![Subasumstat and CFZ combination increases cellular stress response and apoptosis. (A) Top: OPM2, JJN3, and AMO1 cells were treated for 4 hours with dimethyl sulfoxide (DMSO), 250 nM subasumstat, 5 nM CFZ, or the combination thereof and subsequently analyzed by RNA-sequencing (n = 3 biological replicates). Bottom: Gene set enrichment analysis using the fgsea package of the combination treatment (4 hours) vs DMSO control shows enriched apoptosis signatures of the Hallmark set from the molecular signature database. fgsea P values and adjusted P values (P-adj; false discovery rate [FDR]) are indicated. (B) Top: OPM2 cells were treated for 4 hours with DMSO, 250 nM subasumstat, 5 nM CFZ, or the combination thereof and analyzed by quantitative proteomics. Bottom: Enriched Gene Ontology signatures in CFZ vs DMSO-treated OPM2 cells are displayed. (C) Graphical representation of quantitative proteomics data of OPM2 cells that are treated for 4 hours with a combination of subasumstat and CFZ over DMSO-treated control (Ctrl) cells. Proteins are ranked in a volcano plot according to their statistical P value (y-axis) and their relative abundance ratio (log2 fold change [log2FC], x-axis). (D) Top: Significantly synergistically induced genes from transcriptomic data (log2FC > 0, panel A) and proteins significantly induced in the combination treatment of subasumstat and CFZ (log2FC > 0.5; indicated in panel C) were extracted, and matching genes and proteins (n = 19) were subsequently analyzed in all 3 indicated cell lines. Bottom: Heatmap of the fold change of treatment vs control of the identified indicated messenger RNA expression in the cell lines JJN3, OPM2, and AMO1. (E) Immunoblots on JJN3, OPM2, and AMO1 cell lysates treated for 4 hours with 250 nM subasumstat, 5 nM CFZ, or the combination thereof. Protein expression of gH2AX (S139), p-CHK1 (S345), and pan-CHK1 to determine DDR, XBP1 (unfolded protein response), and the apoptosis markers cleaved poly(ADP-ribose) polymerase (Cl. PARP) and cleaved caspase-3 (Cl. Casp-3) have been analyzed. β-Actin served as loading control. (F) Fluorescence-activated cell sorting analysis of JJN3, OPM2, and AMO1 cells stained with Annexin V and 4′,6-diamidino-2-phenylindole to measure apoptosis after treatment for 48 hours with 250 nM subasumstat, 5 nM CFZ, or the combination thereof. P values were determined by one-way analysis of variance. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. ER, endoplasmic reticulum. Suba, subasumstat.](https://ash.silverchair-cdn.com/ash/content_public/journal/bloodadvances/7/4/10.1182_bloodadvances.2022007875/7/blooda_adv-2022-007875-gr4.jpeg?Expires=1720851841&Signature=aa4pgI2na9Jjs1o1wok95rXrbfJHqi8Ehf4d5EXyid2lkj177HfTyBmI~34ieJd4QOqrWnut9OvafSdZoVXQfcfvYn2TehbJfsG-m1ZrD6rP4GYPPkV0tf-aW3METYWE54-3X41AS4q8utH6E-3pnLtTw5Hmg4oxm64zchCNOZZk8dv2pWzaGHk4xc4lLVJ6~h34la5NLhDdeiQM-DK0Pwu5O0tGH8RKEXVrEFvNQib9X~PZe2P~yWtBv~kyf7njzSNkJ~~nXey7V2vconcJP3vPFAgo53A5IuoLOXU6~Os3Xy3MScBV2GXICe6BLy-9IfNIpRjC7JrxM54r4tqagQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Subasumstat and CFZ combination increases cellular stress response and apoptosis. (A) Top: OPM2, JJN3, and AMO1 cells were treated for 4 hours with dimethyl sulfoxide (DMSO), 250 nM subasumstat, 5 nM CFZ, or the combination thereof and subsequently analyzed by RNA-sequencing (n = 3 biological replicates). Bottom: Gene set enrichment analysis using the fgsea package of the combination treatment (4 hours) vs DMSO control shows enriched apoptosis signatures of the Hallmark set from the molecular signature database. fgsea P values and adjusted P values (P-adj; false discovery rate [FDR]) are indicated. (B) Top: OPM2 cells were treated for 4 hours with DMSO, 250 nM subasumstat, 5 nM CFZ, or the combination thereof and analyzed by quantitative proteomics. Bottom: Enriched Gene Ontology signatures in CFZ vs DMSO-treated OPM2 cells are displayed. (C) Graphical representation of quantitative proteomics data of OPM2 cells that are treated for 4 hours with a combination of subasumstat and CFZ over DMSO-treated control (Ctrl) cells. Proteins are ranked in a volcano plot according to their statistical P value (y-axis) and their relative abundance ratio (log2 fold change [log2FC], x-axis). (D) Top: Significantly synergistically induced genes from transcriptomic data (log2FC > 0, panel A) and proteins significantly induced in the combination treatment of subasumstat and CFZ (log2FC > 0.5; indicated in panel C) were extracted, and matching genes and proteins (n = 19) were subsequently analyzed in all 3 indicated cell lines. Bottom: Heatmap of the fold change of treatment vs control of the identified indicated messenger RNA expression in the cell lines JJN3, OPM2, and AMO1. (E) Immunoblots on JJN3, OPM2, and AMO1 cell lysates treated for 4 hours with 250 nM subasumstat, 5 nM CFZ, or the combination thereof. Protein expression of gH2AX (S139), p-CHK1 (S345), and pan-CHK1 to determine DDR, XBP1 (unfolded protein response), and the apoptosis markers cleaved poly(ADP-ribose) polymerase (Cl. PARP) and cleaved caspase-3 (Cl. Casp-3) have been analyzed. β-Actin served as loading control. (F) Fluorescence-activated cell sorting analysis of JJN3, OPM2, and AMO1 cells stained with Annexin V and 4′,6-diamidino-2-phenylindole to measure apoptosis after treatment for 48 hours with 250 nM subasumstat, 5 nM CFZ, or the combination thereof. P values were determined by one-way analysis of variance. ∗P ≤ .05, ∗∗P ≤ .01, ∗∗∗P ≤ .001, ∗∗∗∗P ≤ .0001. ER, endoplasmic reticulum. Suba, subasumstat.
