamustine and Rituximab for relapsed CLL patients(N1087, October 2011). Acknowledgments: This study was funded by the NCI-K23, NCCTG and CLL Global Foundation. Disclosures: Shanafelt: Cephalon: Research Funding; Genentech: Research Funding. Kumar:Genzyme: Research Funding; Novartis: Research Funding; Celgene: Consultancy, Research Funding; Millennium: Research Funding; Merck: Consultancy, Honoraria. Kay:Celgene: Research Funding.

1043 Background: In estrogen receptor (ER) positive MBC, mutations (e.g. ESR1), identified from tumor biopsies or cfDNA, confer resistance. The concordance between mutations observed in tumor and cfDNA and the implications for response to Z-endoxifen, a potent anti-estrogen, are unknown. Methods: We previously conducted a phase I trial of Z-endoxifen in endocrine refractory, ER positive MBC. Seven dose levels were considered ranging from 20 to 160 mg/day followed by expansion cohorts (EC) of 40, 80, and 100 mg/day. Pretreatment blood samples (all pts) and fresh tumor biopsies (EC) were collected prospectively. Tumor and cfDNA were evaluated by targeted NGS. Results: 41 pts (38 evaluable) were enrolled. Prior endocrine therapy included aromatase inhibitors (37/38, 97%), fulvestrant (22/38, 58%) and tamoxifen (26/38, 68%). Substantial endoxifen exposure without DLTs at doses above 80 mg/day led to a halt in dose escalation and opening of the EC. Overall clinical benefit (stable > 6 months [7 pts.] or partial response by RECIST criteria [3 pts.]) rate was 26.3% (95%CI: 13.4-43.1%). cfDNA was obtained from 36 pts and mutations were identified in 13 (36%) including ESR1 [ Y537N or D538G] (5), PIK3CA [ H1047R or E542K] (8), TP53 [ K132R, R248Q, R267Q, or H179Y] (4), AKT ( Q79K) (1), and KRAS ( G12D) (1). In 5 pts with cf ESR1 mutations, 4 had additional cfDNA mutations including PIK3CA (3), and TP53 (1). PFS was shorter in pts with cfDNA mutations relative to those without (median: 61 vs.132 days; log-rank p = 0.021). Discordance was observed between tumor and cfDNA mutations where 3/7 PIK3CA tumor mutations were detected by cfDNA, 1/2 TP53 tumor mutations were detected by cfDNA, and 0/1 AKT tumor mutations were detected by cfDNA. Conversely, 2 pts had cfDNA mutations (either ESR1, TP53 or AKT) undetected in tumor. Conclusions: The absence of cfDNA mutations in patients with endocrine resistant, MBC treated with Z-Endoxifen was associated with significantly longer PFS. Given the discordance between tumor and cfDNA sequence data, future studies must determine which approach maximizes prognosis and prediction of benefit for estrogen-targeted therapy. Clinical trial information: NCT01327781.