Additional file 3 of Targeting the methyltransferase SETD8 impairs tumor cell survival and overcomes drug resistance independently of p53 status in multiple myeloma
Creators: name: Herviou, Laurie
name: Ovejero, Sara
name: Izard, Fanny
name: Karmous-Gadacha, Ouissem
name: Gourzones, Claire
name: Bellanger, Celine
name: De Smedt, Eva
name: Ma, Anqi
name: Vincent, Laure
name: Cartron, Guillaume
name: Jin, Jian
name: De Bruyne, Elke
name: Grimaud, Charlotte
name: Julien, Eric
name: Moreaux, Jérôme
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Cancer
subject: Hematology

description: Additional file 3. Supplementary Figure S1: SETD8 expression status in MM patients (A) Gene expression profiling of MMCs of the patients of UAMS-TT2 cohort were used. PR: proliferation, LB: low bone disease, MS: MMSET, HY: hyperdiploid, CD1: Cyclin D1-Cyclin D3, CD2: Cyclin D1-Cyclin D3, MF: MAF, MY: myeloid. (B) SETD8 expression in 186 patients of the UAMS-TT2 cohort showing Ch1q21 copy number aberration. (C) SETD8 expression in MMCs (patients at diagnosis) presenting low, medium or high gene expression-based proliferation index (GPI). (D) Correlation between SETD8 expression and malignant plasma cell labeling index. Plasma cell labeling index was investigated using BrdU incorporation and flow cytometry in 101 patients at diagnosis. Supplementary Figure S2: Gene Signature of MM patients with high SETD8 expression. GSEA enrichment plots with the absolute enrichment p value and the normalized enrichment score of the gene set. Supplementary Figure S3: SETD8 is more expressed in patients at relapse compared to diagnosis. Boxplot showing SETD8 expression in MM cells of patients at relapse (n=47) compared to diagnosis (n=205). Supplementary Figure S4: UNC-0379 inhibits SETD8 and affects MM cells survival and cell cycle. (A) Immunoblot analysis of SETD8, Histone H4 and H4K20me1 protein levels in XG7 and XG25 cells untreated or treated with 3 μM of UNC-0379 for 24 hours. (B) Quantitation of cell-cycle distribution of control (untreated) and UNC-0379-treated XG7 and XG25 HMCLs 48 hours after treatment. After short-pulse of BrdU incorporation, cell-cycle was analyzed by FACS using DAPI and anti-BrdU antibody. (C) Quantitation of apoptosis in control and UNC-0379-treated XG7 and XG25 HMCLs by flow cytometry with AnnexinV-PE staining and 96h after UNC-0379 treatment. Data shown are mean values ± SD of 4 separate experiments. Statistical analysis was done with a paired t-test. (*) indicates a significant difference compared to control cells using a Wilcoxon test for pairs (P ≤ 0.05). Supplementary Figure S5: UNC-0379 treatment did not affect in vitro differentiation of human memory B cells. In vitro differentiation of memory B cells into plasma cells from 2 different donors. Cells were treated at Day 7 with UNC-0379 at the indicated concentrations and analyzed at Day 10. (A) Total cell number was visually quantified. Dead cells were visualized by trypan blue staining and viability was calculated as the ratio of living cells vs total cells. Annexin V+ cells were quantified by FACS. (B) Percentage of each population at day 10 (MBC: memory B cells, PrePB: preplasmablasts, PB: plasmablasts, PC: plasma cells). Mock is treated with the same percentage of DMSO as 5 μM sample. Supplementary Figure S6: UNC-0379 affects murine MM cells viability. Measure of viability and apoptosis in primary murine 5T3vv cellular models untreated (cnt) or treated with growing concentrations (from 2.5 to 40 μM) of UNC0379 for 24 hours. Supplementary Figure S7: UNC-0379 treatment deregulates gene expression in HMCLs. (A) Heatmap of RNA-sequencing rlog expression data. Genes deregulated by UNC-0379 and shSETD8 in XG7 and XG25 HMCLs are represented. Expression scale shows low expression levels in blue and high expression levels in red. (B) Bar-plot representing the fold expression (UNC-0379 condition over control) of genes related to Fig 1B chart pathways. (C) Bar-plot representing the fold expression (UNC-0379 condition over control) of genes related to Fig 1B chart pathways. Supplementary Figure S8: SETD8 expression correlation with p53 expression or status. (A) Comparison of SETD8 expression according to HMCLs TP53 status. (B) Correlation between TP53 and SETD8 expression in HMCLs. TP53 and SETD8 expression was obtained from Affymetrix microarrays data previously published. Supplementary Figure S9: Correlation between TP53 expression and drug response to UNC-0379 in HMCLs. IC50 was determined using CTG-based growth assay, TP53 expression was obtained from Affymetrix microarrays data previously published. Supplementary Figure S10: UNC-0379 induces genomic instability in XG1 p53-mutant cell line. (A) Immunoblot analysis of indicated proteins in total lysates from UNC-0379-treated (5µM) or untreated XG1 HMCL. β-actin and H2A.X were used as loading controls. (B) Cell cycle of control and 48h UNC-0379-treated (5µM) XG1 HMCL was analyzed by flow cytometry using DAPI, BrdU incorporation and labelling with an anti-BrdU antibody. Results are representative of three independent experiments. * indicates a significant difference compared to control cells using a Wilcoxon test for pairs (P ≤ 0.05). (C) Apoptosis induction was investigated using AnnexinV-PE staining by flow cytometry. Supplementary Figure S11: UNC-0379 did not alter the steady states levels of nucleolar proteins and the nucleolar localization of fibrillarin. (A) immunoblot analysis of indicated proteins in shRNA control and p53-depleted XG7 cells treated with the vehicle DMSO or 3 μM of UNC-0379 for 24 hours. Ponceau staining is used as loading control (B) immunofluorescence microscopy of fibrillarin localization and DNA (DAPI staining) in XG7 cells depleted or not for p53 at low and higher magnitude as indicated. Scale bar : 10 µM. Supplementary Figure S12: The lethal synergy between UNC-0379 and melphalan is caused by SETD8 inhibition but does not depend on p53 activation. (A) immunoblot analysis of SETD8 and tubulin protein levels in total lysates of XG7 cells expressing a doxycycline-inducible shRNA targeting SETD8 mRNA and treated or not with doxycycline (1µg/ml) for 4 days. (B) The graph shows one representative from 3 independent experiments. Cell proliferation analysis was performed by CTG, 4 days after co-treatment with Doxycycline (1ug/ml) to silence SETD8 and various concentrations of melphalan as indicated. (C) IC50 values from 3 independent experiments performed as in (B). p-value = 0.0196. (D) Measure by flow cytometry of apoptosis induction (AnnexinV-PE staining) in XG7-shControl and XG7-shTP53 HMCLs after 96h of treatment with Melphalan (5μM), UNC-0379-treated (3μM) or the combination of the two drugs. Supplementary Figure S13: Immunoblot analysis of histone H2AX and its phosphorylated form γH2A.X (marker of DNA damage) in total lysates from melphalan-resistant XG7 HMCLs. Cells were treated with melphalan (5μM), UNC-0379-treated (3μM) or the combination of the two drugs for 24h. β-actin was used as loading control.