Different FLT3-ITD Integration Sites Are Associated with Differential Sensitivity to Tyrosine Kinase Inhibitors (TKI) In Vitro.

Currently, small molecule FLT3 tyrosine kinase inhibitors (TKIs) are promising therapeutic approaches to overcome the dismal prognosis of AML patients harbouring FLT3-ITD mutations. However, up to 30% of these patients show primary resistance to FLT3-TKIs. Recently, we uncovered a novel mechanism of primary resistance to FLT3 TKIs in a patient displaying an atypical integration site of ITD within the beta2-sheet (ITD_A627E). The data suggested that atypical integration sites of ITDs within the tyrosine kinase domain-1 (TKD1) of FLT3 (beta1-sheet, nucleotide binding loop and beta2-sheet) are associated with rewired signaling and differential responsiveness to TKIs. Here, we characterized response to FLT3-TKIs employing various TKD1-ITDs of the beta1-sheet and nucleotide binding loop, respectively in a cellular reconstitution model.

FLT3 TKD1-ITDs isolated from patient material, were sequenced, subcloned and stably transfected into growth-factor dependent hematopoietic Ba/F3 cells. Constitutive FLT3 phosphorylation and activation of downstream signaling was analyzed by Western-blotting. Transformation potential was analyzed by colony formation in methylcellulose medium and by withdrawal of IL-3. Induction of apoptosis in response to TKI (midostaurin/sorafenib) was measured by FACS analysis.

Biological characteristics of FLT3-ITDs from two structural domains of FLT3-TKD1 were characterized: (1) beta1-sheet-ITDs E611V (96nt) and Q613E (99nt) and (2) nucleotide binding loop-ITD A620V (84nt). Ba/F3 cells expressing these ITDs showed colony formation in methylcellulose assays and growth-factor independent proliferation upon IL-3 withdrawal indicating transformation. Western-blotting revealed constitutive phosphorylation of FLT3 and of downstream signaling nodes (STAT5/AKT/ERK). As compared to a typical juxtamembrane domain (JMD) ITD (ITD598, 36nt), we observed significantly less induction of apoptosis in TKD1-ITDs investigated at 24h of incubation across all concentrations of midostaurin used. Similar results were observed when TKD1-ITDs were treated with different concentrations of sorafenib for 24h. However, this difference in sensitivity gradually decreased when incubating with midostaurin or sorafenib for longer periods of time as 36h and 48h. Currently, additional FLT3-TKIs are being investigated and results will be presented.

Our results employing representative TKD1-ITDs from beta1-sheet and nucleotide binding loop revealed that TKD1-ITDs mediate constitutive activation of FLT3 receptors leading to transformation of hematopoietic cells. In comparison to a typical JMD-ITD, TKD1-ITDs analyzed revealed differential responsiveness in induction of apoptosis to midostaurin and sorafenib. Thus, our in vitro data suggest that different ITD integration sites may be associated with differential sensitivity to midostaurin and sorafenib in vivo and provides a rationale to prospectively analyze not only the FLT3-ITD mutation status but also the ITD integration site in ongoing/future clinical trials using FLT3-TKIs.

No relevant conflicts of interest to declare.