The combination of FLT3 and DNA methyltransferase inhibition is synergistically cytotoxic to FLT3/ITD acute myeloid leukemia cells

Abstract

Effective treatment regimens for elderly acute myeloid leukemia (AML) patients harboring internal tandem duplication mutations in the FMS-like tyrosine kinase-3 (FLT3) gene (FLT3/ITD) are lacking and represent a significant unmet need. Recent data on the effects of FLT3 tyrosine kinase inhibitors on FLT3/ITD(+) AML showed promising clinical activity, including in elderly patients. DNA methyltransferase (DNMT) inhibitors such as decitabine (5-aza-2-deoxycytidine, DEC) and 5-azacitidine (AZA) demonstrated clinical benefit in AML, are well tolerated and are associated with minimal increases in FLT3 ligand, which can represent a potential resistance mechanism to FLT3 inhibitors. In addition, both FLT3 and DNMT inhibition are associated with the induction of terminal differentiation of myeloid blasts. Consequently, there is a strong theoretical rationale for combining FLT3 and DNMT inhibition for FLT3/ITD(+) AML. We therefore sought to study the anti-leukemic effects of DEC, AZA and FLT3 inhibitors, either as single agents or in combination, on AML cell lines and primary cells derived from newly diagnosed and relapsed AML patients. Our studies indicate that combined treatment using FLT3 inhibition and hypomethylation confers synergistic anti-leukemic effects, including apoptosis, growth inhibition and differentiation. The simultaneous administration of AZA and FLT3 inhibition appears to be the most efficacious combination in this regard. These drugs may provide a novel therapeutic approach for FLT3/ITD(+) AML, in particular for older patients.

Figure 1. Combined treatment results in synergistic anti-leukemic effects in the presence and absence of stroma

Molm14 cells were cultured in drug supplemented medium at the indicated concentrations, both in the presence and absence of stroma. Growth inhibition was assessed after 48 hours using MTT cell viability assays (A). Combination Index (CI) values are shown for each experiment. The mean ± SEM is based on replicate experiments (n= 3-12). Significant changes in the percentage of growth inhibition are indicated (*p<.05), (**p<.01), (***p<.001). Flow cytometry was used to measure apoptosis in these cells via Annexin V/propidium iodide and quantified as shown in (B).

Figure 1. Combined treatment results in synergistic anti-leukemic effects in the presence and absence of stroma

Molm14 cells were cultured in drug supplemented medium at the indicated concentrations, both in the presence and absence of stroma. Growth inhibition was assessed after 48 hours using MTT cell viability assays (A). Combination Index (CI) values are shown for each experiment. The mean ± SEM is based on replicate experiments (n= 3-12). Significant changes in the percentage of growth inhibition are indicated (*p<.05), (**p<.01), (***p<.001). Flow cytometry was used to measure apoptosis in these cells via Annexin V/propidium iodide and quantified as shown in (B).

Figure 2. Combined treatment confers additive to synergistic growth inhibitory effects on primary FLT3 positive AML cells

Primary blasts derived from newly diagnosed (A, B) and relapsed (C, D) FLT3/ITD⁺ AML patients were incubated in drug supplemented medium at the indicated concentrations in the presence of stroma. Growth inhibition was assessed after 48 hours using the MTT assay. The mean ± SEM is based on replicate experiments (n= 3). Significant changes in the percentage of growth inhibition are indicated (*p<.05), (**p<.01), (***p<.001).

Figure 3. The sequence of administration for combining FLT3 inhibitors and DNMT inhibitors is crucial

Molm14 cells were cultured in drug supplemented medium in suspension. The different drugs (the FLT3 inhibitor quizartinib and the two DNMT inhibitors) were given in either sequentially or simultaneously: white bars denote sequential treatment with Quiz first followed by DEC/AZA, black bars represent sequential treatment with DEC/AZA first followed by Quiz, and gray bars denote simultaneous administration of both types of drugs. Following pretreatment, cells were incubated for an additional 48 hours before being harvested for flow cytometry analysis with Annexin V/propidium iodide staining. The mean ± SEM is based on replicate experiments (n= 3). Significant changes in the percentage of growth inhibition are indicated (*p<.05), (**p<.01), (***p<.001).

Figure 4. Treating FLT3/ITD blasts with a FLT3 inhibitor, a DNMT inhibitor, or both simultaneously induces differentiation

FLT3/ITD primary blasts co-cultured on stroma were treated with drug at the specified concentrations. Cells were harvested on Day 12 after starting drug treatment, stained for the cell-surface markers CD34, CD117, and CD11b, and analyzed via flow cytometry (A). The remaining cells were lysed and analyzed via immunoblotting (B).

Figure 4. Treating FLT3/ITD blasts with a FLT3 inhibitor, a DNMT inhibitor, or both simultaneously induces differentiation

FLT3/ITD primary blasts co-cultured on stroma were treated with drug at the specified concentrations. Cells were harvested on Day 12 after starting drug treatment, stained for the cell-surface markers CD34, CD117, and CD11b, and analyzed via flow cytometry (A). The remaining cells were lysed and analyzed via immunoblotting (B).

Figure 5. Peripheral blood absolute blast and neutrophil count of patient treated with 5-azacitidine and sorafenib

The patient was treated continuously with sorafenib 400 mg twice daily beginning on Day 1, and with 5-azacitidine 75 mg/meter-squared IV daily for 7 days during the time periods indicated by black bars. At each of the indicated time points, the cellular fraction from the peripheral blood was analyzed for the presence of the FLT3/ITD mutation.