Figure 7.
Reconsideration of PIM2 as an effective target in MM. (A) PIM2 gene expression in cell lines derived from various solid and blood cancers (https://depmap.org/portal/). (B) CRISPR screening data from the DepMap Project show that PIM2 presents the highest dependency score for the 20 tested MM cell lines, relative to other cell lines. (C) Immunohistofluorescence staining of PIM2 (green) and CD138 (orange) in paraffin-embedded BM from patients with MM. 4′,6-diamidino-2-phenylindole (blue) stains the nucleus. Scale bars: 20 µm (top); 50 µm (bottom). (D) Overall survival of patients from the CoMMpass cohort as a function of PIM2 expression (PIM-high group, n = 539; PIM-low group, n = 227). The expression cutoff was determined by applying the MaxStat package (cutoff = 313.02). (E) Effects of BH3 mimetics and PIMi on apoptosis. BH3 mimetics (eg, MCL1i, venetoclax) inhibit the binding of antiapoptotic molecules (MCL1 and BCL2, respectively) to the pro-apoptotic effectors BAK and BAX; this leads to mitochondrial depolarization and, ultimately, cell apoptosis. PIMi enhances the action of BAD: BAD binds to BCL2 and BCL-XL and therefore acts independently of MCL1i. The combination of these 2 compounds might result in greater activation of BAX and BAK and may increase the likelihood of cell death. (F-G) Left: The results of a cell viability assay and a synergy analysis after treatment with increasing concentrations of AZD1208 (PIMi) and AZD5991 (MCL1i) in XG7 cells (F) and U266 cells (G). Right: Flow cytometry assessment of apoptosis using 4′,6-diamidino-2-phenylindole (DAPI)/CaspGlow staining and immunoblotting for the detection of caspase 3 and PARP cleavage, after treatment of XG7 cells (panel F) and U266 cells (panel G) with selected doses of inhibitors. The bar plot shows the percentage of caspase 3–active positive cells. Data are presented as the mean ± SD, n = 5. (H) Flow cytometry assessment of viability for primary cells from 6 patients with MM. The bar plot shows the percentage of viable CD138+ MM cells and CD138– cells, relative to a control (set to 100%) after treatment with the selected doses of inhibitor. Statistical significance was evaluated by using the Mann-Whitney U test in panels F, G, and H. **P < .01. See also supplemental Figure 7.

Reconsideration of PIM2 as an effective target in MM. (A) PIM2 gene expression in cell lines derived from various solid and blood cancers (https://depmap.org/portal/). (B) CRISPR screening data from the DepMap Project show that PIM2 presents the highest dependency score for the 20 tested MM cell lines, relative to other cell lines. (C) Immunohistofluorescence staining of PIM2 (green) and CD138 (orange) in paraffin-embedded BM from patients with MM. 4′,6-diamidino-2-phenylindole (blue) stains the nucleus. Scale bars: 20 µm (top); 50 µm (bottom). (D) Overall survival of patients from the CoMMpass cohort as a function of PIM2 expression (PIM-high group, n = 539; PIM-low group, n = 227). The expression cutoff was determined by applying the MaxStat package (cutoff = 313.02). (E) Effects of BH3 mimetics and PIMi on apoptosis. BH3 mimetics (eg, MCL1i, venetoclax) inhibit the binding of antiapoptotic molecules (MCL1 and BCL2, respectively) to the pro-apoptotic effectors BAK and BAX; this leads to mitochondrial depolarization and, ultimately, cell apoptosis. PIMi enhances the action of BAD: BAD binds to BCL2 and BCL-XL and therefore acts independently of MCL1i. The combination of these 2 compounds might result in greater activation of BAX and BAK and may increase the likelihood of cell death. (F-G) Left: The results of a cell viability assay and a synergy analysis after treatment with increasing concentrations of AZD1208 (PIMi) and AZD5991 (MCL1i) in XG7 cells (F) and U266 cells (G). Right: Flow cytometry assessment of apoptosis using 4′,6-diamidino-2-phenylindole (DAPI)/CaspGlow staining and immunoblotting for the detection of caspase 3 and PARP cleavage, after treatment of XG7 cells (panel F) and U266 cells (panel G) with selected doses of inhibitors. The bar plot shows the percentage of caspase 3–active positive cells. Data are presented as the mean ± SD, n = 5. (H) Flow cytometry assessment of viability for primary cells from 6 patients with MM. The bar plot shows the percentage of viable CD138+ MM cells and CD138– cells, relative to a control (set to 100%) after treatment with the selected doses of inhibitor. Statistical significance was evaluated by using the Mann-Whitney U test in panels F, G, and H. **P < .01. See also supplemental Figure 7.

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