Abstract Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, remains an incurable B-cell malignancy. It is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen and bone marrow. CLL cells display up-regulated B cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) and Bruton's tyrosine kinase (BTK) pathways. Small molecules that target such kinases in the BCR pathway have shown significant clinical activity in CLL patients. The PI3K p110δ inhibitor, idelalisib, was granted a breakthrough therapy designation by the FDA in relapsed CLL and the BTK inhibitor, ibrutinib, has received approval by FDA for treatment of relapsed CLL. However, patients still relapse on these therapies. Here we use pharmacologic and genetic approaches to further characterize the role of PI3K signaling in the leukemia pathogenesis in the CLL cell and in the microenvironment. We describe that a PI3K p110δ and p110γ inhibitor, IPI-145, which is in late stage clinical development, attenuates pro-survival signals in the OSU-CLL cell line and primary human and murine CLL cells and promotes apoptosis and downstream pathway inactivation in primary human and murine CLL cells in a dose- and time-dependent fashion. To examine the cytotoxicity of IPI-145 in normal immune cells, we incubated whole blood from CLL patients with 0.25-5 μM IPI-145 for 48 hours and analyzed by flow cytometry for absolute count of live CD3+ T cells, CD56+ NK cells and CD19+ B cells. T cells and NK cells were sensitive to IPI-145, displaying about 20% decrease in viability at concentrations greater than 0.5μM, however the B cell population showed about 50% decrease in viability. To specifically examine normal B cells, we isolated CD19+ B cells from healthy volunteer blood and incubated with 1 μM IPI-145 for 48 hours and observed no cytotoxicity, despite observing a significant decrease in CLL cells viability under the same conditions. Additionally, IPI-145 is highly effective at reducing downstream PI3K signaling in a B cell line with the ibrutinib resistance conferring BTK C481S mutation. Genetically we show that the PI3K p110δ-inactivating and the TCL1 leukemia murine models can be utilized to further explore the differential role of PI3K p110δ in leukemic cell and microenvironment. Our study indicates that systemic disruption of PI3K p110δ function in the TCL1 mouse significantly prevents spontaneous leukemia development, indicating that PI3K p110δ is a critical kinase for CLL disease initiation and expansion. Moreover, inactivation of PI3K p110δ in the microenvironment showed a dose dependent effect in delaying leukemia engraftment. This suggests that PI3K p110δ activity is also critical in the non-B cell compartment for leukemia progression. While our group has focused on the role of PI3K p110δ, we continue to examine the role of PI3K p110γ using the PI3K p110δ and p110γ inhibitor, IPI-145. This study provides solid rationale for ongoing clinical development of IPI-145 in CLL patients. Additionally, as IPI-145 can continue to inhibit the PI3K/AKT pathway in the setting of a C481S BTK mutation, it provides justification for clinical studies of this agent in this subset of patients. Citation Format: Amy J. Johnson, Shuai Dong, Daphne Guinn, Jason A. Dubovsky, Yiming Zhong, Amy Lehman, Jeff Kutok, Jennifer A. Woyach, John C. Byrd. Pharmacologic inhibition with IPI-145 and genetic inhibition of PI3K p110δ antagonizes intrinsic and extrinsic survival signals in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B44.