Abstract 1893: Activating NRAS mutations are associated with FLT3-independent resistance to the clinically active FLT3 inhibitor AC220 in vitro
Authors: Lasater Elisabeth A. , Smith Catherine C. , Shah Neil P. 
Published: August 2012

Background: Clinical outcomes associated with acute myeloid leukemia (AML) have not improved substantially in decades. Activating FMS-like tyrosine kinase-3 (FLT3) mutations are detected in ∼30% of AML cases with internal tandem duplication (ITD) mutations conferring worse prognosis. Recently, the potent FLT3 inhibitor AC220 has achieved composite complete remission in 45% of patients with relapsed/refractory FLT3-mutant AML (Cortes et al, EHA 2011). Translational studies from our laboratory implicate reactivation of FLT3-ITD through kinase domain mutations in the majority AC220 relapse cases (Smith et al, ASH 2011). Given that less than half of treated patients initially achieve deep remissions on AC220, we hypothesize that off-target (non-FLT3 mediated) resistance is responsible for primary resistance to AC220, and may mediate loss of response in a subset of patients. Results: To model and identify putative mechanisms of off-target resistance to AC220 in vitro, we cultured the FLT3-ITD+ patient derived cell lines MV4;11 and Molm14 in escalating concentrations of AC220. To date, we have isolated 8 Molm14 subclones that are resistant to at least 20nM AC220, which is &gt;60-fold the IC50 of the parental line (0.3nM), while efforts to generate resistant MV4;11 cells have repeatedly failed. Analysis of FLT3 signaling revealed persistent FLT3 phosphorylation in 5 clones in the presence of 10nM AC220. Sequencing of the FLT3 kinase domain identified acquired secondary activation loop mutations suggesting on-target acquired resistance. The remaining 3 clones demonstrated off-target resistance as evidenced by the absence of a FLT3 mutation and loss of FLT3 phosphorylation with 10nM AC220 treatment. Despite FLT3 inhibition, the clones failed to undergo apoptosis and maintained ERK phosphorylation when treated with AC220. Sequencing of K- and NRAS revealed an NRAS G12C mutation in 2 clones and an NRAS Q61K mutation in the third clone, both of which are known activating RAS mutations. The AC220 IC50 values for the G12C mutant clones were determined to be 134nM and 329nM AC220 and the Q61K mutant was one of the most resistant of all clones with an IC50 of &gt;1000nM AC220. Further, all 3 NRAS mutant clones showed increased sensitivity to the MEK inhibitor PD0325901 compared to the parental cells and on-target resistant clones. Conclusions: Generation of 8 independent AC220-resistant AML cell lines revealed evidence for on- or off-target resistance mechanisms. We have identified oncogenic RAS as a putative mechanism of acquired AC220 off-target resistance in vitro, suggesting that pathologic activation of the RAS/ERK pathway may result in failure of clinically active FLT3 inhibitor therapy in AML patients. Assessment for activating RAS mutations in patient samples with primary and acquired resistance to AC220 and other clinically effective FLT3 inhibitors is currently ongoing.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1893. doi:1538-7445.AM2012-1893