We report here on a novel pro-leukemogenic role of FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) that interferes with microRNAs (miRNAs) biogenesis in acute myeloid leukemia (AML) blasts. We showed that FLT3-ITD interferes with the canonical biogenesis of intron-hosted miRNAs such as miR-126, by phosphorylating SPRED1 protein and inhibiting the "gatekeeper" Exportin 5 (XPO5)/RAN-GTP complex that regulates the nucleus-to-cytoplasm transport of pre-miRNAs for completion of maturation into mature miRNAs. Of note, despite the blockage of "canonical" miRNA biogenesis, miR-155 remains upregulated in FLT3-ITD+ AML blasts, suggesting activation of alternative mechanisms of miRNA biogenesis that circumvent the XPO5/RAN-GTP blockage. MiR-155, a BIC-155 long noncoding (lnc) RNA-hosted oncogenic miRNA, has previously been implicated in FLT3-ITD+ AML blast hyperproliferation. We showed that FLT3-ITD upregulates miR-155 by inhibiting DDX3X, a protein implicated in the splicing of lncRNAs, via p-AKT. Inhibition of DDX3X increases unspliced BIC-155 that is then shuttled by NXF1 from the nucleus to the cytoplasm, where it is processed into mature miR-155 by cytoplasmic DROSHA, thereby bypassing the XPO5/RAN-GTP blockage via "non-canonical" mechanisms of miRNA biogenesis.

Supplementary Figure 9 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT+ Cancers

The USP9X inhibitor WP1130 enhances induction of apoptosis of Ba/F3-ITD and MV4-11 cells by quizartinib in a concentration-dependent manner.

Supplementary Figure 6 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT+ Cancers

Pim kinase and FLT3 inhibition does not enhance induction of apoptosis of cells with FLT3-WT.

Supplementary Figure 6 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT+ Cancers

FIGURE S3. Graphs of correlations between endogenous mRNA expression levels

Supplementary Figure 10 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT+ Cancers

Range values of controls for the indicated experiments

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.

Supplementary Figure 5 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT+ Cancers

Supplementary Figure Legends 1-10 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT+ Cancers

PDF file - 325K, Supplemental Table 1: Particle Size Distribution and Zeta Potential; Supplemental Figure 1: Transferrin receptor (CD71) expression on AML patient blasts; Supplemental Figure 2: miR entrapment efficiency; Supplemental Figure 3: Relative expression of miR-29b in AML cell lines, patient blasts and bone marrow samples from healthy donors; Supplemental Figure 4: Expression of pri-miR-29b-1 and pri-miR-29b-2 in AML cell lines and patient blasts following Tf-NP-miR-29b treatment; Supplemental Figure 5: Safety profile of Tf-NPs treatment in immune competent mice.

Supplementary Figure 3 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT+ Cancers

<p>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.</p>

Table S2. Predicted C-MYC-regulated MicroRNA Binding Sites in the Coding Region and 3'UTR of LIG3 and PARP1.

Supplementary Methods for Figure 1 and Table 1 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT+ Cancers

<p>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.</p>

Supplementary Figure 4 from Essential Requirement for PP2A Inhibition by the Oncogenic Receptor c-KIT Suggests PP2A Reactivation as a Strategy to Treat c-KIT+ Cancers

Post-transcriptional regulation of Mcl-1 expression.