Background: The neoplastic B cells of indolent non-Hodgkin lymphoma (iNHL) and chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) rely upon the support of non-neoplastic cells within their microenvironment for proliferation and survival. Support cells include T cells, myeloid-derived cells, and mesenchymal stromal cells, which provide phosphoinositide-3 kinase (PI3K)-dependent survival and growth signals for the neoplastic cells, as well as signals that maintain the tumor microenvironment (TME). Duvelisib (IPI-145) is an oral inhibitor of PI3K-δ,γ in clinical development for iNHL and CLL/SLL. To better understand the roles of the PI3K-δ and PI3K-γ isoforms in mediating signaling between the tumor and TME cells in B-cell malignancies, Infinity’s highly potent (low nM) PI3K isoform-selective compounds that target either PI3K-δ or PI3K-γ with >100-fold selectivity over the other PI3K isoforms were utilized in in vitro experiments.
Methods/Results: A mixture of cytokines (CD40L/IL-2/IL-10) was utilized in an assay that recapitulated TME-induced malignant B-cell proliferative responses. Duvelisib inhibited CD40L/IL-2/IL-10-induced proliferation of primary CLL cells with an average IC50 in the sub-nanomolar range. The use of PI3K isoform-selective inhibitory compounds revealed these proliferative signals are PI3K-δ dependent, as the PI3K-δ-selective inhibitor was more active than the PI3K-γ-selective inhibitor.
While these experiments established the direct PI3K-δ dependence of TME-derived cytokines on CLL cell proliferation, the role of PI3K-γ in key functions such as the directed migration of normal immune cells of the TME was also tested. We hypothesized that chemokines that recruit immune cells to the TME would signal through G-protein coupled receptors linked to PI3K-γ. The stromally-derived chemokine CXCL12 resulted in upregulation of phospho (p)-AKT in both the CD3+ T-cell and CLL-cell populations in CLL patient total peripheral blood mononuclear cells (PBMCs). Using isoform-selective inhibitors, the increase in CXCL12-induced pAKT in CD3+ T cells was found to be mediated by PI3K-γ. Interestingly, within the malignant B-cell population, the increase in CXCL12-induced pAKT was PI3K-δ dependent, suggesting that CXCL12 signals through different PI3K isoforms in these varying cell types. Chemotactic assays demonstrated reduced migration of total CLL PBMCs towards CXCL12 in the presence of combined PI3K-δ and PI3K-γ inhibition by duvelisib. Flow cytometric analyses of the migrating populations revealed that the greatest effect of duvelisib on CXCL12-induced migration occurred primarily within the T-cell population. Utilizing PI3K isoform-selective compounds, the inhibition of T-cell migration toward CXCL12 was found to be a PI3K-γ mediated process, as the PI3K-γ-selective inhibitor was more potent than the PI3K-δ-selective inhibitor in blocking T-cell migration.
Myeloid-derived cells and mesenchymal stromal cells can also support CLL cell survival as components of the TME. Recent reports suggest that CLL cytoprotective nurse-like cells may have an M2 polarization and be similar to the immunosuppressive myeloid-derived suppressor cells found in some solid tumors [Gianonni et al. Haematologica 2014, 99(6)]. To model these TME components, mouse bone marrow cells were differentiated into macrophages with murine MCSF and IL-4 (M2–polarized). CXCL12-induced pAKT in these M2 cells, which express CXCR4, was more potently inhibited by duvelisib and the PI3K-γ-selective inhibitor than the PI3K-δ-selective inhibitor. Finally, co-cultures of M2 macrophages with CLL cells led to extended CLL cell survival. These data show that CXCL12 mediated-M2 activation is dependent upon PI3K-γ and that M2-cells can act to support CLL cell survival.
Conclusions: T cells and myeloid cells provide a survival and proliferative advantage to malignant CLL cells within the TME. The role of PI3K-γ in the migration and activation of these cells supports the potential for therapeutic benefit from inhibition of PI3K-γ. By inhibiting both the PI3K-δ and PI3K-γ isoforms, duvelisib is uniquely positioned to inhibit key signals important in the pathogenesis of B-cell malignancies.
Peluso:Infinity Pharmaceuticals, Inc.: Employment. Faia:Infinity Pharmaceuticals, Inc.: Employment. Winkler:Infinity Pharmaceuticals, Inc.: Employment. Patel:Infinity Pharmaceuticals, Inc.: Employment. Brophy:Infinity Pharmaceuticals, Inc.: Employment. White:Infinity Pharmaceuticals, Inc.: Employment. Douglas:Infinity Pharmaceuticals, Inc.: Employment. Stern:Infinity Pharmaceuticals, Inc.: Employment. Palombella:Infinity Pharmaceuticals, Inc.: Employment. McGovern:Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment.
Asterisk with author names denotes non-ASH members.