Chronic lymphocytic leukemia (CLL) is a B-cell malignancy influenced by oncogenic mutations and the factors of the microenvironment such as BCR signaling that promote B-cell survival and proliferation. BCR signaling cascade activates multiple downstream effector kinases including Phosphatidylinositol 3-kinase (PI3K) pathway that is critically important in the clonal expansion of B-CLL cells. Of the four p110 catalytic subunit isoforms, p110 delta isoform is over-expressed in many B-cell malignancies, including CLL. Idelalisib (I) is a small molecule highly selective PI3K delta inhibitor. I promotes apoptosis in B-cell malignancies by disrupting the molecular pathways related to BCR signaling, leukemia cell migration and microenvironment. Importantly, I inhibits BCR derived PI3K signaling, which ultimately dampens AKT activation. Bendamustine (B), an alkylating agent that induces DNA damage and repair response, is recently approved for CLL therapy. Kinases that are important for CLL cell survival may also be activated during this DNA damage response. Based on this background, we hypothesize that the treatment of CLL cells with I and B may modulate the repair mechanisms and sensitize B-CLL cells to enhanced apoptosis. Our model system includes primary CLL patient samples with or without BCR activation. Our findings show that compared to time-matched DMSO control, treatment with I or B as a single agent resulted in 5-15% and 5-20% dose-dependent cell death, respectively. In contrast, combined treatment (I+B at 0.5+5, 1+10, 3+15, 5+20, 10+30 µM) of CLL cells for 24 h resulted in 10-40% of apoptosis as measured by annexin/PI (n=6). This was synergistic with a combination index of <0.8 (Chou-Chou, CalcuSyn method). Moreover, at these five concentrations, I+B combination resulted in 20-60% decline in phospho-AKT (S473) but not total AKT. In contrast, B (up to 15 µM) resulted in 40-60% increase in phospho-AKT levels. Phosphorylation of H2Ax, a measure of DNA damage response, is induced with combination treatment in comparison to single agent treatments. We next investigated the molecular targets downstream of BCR signaling. Primary CLL cells treated with I, in assays with BCR (IgM) stimulation show a time-dependent decrease in phospho-AKT except at 24 h (n=4). For phospho-GSK3β, a downstream target of AKT, results were heterogeneous. In contrast, at 24 h, I treatment consistently resulted in a decrease in total Mcl-1; without any change in Bcl-2 protein levels. The decline in Mcl-1 protein level may be due to a decrease in transcription as total RNA synthesis was decreased by at least 30% after treatment with I. Transcript levels and post-translational modification of Mcl-1 will elucidate the mechanism of I+B cytotoxicity. The emerging role of kinase inhibitors in combination with chemotherapeutic agent provides a new modality of treatment for CLL patients.

Citation Format: Prexy Shah, Kumudha Balakrishnan, William Wierda, Varsha Gandhi. Mechanism-based combination therapy of PI3 kinase delta-specific inhibitor Idelalisib with Bendamustine in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4530. doi:10.1158/1538-7445.AM2014-4530