Several PI3Kδ inhibitors are approved for the therapy of B cell malignancies, but their clinical use has been limited by unpredictable autoimmune toxicity. We have recently reported promising efficacy results treating chronic lymphocytic leukemia (CLL) patients with combination therapy with the PI3Kδγ inhibitor duvelisib and fludarabine cyclophosphamide rituximab (FCR) chemoimmunotherapy, but approximately one-third of patients develop autoimmune toxicity. We show here that duvelisib FCR treatment in an upfront setting modulates both CD4 and CD8 T cell subsets as well as pro-inflammatory cytokines. Decreases in naïve and central memory CD4 T cells and naïve CD8 T cells occur with treatment, while activated CD8 T cells, granzyme positive Tregs and Th17 CD4 and CD8 T cells all increase with treatment, particularly in patients with toxicity. Cytokines associated with Th17 activation (IL-17A and IL-21) are also relatively elevated in patients with toxicity. The only CLL feature associated with toxicity was increased priming for apoptosis at baseline, with a significant decrease during the first week of duvelisib. We conclude that an increase in activated CD8 T cells with activation of Th17 T cells, in the context of lower baseline Tregs and greater CLL resistance to duvelisib, is associated with duvelisib-related autoimmune toxicity.

Covalent inhibitors of Bruton tyrosine kinase (BTK) have transformed the therapy of chronic lymphocytic leukemia (CLL), but continuous therapy has been complicated by the development of resistance. The most common resistance mechanism in patients whose disease progresses on covalent BTK inhibitors (BTKi) is a mutation in the BTK 481 cysteine residue to which the inhibitors bind covalently. Pirtobrutinib is a highly selective, non-covalent BTK inhibitor with substantial clinical activity in patients whose disease has progressed on covalent BTKi, regardless of BTK mutation status. Using in vitro ibrutinib-resistant models and CLL patient cells we show that pirtobrutinib potently inhibits BTK-mediated functions including BCR signaling, cell viability and CCL3/CCL4 chemokine production in both BTK wild-type (WT) and C481S mutant CLL cells. We demonstrate that primary CLL cells from responding patients on the pirtobrutinib trial show reduced BCR signaling, cell survival and CCL3/CCL4 chemokine secretion. At time of progression, these primary CLL cells show increasing resistance to pirtobrutinib in signaling inhibition, cell viability and cytokine production. We employed longitudinal whole exome sequencing on two patients whose disease progressed on pirtobrutinib and identified selection of alternative-site BTK mutations, providing clinical evidence that secondary BTK mutations lead to resistance to non-covalent BTKi.