Methylation of HSP70 orchestrates its binding to and stabilization of BCL-2 mRNA and renders pancreatic cancer cells resistant to therapeutics Liang Wang1,3, Zhiliang Jia1, Dacheng Xie1,4, Tiansuo Zhao1, Zhi Tan2,3, Shuxing Zhang2,3, Fanyang Kong1, Daoyan Wei1, Keping Xie1,3,4 Departments of 1Gastroenterology, Hepatology and Nutrition, 2Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, Texas; 3The University of Texas MD Anderson Cancer Center and UTHealth Graduate School of Biomedical Sciences, Houston, Texas; and 4The Precision Institutes of Medicine and Oncology, Houston, Texas Supplemental Information 1. Supplemental Figures and Legends 2. Supplemental Tables 3. Supplemental Materials and Methods 1 1. Supplemental Figures 2 Figure S1. PRMT1 is overexpressed in multiple gastrointestinal tumors and is associated with patient overall survival. (A) PRMT1 protein expression level in two pairs of isogeneic pancreatic cancer cell lines. (B) Survival analysis of The Cancer Genome Atlas (TCGA) data for various tumor types. Patients were divided into low and high groups based on PRMT1 mRNA expression level. Five-year overall survival rates and log-rank P values are shown. (C) Immunohistochemical staining demonstrating markedly higher PRMT1 expression in gastric, colon, and liver tumor tissues than in adjacent normal tissues. 3 4 Figure S2. Genetic alterations and inflammatory factors contribute to increased PRMT1 expression during pancreatic cancer (PDA) progression. (A) Representative immunohistochemical staining of PRMT1 expression in pancreatic tissue sections from mouse pancreatic cancer models. In the lower panels, the red arrow indicates a pancreatic intraepithelial neoplasia (PanIN) lesion and the black arrowhead indicates a PDA lesion. (B) PRMT1 staining scores were higher in mouse PanIN and PDA lesions. Error bars indicate mean ± SEM. (C) Double immunofluorescence staining of PRMT1 and Ki67 in two KPC mouse pancreatic tissue sections. (D) Percentage of the pancreatic area that is stained positive for CK19 in PBS and DB75 treated groups. (E) Copy number change of the PRMT1 gene locus in 105 human PDA specimens. (F) TNF-α dose-dependently increased PRMT1 expression in human and mouse PDA cells. 5 6 Figure S3. Altered expression and activity of PRMT1 impacts pancreatic cancer (PDA) cell migration, invasion, and growth. (A) Transfected cells were photographed at 0 hours and 12 hours after transfection, and wound areas are outlined by dashed lines (upper and middle panels). Boyden chamber invasion assays were performed, and photos of invaded cells stained by hematoxylin and eosin are shown (bottom panels). (B) Quantification of cell migration and invasion assay results, which were conducted in triplicate. (C) PRMT1 was knocked down in PANC-1 cells, and the growth of tumor xenografts in nude mice was slower when compared with mock cells. (D) L3.7 cells were treated with TC-E5003 (Low=6.25M, High=12.5M) for 48 hours and cell apoptosis was observed (left panel). MiaPaCa-2 cells were treated with DB75 (Low=20M, High=50M) for 48 hours and cell apoptosis (black arrows) was observed. (E) MiaPaCa-2 cells were treated with either DB75 or TC-E5003 for 36h. asymmetric dimethyl- arginine (ASYM24) and asymmetric demethylation of histone H4R3 were tested by Western blot. (F) Human pancreatic cancer MDA28 cells were injected subcutaneously into nude mice to grow for 3 weeks. Phosphate-buffered saline (PBS) or low-dose (5 mg/kg) and high-dose (20 mg/kg) DB75 was administrated intraperitoneally twice per week. Mice were euthanized at day 21. Xenograft tumors were photographed and weighed (left panel). Tumors tissues were used to detect PRMT1 protein expression and protein methylation level by Western blot (right panel). (G) Human pancreatic stellate cells were treated with either DB75 or TC-E5003 at indicated concentrations and cell viability is determined. *P < 0.05, ***P < 0.001. All bar plot data are means ± SD. 7 8 Figure S4. PRMT1 and HSP70 proteins exhibit subcellular co-localization and binding motifs of PRMT1-HSP70 interaction are evident. (A) HSP70 (green) and PRMT1 (red) were detected by immunofluorescence microscopy using HSP70- and PRMT1-specific antibodies in mouse embryonic fibroblasts (MEFs) under normal culture conditions and after heat shock treatment (1 hour at 43°C and then recovery for 6 hours at 37°C). White arrows indicate cells undergoing apoptosis after heat shock, exhibiting cell shrinkage and condensed nuclei. Merge I is merged green and red fluorescence and Merge II includes all three fluorescence colors. (B) Deletion mutants of HSP70 used in interaction assays. Blue boxes indicate the nucleotide binding domain. Yellow boxes indicate the substrate binding region. Green boxes indicate the helical lid region. FL, full length. (C) Deletion mutants of human PRMT1 used in interaction assays. Red boxes indicate the variable N terminal helix. Green boxes indicate the S-adenosyl methionine binding domain. Yellow boxes indicate the β-barrel structure. Blue boxes indicate the dimerization arm. (D) HA-tagged full-length (FL) or deletion mutants of human PRMT1 and Myc-tagged HSP70 were co-transfected into 293T cells, and cell lysates were subjected to SDS-PAGE and then blotted with anti-Myc and anti-HA antibodies. WCL, whole cell lysate. 