Figure 4.
Differential treatment response is associated with subclone-specific BME interaction. (A) (Left) Bar plot depicting the proportion of subclones per time point for patient RRMM6, detected via scATAC-seq. The individual stacks are colored according to the respective subclone identity. (Right) Phylogenetic tree for patient RRMM6. Top node at baseline denotes the most recent common ancestor with subsequent decedents being shown according to the combined CNA and mtDNA mutation subclone assignment. (B) Viability of the MM cell line AMO-1 based on normalized CellTiter-Glo luminescence reads on exposure to increasing concentrations of MCL-1 inhibitor MIK665 (Novartis) for 24 hours in cells with wt TP53 or biallelic TP53 (TP53 del/mut [R175H]). Data are represented as means ± standard deviation; n = 3. Welch two-sample t test: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. (C) Volcano plot summarizing the global changes in gene expression levels between subclones 1 and 2 for patient RRMM6. An absolute Padj value of .05 is indicated by a dashed line (Wilcoxon rank sum test). Surface markers are labeled. (D) Chromatin accessibility at the CD44 promoter plus 50 kb upstream and downstream in patient RRMM6. The CD44 promoter peak is highlighted in light blue. (Top) Aggregated pseudobulk scATAC-seq tracks at both timepoints. (Right) Violin plots showing normalized CD44 expression from scRNA-seq data per timepoint with individual cells plotted as dots. (Middle) Peaks are colored based on the location of the peak in either promoter (orange), distal (red), exonic (blue), or intronic (green) regions; (bottom) peak coaccessibility in the CD44 region at both timepoints colored based on the Pearson correlation coefficient. (E) CD44 expression based on bulk RNA-seq data in a set of 46 patients with RRMM with biallelic-inactivated (n = 8), monoallelic-depleted (n = 11) or wt TP53 (n = 27). (F) CD44 expression in genetically engineered biallelic TP53 del/mut (R175H) and wt/wt AMO-1 cell lines. (Top) Publicly available bulk RNA-seq data (GSE13234052,53) were analyzed for CD44 expression. Box plot of normalized expression values for n = 3 replicates. (Bottom) Western blot analysis of CD44 protein levels in the indicated MM cell lines. Tubulin was used as a loading control. This blot is representative of 3 independent experiments. (G) Violin and box-whisker plots showing normalized CD44 expression per timepoint and subclone for patient RRMM6. (H) Circos plot and chord interaction diagram of LGALS9-CD44–mediated interactions between subclones of patient RRMM6 and BME cells. The links start at a sender and end at a receiver. Sender-receiver interactions seen in all subclones as well as between BME cells are not indicated.

Differential treatment response is associated with subclone-specific BME interaction. (A) (Left) Bar plot depicting the proportion of subclones per time point for patient RRMM6, detected via scATAC-seq. The individual stacks are colored according to the respective subclone identity. (Right) Phylogenetic tree for patient RRMM6. Top node at baseline denotes the most recent common ancestor with subsequent decedents being shown according to the combined CNA and mtDNA mutation subclone assignment. (B) Viability of the MM cell line AMO-1 based on normalized CellTiter-Glo luminescence reads on exposure to increasing concentrations of MCL-1 inhibitor MIK665 (Novartis) for 24 hours in cells with wt TP53 or biallelic TP53 (TP53 del/mut [R175H]). Data are represented as means ± standard deviation; n = 3. Welch two-sample t test: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. (C) Volcano plot summarizing the global changes in gene expression levels between subclones 1 and 2 for patient RRMM6. An absolute Padj value of .05 is indicated by a dashed line (Wilcoxon rank sum test). Surface markers are labeled. (D) Chromatin accessibility at the CD44 promoter plus 50 kb upstream and downstream in patient RRMM6. The CD44 promoter peak is highlighted in light blue. (Top) Aggregated pseudobulk scATAC-seq tracks at both timepoints. (Right) Violin plots showing normalized CD44 expression from scRNA-seq data per timepoint with individual cells plotted as dots. (Middle) Peaks are colored based on the location of the peak in either promoter (orange), distal (red), exonic (blue), or intronic (green) regions; (bottom) peak coaccessibility in the CD44 region at both timepoints colored based on the Pearson correlation coefficient. (E) CD44 expression based on bulk RNA-seq data in a set of 46 patients with RRMM with biallelic-inactivated (n = 8), monoallelic-depleted (n = 11) or wt TP53 (n = 27). (F) CD44 expression in genetically engineered biallelic TP53 del/mut (R175H) and wt/wt AMO-1 cell lines. (Top) Publicly available bulk RNA-seq data (GSE13234052,53) were analyzed for CD44 expression. Box plot of normalized expression values for n = 3 replicates. (Bottom) Western blot analysis of CD44 protein levels in the indicated MM cell lines. Tubulin was used as a loading control. This blot is representative of 3 independent experiments. (G) Violin and box-whisker plots showing normalized CD44 expression per timepoint and subclone for patient RRMM6. (H) Circos plot and chord interaction diagram of LGALS9-CD44–mediated interactions between subclones of patient RRMM6 and BME cells. The links start at a sender and end at a receiver. Sender-receiver interactions seen in all subclones as well as between BME cells are not indicated.

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