The Combination Of FLT3 Inhibition and Hypomethylation Confers Synergistic Anti-Leukemic Effects On FLT3/ITD Positive AML Cell Lines and Primary Cells
Authors: Konig Heiko , Levis Mark 
Published: October 2019

Abstract

Background
Effective treatment regimens for elderly FLT3/ITD+ AML patients are lacking. Recent data on the effects of the FLT3 tyrosine kinase inhibitor quizartinib (QUIZ) on FLT3/ITD+ AML showed promising clinical activity, including in elderly patients. Hypomethylating agents (DNMTis), such as Decitabine (DEC) and 5-azacytidine (AZA), demonstrated clinical benefit in AML and are well tolerated in elderly patients, and are associated with minimal increases in FLT3 ligand, which can represent a potential resistance mechanism to FLT3 inhibitors. In addition, both FLT3 inhibition and hypomethylation therapy are associated with the induction of terminal differentiation of myeloid blasts. Therefore, there is a strong theoretical rationale for combining FLT3 inhibition and hypomethylation for FLT3/ITD AML, particularly in the elderly. Here, we investigated whether the combination of DEC or AZA with QUIZ bears synergistic anti-leukemic potential, and if the sequence of administration was important.

Methods
Molm14 (M14) and primary cells from elderly, newly diagnosed (ND) and relapsed (REL) FLT3/ITD+ AML patients were incubated in culture medium. QUIZ, DEC or AZA were added either as single agents or in combination (simultaneous vs. sequential administration) at the indicated concentrations, in the presence (M14 and primary cells) and absence of stroma (M14). After 48 to 72 hours, cells were assessed for proliferation and apoptosis using MTT assays, annexin-V staining and FACS analysis. A combination index (CI) was calculated using Calcusyn Software.

Results
We found that the presence of stromal cells significantly blunted the cytotoxic effects of each compound, both when administered as single agent and in combination. In M14, CI values indicated that combined treatment resulted in synergistic effects that were more pronounced in the presence as compared to the absence of stroma (CIstroma: 0.16 [QUIZ 1nM+DEC 1μM] and 0.2 [QUIZ 1nM+AZA 1μM]; CIno stroma: 0.43 [QUIZ 1nM+DEC 1μM] and 0.87 [QUIZ 20nM+AZA 1μM]). Furthermore, a trend towards greater growth inhibition was observed when the drugs were administered simultaneously rather than in a sequential mode. While the pro-apoptotic effects of DEC were less marked, combined treatment (QUIZ +DEC or AZA) did not further induce apoptosis as compared to QUIZ alone. In primary FLT3/ITD+ AML cells, single agent QUIZ, DEC and AZA demonstrated dose dependent, growth inhibitory effects. While AZA was significantly more potent than DEC in ND (n=3) and REL (n=3) cells, REL cells were generally more drug sensitive (ND: 48.7±10.6% [4μM AZA] vs. 6.0±7.9% [4μM DEC]; REL: 71.3±2.7% [4μM AZA] vs. 5.5±3.1% [4μM DEC]; p<.005). Consistent with greater FLT3-addiction, QUIZ was significantly more potent in REL than in ND cells (5nM: 13.5±4.9% [ND] vs. 28.5±5.0% [REL], p<.05; 10nM: 21.8±4.0% [ND] vs. 41.5±4.8% [REL], and 20nM: 24.9±3.0% [ND] vs. 49.2±6.5% [REL]; p<.005). Combined treatment with QUIZ and AZA resulted in significant, additive to synergistic growth inhibition compared to QUIZ alone in ND and REL cells. QUIZ and DEC demonstrated synergistic growth inhibition only in REL (46.0±6.8% [20nM AC], 15.5±3.1% [DEC 4μM], 55.5±6.3% [QUIZ 20nM+ DEC 1uM], CI=0.68) but not in ND cells. The combination of QUIZ/AZA induced a modest degree of apoptosis in both ND and REL primary cells, whereas DEC with or without QUIZ did not induce any meaningful degree of apoptosis.

Conclusions
1. Not surprisingly, stroma blunted the cytotoxic effect of all treatments
2. On stroma, 5-azacytidine was considerably more cytotoxic than decitabine, at least from the standpoint of inducing growth inhibition or apoptosis.
3. There was synergistic cytotoxicity in blasts on stroma with quizartinib/5-azacytidine, less so with quizartinib/decitabine.
4. Simultaneous administration was more effective than sequential.
5. We are currently studying the efficacy of these combinations at inducing terminal myeloid differentiation in these FLT3/ITD AML samples.
6. The combination of a hypomethylating agent and FLT3 inhibition is a potentially important new therapeutic regimen for FLT3/ITD AML, particularly for older patients.
A current Phase 1 trial with the combination of AZA and QUIZ in newly diagnosed and relapsed patients is currently being explored.

Disclosures:
Levis: Ambit Biosciences: Consultancy.