Supplemental Figure Legends
Supplemental Figure S1. Compared to BET inhibitors JQ1 and OTX015 which induce BRD4, treatment with ARV825 depletes BRD4 expression in cultured sAML cells. A. SET2 and HEL92.1.7 cells were treated with the indicated concentrations of JQ1 for 18 hours. At the end of treatment, total cell lysates were prepared and immunoblot analyses were conducted as indicated. The expression levels of β-Actin in the cell lysates served as the loading control. B. HEL92.1.7 and SET2 cells were treated with the indicated concentrations of pomalidomide for 18 hours. Then, total cell lysates were prepared and immunoblot analyses were conducted as indicated. The expression levels of GAPDH in the cell lysates served as the loading control. C. SET2 cells were treated with the indicated concentrations of ARV825 and/or pomalidomide for 18 hours. Then, total cell lysates were prepared and immunoblot analyses were conducted as indicated. The expression levels of GAPDH in the cell lysates served as the loading control.
Supplemental Figure S2. Treatment with ARV825 causes more significant mRNA expression changes than OTX015 in cultured sAML cells. A-B. SET2 cells were treated with the 1 µM of ARV825 or OTX015 for 4 hours. At the end of treatment, RNA was isolated; libraries were prepared in the MD Anderson Cancer Center DNA Sequencing and Microarray core facility and sequenced on an Illumina HiSeq 4000. Each library yielded 30-40 million read pairs. Data was mapped using TopHat2 onto the human genome build UCSC hg19 (NCBI 37) for human data. Gene expression was assessed using Cufflinks2, then variance stabilization and quantile normalization were applied. Significantly altered genes were determined using the limma package in R. The table indicates the fold change (drug treated divided by control) of selected targets and the corresponding p-value for ARV825 and OTX015-treated cells.
Supplemental Figure S3. Gene set enrichment analysis of positively enriched pathways in
sAML cells treated with ARV825 or OTX015 for 4 hours. A. Gene Set enrichment analysis
(GSEA) evaluating the transcriptome footprint of ARV825 or OTX015 treatment in SET2 cells against the REACTOME pathway compendium. Shown are positively enriched pathways. The table reflects the normalized enrichment score (NES) for each pathway in ARV825 and OTX015 treated cells. All q-values are less than 0.01. B-C. GSEA plot comparing SET2 cells against the gene signature REACTOME pathway RNA Pol II transcription. All q-values are less than 0.01
Supplemental Figure S4. Gene set enrichment analysis of negatively enriched pathways in sAML cells treated with ARV825 or OTX015 for 4 hours Gene Set enrichment analysis (GSEA) comparing the transcriptome changes induced by ARV825 and OTX015 treatment of SET2 cells against the REACTOME pathways collection. Shown are negatively enriched pathways. The table reflects the normalized enrichment score (NES) for each pathway in ARV825 and OTX015 treated cells. All q-values are less than 0.01.
Supplemental Figure S5. Treatment with ARV825 causes more significant mRNA expression changes than OTX015 in patient-derived CD34+ sAML cells. Patient-derived CD34+ cells were treated with 1 µM of ARV825 or OTX015 for 8 hours. Samples represent and were prepared as biologic duplicates. At the end of treatment, RNA was isolated; libraries were prepared and sequenced on an Illumina HiSeq4000 next generation sequencer and processed as described above. A. Heat map showing the number of significantly altered mRNAs (up or down 1.33 fold and a p-value of less than 0.05) following treatment with ARV825 or OTX015. B. Fold change (drug-treated divided by control) of selected targets and their corresponding p-value in the ARV825-and OTX015-treated cells. C. Venn diagram representing the expression signature overlap in down and upregulated genes cells following treatment with ARV825 and OTX015 in the PD sAML cells. D-G. Quantitative PCR of PD CD34+ sAML cells treated with the indicated concentrations of ARV825 or OTX015 for 8 hours. The relative mRNA expression of each target was normalized to GAPDH and compared to the untreated control cells. * indicates mRNA values that are significantly depleted compared to the untreated control cells (p <0.05) † indicates mRNA values that are significantly induced compared to the untreated control cells (p <0.05).
Supplemental Figure S6. Treatment with ARV825causes efficient and prolonged depletion of BET protein BRD4, leading to sustained attenuation of BRD4 target genes such as c-MYC in sAML cells. A-B. SET2 cells were treated with the indicated concentrations of ARV825 or OTX015 for 18 hours. Samples were prepared as biologic triplicates. At the end of treatment, cells were harvested and lysed. The resulting cell lysates were utilized for reverse phase protein array (RPPA) analysis. The heatmap shows the protein targets with a greater than or equal to 25% change (up or down regulated) in protein expression and a p-value less than 0.05.
Supplemental Figure S7. Treatment with pomalidomide restored ARV825-mediated reduction of oncogene expressions and inhibited ARV-825-induced apoptosis in sAML cells. A. SET2 cells were treated with the indicated concentrations of ARV825 and/or pomalidomide for 18 hours. Then, total cell lysates were prepared and immunoblot analyses were conducted as indicated. The expression levels of GAPDH in the cell lysates served as the loading control. B. SET2 cells were treated with the indicated concentrations of ARV825 and/or pomalidomide for 48 hours. At the end of treatment, the % annexin-V- positive, apoptotic cells was determined by flow cytometry. Columns, mean of three experiments; Bars, standard error of the mean.
Supplemental Figure S8. Superior activity of ARV-825 compared to OTX-015 in patient-derived (PD) CD34+ stem-like progenitor sAML cells. PD CD34-positive sAML cells were treated with the indicated concentrations of ARV-825 or OTX-015 for 16 hours. At the end of treatment, cells were fixed and labeled with a cocktail of primary antibodies conjugated to rare metal elements. Mass cytometry ‘CyTOF’ analysis was conducted and data were normalized and exported. Data were further analyzed utilizing the SPADE software. A. Heatmap of the annotated clusters based on the expression levels of CD markers associated with AML stem/progenitor cells. B. Heatmap of the protein expression changes in stem-like progenitor cells caused by treatment with ARV-825 or OTX-015, relative to the untreated control cells.
Supplemental Figure S9. In contrast to OTX015, treatment with ARV-771 does not have a significant impact on the mass of mice engrafted with sAML cells. Mice (n=8) engrafted with sAML, HEL92.1.7 cells were treated with vehicle, OTX015 or ARV-771 for three weeks. Mice were monitored and weighed once per week while on treatment. The mean mass of the mice in each cohort is displayed as a line graph + standard deviation.
Supplemental Figure S10. Synergistic lethal activity of co-treatment with ARV825 and ruxolitinib in cultured and patient-derived (PD) CD34+ sAML cells. A-H. SET2, HEL92.1.7 and PD CD34+ sAML cells were treated with ARV825 (dose range: 2.5-500 nM) and ruxolitinib (dose range: 50-1000 nM) for 48 hours. Then, the % of annexin V- and TO-PRO-3-positive, apoptotic cells was determined by flow cytometry. Median dose effect and isobologram analyses were performed utilizing CompuSyn, assuming mutual exclusivity. Combination index (CI) values less than 1.0 indicate a synergistic interaction of the two agents in the combination. Dose, fractional effect and combination index value tables for each combination are shown.