Functional testing of PI3K inhibitors stratifies responders to idelalisib and identifies treatment vulnerabilities in idelalisib-refractory/intolerant chronic lymphocytic leukemia

Purpose: Phosphatidylinositol 3-kinase inhibitors (PI3Ki) are approved for relapsed chronic lymphocytic leukemia (CLL). While patients may show an initial response, development of treatment intolerance or resistance remains a clinically challenging. Prediction of individual treatment responses based on clinically actionable biomarkers is needed to overcome these challenges. Here, we investigated whether ex vivo functional responses to targeted therapies can stratify responders to idelalisib and guide precision medicine in CLL. Experimental design: CLL cells from treatment naive, idelalisib-responding, and idelalisib-refractory/intolerant patients (n=33 in total) were profiled against ten PI3Ki and the Bcl-2 antagonist venetoclax. Cell signaling and immune phenotypes were analyzed by flow cytometry. Cell viability was monitored by detection of cleaved caspase-3 and the CellTiter-Glo assay. Results: Among the ten PI3Ki studied, pan-PI3Ki were most effective at inhibiting PI3K signaling and cell viability, and they showed activity also in CLL cells from idelalisib-refractory/intolerant patients. The pan-PI3Ki copanlisib, but not the p110delta inhibitor idelalisib, inhibited PI3K signaling in CD4+ and CD8+ T cells in addition to CD19+ B cells, while it did not significantly affect T cell numbers. Combination treatment with a PI3Ki and venetoclax resulted in synergistic induction of apoptosis. Based on ex vivo drug sensitivity testing, a relapsed CLL patient was treated with idelalisib plus venetoclax, and the patient achieved a partial response. A more systematic analysis revealed that CLL cells from patients with a long-term response to idelalisib showed significantly higher drug sensitivities to 73 drug combinations at baseline compared to short-term responders. Conclusions: Our findings suggest novel treatment vulnerabilities in idelalisib-refractory/intolerant CLL, and demonstrate that ex vivo functional profiling may guide precision medicine and predict treatment responses of individual CLL patients. ### Competing Interest Statement A.J.A. has received travel grants from Astra Zeneca. F.B. has received institutional research funds from Acerta, ADC Therapeutics, Bayer AG, Cellestia, CTI Life Sciences, EMD Serono, Helsinn, ImmunoGen, Menarini Ricerche, NEOMED Therapeutics 1, Nordic Nanovector ASA, Oncology Therapeutic Development, PIQUR Therapeutics AG; consultancy fee from Helsinn, Menarini; expert statements provided to HTG; travel grants from Amgen, AstraZeneca, Jazz Pharmaceuticals, PIQUR Therapeutics AG. A.R.M. has received grants, personal fees, DSMB membership, and other funds from TG Therapeutics; grants and personal fees from AbbVie, Adaptive, AstraZeneca, Genentech, Janssen, Pharmacyclics; grants and other funds from Celgene; grants from DTRM Biopharm, Loxo, Regeneron, Sunesis; and personal fees from BeiGene. E.N. reports employment and ownership of stock with TG Therapeutics. J.R.B. has served as a consultant for AbbVie, Acerta/AstraZeneca, BeiGene, Bristol-Myers Squibb/Juno/Celgene, Catapult, Genentech/Roche, Eli Lilly, Janssen, MEI Pharma, Morphosys AG, Nextcea, Novartis, Pfizer, Rigel; received research funding from Gilead, Loxo/Lilly, Verastem/SecuraBio, Sun, TG Therapeutics; and served on the data safety monitoring committee for Invectys. S.S.S. has received honoraria from AbbVie and AstraZeneca, and research support from BeiGene and TG Therapeutics. The other authors declare no competing financial interests.