PKT16 induces PCD in CLL cells with no detected resistance. (A) Patients from supplemental Table 1 were classified into IGVH mutated (IGHV-M) or unmutated (IGHV-UM; mutated-VH3-21 gene [V3-21*], conferring worse prognosis, has been included in this group) and absence of del17p (No del17p) or del17p and incubated with PKT16 (100 µM, 6 hours). Cell viability was measured by Annexin-V/PI colabeling. After accounting for spontaneous PCD, the percentages of Annexin-V positive cells were recorded and expressed as a histogram. Box plots represent the mean of cell death in each population with minimum to maximum values. (B) B cells from patients with CLL with del13q alone (n = 14), tri12 (n = 3), del11q (n = 7), del17p (n = 6), 2p gain (n = 3), complex karyotype (CK; n = 7), or without any of these cytogenetic abnormalities (Abs; n = 8) were untreated or treated with PKT16 (100 µM, 6 hours), and cell death, measured by Annexin-V/PI costaining, was recorded and graphed. Box plots represent the mean of cell death in each population with minimum to maximum values. (C) Cell death was measured in CLL cells presenting in vitro resistance to at least 1 drug used in the treatment of CLL. Cells were incubated either with the combination FCR (fludarabine 10 µM, cyclophosphamide 3 mM, rituximab 10 µg/mL), ibrutinib (15 µM), idelalisib (100 µM), venetoclax (4 nM), or PKT16 (100 µM) over the course of 24 hours, and cytotoxicity was assessed by flow cytometry by Annexin-V/PI colabeling. The percentages of Annexin-V-positive cells were recorded and expressed as a dot plot. The points represent the results obtained in the CLL cells from each individual, and the lines indicate the mean of the 8 patients tested. (D) CLL cells were left untreated (Control) or incubated over the course of 24 hours, either with low doses of FCR (fludarabine 1 µM, cyclophosphamide 2 mM, and rituximab 10 µg/mL), ibrutinib (10 µM), idelalisib (50 µM), venetoclax (2 nM), PKT16 (50 µM), or the combination of PKT16 and the different drugs, all at the above low doses. The PCD induced was assessed by Annexin-V/PI colabeling and the percentages of Annexin-V-positive cells were recorded and expressed as a histogram (n = 6). Note that the combination of PKT16 with each individual drug induced an additional PCD rate. (E) Cell death was evaluated as above in CLL cells untreated (Control) or treated with venetoclax (4 nM) or PKT16 (100 µM, 6 hours) in the absence (−) or presence of interleukin 4 (50 ng/mL) and CD40L (100 ng/mL; n = 5). Statistical significance was calculated in panels A and C by the Student t test and in panels B, D, and E by Mann-Whitney U test. Symbols and bars represent mean ± SD.