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K. Seipel et al.
FLT3-ITD/TP53wt/NPM1wt and TP53mut cells were minimally susceptible to NVP-HDM201 with 1-5% reduced viability when treated with 1 mM compound (Figure 2B).
In single agent treatments with 50nM compound, FLT3- ITD/TP53wt/NPM1wt cells were significantly more sus- ceptible to midostaurin and NVP-HDM201 than FLT3- ITD/TP53wt/NPM1mut and FLT3wt cells (Figure 2C,D). FLT3-ITD/TP53wt/NPM1wt cells lost more than 20% cell viability within 24hours at 50nM PKC412 while all other AML cells and normal peripheral blood monocytes were unaffected by this low dose treatment (Figure 2C). Similarly, FLT3-ITD/TP53wt/NPM1wt cells lost 30% cell viability within 24 hours at 50nM NVP-HDM201 while all other AML cells were significantly less affected by this low dose treatment and TP53mut AML cells as well as normal peripheral blood monocytes were unaffected (Figure 2D).
These data propose that the ideal target population for the treatment with midostaurin and NVP-HDM201 are FLT3-ITD NK-AML cells with a high allelic ratio of FLT3- ITD that are wild type for TP53 and NPM1.
Specificity and efficacy of combined HDM201 and midostaurin against FLT3-ITD/TP53wt/NPM1wt AML cells
The response of AML cells to 50nM midostaurin in combination with conventional induction therapy (CI, 20nM cytarabine and 20nM idarubicin) or in combination with 50nM NVP-HDM201 was determined by in vitro cytotoxicity assays. Similar to the single agent treatments reported above, FLT3-ITD/TP53wt NK-AML cells were most susceptible to the combined treatment whereas FLT3-ITD/TP53mut cells turned out to be resistant and FLT3-ITD/TP53wt cells showed intermediate responses (Figure 2E,F).
The combination of midostaurin with conventional induction treatment had significant effects on TP53wt AML cells with 30-40% median loss of cell viability in FLT3-ITD cells and 15-25% reduction in FLT3wt cells exposed to 20nM CI and 50nM midostaurin for 24 hours (Figure 2E). TP53mut AML cells and normal peripheral blood monocytes were affected with 5-10% median loss- es in cell viability. The effects of conventional induction treatment on cell viability were enhanced by the addition of midostaurin in FLT3-ITD/TP53wt cells independent of their NPM1 status (Figure 2E).
The combination of midostaurin with NVP-HDM201 was as effective as the combination of midostaurin with standard induction therapy. As in the single agent treat- ments, FLT3-ITD/TP53wt/NPM1wt cells were most sus- ceptible to this combination with 40% median loss of cell viability in 24 hours to 50nM NVP-HDM201 and 50nM midostaurin (Figure 2F). FLT3-ITD/TP53wt/NPM1mut and FLT3wt/TP53wt/NPM1wt AML cells were less sus- ceptible with a median reduction of 20% cell viability in this combination treatment. FLT3wt/TP53wt/NPM1mut and TP53mut AML cells as well as normal peripheral blood monocytes were least affected with 3% median losses in cell viability. The combination of NVP-HDM201 and midostaurin had synergistic effects on cell survival of FLT3-ITD positive AML cells with a combination index of 0.63 in the relapsed AML patient #13 (Figure 3A), and moderate synergistic effects in FLT3-ITD positive primary AML cells (Figure 3B,C). FLT3-ITD/TP53wt/NPM1wt pri-
mary AML cells (patient #16) were most susceptible to this combination treatment (Figure 3B) with reduced responses in FLT3-ITD/TP53wt/NPM1wt (patient#20) pri- mary AML (Figure 3C).
To confirm p53 activation in the presence of MDM2 inhibitors we determined the expression levels of the tumor suppressor protein p53 and of the p53 target genes CDKN1A and MCL1 in AML cells treated for 24 hours with single compounds and with combined treatment. Protein p53 was stabilized and p53 levels were increased in AML cells treated with 200nM NVP-HDM201, with three- to eightfold induction in MV4-11, MOLM-13 and OCI-AML3 cells, while OCI-AML2 cells had a high p53 level with a maximal 20% increase (Figure 4 A, B, C, D). CDKN1A gene expression was significantly induced in FLT3-ITD/TP53wt/NPM1wt AML cells (MOLM-13, MV4-11, patient#13) and in FLT3wt/TP53wt/NPM1wt cells (OCI-AML2) treated with 50nM NVP-HDM201 (Figure 4E), and in FLT3wt/TP53wt/NPM1mut cells (OCI- AML3) treated with 500nM NVP-HDM201, but not in FLT3wt/TP53mut cells (MOLM-16). To reach the same level of p53 target gene expression induced by 50nM NVP- HDM201 in FLT3-ITD/NPM1wt (MOLM-13, MV4-11, pat#13) and FLT3wt/NPM1wt (OCI-AML2) cells, ten times more MDM2 inhibitor was required in FLT3wt/NPM1mut (OCI-AML3) cells. Yoshimoto et al. 2009 showed that FLT3-ITD up-regulates the apoptosis inhibitor MCL-1 to promote survival of stem cells in acute myeloid leukemia. They analyzed the function of MCL-1 in FLT3-ITD AML and showed that the enforced expres- sion of MCL-1 prevented MV4-11 cells from apoptosis in the presence of 100nM midostaurin. Inhibition of MCL-1 by shRNA resulted in apoptosis of MV4-11 cells. To eluci- date the mechanism of apoptosis induction by NVP- HDM201 and midostaurin we analyzed MCL-1 expres- sion in a variety of AML cells (Figure 4F). MCL-1 gene expression was repressed in the presence of 50nM NVP-HDM201 or 50nM midostaurin in FLT3-ITD/TP53wt/NPM1wt AML cells (MOLM-13, MV4-11, patient#13), with enhanced effects in the combi- nation treatments. MCL-1 gene expression was repressed in the presence of 50nM NVP-HDM201 in FLT3wt/TP53wt/NPM1wt cells (OCI-AML2) and by 500nM NVP-HDM201 in FLT3wt/TP53wt/NPM1mut cells (OCI-AML3), but not by midostaurin. There was no repression of MCL-1 gene expression in FLT3wt/TP53mut cells (MOLM-16). In the susceptible FLT3-ITD cell lines MV4-11 and MOLM-13 as well as in the relapsed FLT3- ITD AML sample (patient#13) both compounds led to a significant reduction in MCL-1 gene expression with enhanced reduction in the combination treatments (Figure 4F). The effect of NVP-HDM201 and midostaurin treat- ment on MCL-1 gene repression appeared to be strongly synergistic with a combination index of 0.25. To further assess pro-apoptotic effects in AML cells treated with midostaurin and with the MDM2 inhibitor NVP- HDM201, cells were stained with AnnexinV and DAPI and analyzed on a cell imager. Apoptosis and cell death were induced in FLT3-ITD/TP53wt/NPM1wt cells in a dose dependent manner by both inhibitors in single com- pound and combination treatments. There was a signifi- cant increase in dead cells with subG1 DNA content, and a concomitant loss of cells in defined cell cycle stages, most prominently a reduction of cells with G0/G1 phase DNA content, but also of cells with S-phase and G2 phase
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haematologica | 2018; 103(11)