Mutations in FLT3/ITD Produce Varying Levels of Resistance to FLT3 Tyrosine Kinase Inhibitors.

Abstract Abstract 3776 Poster Board III-712 Mutations in the receptor tyrosine kinase FLT3 generally map to the kinase domain as point mutations or to the juxtamembrane domain as internal tandem duplications (ITDs). Both types of mutations lead to constitutive activation of the receptor and transformation of hematopoietic cell lines to cytokine independence. About 30% of acute myeloid leukemia (AML) cases harbor FLT3 mutations, which are now targets for inhibition by numerous small molecule tyrosine kinase inhibitors (TKIs). However, molecularly targeted inhibition of kinases in patients often leads to the emergence of clones that contain mutations within the targeted kinase that impair drug binding. This has been repeatedly demonstrated for BCR-ABL-directed inhibition by imatinib in chronic myeloid leukemia, in which numerous mutations have now been documented. Treatment for FLT3-driven leukemias may well follow suit as is evidenced by the detection of the N676K FLT3/ITD mutation in one patient that resulted in PKC412 resistance. In this study, we tested multiple FLT3 TKIs, some of which are currently in clinical trials for treatment of AML, against a panel of FLT3 mutations that were previously reported to confer resistance to a more limited panel of inhibitors. We also sought to identify new mutations in FLT3 that might impart some level of resistance to various inhibitors. To this end, we utilized a non-biased approach in which the XL1-Red E. coli mutator strain randomly generated mutations in the FLT3/ITD clone. After selecting cells for cytokine independence, mutants were grown in methylcellulose for two weeks in the presence of an inhibitor to select for resistance. The resulting cell clones were analyzed by Western blotting for their resistance to FLT3 inhibition as well as for increases in the IC50 to a variety of FLT3 TKIs in the MTT cell proliferation assay. After sequencing to uncover mutations within the rescued FLT3/ITD clone, site-directed mutagenesis was used to generate the mutations to confirm that they recapitulate the pattern of resistance seen in the selected resistant cell clones. BaF3 cells were nucleofected with the candidate FLT3 mutation and analyzed for their resistance profile. Our screen revealed three mutations within the drug binding cleft of FLT3/ITD, including F621L, A627P and F691L. In addition, one mutation in the second half of the kinase domain was identified, Y842C. TheY842C/ITD mutant produced high levels of resistance to sunitinib, sorafenib and AGL2043, but it retained sensitivity to CEP701 and PKC412. The F691L/ITD produced a resistance profile similar to the Y842C/ITD mutant with modest resistance to CEP701 and PKC412. The F621L mutant exhibited low levels of resistance to sunitinib and AGL2043 and was even more sensitive to inhibition by sorafenib, lestaurtinib and PKC412 than parental BaF3/ITD cells. The A627P mutant expressed lower levels of FLT3/ITD but displayed resistance to all inhibitors, similar to the resistance profile published for the A627E FLT3/ITD mutation found in one patient. Interestingly, the inhibitors that were most commonly affected by mutations were sunitinib>AGL2043>sorafenib> PKC412≥lestaurtinib. The results of our random mutagenesis screen indicate that there are still undiscovered mutations in FLT3 that confer resistance to TKIs in vitro and may arise during treatment of leukemia patients using FLT3 inhibitors. While lestaurtinib and PKC412 were more effective than the other inhibitors tested against this set of FLT3/ITD mutants, even a slight increase in their IC50 may preclude their use against some FLT3 mutants. The resistance profiles for these mutants will be presented as described in addition to other inhibitors that may soon become available. Disclosures: Levis: Cephalon: Member, clinical advisory board.