9 10 Figure S5. Arginine methylation sites of HSP70 are identified. (A) PRMT1-overexpressed and knockdown cell lysates were separated by SDS-PAGE and immunoblotted (IB) with ASYM24 antibody (left panel), and Ponceau S staining showed equal loading (right panel). (B) HSP70 protein was immunopurified from PRMT1 overexpression and control cells, and HSP70 methylation was detected by ASYM24 antibody. IP, immunoprecipitation. (C) Myc-HSP70 was immunopurified from 293T cells and the gel was stained with Coomassie brilliant blue. The HSP70 band was excised and subjected to mass spectrometry. (D and E) Protein sequence alignment of the regions surrounding 410-460 binding motif in 13 human HSP70 family members (D) or in paralogous HSP70 genes in various organisms (E). 11 12 Figure S6. Arginine methylation of HSP70 protects cells against various stresses. (A) HSP70 knockout (KO) was confirmed by Western blot analysis and DNA sequencing. Left panel, expression of HSP70 was determined by Western blot analysis in 14 MiaPaCa-2 cell clones, including 6 control clones and 8 knockout clones. Right panel, DNA sequencing result of KO clone 20. The CRISPR target region revealed different indel mutations in two alleles, indicated as dual peaks. (B) HSP70 control (WT) and knockout (KO) MiaPaCa-2 cell clones were treated with gemcitabine at different concentrations for 24 hours. Cell growth inhibition was measured using the CCK-8 method. HSP70 WT group is pooled cells of 6 control clones from (A). (C) HSP70 knockout (KO) MiaPaCa-2 cell clones 20 and 41 and PATU-8902 (8902) cells were transfected with HSP70-WT, single mutant (R416A) or double mutant (Mut) HSP70, and then incubated for 3-4 days. Relative cell growth was measured using the CCK-8 method (expressed as “OD” values). (D) Western blot analysis of HSP70 protein in knockout cells with exogenous HSP70 reconstitution. (E) HSP70 wild-type and knockout MiaPaCa-2 cells were subcutaneously injected into nude mice. Xenograft tumors were photographed and weighed after 2 weeks. **P < 0.01. All bar plot data are means ± SD. 13 14 Figure S7. Arginine methylation of HSP70 promotes its mRNA binding and stabilization activity. (A) HSP70 was knocked down or overexpressed in MDA28 cells, and BCL-2 protein levels were determined by Western blot analysis. Red and black arrows indicate exogenous and endogenous HSP70 protein respectively. (B) Four PDA cell lines were transfected with pcDNA3.0, HSP70 wild-type (WT), and HSP70 methylation-deficient mutant (Mut) vectors, and BCL-2 mRNA levels were determined by quantitative real-time PCR. (C) Upper panel: two pancreatic cancer cell lines were treated with gemcitabine (Gem) for 24 hours and BCL-2 expression was determined by Western blot analysis. Bottom panel: two pancreatic cancer cell lines were treated in the presence or absence of 1 μM venetoclax (VENE); 20 μM gemcitabine (GEM), or both (V+G), and cytotoxicity was determined by CCK-8. CTRL, vehicle control. (D) The human and mouse 3′- untranslated regions (UTR) of BCL-2 mRNA were analyzed. There are three typical AU-rich elements (AREs), two pentamers (black boxes), and one nonamer (red box) in the first 500 base pairs (bp) of the BCL-2 3′-UTR. (E) The ribonucleoprotein immunoprecipitation (RNP-IP) assay was conducted in 293T cells transfected with wild-type or mutant HSP70. mRNAs of BCL-2 (lanes 2 and 6 for primer pair 1 and lanes 3 and 7 for primer pair 2), VEGFA (lanes 4 and 8), and GAPDH (lanes 5 and 9) were determined by reverse-transcriptase PCR. Lane 1, 100 bp DNA ladder. (F) The 557bp 3′-UTR of BCL-2 containing three AREs was cloned into a pMIR-REPORT vector (pMir-557WT), and the ATTTA conserved sequences were mutated to AGGAA to generate pMir- 557Mut vector. (G) HSP70 failed to stabilize mRNA without AREs. The pMir-557 Mut vector was co-transfected with wild-type or mutant HSP70 for 36 hours and luciferase activity was measured. ***P < 0.001, NS non-significant. All bar plot data are means ± SD. 15 16 Figure S8. Arginine methylation of heat shock proteins mediates therapeutic resistance in pancreatic cancer. During PDA development, PRMT1 expression is gradually increased. Two conserved arginine residues of HSP70 are methylated by PRMT1, and methylated HSP70 binds to BCL-2 mRNA through AREs in its 3’-UTR. BCL-2 mRNA stabilization promotes BCL-2 protein expression and increases drug resistance and anti-apoptosis of cancer cells. PRMT1 inhibitors may interrupt this pathway and sensitize cancer cells to chemotherapy. 17 2. Supplemental Tables Table S1. Clinicopathologic parameters and PRMT1 expression in the pancreatic cancer patients Table S2. HA-PRMT1 and HA-ctrl binding proteins identified in mass spectrometry Table S3.