PI3Kδ inhibition elicits anti-leukemic effects through Bim-dependent apoptosis

PI3Kδ plays pivotal roles in the maintenance, proliferation and survival of malignant B-lymphocytes. Although not curative, PI3Kδ inhibitors (PI3Kδi) demonstrate impressive clinical efficacy and, alongside other signaling inhibitors, are revolutionizing the treatment of hematological malignancies. However, only limited in vivo data are available regarding their mechanism of action. With the rising number of novel treatments, the challenge is to identify combinations that deliver curative regimes. A deeper understanding of the molecular mechanism is required to guide these selections. Currently, immunomodulation, inhibition of B-cell receptor signaling, chemokine/cytokine signaling and apoptosis represent potential therapeutic mechanisms for PI3Kδi. Here we characterize the molecular mechanisms responsible for PI3Kδi-induced apoptosis in an in vivo model of chronic lymphocytic leukemia (CLL). In vitro, PI3Kδi-induced substantive apoptosis and disrupted microenvironment-derived signaling in murine (Eμ-Tcl1) and human (CLL) leukemia cells. Furthermore, PI3Kδi imparted significant therapeutic responses in Eμ-Tcl1-bearing animals and enhanced anti-CD20 monoclonal antibody therapy. Responses correlated with upregulation of the pro-apoptotic BH3-only protein Bim. Accordingly, Bim−/− Eμ-Tcl1 Tg leukemias demonstrated resistance to PI3Kδi-induced apoptosis were refractory to PI3Kδi in vivo and failed to display combination efficacy with anti-CD20 monoclonal antibody therapy. Therefore, Bim-dependent apoptosis represents a key in vivo therapeutic mechanism for PI3Kδi, both alone and in combination therapy regimes

Induction of p16INK4a is the major barrier to proliferation when Epstein-Barr virus (EBV) transforms primary B cells into Lymphoblastoid cell lines

To explore the role of p16INK4a as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding p16INK4a and p14ARF. Using recombinant EBV-BAC viruses expressing conditional EBNA3C (3CHT), we developed a system that allows inactivation of EBNA3C in lymphoblastoid cell lines (LCLs) lacking active p16INK4a protein but expressing a functional 14ARF-fusion protein (p14/p16). The INK4a locus is epigenetically repressed by EBNA3C – in cooperation with EBNA3A – despite the absence of functional p16INK4a. Although inactivation of EBNA3C in LCLs from normal B cells leads to an increase in p16INK4a and growth arrest, EBNA3C inactivation in the p16INK4a-null LCLs has no impact on the rate of proliferation, establishing that the repression of INK4a is a major function of EBNA3C in EBV-driven LCL proliferation. This conditional LCL system allowed us to use microarray analysis to identify and confirm genes regulated specifically by EBNA3C, independently of proliferation changes modulated by the p16INK4a-Rb-E2F axis. Infections of normal primary B cells with recombinant EBV-BAC virus from which EBNA3C is deleted or with 3CHT EBV in the absence of activating ligand 4-hydroxytamoxifen, revealed that EBNA3C is necessary to overcome an EBV-driven increase in p16INK4a expression and concomitant block to proliferation 2–4 weeks post-infection. If cells are p16INK4a-null, functional EBNA3C is dispensable for the outgrowth of LCLs