Dual Inhibition of PI3K-δ and PI3K-γ By Duvelisib Eliminates CLL B Cells, Impairs CLL-Supporting Cells, and Overcomes Ibrutinib Resistance in a Patient-Derived Xenograft Model
Authors: Chen Shih-Shih , Kutok Jeffery Lorne , Ferrer Gerardo , Ravichandran Priyadarshini , Ibrahim Michael , Kieso Yasmine , Barrientos Jacqueline C , Weaver David T. , Pachter Jonathan A , Rai Kanti R , Chiorazzi Nicholas 
Published: October 2019

Abstract
Novel agents targeting the B-cell receptor signaling pathway, such as ibrutinib and idelalisib, are effective in patients with chronic lymphocytic leukemia (CLL), but complete responses are infrequent and residual disease often remains. Patients may discontinue ibrutinib because of unacceptable adverse events or if they develop ibrutinib resistance due to Bruton tyrosine kinase (BTK) or phospholipase C gamma 2 (PLCG2) mutations or other less-well-defined mechanisms. Therefore, alternative therapies that can overcome ibrutinib intolerance and resistance are needed. Duvelisib (DUV) is an oral dual phosphatidylinositol 3-kinase (PI3K)-δ and -γ inhibitor with activity in patients with relapsed/refractory CLL and a manageable safety profile. Preclinical studies point to distinct roles of PI3K-δ and -γ in CLL biology, suggesting that dual isoform inhibition may enhance efficacy by targeting both CLL and CLL-supporting cells. Importantly, DUV potently inhibits PI3K-δ and -γ in human peripheral blood mononuclear cells and CLL cells. Here, we further characterize the distinct functions of PI3K-δ and -γ in CLL and examine the mechanisms of action of DUV in vitro and in a patient-derived xenograft (PDX) model, highlighting the ability of DUV to overcome ibrutinib resistance.
We first assessed the in vivo efficacy of PI3K-δ and/or PI3K-γ inhibition on CLL B cells using a PDX mouse model in which activated patient-derived T cells and CLL B cells were injected into alymphoid NOD-scid IL-2Rγnull (NSG) mice. Cells from 4 patients were transferred and allowed to expand for 2 weeks, after which mice were treated with DUV, a PI3K-δ inhibitor, or a PI3K-γ inhibitor for 3 weeks. DUV and the PI3K-δ inhibitor, but not the PI3K-γ inhibitor, significantly decreased the number of CLL B cells in the spleens of mice. In another set of experiments, combination treatment with the PI3K-δ and -γ inhibitors more potently impaired CLL B-cell survival than the PI3K-δ inhibitor alone.
Next, the effects of PI3K-δ or -γ inhibition on CLL-supporting cells, ie, autologous T cells and murine macrophages, were examined in the PDX model using the same 4 patient samples. Significant decreases in both patient-derived T cells and murine macrophages were observed in the spleens of mice treated with DUV or the PI3K-γ inhibitor but not with the PI3K-δ inhibitor. Therefore, the function of PI3K-δ and -γ in macrophages and macrophage-supported CLL cell survival were examined in vitro. Murine bone marrow-derived macrophages were polarized via interleukin 4 and macrophage colony stimulating factor in the presence of DUV, a PI3K-δ inhibitor, or a PI3K-γ inhibitor. Both DUV and the PI3K-γ inhibitor impaired M2 polarization, assessed by arginase 1 (ARG1) mRNA expression. Culture with M2 macrophages increased CLL B-cell viability, and the addition of DUV inhibited this survival-promoting activity more than the PI3K-δ or -γ inhibitors alone.
To assess whether DUV could inhibit CLL B cells from ibrutinib-unresponsive patients, activated T cells and CLL B cells from 2 patients who progressed on ibrutinib (1 with a BTK C481S mutation and 1 without a BTK mutation) were transferred into NSG mice and allowed to expand for 2 weeks, and then mice were treated with DUV or ibrutinib for 3 weeks. For both patient samples, there was a > 10-fold reduction in the number of CLL B cells recovered from the spleens of mice treated with DUV, as well as a significant reduction in the percentage of proliferating CLL B cells. In contrast, ibrutinib did not have significant impact on CLL B-cell numbers or proliferation in the spleen.
In conclusion, DUV inhibits the in vivo survival and proliferation of leukemic B cells from CLL patients, including those who have progressed on ibrutinib. Dual PI3K-δ and -γ inhibition is more effective at inhibiting CLL B cells in vivo than PI3K-δ inhibition alone. Moreover, PI3K-γ inhibition shifts macrophage polarization away from a CLL-supportive M2 phenotype. Thus, DUV exerts inhibitory effects on CLL B cells and on CLL-supporting T and myeloid cells. Overall, these findings elucidate the non-redundant roles of PI3K-δ and -γ in CLL and demonstrate the potent antitumor activity of dual PI3K isoform inhibition by DUV in ibrutinib-resistant patient CLL cells in vivo. Further investigation of DUV as a therapeutic option for patients who are refractory to or intolerant of ibrutinib or other BTK inhibitors is ongoing in a phase 2 clinical trial (BRIO; NCT03370185).

Disclosures
Chen: Janssen: Research Funding; ArgenX: Research Funding; Beigene: Research Funding; Pharmacyclics: Research Funding; Verastem: Research Funding. Kutok:Infinity Pharmaceuticals: Employment, Equity Ownership. Barrientos:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Weaver:Verastem Oncology: Employment, Other: Stockholder; Agios Pharmaceuticals: Employment; Femto Dx: Equity Ownership. Pachter:Verastem: Employment, Other: Stockholder. Rai:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.