Persistent ERK Activation in Bone Marrow Blasts May Account for the Difference in Bone Marrow Versus Peripheral Blood Response to FLT3 Inhibition in FLT3/ITD AML
Authors: Yang Xiaochuan , Sexauer Amy , Levis Mark J. 
Published: October 2019

Abstract
Abstract 736
Given the poor prognosis of AML patients with FLT3/ITD mutations, a number of small molecule FLT3 inhibitors are being investigated in clinical trials for these patients. When administered as monotherapy, these agents induce clearance of peripheral blood blasts, but usually have only minimal effects on bone marrow blasts. For example, treatment with sorafenib, while effective in clearing the peripheral blood of blasts, has resulted in only modest marrow blast reductions (Borthakur et al, Haematologica 2010). In contrast, AC220 (quizartinib), a more recently developed FLT3 inhibitor, has induced a much higher rate of marrow responses in an ongoing clinical trial (Cortes et al, 2011 EHA, abstract #1019). We wished to investigate the difference in response to FLT3 inhibition between peripheral blood and bone marrow blasts, and to better understand the basis for the difference in apparent efficacy between sorafenib and AC220. In our approach, we used a FLT3/ITD cell line (Molm14) and primary blasts from FLT3/ITD AML patients treated with sorafenib and AC220 either in suspension or in co-culture with human bone marrow stromal cells. We found that both sorafenib and AC220 could successfully inhibit FLT3, STAT5, AKT, and ERK signaling in Molm14 cells in suspension at concentrations below 100nM, with apoptosis induced in a dose-dependent fashion. The IC50 for inducing apoptosis was 0.4 nM for AC220 and 3.3 nM for sorafenib. The co-culture of Molm14 with bone marrow stroma did not affect the ability of AC220 and sorafenib to inhibit FLT3 and STAT5. However, downstream signaling of AKT and ERK was augmented by co-culture with stroma, and, in comparison with suspension cultures, 3-fold higher concentrations of sorafenib and AC220 were required to inhibit them. The addition of exogenous FL (2.5 ng/mL) to Molm14 cells co-cultured with stroma further enhanced the phosphorylation of FLT3, Akt and ERK, but not of STAT5. Cell survival (as determined by an apoptosis assay) correlated tightly with Akt and ERK phosphorylation. By comparison, treatment with another cytokine, IL-3, had no effect on the response to FLT3 inhibitors on or off stroma. Thus, in the Molm14 model, the resistance to FLT3 inhibitors conferred by both FL and bone marrow stroma could be overcome by increasing the dosage of drugs. In Molm14 cells co-cultured with stroma and with FL, the IC50 for inhibition of ERK was 20nM and 100nM for AC220 and sorafenib, respectively. In primary FLT3/ITD AML blasts, STAT5 activation did not appear to be affected by FLT3 inhibition, either on or off stroma. Strikingly, ERK was strongly activated in these blasts when they were co-cultured with stroma, and this activation was unaffected by FL or by FLT3 inhibition. As reported previously, FLT3/ITD AML blasts cultured in suspension underwent apoptosis in response to FLT3 inhibition. However, when cultured for several days on stroma, FLT3 inhibition did not induce apoptosis. We conclude that bone marrow stroma provides survival signals to FLT3/ITD AML blasts that allow them to survive in the presence of even very potent FLT3 inhibition. We have previously presented data that FLT3 inhibition by AC220 induces terminal differentiation of bone marrow blasts (rather than apoptosis) in vivo (Levis et al, 2010 ASH, abstract #660). Our data suggests that FLT3 inhibition needs to be extremely potent in order to induce a meaningful affect on leukemia cells in the bone marrow, explaining the difference in responses between sorafenib and the more potent inhibitor AC220. The persistent activation of ERK in blasts on stroma irrespective of the activation of the FLT3 signaling pathway suggests that this represents an important survival signal conferred by the bone marrow stroma that may account for the difference in response to FLT3 inhibition observed between peripheral blood and bone marrow blasts.

Disclosures:
Levis: Ambit Biosciences, Inc: Consultancy.