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K. Seipel et al.
translocations with MLL gene rearrangements. Some cell lines contained additional mutations in driver genes such as DNMT3A (OCI-AML2, OCI-AML3) and RAS genes (OCI-AML3, PL-21). DNMT3a and RAS gene mutations may influence sensitivity to MDM2 or FLT3 inhibitors. The MDM2 inhibitors included idasanutlin (RG7388), NVP-CGM097 and NVP-HDM201. The FLT3 inhibitors included the 1st, 2nd and 3rd generation inhibitors midostau- rin (PKC412), quizartinib (ACC220) and gilteritinib (ASP2215). The FLT3-ITD positive and TP53 wild type cell lines MOLM-13 and MV4-11 were most susceptible to all three FLT3- and all three MDM2-inhibitors (Figure 1). The effects on MOLM-13 cell survival induced by the three FLT3 inhibitors were consistent with IC50 values of 200nM (Figure 1A,B,C). Quizartinib had a greater potency with 70% cell viability after treatment with 10nM com- pound, while midostaurin and gilteritinib had comparable potencies with 90% cell viability at 10nM compound. OCI-AML2 and PL-21 cells showed some response to midostaurin while OCI-AML3, MOLM-16 and HL-60 cells were rather resistant to midostaurin and quizartinib. With respect to gilteritinib, however, OCI-AML3, PL-21 and HL-60 cells showed some response and only MOLM- 16 cells were resistant. The effects on MOLM-13 cell sur- vival varied for the MDM2 inhibitors with IC values of
NPM1 mutant and wild type, as well as TP53 mutant and wild type cells (Online Supplementary Table S1). Most of the FLT3-ITD AML cells had a high allelic ratio of FLT3-ITD (>0.5). Only few of the patient samples contained addi- tional mutations in driver genes, one with DNMT3A, one with RAS mutations. Samples of AML blast cells were grouped according to the major molecular subtypes (FLT3/TP53/ NPM1) and comprised at least four samples per molecular genetic combination, with median 84% blast cells ranging from 25 to 95% (Online Supplementary Table S1). Both midostaurin and NVP-HDM201 used as single agent treatment induced varying levels of loss in cell viability with correlation to certain AML subsets (Figure 2). Data for MOLM-13 and MV4-11 cell lines were includ-
Figure 3. Synergistic responses in FLT3-ITD AML blast cells to midostaurin and HDM201. Cell viability was determined in individual AML patient cells treated with increasing dosage of NVP-HDM201 as single compound and in combina- tion treatment with midostaurin (PKC412) in FLT3-ITD/TP53wt/NPM1wt relapsed AML (patient 13) (A), FLT3-ITD/TP53wt/NPM1wt primary AML (patient 16) (B) and in FLT3-ITD/TP53wt/NPM1mut primary AML (patient 20) (C). Combination indexes were calculated according to Chou Talalay.42
A
B
C
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300nM NVP-HDM201, 1 mM RG7388 and 10 mM NVP-
CGM097 (Figure 1D,E,F). MOLM-13 cells were most sus- ceptible to MDM2 inhibitor idasanutlin with IC50 at 1 mM RG7388. MV4-11 and OCI-AML2 cells showed some response to idasanutlin with IC50 of 10 mM RG7388 while OCI-AML3, PL-21, HL-60 and MOLM-16 cells were rather resistant to idasanutlin. With respect to the MDM2 inhibitors NVP-CGM097 and NVP-HDM201 MOLM-13, MV4-11 and OCI-AML2 cells showed consistent suscepti- ble responses while OCI-AML3 and PL-21 cells were less susceptible and MOLM-16 and HL60 cells were resistant. In order to define the most effective treatment combina- tion we focused our studies on the latest and most potent MDM2 inhibitor NVP-HDM201 and tested its effects in single agent treatment and together with the three FLT3- inhibitor compounds in MOLM-13 cells. The combination of NVP-HDM201 and midostaurin had excellent synergis- tic effects on cell survival with a combination index of 0.4, while the combination of NVP-HDM201 with quizartinib or gilteritinib had only moderate synergistic effects with combination indexes of 0.7 and 0.8 (Figure 1H). To deter- mine the relevance of the order of addition, midostaurin and NVP-HDM201 were tested as direct combination or sequential treatment and found to be effective independ- ent of sequence of application. NVP-HDM201 pretreat- ment followed by midostaurin treatment had similar effects on cell viability as midostaurin pretreatment fol- lowed by NVP-HDM201 treatment. Moreover, both sequential treatments had comparable effects on cell via- bility as direct combination treatment (Online Supplementary Figure S1).
Sensitivity of AML patient cells to the MDM2 inhibitor HDM201 and the FLT3 inhibitor midostaurin
To determine the sensitivity of NK-AML blast cells to HDM201 and midostaurin, mononuclear cells isolated from peripheral blood or bone marrow of NK-AML patients were subjected to in vitro cytotoxicity assays. The NK-AML cells covered the major morphologic and molec- ular subtypes including FLT3-ITD and FLT3 wild type,
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