CAL-101, a Specific Inhibitor of the p110δ Isoform of Phosphatidylinositide 3-Kinase Induces Cytotoxicity in Multiple Myeloma (MM)
Authors: Ikeda Hiroshi , Hideshima Teru , Okawa Yutaka , Vallet Sonia , Pozzi Samantha , Santo Loredana , Gorgun Gullu , Fulciniti Mariateresa , Raje Noopur S. , Perrone Giulia , Munshi Nikhil , Richardson Paul , Lannutti Brian J , Puri Kamal D. , Giese Neill A. , Anderson Kenneth C. 
Published: April 2020

Abstract
The phosphatidylinositide 3-kinase (PI3K)/Akt pathway mediates cell proliferation, cell cycle regulation, apoptosis, and autophagy. Of the 8 distinct mammalian isoforms of PI3K, it is the class I PI3Ks (p110α, p110β, p110δ, and p110γ) that are responsible for Akt activation. Recently, p110δ expression has been shown in colon and bladder carcinoma, glioblastoma, and acute myeloid leukemia cells. In this study, we demonstrate expression and examined biologic sequelae of p110δ signaling in MM using a novel specific inhibitor CAL-101. Overexpression of p110δ protein was observed in 24/24 primary tumor cells from MM patients. High level of p110δ expression was detected in two (INA-6 and LB) of the 11 MM cell lines examined. Treatment of INA-6 and LB cells with CAL-101 (1.0 μM for 1.5, 3, 6 hr) significantly inhibited phosphorylation of Akt at serine 473 and threonine 308, suggesting that p110δ is the isoform responsible for PI3K signaling in these cells. Furthermore, CAL-101 induced cytotoxicity in INA-6 cells in a dose-dependent fashion, with an IC50 of 0.625 μM-1.25 μM at 48 h. In contrast, minimal cytotoxicity was observed in p110δ-negative cell lines. Importantly, CAL-101 (0.625 μM) triggered cytotoxicity against tumor cells from MM patients without affecting the survival of normal peripheral blood mononuclear cells at concentrations as high as 20 μM. The specificity of CAL-101 towards p110δ was confirmed by the observation that p110δ small interfering RNA knock-down, but not control, caused inhibition of cell growth and cytotoxicity that was consistent with CAL-101 treatment, further confirming the key role of p110δ in MM cells. Interleukin 6 (IL-6) and insulin-like growth factor I (IGF-I) and cell-cell contact with stromal cells have been shown to induce proliferative and anti-apoptotic responses in MM cells that abrogate cytotoxic effects of conventional therapies. Importantly, neither either IL-6 and IGF-1 treatment nor co-culture of MM cells with bone marrow stromal cells abrogated CAL-101-induced MM cell growth inhibition. Moreover, CAL-101 inhibited adherence of MM cells to bone marrow stromal cells. Since the PI3K/Akt pathway is involved in autophagy, we also examined whether CAL-101 induced autophagic cell death in MM cells. CAL-101 (6 hr treatment) significantly increased LC3-II expression, assessed by western blot and immunofluorescent analyses; conversely, autophagy inhibitor 3-MA (100 μM) blocked LC3-II expression induced by CAL-101. Acridine orange staining and electron microscopic analysis to assess the relative number of autolysosomes further confirmed induction of autophagy by CAL-101. Finally, combined CAL-101 with bortezomib showed synergistic cytotoxicity against MM cells (combination index = 0.64). In conclusion, our studies showed that p110δ is a novel therapeutic target in MM and provide the basis for clinical evaluation of CAL-101 in ongoing Phase 1/2 trial in hematologic malignancies including MM.