Mechanism-Based Combinatorial Strategy for Overcoming FLT3-Inhibitor Resistance in Dual FLT3 ITD and TKD Point Mutations
Authors: Ly Charlie , Zhang Weiguo , Ruvolo Vivian , Shah Neil P. , Andreeff Michael 
Published: October 2019

Abstract
One-third of acute myeloid leukemias (AML) harbor fms-like tyrosine kinase 3 (FLT3) gene mutations including internal tandem duplication (ITD) and D835 mutations (1). ITD mutations are associated with very poor prognosis (2). Targeted therapy directed against FLT3-ITD mutations has been shown to induce responses in AML patients, albeit only transiently (3). We and others have reported that secondary point mutations, in addition to ITD mutations and aberrant pro-survival signaling pathways such as MAPK or PI3K, are related to the development of resistance (4). To further address the mechanism of FLT3 inhibitor resistance, we generated several murine AML cell lines by introducing dual ITD and point mutations of the tyrosine kinase domain (TKD) 2 of FLT3. We hypothesized that concomitantly targeting FLT3 and MAPK/PI3K may act as a promising approach for overcoming resistance.
Previously we showed that E6201, a dual FLT3/MEK inhibitor, demonstrated encouraging anti-leukemia effectiveness in FLT3-ITD and D835G mutated cell lines (5). However, it was less sensitive in triggering cell death in D835Y and ITD/TKD2 dual mutation cells (e.g. ITD plus F691L, Y842C or D835Y etc.). Further investigations demonstrated that those mutated cells have high basal levels of either phospho-FLT3, -ERK, or -AKT. Interestingly, the phosphorylation levels of these proteins were persistently high during long-term exposure to low doses of E6201 (up to 7 to 11 days). As result, these cells became more resistant to subsequent E6201 treatment compared to naïve cells (EC50s were 0.92 vs 0.57 µM and 3.74 vs 1.08 µM in ITD+691 and D835Y mutated cells, respectively). However, concomitant targeting of FLT3/MEK and PI3K/mTOR with E6201 and voxtalisib (XL765) triggered pronounced synergistic pro-apoptotic effects in these pretreated cells (CIs were 0.83 and 0.16 in pretreated ITD+691 and D835Y mutated cells vs. 0.39 and 0.61 in naïve ITD+691 and D835Y mutated cells, respectively; suggesting that this combinatorial strategy may eliminate leukemic cells which have developed resistance during long-term E6201 monotherapy ..
Importantly, this synergistic effect was observed even under physiological hypoxic conditions (CI was 0.06 ± 0.01) and in the presence of mesenchymal stromal cells (MSC) (CI was 0.06 ± 0.04) as well, which are considered hallmark conditions of the bone marrow microenvironment. Since CXCR4-SDF-1a signaling axis is associated with MSC-mediated leukemia cell protection, we further tested the combination regime in the presence of CXCR4 antagonist plerixafor. Results demonstrated massive apoptosis induction in MSC co-culture systems (apoptosis induction was 23.9% vs. 91.8% in the absence vs. presence of plerixafor).
To minimize combination regime-induced toxicity, we further tested sequential treatment by E6201 and voxtalisib individually. Treatment with voxtalisib followed by E6201 demonstrated identical efficacy as concomitant voxtalisib/E6201 treatment, suggesting that this sequential approach may be beneficial by reducing putative toxicity and enhancing tolerance of the targeted drugs in clinical FLT3-targeted therapy. Conclusion: targeting FLT3/MEK and PI3K/mTOR with E6201 and voxtalisib has potential of preventing the development of FLT3-inhibitor resistance.

Disclosures
No relevant conflicts of interest to declare.