Figure 5.

Induction of apoptosis via enhanced BCL-2 dependence in FLT3-ITD+ AML cells with diverse co-existing somatic mutations by in vivo kinase inhibition

Overall, treatment with RK-20449 resulted in significant reduction of AML cells in BM, spleen, and PB of recipients (p < 1E-19 for each; data tabulated in table S3). To document patient-to-patient variability, RK-20449 responses were classified as follows: Complete response (A), if all recipients treated showed residual BM human CD45+ chimerism < 5%; good response (B), if the case did not meet the criterion for complete responder but all recipients showed < 50% residual BM human CD45+; partial response (C), if at least one of the recipients showed greater than 50% residual BM human CD45+. For each response group, PB time-course of human AML chimerism (leftmost panels) for RK-20449 treated recipients and final BM (middle panels) and spleen (rightmost panels) human AML chimerism of RK-20449 treated and untreated recipients are shown. Pre-treatment PB human AML cell chimerism is shown at week 0. The numbers of recipients for each patient/each treatment group and pre-/post-treatment AML chimerism are shown in table S3. In all response groups, BM, spleen, and PB chimerism was significantly reduced with RK-20449 treatment. For BM and spleen, final chimerism for recipients in each treatment group was compared. For PB, pre- and post-treatment chimerism for RK-20449-treated recipients was compared. p < 5.8E-5 by two-tailed t-test for all comparisons. In each scatter graph, dotted lines are drawn at 50%, 5%, and 0%.

(D) Dynamics of apoptotic response to BIM peptide in the presence of RK-20449 was measured for six AML cases. Bars represent the BIM IC50 of cytochrome C loss in RK-20449-treated human CD45+ cells as percentages of IC50 in cells treated with DMSO alone. Enhancement of apoptotic response to BIM by RK-20449 showed substantial patient-to-patient variability.

(E) Dynamics of apoptotic response to BH3-only peptides BAD, HRK, and NOXA in the presence of RK-20449 or DMSO alone was measured for seven AML cases. Bars represent the percentage reduction of IC50 in the presence of RK-20449 compared with DMSO alone. RK-20449 enhanced apoptotic response to BAD and HRK peptides with substantial patient-to-patient variability, whereas apoptotic response to NOXA peptide was not substantially enhanced by RK-20449.

(F) Apoptotic response to a BCL-2 inhibitor ABT-199 was enhanced by RK-20449 in seven AML cases. Bars show increased cytochrome C loss in human AML cells treated with ABT-199 and RK-20449 compared with DMSO alone-treated human AML cells.