Abstract 4737: Saturation mutagenesis of FLT3-ITD: AC220-resistance-conferring kinase domain mutations are restricted to a limited number of residues and are cross-resistant to sorafenib in vitro
Authors: Smith Catherine C. , Damon Lauren , Salerno Sara , Shah Neil P. 
Published: June 2012

Abstract
Activating mutations in FLT3 are detected in approximately 30 percent of adult acute myeloid leukemia (AML) cases, most commonly involving in-tandem duplication (ITD) events. AC220 is a promising potent and selective inhibitor of FLT3 that has notably achieved morphologic bone marrow remissions in a substantial proportion of relapsed and refractory AML patients in a previous phase I study (Cortes et al, ASH 2009). The ability of AC220 to achieve deep remissions in AML patients suggests that FLT3 activity is essential for survival of malignant myeloblasts in some cases. Although some responses observed in phase I have been durable, many patients ultimately suffered relapsed disease. AC220 is currently being evaluated in a multinational phase II study of AML patients who harbor FLT3 ITD mutations and have failed prior chemotherapy.
As has been previously observed with BCR-ABL kinase inhibitors, it is anticipated that loss of response to AC220 may occur through selection of leukemic cells that harbor resistant kinase domain mutations in FLT3. In an effort to identify substitutions in FLT3 that are capable of conferring AC220 resistance in vitro, we have employed a well-validated in vitro mutagenesis approach (Azam et al, Cell, 2003) of FLT3-ITD using the DNA repair deficient E. coli strain XL1-Red, with subsequent selection for resistance in Ba/F3 cells grown in soft agar (devoid of IL-3) containing 20 nM AC220 (approximately 20x the inhibitory concentration 50 (IC50) of unmutagenized FLT3-ITD). Through sequence analysis of over 100 resistant clones, we have identified at least two independent isolates of substitutions occurring at four individual residues scattered throughout the kinase domain. The most commonly isolated mutations involved the “gatekeeper” residue F691 and activation loop residues D835 and Y842, which have also been implicated in preclinical resistance to other FLT3 inhibitors. Each of the kinase domain mutations is sufficient to confer AC220-resistant growth when re-introduced into Ba/F3 cells. Western immunoblot analysis reveals that these mutations confer varying degrees of biochemical resistance to AC220 that correlate with the degrees of resistance observed in cell growth assays. Notably, AC220-resistant mutations FLT3-ITD kinase domain confer in vitro cross-resistance to sorafenib, which has recently been reported to harbor activity in a small number of FLT3-ITD AML cases assessed. Sequence analysis of the FLT3 kinase domain in relapsing AML patients will be required to ascertain the importance of kinase domain mutation in clinical resistance, and is presently ongoing.
These studies may inform the development of novel FLT3 inhibitors capable of treating AML that has acquired resistance to AC220 or sorafenib through selection of resistant FLT3 kinase domain mutations.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4737. doi:10.1158/1538-7445.AM2011-4737