Overactivation of STAT Pathways and Overexpression of Survivin Confer Resistance to FLT3 Inhibitors and Could Be Therapeutic Targets in AML.

Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) receptor plays an important role in the pathogenesis of acute myeloid leukemia (AML). A number of small molecule kinase inhibitors are currently proceeding in different phases of clinical trials. As with imatinib in CML, leukemic cells could develop resistance to these RTK inhibitors when used as monotherapy. Mutations in the ATP-binding pocket have been identified through PCR-based mutagenesis screening in Ba/F3-FLT3-ITD cells and selected for growth in the presence of PKC412, or in a resistant Ba/F3-FLT3-ITD cell line developed by cocultured with SU5416. Resistance to PKC412 resulting from the N676K point mutation in the FLT3 kinase domain has been described in a clinical trial patient. Selection of activating Ras mutations has been found in 2 out of 6 FLT3 inhibitor resistant cell lines, but no point mutation of FLT3 kinase domain was found in all 6 resistant cell lines. To further investigate other potential mechanisms of resistance to FLT3 inhibitors, we developed a resistant cell line by long-term culture of MV4-11 cells with ABT-869, designated as MV4-11-R (IC₅₀: 52 vs 6 nM for the parental MV4-11 cell line), which is also cross resistant to other structurally unrelated inhibitors including SU5416, AG1296 and a FLT3 inhibitor III (MERCK). No point mutation in the kinase domain of FLT3 was found in MV4-11-R cells. Western blot and FACS analysis excluded overexpression of p-FLT3, FLT3 or 3 multidrug resistance related proteins (MDR, MRP1 and LRP) in this resistant line. Gene expression profiling revealed up-regulation of FLT3 ligand (FLT3LG) (2.4 fold) and Survivin (2 fold), while down-regulation of SOCS1, SOCS2, and SOCS3 was observed in MV4-11-R compared to MV4-11 parental cells. Overexpression of FLT3LG and Survivin was also demonstrated at the protein level. Survivin is a unique member of the inhibitor of apoptosis proteins (IAP) family and a known target of the STAT3 pathway. Down-regulation of suppressor of cytokine signaling (SOCS) proteins (negative regulators of STAT pathways) was observed even in the presence of overactivation of the STAT1, STAT3 and STAT5 pathways in the MV4-11-R line. We screened a panel of small molecule inhibitors including a STAT inhibitor (indirubin derivative IDR E804), 3 JAK inhbitors (Tyrene CR4, AG490, and JAK3 Inhibitor II), and a CDK/survivin inhibitor (NU6140). We found that MV4-11-R is most sensitive to IDR E804 (an inhibitor of CDKs and the SRC-STAT3 pathway). The IC₅₀ value of ABT-869 in MV4-11-R was decreased from 52 to 6.2 nM in the presence of 2 nM of IDR E804. Further validation of the therapeutic effect of IDR E804 in combination with ABT-869 in the MV4-11-R mouse xenograft model is ongoing. Targeting Survivin by shRNA and a dominant-negative vector (survivin-T34A) induced MV4-11-R to undergo apoptosis. Taken together, these results demonstrate that overactivation of STAT pathways and overexpression of survivin are the main mechanism of resistance to ABT-869; suggesting that the STAT pathways and survivin could be potential targets for the treatment of patients who develop resistance to FLT3 inhibitors.