Genetic Inhibition of PI3K p110delta Antagonizes Survival Signals and Induces Immune Activation in Chronic Lymphocytic Leukemia (CLL)

Background: Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the western world, is characterized by the accumulation of malignant mature B cells in the blood, lymph nodes, spleen and bone marrow. CLL cells display up-regulated B cell receptor (BCR) activation, which maintains B cell survival and proliferation through transmitting microenvironmental stimuli. Due to aberrant regulation of the BCR, CLL cells display constitutively activated survival and proliferation pathways, such as phosphoinositide-3 kinase (PI3K) p110δ pathway. In addition, immune tolerance, a critical mechanism for cancer immune invasion, remains a major barrier for effective anti-cancer therapy. PI3K p110δ also plays critical roles in T cell development and function. Studies using genetic p110δ kinase-dead mice have showed that p110δ inhibition can reverse the tumor induced immune tolerance, impairs the expansion of regulatory T cells (Treg) thereby enhancing the cytotoxic CD8⁺T cells to induce tumor regression in various solid tumor models.

Methods: Here we use a PI3K p110δ kinase-dead (δ^D910A/D910A) mouse model and a well-characterized Eμ-TCL1 mouse CLL model to delineate the role of p110δ signaling in CLL leukemia pathogenesis and microenvironmental immune tolerance. The δ^D910A/D910A has a point mutation on the p110δ kinase domain, which results in the complete inactivation of p110δ activity without affecting the expression of p110δ or the other isoforms. TCL1 mice exhibit B-cell leukemia similar to that in humans beginning around 4-6 months. Adoptive transfer experiments are performed by tail-vein injection of 5e⁶ splenic lymphocytes from a leukemic Eμ-TCL1 mouse. Leukemia progression is measure by flow cytometry for the CD5⁺CD19⁺% lymphocytes in the peripheral blood. Whole blood cytotoxicity assay was performed by incubating whole blood from recipient mice with cryopreserved cells from the original donor to assess the their cytotoxic effects towards the donor cells.

Results: δ^D910A/D910A mice were crossed with TCL1 mice to examine the effect of systemic p110δ inactivation in CLL. We show that global inactivation of PI3K p110δ significantly prevented spontaneous leukemia development. Both δ^WT/TCL1 and δ^WT/D910A/TCL1 mice developed CLL like disorder starting 4 months whereas δ^D910A/D910A/TCL1 mice did not show any sign of disease up to 14 months. This indicates that p110δ is a critical kinase for CLL disease initiation and expansion.

We then performed adoptive transfer of TCL1 leukemic cells into p110δ mice to examine the role of p110δ in the microenvironment. We show that inhibiting p110δ in the microenvironment impairs leukemia engraftment. On day 31 after engraftment, 7 out of 10 mice from the δ^WT group developed leukemia while no mice from δ^D910A/D910A group had detectable leukemia. On day 43, δ^D910A/D910A group started to have leukemia incidence (2 out of 7) when δ^WT group continued to progress. δ^WT/D910A group showed intermediate leukemia incidence and disease development, which were different from the phenotype we saw in δ^WT/D910A/TCL1. This suggests that p110δ plays a different role outside of the leukemia compartment.

To identify potential effector cells, we performed adoptive transfer of ovalbumin (OVA) –expressing TCL1 leukemia cells into p110δ mice. The TCR (OT1) specific SIINFEKL peptide of OVA is used to induce the expansion of OVA antigen specific CD8⁺ T cells. Interestingly, upon engraftment, δ^WT and δ^WT/D910A mice showed similar levels (4% and 3.5%, respectively) of OVA specific CD8⁺ T cells whereas the δ^D910A/D910A mice only have 2%. This appears to be contradicting to the less leukemia development we observed previously. However, in vitro whole blood killing assay showed that whole blood from δ^D910A/D910A mice exhibited higher cytotoxicity to TCL1 leukemia cells despite their impaired development of the antigen specific anti-tumor CD8⁺ T cells.  

Conclusions: This study demonstrates that blocking p110δ in CLL not only abrogates survival signals in leukemic cells but also has the potential to reverse immune suppression in the micoenvironment. Studies are ongoing to pinpoint the effector cell subsets. The finding from this study will suggest that idelalisib, the PI3K p110δ inhibitor recently approved by FDA for the treatment of CLL, or other inhibitors of this pathway can potentially be used to treat a wide range of cancers by unleashing host anti-tumor immunity.