Location of FLT3-ITDs within the beta1-Sheet of FLT3 Kinase Confers Resistance to Tyrosine Kinase Inhibitors (TKI) in Vitro.

In AML, the recently described tyrosine kinase domain-1 (TKD1)-ITDs of FLT3 (Breitenbuecher et al., Blood 2009, Kayser-S et al., Blood 2009) are located within the beta1-sheet, nucleotide binding loop and beta2-sheet of tyrosine kinase domain 1 (TKD1), respectively. Multivariate analysis of clinical data revealed that location of FLT3-ITDs within the beta1-sheet of TKD1 is an unfavorable prognostic factor (Kayser-S et al., Blood 2009). Recently, we uncovered a novel mechanism of primary resistance to FLT3 tyrosine kinase inhibitors (TKIs) in a patient displaying an atypical localization within the beta2-sheet-ITD (A627E). Here, we characterized in-vitro sensitivity to FLT3-TKI in growth factor dependent hematopoietic cell lines expressing a representative panel of FLT3-ITDs isolated from patient material. In particular, we compared sensitivity of beta1-sheet ITDs with typical ITDs located in the juxtamembrane domain of FLT3.

FLT3-ITDs isolated from patient material, were sequenced, subcloned in parallel into two expression vectors (pAL and pMSCV-Puromycin-IRES-GFP), and stably expressed in growth-factor dependent hematopoietic Ba/F3 cells and in parallel in 32D cells. Constitutive phosphorylation of FLT3-ITD receptors and of downstream signaling pathways was analyzed by Western-blotting. Transformation of Ba/F3 and 32D cells was investigated by colony formation assays and by withdrawal of IL-3. Induction of apoptosis in response to various concentrations of FLT3-kinase inhibitors midostaurin (PKC412) and quizartinib (AC220) was measured by flow-cytometry at 24h and 48h, respectively. For statistical analysis of replicates t-test was employed.

Biological characteristics of FLT3-ITD mutations located in two different structural domains of FLT3-kinase were characterized: (1) beta1-sheet-ITDs E611V(96nt) and Q613E(99nt) and (2) nucleotide binding loop-ITD A620V(84nt). (Our nomenclature used for description of ITDs indicates position of amino-acid where ITD is located, possible exchange of amino-acid residues at this position and nucleotide length of ITD) Hematopoietic cells (32D and Ba/F3) expressing these ITD mutations showed colony formation in methylcellulose medium, growth-factor independent proliferation upon IL-3 withdrawal and constitutive phosphorylation of FLT3 signaling. Three ‘typical’ juxtamembrane domain (JMD) ITDs were used as controls; ITD598/599(36nt), ITD598/599(66nt) and ITDK602R(21nt). As compared to these JM-ITDs, we observed significantly less apoptosis of beta1-sheet-ITDs at all concentrations of FLT3-kinase inhibitors applied. However, this difference in sensitivity gradually decreased when incubating with midostaurin or quizartinib for longer periods of time as 48h. Of note, length of FLT3-ITD mutations did not appear to influence sensitivity to TKI treatment.

Investigating representative FLT3-ITD-mutations located within the TKD1 revealed that beta1-sheet-ITDs mediate constitutive activation of FLT3 receptors leading to transformation of hematopoietic cell lines 32D and Ba/F3. In comparison to typical JMD-ITDs, beta1-sheet-ITDs analyzed here revealed resistance to FLT3-inhibitors as midostaurin and quizartinib across two cellular reconstitution models (32D and Ba/F3) and using two different expression vectors (pAL and pMSCV). Our results indicate that differential sensitivity is rather an effect of ITD-localization within a functional domain of FLT3 and not necessarily conferred by the length of ITDs. Taken together, our data provide a rationale to prospectively analyze not only the FLT3-ITD mutation status or FLT3-ITD allelic ratio but also location of ITD-mutations in ongoing clinical trials as this may have direct impact on response to therapy (tyrosine kinase inhibitors, chemotherapy, allogeneic stem cell transplantation) in FLT3-ITD positive AML.

Heidel:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Fischer:Novartis: Honoraria.