FLT3 inhibition has elicited encouraging responses in acute myeloid leukemia (AML)
FLT3 inhibition has elicited encouraging responses in acute myeloid leukemia (AML) therapy. and during co-culture with mesenchymal stem cells that mimic the AML microenvironment. Furthermore, E6201 markedly reduced leukemia burden and improved the survival of mice in a human FLT3-mutated AML model. Collectively, our data provide a preclinical basis for the clinical evaluation of E6201 in AML patients harboring FLT3 mutations, including those who relapse following FLT3-targeted monotherapy. genes are common in patients with acute myeloid leukemia (AML) with normal cytogenetics, and are present in 24 to 30% (1, 2) and 10 to 15% (3), respectively. internal tandem duplication mutations (mutation is usually more frequent than mutation in AML. These mutations in turn lead to aberrant activation of FLT3 and/or RASCmitogen-activated protein kinase (MAPK) pathways. Responses to single agent tyrosine kinase inhibitors of FLT3 (e.g., quizartinib and sorafenib) (4, 5) or MAPK kinase (MEK1/2) (e.g., GSK1120212) (6) have been mostly restricted to patients with the corresponding mutations, which confirm that these mutations are valid targets. However, the treatment responses obtained with these brokers have been unsustainable. Purchase of point mutations, for which novel inhibitors are in early development, has emerged as an important mechanism of resistance to FLT3 inhibitors. However, this mechanism does not account for all of the acquired resistance processes that have been reported (7). Indeed, the Mouse monoclonal to CSF1 aberrant activation of parallel signaling pathways such as MAPK and AKT may also contribute to acquired resistance (8). In our clinical trials with sorafenib, the upregulation of phospho-ERK was observed in AML cells from patients with disease relapse, suggesting that MAPK activation takes place even when FLT3 phosphorylation remained suppressed (5). In addition, we have developed sorafenib-resistant cells by introducing clinically-relevant point mutations of Celiprolol HCl IC50 into murine leukemia cells (e.g., Ba/F3-ITD+842 and Ba/F3-ITD+676/842) and the upregulation of phospho-ERK was also observed in these cells (9). Unfortunately, comparable data are not available for patients treated with MEK inhibitors. However, Celiprolol HCl IC50 preclinical data suggests that FLT3 is usually upregulated when AML cells are uncovered to an inhibitor of MEK signaling (10). Furthermore, concomitantly targeting FLT3 and MEK signaling pathways has achieved encouraging synergistic anti-leukemia effects in our and studies, suggesting a potential for preventing/overcoming relapse in patients treated with FLT3 inhibitors like sorafenib and quizartinib (9). E6201 is Celiprolol HCl IC50 usually a synthetic small molecule that functions as a non-allosteric tyrosine kinase inhibitor, which inhibits both MEK1 and FLT3 (11). E6201 shows identical affinity and residence time for the active and inactive forms of MEK1 (12), and demonstrates different pharmacologic activities than those of allosteric MEK inhibitors and exerts exclusive effects on targeting acquired MEK1-C121S mutation, which confers resistance to the allosteric MEK inhibitor selumetinib (AZD6244) in melanoma (13). E6201 also has a long occupancy time for FLT3 (11-fold longer than that for MEK). In addition, the backbone structure of E6201 markedly differs from other allosteric FLT3 inhibitors such as sorafenib or quizartinib (Fig S1). Thus, E6201 is usually an attractive clinical compound for effectively targeting leukemic cells with aberrant activation of both FLT3 and MAPK signaling pathways, especially for those resistant to FLT3-inhibitors. Here, we report that E6201 has designated cytotoxic activity against AML cells harboring mutations. E6201 was especially effective in the killing of FLT3-inhibitor resistant cells harboring acquired point mutations of the FLT3 TKD domains. Thus, one-third of AML patients harboring FLT3 mutations may benefit from a dual MAPK/FLT3 inhibitor with potent anticancer effects, including in cells resistant to FLT3 monotherapy. Materials and Methods Reagents and antibodies E6201 was provided by Eisai Inc. (Woodcliff Lake, New Jersey), sorafenib and AC220 (quizartinib) were purchased from Selleckchem (Houston, TX). The chemical structures of these brokers are shown at Supplementary Physique S1. Recombinant human FLT3/FLK2 ligand (FL) was purchased from R&Deb (Minneapolis, MN). Interleukin-3 (IL-3) was purchased from PEPROTECH (Rocky Hill, NJ). The antibodies against human phosphorylated (p)-p44/42 MAPK (ERK1/2)(Thr202/Tyr204), phospho-AKT(Ser473), phospho-FLT3(Tyr589/591), phospho-S6K(Ser240/244), phospho-MEK1/2, AKT, S6K, Bcl-xL, and cleaved-caspase-3 were purchased from Cell Signaling Technology (Danvers, MA), against Bcl-2 from Dako (Carpinteria, CA), against phospho-STAT5 A/W from Upstate (Lake Placid, NY), against ERK2, FLT3, p53 and Mcl-1 from Santa Cruz Biotechnology (Santa Cruz, CA), against Bim and Puma from CalBiochem (San Diego, CA), and against Ki67 was purchased from Abcam (Cambridge, MA). The anti-luciferase antibody was purchased from Promega (Madison, WI). AML cell lines and patient samples The Ba/F3-FLT3, Ba/F3-ITD, Ba/F3-Deb835G and Ba/F3-Deb835Y cell lines (kindly provided by Dr. Donald Small in 2010, Department of Pediatric.