Pharmacological inhibition of phosphatiylinositide-3-kinase (PI3K)-mediated signaling holds great promise for treating chronic lymphocytic leukemia (CLL). Therefore we assessed three structurally related PI3K inhibitors targeting the PI3K-d isoform for their ability to inhibit the survival of freshly isolated CLL cells. The purely PI3K-d-selective inhibitor idelalisib was compared to copanlisib and duvelisib (IPI-145), with isoform target profiles that additionally include PI3K-a or PI3K-c, respectively. The concentrations leading to half-maximal reduction of the survival of CLL cells were more than ten-fold lower for copanlisib than for idelalisib and duvelisib. At concentrations reflecting the biological availability of the different inhibitors, high levels of apoptotic response among CLL samples were attained more consistently with copanlisib than with idelalisib. Copanlisib selectively reduced the survival of CLL cells compared to T cells and to B cells from healthy donors. In addition copanlisib and duvelisib impaired the migration of CLL cells towards CXCL12 to a greater extent than equimolar idelalisib. Similarly copanlisib and duvelisib reduced the survival of CLL cells in co-cultures with the bone marrow stroma cell line HS-5 more strongly than idelalisib. Survival inhibition by copanlisib and idelalisib was enhanced by the monoclonal CD20 antibodies rituximab and obinutuzumab (GA101), while antibody-dependent cellular cytotoxicity mediated by alemtuzumab and peripheral blood mononuclear cells was not substantially impaired by both PI3K inhibitors for the CLL-derived JVM-3 cell line as target cells. Taken together, targeting the a-and d-p110 isoforms with copanlisib may be a useful strategy for the treatment of CLL and warrants further clinical investigation.Chronic lymphocytic leukemia (CLL) is a common B cell malignancy that usually occurs at advanced age and follows a highly variable course. 1 Although management of the disease has been steadily improved, with chemo-immunotherapy by fludarabine, cyclophosphamide and rituximab (FCR) as the current treatment standard for patients without co-morbidity, 2 there is a need for more efficient and less toxic therapies, since CLL can only be cured by allogeneic stem cell transplantation. Therefore, novel targeted agents are being developed, some of which show great promise and might replace chemotherapy regimens in the near future. 3

The antibody-dependent cell-mediated cytotoxicity (ADCC) of the anti-CD20 monoclonal antibodies (mAbs) rituximab and obinutuzumab against the cell line Raji and isolated CLL cells and its potential impairment by kinase inhibitors (KI) was determined via lactate dehydrogenase release or calcein retention, respectively, using genetically modified NK92 cells expressing CD16-176V as effector cells. Compared to peripheral blood mononuclear cells, recombinant effector cell lines showed substantial alloreactivity-related cytotoxicity without addition of mAbs but afforded determination of ADCC with reduced interassay variability. The cytotoxicity owing to alloreactivity was less susceptible to interference by KI than the ADCC of anti-CD20 mAbs, which was markedly diminished by ibrutinib, but not by idelalisib. Compared to rituximab, the ADCC of obinutuzumab against primary CLL cells showed approximately 30% higher efficacy and less interference with KI. Irreversible BTK inhibitors at a clinically relevant concentration of 1 μM only weakly impaired the ADCC of anti-CD20 mAbs, with less influence in combinations with obinutuzumab than with rituximab and by acalabrutinib than by ibrutinib or tirabrutinib. In summary, NK cell line-based assays permitted the sensitive detection of ADCC of therapeutic anti-CD20 mAbs against CLL cells and of the interference of KI with this important killing mechanism.

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To elucidate their mechanism of action, inhibitors of Bruton tyrosine kinase (BTK) and resistant BTK mutants were employed to dissect target-dependent cellular functions. BTK-C481S and -T474I, expressed in Ramos and NALM-6 cells, maintained BTK auto-phosphorylation under treatment with ibrutinib or dasatinib, respectively, which showed only modest cytotoxicity. Retained activity of BTK-T474 partially rescued cell migration from inhibition by dasatinib. Importantly, resistant BTK mutants reconstituted B cell receptor-triggered chemokine secretion in the presence of corresponding inhibitors, demonstrating that BTK activity is connected with cell-intrinsic functions of malignant B cells with importance for their dialogue with the micro-environment.