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AZD1208 and quizartinib co-treatment was well tolerated in the orthotopic model.

Purpose: fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML), and these patients have short disease-free survival. FLT3 inhibitors have limited and transient clinical activity, and concurrent treatment with inhibitors of parallel or downstream signaling may improve responses. The oncogenic serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD and also promotes its signaling in a positive feedback loop, suggesting benefit of combined Pim and FLT3 inhibition.Experimental Design: Combinations of clinically active Pim and FLT3 inhibitors were studied in vitro and in vivo.Results: Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated in vitro growth of FLT3-ITD, but not wild-type FLT3 (FLT3-WT), cell lines. AZD1208 cotreatment increased FLT3 inhibitor–induced apoptosis of FLT3-ITD, but not FLT3-WT, cells measured by sub-G1 fraction, annexin V labeling, mitochondrial membrane potential, and PARP and caspase-3 cleavage. Concurrent treatment with AZD1208 and the FLT3 inhibitor quizartinib decreased growth of MV4-11 cells, with FLT3-ITD, in mouse xenografts, and prolonged survival, enhanced apoptosis of FLT3-ITD primary AML blasts, but not FLT3-WT blasts or remission marrow cells, and decreased FLT3-ITD AML blast colony formation. Mechanistically, AZD1208 and quizartinib cotreatment decreased expression of the antiapoptotic protein Mcl-1. Decrease in Mcl-1 protein expression was abrogated by treatment with the proteasome inhibitor MG132, and was preceded by downregulation of the Mcl-1 deubiquitinase USP9X, a novel mechanism of Mcl-1 regulation in AML.Conclusions: The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. Clin Cancer Res; 24(1); 234–47. ©2017 AACR.

Supplementary figure legends

Figure S6. Selective suppression of MIR300 pro-apoptotic but not anti-proliferative activity by TUG1 lncRNA in quiescent LSCs. A, BloodSpot array-based TUG1 expression levels during normal myelopoiesis and in myeloid neoplams. B, GEO Profiles show TUG1 levels in in lineage-negative (Lin-) and -positive (Lin+) CD34- and CD34human stem/progenitor cells from healthy individuals. C, Experimental data- and current literature-based graphic representation of signaling network controlling CML LSC quiescence and survival through the BMM-C/EBPbeta-MIR300 and BMM-TGFbeta-FoxM1 pathways. Dotted lines indicate inactive pathways, line thickness indicates relevance of the signal for LSC quiescence. Red lines indicate signals increasing MIR300 levels. Black lines signals increasing TUG1 levels. (bottom) effects of different TUG1 levels on CML leukemic stem (LSC) and progenitor (LPC) cell fate.

Figure S4. MIR300 anti-proliferative activity accounts for BMM-induced LSC entry into quiescence. A, Structure of 14q32 DLK1-DIO3 genomic imprinted locus hosting the MEG3-regulated human MIR300. B, MIR300 levels in 5-Aza- or DMSO-treated (24h) Ph+ cells. C, Effect of hypoxia on proliferation of CFSE+CD34+ CML-BC cells. D, left: Effect of MSC (HS-5)-derived CM on LAMA-84 proliferation expressed as fold changes of CFSE mean of fluorescence intensity (MFI)+/-SEM; middle: pro-apoptotic effect of PAD (FTY720; 2.5uM) and DMSO (control) on HS-5-cultured LAMA-84 cells; right: Effect of MSC (HS-5)-derived CM BCR-ABL1 expression (anti-ABL1) and activity anti-PY), phospho-BCR-ABL1, JAK2 expression and activity JAK2 Y1007/1008, PP2A activity (pPP2AY307 inactive form) and GRB2 used as a control (blots are representative of three independent experiments). E, Levels of C/EBPbeta and GRB2 mRNA and protein in HS-5 cells exposed to hypoxia (48h; 1% O2). F, Effect of neutralizing TGFbeta antibody (anti-TGFb Ab; 48h, 1.25 μg/ml) on MIR300 levels in CD34+ CML-BC cells. G, Effect of ectopic C/EBPalpha (MigR1-deltauORF-C/EBPalpha-HA) and C/EBPbeta (MigR1-C/EBPB-ERTAM) on MIR300 levels in K562 cells. Immunoblot shows levels of C/EBPbeta and GRB2 in normoxic and hypoxic K562 cells.

Range values of controls for the indicated experiments

Figure S3. MIR300 acts as master PP2A activator and inhibitor of G1/S transition. A, (left) Additional effects of MIR300 on SET, CCND2 and CDK6 expression; (right) KEGG/GO analysis of MIR300 effects on signal transduction pathways. B, CSmiRTar (filters: bone marrow normal and myeloid leukemia cells) and miRDIP-ComiR integrated analyses show functional clustering of predicted/validated MIR300 targets.

Figure S2. KEGG/GO analysis of MIR300 on PP2A-regulated signal transduction pathways. Cartoon shows the SET-dependent PP2A Inhibitory pathway in CML and the pleiotropic inhibitory effect of PP2A activation on validated and predicted MIR300 targets regulating G1/S cell cycle transition, Wnt-beta-catenin, TGFbeta, JAK-STAT, PI-3K-Akt, RAS-MAPK and Notch signaling pathways.

Figure S5. BMM-induced MIR300 anti-proliferative and PP2A-activating functions impair NK cell immune-response. A, PP2A-dependent regulation of MIR300 and miR-155 validated pathways predicted to occur also in the bone marrow endosteal niche. B, Effect of hypoxia (1% O2, 7 days) on CFSE+NK cell proliferation (% CFSE mean of fluorescence). C, HS-5 exosomal miRNAs reported as negative regulators of NK cell proliferation/activity and their experimentally validated targets. miRNA RNAseq was performed on an Illumina platform using libraries derived from 100 ng RNA/sample from HS-5 exosome purifications (n=3). D, Precursor (pre-miR-155) miR-155 (BIC) levels in resting and IL-12/IL-18 (18h)-stimulated NK cells exposed (48h) to HS-5 exosomes. E, Effect of hypoxia (1% O2) and HS-5 CM (48h) on TUG1 expression in CD56+CD3- primary NK and NK-92 cells. F, Effect of CpG-TUG1-shRNA and CpG-scramble (200-500 nM, 5 days) on IL-2-induced NK cell proliferation (% cell number). Data are represented as mean+/-SEM.

Figure S1. MIR300 activity in quiescent leukemic stem and progenitor cells. CFC-replating assays shows effects of lentiviral-mediated ectopic MIR300 expression, 250 nM and 500 nM CpG-miR-300 on serial replating activity (2nd replating) of leukemic chronic and acute CML and normal UCB CD34+CD38- HSC-enriched cell fractions. Infection with lentiviral empty vector and treatment with CpG-anti-MIR300 and CpG-scramble served as controls.