Session Speaker

Molecular Characterization of Resistance in Multi-Targeted Tyrosine Kinase Inhibitors and Bcl-2 Inhibitors-Implication on Clinical Development of Small Molecular Targeted Therapy
C.S. Chen
USA

We developed resistant cell lines by long-term culture of leukemia cells with targeted ABT-869 and ABT-737, scenario reminiscence to chronic dosing schedule in clinical practice for molecular targeted agents. In ABT-869 resistant lines, MV4-11-R was cross-resistance to structurally unrelated FLT3 inhibitors including FLT3 inhibitor III,

AG1296 and SU5416. Overexpression of FLT3, p-FLT3 receptor or multi-drug resistant related proteins, or mutations in KD were not responsible for resistance to FLT3 inhibitors in MV4-11-R.

Gene profiling reveals up-regulation of FLT3LG (FLT3 ligand) and BIRC5 (survivin), but down-regulation of SOCS1, SOCS2, and SOCS3 in MV4-11-R cells. Hypermethylation of these SOCS genes leads to their transcriptional silencing. Survivin is directly regulated by STAT3. Stimulation of the parental MV4-11 cells with FLT3LG increases the expression of survivin and phosphorylated protein STAT1, STAT3, STAT5. Targeting survivin by shRNA in MV4-11-R cells induce apoptosis and augments ABT-869-mediated cytotoxicity. Flow cytometric analysis revealed that MV4-11 parental cells had a significantly decreased S phase population (6.5% vs 17.8%, p < 0.01), but a dramatically increased G2/M phase population (49.6% vs 20.3%, p < 0.01) as compared to MV4-11-R cells. Furthermore, there were 4.5 times more dead cells in MV4-11 cells than in MV4-11-R cells as determined by the Trypan Blue dye exclusion method at the end of serum depletion 48 hours. Taken together, these results suggest that the overexpression of survivin in MV4-11-R cells leads to accelerate S phase shift and resistance to apoptosis. Sub-toxic dose of indirubin derivative (IDR) E804, an inhibitor of SRCSTAT3 pathway, resensitize MV4-11-R to ABT-869 treatment by inhibiting STAT signaling activity and abolishing survivin expression. Combining IDR E804 with ABT-869 shows potent in vivo efficacy in the MV4-11-R mouse xenograft model.

Taken together, these results, for the first time, demonstrate that enhanced activation 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 reducing resistance developed in patients receiving FLT3 inhibitors. Understanding the mechanism of resistance to FLT3 inhibitors could help develop new antileukemic agents or uncover compelling combinations. Our data strongly support the combination of FLT3 inhibitors with agents targeting STAT pathway or survivin such as small molecular inhibitors or shRNA and may represent a novel strategy to minimize resistance or resensitize resistant cells to FLT3 inhibitors in AML patients with FLT3-ITD mutation.

Distinctive mechanism was also identified when ABT-737 resistant lines were investigated. In addition, we were able to observe the resistance development by morphological evaluation. The data will be presented. The strategies of
identification of resistant mechanisms will be important for clinical development of novel agents which chronic dosing schedule is expected.

Related publications:
Zhou J, Bi C, Jasinghe VJ, Liu S-C, Tay K-G, Poon L-F, Xie Z, Palaniyandi S, Yu H, Chng W-J, Glaser K, Albert D, Davidsen S, Chen CS*. Enhanced activation of STAT pathways and overexpression of survivin confer resistance to FLT3 inhibitors and could be therapeutic targets in AML. Blood 113:4052-4062, 2009.
Zhou J, Goh BC, Albert D, Chen CS*. ABT-869, a promising multi-targeted tyrosine kinase inhibitor: from bench to bedside. Journal of Hematology & Oncology 2009, 2:33 doi:10.1186/1756-8722-2-33.