Abstract 3037: Stromal cell activation of MAPK signaling pathways mediates resistance to MERTK inhibition in acute myeloid leukemia
Authors: Minson Katherine A. , Vasileiadi Eleana , Huey Madeline G. , Wang Xiadong , Frye Stephen V. , Earp H. Shelton , DeRyckere Deborah , Graham Douglas K. 
Published: August 2019

Abstract
MERTK is a TAM family (TYRO-3, AXL, MERTK) receptor tyrosine kinase that is aberrantly expressed in &gt;80% of primary acute myeloid leukemia (AML) samples. MERTK inhibition mediated by the small molecule tyrosine kinase inhibitor (TKI), MRX-2843, induced apoptosis in leukemia cell cultures and prolonged survival in mouse xenograft models of AML, but was not curative. In these models, treatment efficiently reduced peripheral disease burden but was less effective in the bone marrow, suggesting a role for the bone marrow microenvironment in therapeutic resistance. To evaluate the role of the bone marrow stromal niche in resistance to MERTK inhibition by MRX-2843, Kasumi-1 and OCI-AML5 AML cell lines were cultured with the fibroblast-like Hs27 cell line and induction of apoptosis and cell death was measured by flow cytometry after treatment with MRX-2843 or vehicle. Co-culture with Hs27 cells significantly reduced AML cell death in response to MRX-2843 compared to leukemia cells alone (e.g. Kasumi-1 31.9% vs 61.2%, p&lt;0.05). To identify parallel signaling pathways that may be activated under cytokine rich bone marrow stromal conditions and mediate resistance to MRX-2843, a unbiased phospho-kinase array was performed to identify targets differentially activated by co-culture and/or MRX-2843. Kasumi-1 cells were treated with MRX-2843 in the presence or absence of stromal co-culture, then cell lysates were incubated with human phospho-kinase arrays and proteins were quantitated by densitometry. Five kinases were differentially inhibited by &gt;1.5-fold in the presence or absence of stromal cells, including numerous MAPK pathway components - p38α, ERK1/2, EGFR, TOR, and HSP27. Changes in ERK1/2 phosphorylation (pERK) were confirmed by immunoblot in Kasumi-1 and OCI-AML5 cell cultures. In the absence of co-culture MRX-2843 potently inhibited pERK at doses as low as 100nM, while in co-culture even a 3-fold higher dose did not fully inhibit pERK, consistent with a potential role in resistance. To determine whether the observed upregulation and persistent activation of ERK1/2 has functional significance, Kasumi-1 cells were treated with MRX-2843 and the MEK1/2 TKI pimasertib in the presence or absence of stromal co-culture. Treatment with MRX-2843 or pimasertib alone did not significantly affect survival of AML cells in co-culture (31.3% MRX-2843, 28.1% pimasertib vs 22.7% vehicle, p=ns), but treatment with a combination of MRX-2843 and pimasertib resulted in significant induction of apoptosis compared to vehicle or single TKIs (56.7%, p&lt;0.05). Similar results were observed in OCI-AML5 cell cultures and in combination with the MEK1/2 inhibitor PD0325901. Together these results indicate a critical role for stromal cell-mediated activation of ERK1/2 in resistance to MRX-2843 and demonstrate the utility of translational strategies targeting both MERTK and MAPK pathways to overcome resistance.
Citation Format: Katherine A. Minson, Eleana Vasileiadi, Madeline G. Huey, Xiadong Wang, Stephen V. Frye, H. Shelton Earp, Deborah DeRyckere, Douglas K. Graham. Stromal cell activation of MAPK signaling pathways mediates resistance to MERTK inhibition in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3037.