Abstract 1711: Proteasomal alterations in a newly created model of FLT3-ITD positive AML with acquired pan-TKI resistance
Authors: Wilson Katie , Irwin Mary E. , Chandra Joya 
Published: May 2015

Abstract
Acute myeloid leukemia (AML) is an aggressive hematologic cancer. Thirty percent of AMLs express mutations in the FMS-like tyrosine kinase receptor-3 (FLT3) that render it constitutively active. The internal tandem duplication (ITD) mutation of FLT3 is associated with poor clinical prognosis as it results in aberrant signaling leading to proliferation and survival. As such, tyrosine kinase inhibitors (TKI) directed against FLT3 are being developed. However, as with most TKI therapies, de novo and acquired resistance occurs. We have developed an isogenic cell line model of acquired resistance to FLT3 TKIs by treating BaF3/FLT3-ITD cells (FLT3-ITD) with lestaurtinib over time (FLT3-ITDR cells). Challenge of FLT3-ITDR cells with lestaurtinib showed &gt;3-fold resistance to this TKI as measured by proliferation and propidium iodide sub-diploid analysis. In addition, we tested FLT3-ITDR cells for sensitivity to quizartinib, a more potent and selective inhibitor of FLT3, and found that FLT3-ITDR cells were 7-fold more resistant, suggesting pan-FLT3-TKI resistance. To confirm this resistance was specific for TKI, FLT3-ITDR and FLT3-ITD cells were treated with the histone deactylase inhibitor vorinostat and the anthracycline doxorubicin. Similar sensitivities were noted between the two cell lines to both of these drugs suggesting that resistance of FLT3-ITDR cells to FLT3-directed TKI is specific. Combination therapy is often utilized as a mechanism to overcome clinical resistance. Recently, proteasome inhibitors have been explored as an option for combinatorial therapeutics in leukemia. To determine if the proteasome may be a good target for combination therapy in FLT3-ITDR cells, the proteasomal chymotrypsin-like activity was measured in FLT3-WT, FLT3-ITD, and FLT3-ITDR cells. Chymotrypsin-like proteasome activity was significantly reduced in both FLT3-ITD (41%) and FLT3-ITDR (59%) vs. FLT3-WT cells. This activity stems from activation of the β5 subunit of the proteasome; therefore we performed western blotting with antibodies directed against β5. As seen with chymotrypsin-like activity, FLT3-ITD cells had a significant decrease in β5 protein expression (58%) compared to FLT3-WT. However, despite decreased proteasome activity, FLT3-ITDR had no decrease in β5 protein compared to FLT3-WT cells. One potential explanation for this result is that FLT3-ITDR cells have increased levels of inactive free β5 subunits compared to FLT3-WT. Future studies will examine the implication of these alterations. Despite these changes in proteasome sub-unit regulation and activity, all three cell lines were sensitive to single agent treatment with the reversible FDA-approved proteasome inhibitor bortezomib and its irreversible counterpart marizomib. As such, we will continue to explore the efficacy of proteasome inhibitors in combination therapy for TKI resistant FLT3-ITD-positive AML.
Citation Format: Katie Wilson, Mary E. Irwin, Joya Chandra. Proteasomal alterations in a newly created model of FLT3-ITD positive AML with acquired pan-TKI resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1711. doi:10.1158/1538-7445.AM2014-1711