Polycomb-Like Protein 3 Induces Proliferation and Drug Resistance In

Author Manuscript Published OnlineFirst on April 20, 2020; DOI: 10.1158/1541-7786.MCR-19-0852 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 Polycomb-like Protein 3 induces proliferation and drug resistance in multiple 2 myeloma and is regulated by miRNA-15a 3 4 Tengteng Yu 1,2, Chenxing Du1, Xiaoke Ma3, Weiwei Sui1, Zhen Yu1, Lanting 5 Liu1, Lei Zhao6, Zhongqing Li1, Jie Xu1, Xiaojing Wei1, Wen Zhou4, 6 Shuhui Deng1, Dehui Zou1, Gang An1, Yu-Tzu Tai2, Guido Tricot5, Kenneth C 7 Anderson2, Lugui Qiu1, Fenghuang Zhan5, Mu Hao1 8 9 Author Information 10 1. State Key Laboratory of Experimental Hematology, National Clinical 11 Research Center for Hematological Disorders, Institute of Hematology and 12 Blood Diseases Hospital, Chinese Academy of Medical Sciences and 13 Peking Union Medical College, Tianjin 300020, China; 14 2. Jerome Lipper Multiple Myeloma Center, LeBow Institute for Myeloma 15 Therapeutics, Dana-Farber Cancer Institute, Harvard Medical School, 16 Boston 02115 MA, USA; 17 3. School of Computer Science and Technology, Xidian University, Xi’an, 18 China; 19 4. Key Laboratory of Carcinogenesis, Ministry of Health, Key Laboratory of 20 Carcinogenesis and Cancer Invasion, Ministry of Education, Xiangya 21 Hospital; Cancer Research Institute, School of Basic Medicine, Central 22 South University, Changsha, Hunan, China; 23 5. Myeloma Center, University of Arkansas for Medical Sciences, Little Rock, 24 AR, USA; 25 6. Department of Biophysics and Molecular Physiology, the University of Iowa, 26 Roy J and Lucille A. Carver College of Medicine, Iowa City, IA, USA. 27 28 * Tengteng Yu, Chenxing Du and Xiaoke Ma contributed equally to this work. 29

Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 20, 2020; DOI: 10.1158/1541-7786.MCR-19-0852 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

30 Correspondence: Mu Hao, State Key Laboratory of Experimental 31 Hematology, National Clinical Research Center for Hematological Disorders, 32 Institute of Hematology and Blood Diseases Hospital, Chinese Academy of 33 Medical Sciences and Peking Union Medical College, 288 Nanjing Road, 34 Tianjin 300020, China; phone 86-22-2390-9025; email: [email protected]. 35 36 Financial Support: This work was supported by Natural Science Foundation 37 of China (81570181 & 81400174 to M.H. and 81630007 & 81920108006 to 38 L.Q.); Chinese Academy of Medical Sciences (CAMS) Innovation Fund for 39 Medical Sciences CAMS-2016-I2M-3-031, CAMS-2017-I2M-1-005 and CAMS 40 2017-I2M-1-015 (to M.H. and L.Q.); Tianjin Science and Technology 41 Supporting Program (17JCYBJC27900 to M.H.) and the Non-profit Central 42 Research Institute Fund of Chinese Academy of Medical Sciences 43 (2018RC320012, to M.H.).

45 Declaration of Interests: Kenneth. C Anderson: Celgene: Consultancy, 46 Speakers Bureau; Sanofi-Aventis: Other: Advisory Board; Bristol-Myers Squibb: 47 Other: Scientific Founder; Oncopep: Other: Scientific Founder; Amgen: 48 Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; 49 Takeda: Consultancy, Speakers Bureau. Fenghuang Zhan: BIPHARM LLC: 50 Consultancy, Other: % Allocation of Profit. The other authors declare no 51 potential conflicts of interest. 52 Word count for text: 4981 53 Abstract word count: 233 54 Numbers of Figures: 7 55 Numbers of Supplementary Figures: 8 56 Numbers of References: 36 57 Running title: PHF19 promotes MM progression 58 Key Words: PHF19; drug resistance; phosphorylation EZH2; miR-15a; 59 multiple myeloma. 2

