Abstract A24: The dual PI3K δ/γ inhibitor, RP6530, in combination with ibrutinib or fludarabine, synergistically enhances cytotoxicity in primary CLL cells in vitro. (2015)

Abstract Background: The PI3K pathway is a central pro-survival mechanism in chronic lymphocytic leukemia (CLL). While the delta isoform of PI3K is largely associated with B-cell signaling, gamma is involved in cytokine trafficking and modulation of the tumor microenvironment. Selective pharmacologic targeting of PI3K δ/γ may therefore have therapeutic potential. RP6530 is a novel, potent, and selective PI3K δ/γ inhibitor. RP6530 demonstrates high potency against PI3Kδ (IC50=25 nM) and γ (IC50=33 nM) enzymes with selectivity over α (&amp;gt;300-fold) and β (&amp;gt;100-fold) isoforms. In this study, the potential of RP6530 in inducing primary CLL cell cytotoxicity, both as a single agent and in combination with standard CLL drugs, was assessed. Methods: Blood from treatment-naïve CLL patients seen at the Duke Center for CLL and enrolled in IRB approved protocols at the Duke University and Durham VA Medical Centers was collected. Cells were enriched using Rosette-Sep B cells from Stem Cell Technology, generally giving purity of &amp;gt; 97% of B cells/CLL cells. 2.5 x 105 primary CLL cells were incubated in serum free medium (SFM) with serial dilutions of RP6530, ibrutinib, fludarabine, bendamustine, and chlorambucil, either alone or in combination, in 96 well plates in triplicate. After three days, cytotoxicity was determined using the MTS assay. Fractional cytotoxicity was calculated by comparing absorbance for each experimental condition to absorbance from wells containing CLL cells cultured in SFM alone. For cytotoxicity results of single drugs, a 2-parameter log-logistic model was used to plot the data. For cytotoxicity results of drugs in combination, a 4-parameter log-logistic model was used to plot the data. Modeling and graphing was performed using the drc package in the statistical environment R. Results: The efficacy of RP6530 in CLL lymphocytes collected from five patients was evaluated; 4 with normal FISH cytogenetics, and 1 with 13q deletion. IGHV was mutated in three of the five samples including the one with 13q deletion. Data demonstrate that RP6530 was highly effective at killing primary CLL cells in vitro. Calculated IC50 for RP6530 (362 nM) was lower that the Burton tyrosine kinase inhibitor, Ibrutinib (567 nM), as well the other conventional chemotherapy agents such as fludarabine (14.8 μM), bendamustine (382 μM), and chlorambucil (152 μM). Combination with RP6530 with any of the four other drugs demonstrated potentiation of cytotoxicity. Combination of very low concentrations of ibrutinib and RP6530 (3 nM of each) resulted in a &amp;gt;50% cell kill indicating a dramatic synergistic effect. Likewise, the addition of low concentrations of fludarabine (3 μM) potentiated the cytotoxic effect by causing a 10-fold increase in potency of RP6530 (IC50 = 32 nM). On the other hand, addition of only the higher concentrations of bendamustine (500 μM) or chlorambucil (60 μM) potentiated the cytotoxicity due to RP6530 (IC50 of &amp;lt;3 nM and 60 nM respectively), indicating that the combinatorial effect is most likely to be additive. Conclusions: RP6530 is a potent PI3K δ/γ inhibitor that induces concentration-dependent cytotoxicity in primary CLL lymphocytes both as a single agent as well as in combination with standard CLL therapy. The extreme synergism demonstrated by RP6530 and Ibrutinib even at the lowest concentrations tested warrant evaluation of the efficacy of the combination in CLL patients. Citation Format: Swaroop Vakkalanka, Kumar V. Penmetsa, Dawn Chasse, Youwei Chen, Srikant Viswanadha, Daphne R. Friedman, J Brice Weinberg. The dual PI3K δ/γ inhibitor, RP6530, in combination with ibrutinib or fludarabine, synergistically enhances cytotoxicity in primary CLL cells in vitro. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A24.