FLT3 Inhibitor Resistant Cells Show Different Downstream Signaling Pathway Changes.

Receptor tyrosine kinase FLT3 plays an important role in leukemogenesis, especially in acute myeloid leukemia (AML). Tyrosine kinase inhibitors (TKI) targeting wild-type and mutant FLT3 have been developed and shown to have activity in clinical trials. However, as seen with Gleevac in CML, prolonged incubation with TKIs can select for resistant clones that may contribute to disease progression. To study resistance to TKIs against FLT3 we developed FLT3 inhibitor resistant cell lines by co-culturing MOLM14 and BaF3/ITD cells, expressing FLT3/ITD mutants with increasing concentrations of the FLT3 inhibitor CEP-701. The resulting cell lines, MOLM14(R) and BaF3/ITD(R) are resistant to CEP-701 induced cytotoxicity. MOLM14(R) is also resistant to other selective FLT3 TKIs including CEP-5214 and PKC412. In contrast, BaF3/ITD(R) cells were still sensitive to CEP5214 and PKC412. Western blot analysis reveals that CEP-701, CEP-5214 and PKC412 all still inhibit FLT3 in MOLM14(R) cells implying selection of a clone no longer dependent on FLT3 signaling. FLT3 phosphorylation is not inhibited by CEP-701 in BaF3/ITD(R) cells but is still inhibited by CEP-5214 and PKC412. Thus the BaF3/ITD(R) cells appear to remain FLT3-dependent. Sequencing of FLT3 from the resistant clones showed that the resistance was not the result of drug resistance mutations in FLT3/ITD. To investigate possible mechanisms of resistance in FLT3-dependent and FLT3-independent FLT3 inhibitor resistant cells, we examined pathways downstream of FLT3. Previously, we and others reported that constitutive FLT3 activation results in specific changes in gene expression in myeloid leukemic cells. As expected for cells with continued FLT3/ITD activation, Western blot analysis of BaF3/ITD(R) cells treated with CEP-701 show that they maintain activation of Erk/MAPK, Akt, and STAT5 pathways and induction of FLT3 dependent genes including Pim-1 and cMyc. In the apparently FLT3-independent MOLM-14(R) clones, inhibition of FLT3 activity resulted in decreased phosphorylation of downstream Akt and Stat5. However, we found Erk/MAPK phosphorylation and cMyc expression were not decreased in response to FLT3 TKI. This implies that whatever pathway has been selected for the ability to grow in this inhibitor is still feeding into this part of the downstream signaling pathway normally activated by FLT3/ITD. Thus, BaF3/ITD(R) FLT3-dependent and MOLM-14(R) FLT3 independent cells differ in response to several FLT3 inhibitors that results from the differences in their mechanisms of resistance.