INTRODUCTION For fit patients (pts) with acute myeloid leukemia (AML), conventional chemotherapy (chemotx) with DNA-damaging agents remains the mainstay of treatment. These agents damage DNA by targeting cells during DNA replication. Chromatin structure is highly influential in the efficacy of chemotx agents, as areas of compacted chromatin are more resistant to DNA-damaging effects. Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 which methylates H3K27 to produce a di- or trimethylated state (H3K27me2/3). Areas of DNA marked with H3K27me3 are transcriptionally repressed and exhibit reduced accessibility to DNA-damaging agents. In preclinical studies, AML cell lines treated with the combination of an EZH2 inhibitor (EZH2i) and doxorubicin exhibited increased DNA damage and apoptosis compared to those treated with doxorubicin alone. This effect was enhanced by temporary pre-treatment of AML cells with palbociclib (PALB), a CDK4/6 inhibitor, by significantly increasing the number of cells in S phase prior to treatment with the EZH2i and doxorubicin combination. These findings were reproduced in vivo in mice injected with AML cell lines and primary AML cells. Mice treated with PALB followed by EZH2i + chemotx had prolonged survival compared to those treated with EZH2i + chemotx, PALB + chemotx and each drug as monotherapy. METHODS This is a single-center, 2-part, phase 1 dose escalation study of EZH2i tazemetostat (TAZ) in combination with CPX-351 induction chemotx (Part 1) and of PALB pre-treatment followed by CPX-351 (Part 2) in pts with relapsed or refractory (R/R) AML. Pending completion of Parts 1 and 2, the protocol will be amended to add Part 3 to evaluate safety of the 3-drug combination. All pts will receive CPX-351 at standard dose on days 1, 3 and 5. Dose escalation of TAZ (Part 1) and PALB (Part 2) follows a 3+3 design. In Part 1, escalating doses of TAZ are administered on days -1 to 6. Key eligibility criteria include age ≥18 years, non-acute promyelocytic R/R AML who have received at least 1 prior line of therapy and have adequate performance status and organ function and who are not at excess risk of anthracycline-induced cardiotoxicity. The primary endpoint of Part 1 is to determine the maximum tolerated dose and recommended Part 3 dose of TAZ in combination with CPX-351. Key secondary endpoint for Parts 1 is preliminary assessment of efficacy. Correlative studies to evaluate the on-target effect of TAZ on H3K27me3 and DNA-damage are performed at protocol-specified time points. RESULTS Ten pts were treated in Part 1, 7 at dose level 1 (1 subject was not evaluable due to not taking the required number of doses of TAZ), and 3 at dose level 2. A total of 1 DLT was observed in the 6 evaluable pts in dose level 1. One DLT was observed in the 3 pts treated at dose level 2. The study was then suspended to review the aggregate safety, efficacy and correlative data. Regarding clinical efficacy, the overall response rate with TAZ + CPX-351 is 2/10 (20%). Of the 2 responding pts, 1 pt at dose level 2 achieved a complete response (CR) and 1 pt at dose level 1 initially achieved a morphologic leukemia free state and subsequently achieved a CR with incomplete hematologic recovery. Six pts had refractory disease and 2 pts were not evaluable. Of the 2 responding pts, both went on to receive consolidative cellular therapies (1 allogeneic stem cell transplant and 1 donor lymphocyte infusion) and 1 is still alive over 1 year after study enrollment. Evaluation of levels of DNA damage (as measured by γH2AX foci) and H3K27me3, the histone H3 mark added by EZH2, (as measured by single-cell proximity ligation amplification) in pts treated with TAZ + CPX-351 were performed. The available samples from 9 evaluable pts revealed similar measures of DNA damage and H3K27me3 between those treated at dose level 1 and dose level 2. CONCLUSIONS The combination of TAZ + CPX-351 yielded similar tolerability compared to CPX-351 alone. Similar levels of H3K27me3 and DNA-damage were observed at TAZ dose levels 1 and 2, suggesting that no additional biologic activity was observed at the higher dose level. Dose level 1 has been selected as the recommended Part 3 dose.

Enhanced apoptosis of Ba/F3-ITD and 32D/ITD cells treated with FLT3 inhibitors at serial concentrations in the presence of AZD1208.

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

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

AZD1208 and quizartinib co-treatment does not increase celluar ROS generation, but increases mitochondrial ROS generation.

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.

<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>

<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>

Post-transcriptional regulation of Mcl-1 expression.

<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>

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 S7. Bioinformatic analysis of TUG1-sponged miRNAs and mRNA targets in CML. A, (left) Integration of validated TUG1-sponged miRNAs with RNAseq data (heatmaps) from BM CD34+CD38- and CD34+CD38+ CML (CP and BC) and NBM (n=3/group). Effect of TUG1-sponged miRNA on PP2A inhibitors (Venn diagram); Functional integration of TUG1-sponged miRNAs and their mRNA targets into regulatory network KEGG/GO analysis; (right) Circular diagrams show top 4 pathways affected by TUG1-sponged miRNAs. B, Predicted effect of MIR300 and of TUG1-sponged miRNAs on Pluripotent Stem Cell Signaling pathways.

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

Supplementary figure legends

Combined AZD1208 and quizartinib treatment does not abrogate growth of KG-1a cells, expressing FLT3-WT, in a xenograft model.

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.

Quizartinib, sorafenib, crenolanib and gilteritinib cytotoxicity in cell lines with FLT3-ITD or FLT3-WT

<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>

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.