Figure 6. Alteration in protein interactions following MOZ or Menin inhibition.

A, Western blot of parental GIST-T1 cells or those following sgRNA deletion and rescue with a codon optimized MEAF6 construct fused to BirA* (R118G). Western blots include MEAF6 and HA, indicating the endogenous and full-length rescue construct, or actin as loading control. B, Plot of PSM and log₂ signal intensity of proximal proteins identified by MEAF6 BioID. MEAF6-enriched proteins, indicated in blue, show >2-fold intensity enrichment compared to background control (n=243). Select interactors are labeled. C, GO term enrichment for MEAF6 proximal proteins. D, Log₂ ratio of VTP-50469/DMSO or WM-1119/DMSO signal intensity for MEAF6-enriched proteins following 3 days pre-treatment with inhibitors with an additional 24 hour treatment during biotin labeling. The Pearson correlation is shown. E, Heatmap showing unsupervised hierarchical clustering of DMSO-normalized signal intensity of 67 proteins significantly changing in at least one condition in response to VTP-50469, WM-1119 or the combination treatment. F-H, Plots of DMSO-normalized signal intensity for protein interactors enriched with WM-1119 or combination treatment (F), interactors lost with VTP-50469 or combination treatment (G), or showing up- or down-regulation in response to either or both inhibitors (H). I, Heat maps demonstrating spike-in normalized signal of DOT1L at MACS-defined peaks (n=67,769) in GIST-T1 cells treated with DMSO, VTP-50469 or WM-1119 for 3 days. Scaled read densities ± 1.25 kb from the peak center are shown in rows. J-K, Box plots showing spike-in normalized DOT1L (J) or MEAF6 (K) signal at MACS-defined peaks (DOT1L n=67,769, MEAF6 n=22,581). Data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test; compared to DMSO control ***,P<0.001; **, P<0.01; *,P<0.05. L, Tracks showing regions of genomic occupancy of spike-in normalized DOT1L under the indicated treatments, H3K79me2, MEAF6 and H3K27ac at the HAND1 locus. M, Day 21 cell count normalized to DMSO following treatment of GIST-T1 or GIST48B with the indicated concentrations of EPZ-5676 (n=5 per condition). Growth over time experiments were analyzed by two-way ANOVA with Tukey’s post-hoc test, compared to GIST48B; ***,P<0.001; *,P<0.05. N, Heatmap showing Pearson correlation of control normalized RNA-seq data from cells treated for 5 days with the indicated inhibitors. O, Correlation of gene expression changes in expressed transcripts (n=5,000) comparing control-normalized drug treatments with EPZ-5676 and VTP-50469. Pearson correlation was performed with P value and r² shown. P, GSEA plots showing changes in HAND1 regulated genes arising from EPZ-5676 treatment. Q-R, Expression of select genes associated with GIST lineage and transcription factors (n=4 per condition). Data were analyzed by two-tailed t test, compared to DMSO; ***,P<0.001; **,P<0.01; *,P<0.05.