Upregulation of Bcl-2 Confers Resistance to FLT3 Inhibition in FLT3-ITD AML with Secondary Acquired Mutations
Authors: Yamatani Kotoko , Tabe Yoko , Saito Kaori , Yang Haeun , Murakami-Tonami Yuko , Suzuki Koya , Zhang Weiguo , Kinjo Sonoko , Ikeo Kazuho , Mogushi Kaoru , Hosoya Masaki , Yamaguchi Shigeo , Harada Hironori , Miida Takashi , Shah Neil P. , Konopleva Marina Y. , Hayashizaki Yoshihide , Andreeff Michael 
Published: October 2019

Abstract
Internal tandem duplications (ITD) in the juxtamembrane domain of FMS-like tyrosine kinase 3 (FLT3) cause ligand-independent constitutive activation of FLT3 kinase and its downstream signaling. FLT3-ITD mutations confer poor prognosis with high relapse rates in AML patients. FLT3-targeted therapies using tyrosine kinase inhibitors (TKIs) often induce additional point mutations within the tyrosine kinase domains (FLT3-TKD mutations), most commonly found at D835 activation loop. Additional FLT3-TKD mutations that cause secondary resistance emerge in at least 20% of patients with TKI treatment. To elucidate the alterations of transcriptome signatures of FLT3-ITD and TKD double mutations in AML, we performed cap analysis of gene expression (CAGE) sequencing for 26 primary AML samples (14 with FLT3-ITD, 12 with FLT3-ITD/D835). CAGE detects and quantifies the specific transcriptional start site (TSS) transcripts, which enables high-throughput gene expression profiling and promoter usage analysis. Altered transcription of TSS in FLT3-ITD/D835 AML samples were detected by comparison with TSS in FLT3-ITD samples, and upregulation of 310 TSS and downregulation of 22 TSS were mapped (FDR < 0.05, EdgeR). Based on Gene Ontology (GO) analysis, up-regulated genes were enriched in "apoptotic process", "intracellular signal transduction" and "immune system development", including pro-survival BCL2A1 and drug resistance related S100A8 and PRKCH. To validate these transcriptional changes, we utilized isogenic paired Ba/F3 cells transfected with FLT3-ITD or FLT3-ITD/D835. CAGE detected upregulation of 1945 TSS and downregulation of 1470 TSS in FLT3-ITD/D835 compared to FLT3-ITD cells (FDR < 0.05). TSS transcriptions of Bcl-2, Prkca, NF-κB1, Myc, and Cdkn1a (p21) were upregulated in FLT3-ITD/D835 cells. GO analysis consistently highlighted higher activation of NF-κB signaling and its downstream Bcl-2 in FLT3-ITD/D835 than in FLT3-ITD cells both for primary AML samples and Ba/F3 cells. To determine a correlation between activated promoters and transcription factors in FLT3-ITD/D835 cells, we performed an unbiased search for enriched sequence motifs using HOMER software. HOMER revealed that the promoter 4 of BCL2A1 contained a common motif of transcription factor STAT6, known to associate with NF-κB and cooperatively bound to their respective promoter elements. Ingenuity Pathway Analysis also highlighted higher activation of STAT6 in FLT3-ITD/D835 AML cells compared to FLT3-ITD. Immunoblot analysis confirmed higher expression of Bcl-2, c-Myc, p27, and lower expression of Bcl-xL and Mcl-1 in FLT3-ITD/D835 Ba/F3 compared to FLT3-ITD Ba/F3 cells. FLT3-ITD and FLT3-ITD/D835 cells showed similar expression level of Bax and Bid. We found that FLT3-ITD/D835 Ba/F3 cells proliferated slower than FLT3-ITD cells (growth rate; FLT3-ITD/D835 9.8±2.4 fold, FLT3-ITD 19.8±1.2 fold, p=0.003, 48 h) with G0/G1 accumulation (FLT3-ITD/D835 61.5±10.1%, FLT3-ITD 31.3±11.2%, p<0.05, 72 h).
Finally, we hypothesized that targeting Bcl-2 may effectively overcome the acquired resistance of FLT3-ITD/D835 cells. Venetoclax (ABT-199/GDC-0199), a clinically available Bcl-2 selective inhibitor, as single agent was not capable of eliminating FLT3-ITD AML due to altered activation of FLT3 downstream signaling and Mcl-1 upregulation. FLT3-ITD/D835 cells showed higher expression of the Bcl-2 gene and/protein and were less dependent on FLT3 signaling compared to FLT3-ITD cells (Tabe, ASH 2017). As expected, venetoclax caused more profound cell growth inhibition and apoptosis induction in FLT3-ITD/D835 Ba/F3 than in FLT3-ITD Ba/F3 cells (IC50 2.98 μM vs 13.9 μM, ED50 28.8 μM vs 173 μM, 48 h). In conclusion, we identified Bcl-2 transcriptional activation as a novel mechanism by which the acquired D835 mutation in FLT3-ITD AML cells facilitates anti-apoptotic network activation and confers TKI resistance. Bcl-2 inhibition by venetoclax represents a putative therapeutic strategy in FLT3-ITD/TDK double-mutated AML cells.

Disclosures
Shah: Bristol-Myers Squibb: Research Funding; ARIAD: Research Funding. Konopleva:Stemline Therapeutics: Research Funding. Andreeff:AstraZeneca: Research Funding.