Combined Inhibition of Phosphatidylinositol 3-Kinase (PI3K) Isoform α and δ By the Pan-Class I PI3K Inhibitor SAR245409 (XL765) in Primary Chronic Lymphocytic Leukemia Cells Blocks Survival, Adhesion and Proliferation
Authors: Thijssen Rachel , van Bochove Gregor , de Rooij Martin FM , ter Burg Johanna , Spaargaren Marcel , Egile Coumaran , Kersten Marie Jose , Eldering Eric , Kater Arnon P. 
Published: November 2019

Abstract
CLL cells are highly dependent on B- cell receptor (BCR) signaling and on stimuli from the microenvironment for survival and proliferation. New drugs targeting PI3K downstream of BCR signaling have emerged as promising treatment options for patients with CLL. Among four PI3K catalytic subunits, the PI3Kd isoform is crucial for downstream BCR signaling, but the relative importance of the PI3Kα isoform in CLL is less clear. Impressive clinical activity of idelalisib in CLL and indolent NHL patients was recently reported. Idelalisib, a PI3Kd specific inhibitor, inhibits chemotaxis and adhesion of leukemia cells, resulting in rapid lymphocytosis followed by a decrease in lymphadenopathy. However, idelalisib has no direct impact on leukemic cell survival [1], raising the potential risk of residual clones responsible for the development of resistance. In this study, we evaluated the impact of a pan-class I PI3K inhibitor (SAR245409/XL765), a PI3Kα-specific inhibitor (BYL719) and a PI3Kd specific inhibitor (idelalisib) on PI3K/mTOR signaling, apoptosis, cell adhesion, CD40-induced survival and proliferation in primary patient derived leukemic cells.
Phosphorylation of the downstream effector of mTOR, S6RP, was completely blocked by SAR245409 but not by BYL719 or idelalisib. SAR245409 induced apoptosis in unstimulated CLL cells (IC50= 0.86µM) in contrast to BYL719 or idelalisib (IC50 >10µM), demonstrating that targeting multiple PI3K isoforms is required to completely block the PI3K/Akt/mTOR pathway (table 1). Importantly, SAR245409 also induced apoptosis in p53 or ATM dysfunctional CLL samples. SAR245409, as well as idelalisib, and in contrast to BYL719 completely inhibited BCR-mediated adhesion to fibronectin [2]. Similarly, SAR245409 inhibited CD40L-mediated survival [3], and induced upregulation of the pro-apoptotic protein BIM. All 3 PI3K inhibitors inhibited CD40 ligation + IL-21-mediated CLL proliferation [4].
This study revealed that the pan-class I PI3K inhibitor SAR245409 is more cytotoxic to primary CLL cells than PI3Kα or PI3Kd specific inhibitors. Furthermore, combined inhibition of PI3Kα and d can block signaling pathways that are critical for CLL survival, adhesion and proliferation in the LN microenvironment (see table 1). This work provides a rationale for the evaluation of SAR245409 in CLL patients either as monotherapy or in combination therapies.
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[4] Pascutti et al. IL-21 and CD40L signals from autologous T cells can induce antigen-independent proliferation of CLL cells. Blood 2013;(122):3010-3019.
Table 1. The effect of the PI3Kd inhibitor idelalisib, PI3Kα inhibitor BYL719 or pan PI3K inhibitor SAR245409 on CLL cells in functional assays PI3Kd inhibitor PI3Kα inhibitor pan PI3K inhibitor Cytotoxicity (IC50)1 >10µM >10µM 0.86µM Inhibition of adhesion2 48%** 21% 43%** Activation Inhibition of CD40L-induced survival3 14% 0% 54%* Inhibition of CD40L+IL21 induced proliferation4 47%* 35%* 51%* 1 CLL cells were incubated with 0.001-10 μM idelalisib (n=18), BYL719 (n=6) or SAR245409 (n=28) for 48 hours. Viability was assessed by DiOC6/PI staining.2 CLLcells pretreated with 1 µM idelalisib, BYL719, or SAR245409 were stimulated with α-IgM and allowed to adhere to fibronectin-coated surfaces (n=5). 3 CLL cells were cultured on fibroblast expressing CD40L in the absence or presence of 1 µM of idelalisib, BYL719, or SAR245409 for 3 days. Apoptosis was assessed by DiOC6/PI staining (n=8).4 CFSE labelledCLL cells were cultured on fibroblast expressing CD40L with IL-21 and co-treated with 1 µM idelalisib, BYL719, or SAR245409. After 4 days, CFSE was measured by FACS (n=11)2-4 The one sample T test was used to determine the significance of differences between means of treated samples and normalized values of untreated samples (100%). * p <0,05;** p<0,01

Disclosures
Egile: Sanofi: Employment. Kersten:Sanofi: Research Funding. Kater:Sanofi: Research Funding.