PS1311 PI3K‐INHIBITION INCREASES APOPTOTIC PRIMING IN T‐CELL LYMPHOMAS AND SYNERGIZES WITH BH3 MIMETICS.
Authors: Nothnick G. , Becker S. , Trümper L. , Wulf G. , Koch R. 
Published: June 2019

Background:Peripheral T‐cell lymphomas (PTCL) are a heterogeneous group of lymphoid malignancies that are highly underserved by current therapeutic regimens and still associated with poor outcomes. Recently, inhibition of the phosphatidylinositol‐3 kinase (PI3K) emerged as a promising therapeutic strategy for patients with T‐cell lymphomas. As a single agent, the PI3K inhibitor duvelisib showed a response rate of 50% in patients with PTCL1. However, complete responses were seen in less than 20% of the patients and in vitro‐analyses revealed broad cytostatic but limited cytotoxic activity through induction of apoptosis.Aims:Thus, to maximize the efficacy of PI3K inhibitors, further mechanistic insight in the regulation of apoptosis is needed to develop rational combination strategies.Methods:We utilized a previously characterized set of T‐cell lymphoma cell lines2 and assessed the cytotoxic activity of various isoform‐specific PI3K inhibitors, including the PI3K gamma‐delta inhibitor duvelisib and the pan‐PI3K inhibitor copanlisib. Next, we performed dynamic BH3 profiling to functionally dissect the impact of PI3K inhibitors on the regulation of mitochondrial apoptosis. To further address the underlying mechanism, we performed immunoblotting and Co‐IP experiments. Ultimately, we performed in‐vitro combination testing of PI3K inhibitors and BH3‐mimetics as suggested by dynamic BH3‐profiling.Results:The PI3K inhibitors duvelisib and copanlisib induced apoptosis in only a subset of PTCL cell lines. However, dynamic BH3 profiling revealed a broad and rapid enhancement of apoptotic priming in all tested cell lines (n = 9), as assessed by stimulation with the pro‐apoptotic BIM‐peptide. Furthermore, dynamic BH3‐profiling revealed increased susceptibility to blockade of anti‐apoptotic proteins, predominantly MCL1. Importantly, PI3K inhibitors enhanced the baseline dependence on specific anti‐apoptotic BCL2‐family members as recently identified by BH3 profiling3. Mechanistically, immunoblotting of pro‐ and anti‐apoptotic BCL2‐family members showed an upregulation of various pro‐apoptotic proteins. Protein levels of anti‐apoptotic BCL2 and BCL‐xL were affected heterogeneously with downregulation of either protein in 4 of 6 cell lines. In contrast, protein levels of MCL1 were maintained in all tested cell lines. Co‐IP experiments confirmed enhanced availability of the pro‐apoptotic BIM protein, but also retained binding to the anti‐apoptotic protein MCL1. Based on these data, we performed a combination testing of the PI3K inhibitors duvelisib and copanlisib with the BH3 mimetics ABT‐263 (targeting BCL‐2 and BCL‐xL) and AZD5991 (targeting MCL1). Induction of cell death by single agents and combinations was assess by flow cytometry. As predicted by dynamic BH3 profiling, we saw broad synergistic activity of PI3K inhibitors with BH3 mimetics, particularly the MCL1 inhibitor AZD5991 in 8 of 9 tested cell lines.Summary/Conclusion:Despite their limited single‐agent activity in PTCL, PI3K inhibitors induce a broad increase in apoptotic priming of PTCLs and their dependence on anti‐apoptotic BCL2‐family members. While this increase in apoptotic priming does not suffice to induce apoptosis in most cases, it reasons for a combination strategy with BH3 mimetics. Indeed, we found broad synergistic activity of PI3K‐inhibitors and BH3‐mimetics, particularly the MCL1 inhibitor AZD5991.