Fig. 5: Development of efficient therapies for ITD/ITD; p53 KO leukemia.

a Growth inhibition of ITD/ITD, ITD/ITD; p53^+/− and ITD/ITD; p53^−/− leukemic cells treated with crenolanib, midostaurin, and carfilzomib. Of note, ITD/ITD; p53^−/− leukemic cells were highly sensitive to carfilzomib (IC50: 17.1 nM), although ITD/ITD; p53^−/− leukemic cells appeared to have decreased sensitivity to carfilzomib compared with ITD/ITD and ITD/ITD; p53^+/− cells (IC50: 5.2 nM and 6.8 nM, respectively). Apoptosis was analyzed by Annexin V and propidium iodide (PI) staining. Leukemic cells were treated with different concentrations of crenolanib, midostaurin, and carfilzomib for 48 h before flow cytometric analyses. Survival was defined as Annexin V/PI double-negative staining. Apoptosis was defined as Annexin V-positive or PI-positive staining. Each plot and bar represents the mean viability rate ± SD under treatment with the indicated inhibitors. T-ALL cell lines, but no AML cell lines were generated from individual mice. ITD/ITD, n = 6; ITD/ITD; p53^+/−, n = 6; and ITD/ITD; p53^−/−, n = 3. b MV4-11 and MV4-11 treated with Sorafenib were simultaneously subject to an antibody array. Treatment with Sorafentib significantly dephosphorylated FLT3 and induced strong AXL activation. Specificity for the phosphorylation of AXL was confirmed by western blot (Supplementary Fig. 13). c Effects of combined therapies in the MV4-11 AML cell line with FLT3-ITD, showing the 20-fold and 4-fold increased expression of MDM2 and MDM4, respectively. The results presented are the mean ± SD of at least three independent experiments. Combination index: carfilzomib/crenolanib = 0.36, carfilzomib/midostaurin = 0.25, carfilzomib/gilteritinib = 0.39, carfilzomib/R428 = 0.36. d Flow cytometric analyses showing the expression of FLT3 and AXL in patient #016. The negative controls were shown as inserts. AXL expression was also found in five of six myeloid leukemic cell lines (data not shown). e A representative flow cytometric analysis showing the strong induction of apoptosis, induced by the combinations of carfilzomib/midostaurin and carfilzomib/R428 in primary AML cells from patient #016 with FLT3-ITD and up to four-fold increased expression levels of MDM2 and MDM4. f Effects of combined therapies in primary patient samples. The results are presented as the mean ± SD of independent experiments. The combination of carfilzomib/R428 induced more apoptotic cells than any single treatment in all patient samples, whereas for other combined therapies an inferior effect was observed in some patient samples when compared with single treatments. For example, carfilzomib/crenolanib induced fewer apoptotic cells than carfilzomib in five patient samples. Up to 25-fold and 41-fold increased expression levels for MDM2 and MDM4, respectively, were observed in samples from FLT3 + patients by TagMan assays. Only one patient harbored a TP53 mutation, for which no additive/synergistic cytotoxicity was observed in the combined therapies. We took both FLT3-ITD and FLT3 WT AML samples for analyses because proteasome inhibitors can downregulate the protein expression of both FLT3-ITD and FLT3 WT [47], and AXL may play an important role in the pathogenesis of FLT3-ITD and FLT3 WT AML [34]. Carfilzomib: n = 22 patients; crenolanib: n = 22; midostaurin: n = 22; gilteritinib: n = 11; R428: n = 14; carfilzomib/crenolanib: n = 22; carfilzomib/midostaurin: n = 22; carfilzomib/gilteritinib: n = 11; carfilzomib/R428: n = 14. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. g Survival curve of NSG mice transplanted with MV4-11 cells. Therapies were started on day 3 and stopped on day 23 (with the transplantation date representing day 0). *p < 0.05, **p < 0.01, ***p < 0.001.