60 Abstract 61 Multiple myeloma (MM) remains incurable due to the persistence of a minor 62 population of MM cells that exhibit drug resistance, which leads to relapse and 63 refractory MM. Elucidating the mechanism underlying drug resistance and 64 developing an effective treatment are critical for clinical management of MM. 65 Here we showed that promoting expression of the gene for polycomb-like 66 protein 3 (PHF19) induced MM cell growth and multi-drug resistance in vitro 67 and in vivo. PHF19 was overexpressed in high-risk and drug-resistant primary 68 cells from patients. High levels of PHF19 were correlated with inferior survival 69 of MM patients, in the total therapy 2 (TT2) cohort and in the Intergroup 70 Francophone du Myeloma (IFM) cohort. Enhancing PHF19 expression levels 71 increased Bcl-xL, Mcl-1 and HIF-1a expression in MM cells. PHF19 also bound 72 directly with EZH2 and promoted the phosphorylation of EZH2 through 73 PDK1/AKT signaling. miR-15a is a small non-coding RNA that targeted the 74 3’UTR of PHF19. We found that downregulation of miR-15a led to high levels 75 of PHF19 in MM cells. These findings revealed that PHF19 served a crucial 76 role in MM proliferation and drug resistance and suggested that the 77 miR-15a/PHF19/EZH2 pathway made a pivotal contribution to MM 78 pathogenesis, offering a promising approach to MM treatment. 79 80 Implications: Our findings identify that PHF19 mediates EZH2 81 phosphorylation as a mechanism of myeloma cell drug resistance, providing a 82 rationale to explore therapeutic potential of targeting PHF19 in relapsed or 83 refractory MM patients. 84

85 Introduction 86 Multiple myeloma (MM) is the second most prevalent hematologic malignancy 87 in the world (1). The outcomes of newly diagnosed patients with myeloma have 88 greatly improved in recent decades since the introduction of novel agents (such 89 as proteasome inhibitors and immunomodulatory drugs) administered in 90 combination with autologous stem cell transplantation (ASCT) (2). 91 Nevertheless, many patients continue to relapse; and after prolonged salvage 92 treatment, the disease becomes multi-drug resistant and leads to refractory 93 MM and eventual death (3,4). 94 95 MM is a heterogeneous disease, and treatment benefits are not uniform, even 96 in patients that harbor similar cytogenetic abnormalities. Accumulating 97 evidence indicates that dysfunctional epigenetic changes - including in DNA 98 methylation, histone modiﬁcation, and non-coding RNA (such as microRNA) 99 dysregulation - are also involved in the development and drug resistance of 100 myeloma (4-8). 101 102 Polycomb-like protein 3 (PCL3) also called PHF19 is a polycomb-like (PCL) 103 protein. PCL proteins are PRC2-associated factors that form sub-complexes 104 with polycomb repressive complex 2 (PRC2) core components (9) and 105 modulate the enzymatic activity of PRC2. PCL proteins are important 106 components of the PRC2 complex, are major H3K27 methyltransferases and 107 are required for recruiting the PRC2 complex to target genes; thus, they play 108 important regulatory roles in PcG protein-mediated transcriptional repression 109 (10). In normal tissues, PHF19 serves a role in transcriptional regulation of 110 gene expression in embryonic stem cell renewal and differentiation (11-13). 111 Recently, PHF19 was found to be highly expressed in a variety of cancers, 112 including melanoma, hepatocellular carcinoma, glioblastoma, ovarian 113 carcinoma, and multiple myeloma (14-18), and the expression of PHF19 has 114 been found to be correlated with overall tumor progression (19). The role(s) of 4

115 PHF19 in tumor (including MM) pathogenesis and drug resistance, however, 116 needs to be further elucidated. 117 118 Here we demonstrated that PHF19 was highly expressed in MM, especially in 119 relapsed or refractory myeloma cells, and that was associated with inferior 120 outcomes among MM patients. PHF19 overexpression promotes MM cell 121 growth and drug resistance. Knocking down PHF19 expression with use of 122 shRNA increased the sensitivity of MM cells to common chemotherapeutic 123 reagents and enhanced the apoptosis of the MM cells. Our study also 124 documented the pivotal role of PHF19 in promoting phosphorylation-mediated 125 inactivation of EZH2, a core protein of the PRC2 complex, by activating the AKT 126 pathway. PHF19 thus engendered a repressed H3K27me3 level and 127 consequently up-regulated expression of several pro-survival targets involved 128 in MM cell growth and drug resistance. Additionally, we also showed that 129 PHF19 was a direct target of miR-15a, loss of miR-15a led to overexpression of 130 PHF19 in multiple myeloma cells. These findings suggest that the 131 miR-15a/PHF19/pho-EZH2 axis acts as a key mediator of MM proliferation and 132 drug resistance. 133

134 Materials and Methods 135 PHF19 expression and survival analyses in myeloma patients 136 We determined levels of PHF19 mRNA in myeloma patients, with use of 137 Affymetrix U133 Plus 2.0 microarrays (Santa Clara, CA), as described (20). 138 Results are available in the NIH Gene Expression Omnibus (GEO) under 139 accession numbers GSE2658 and GSE31161. Microarray data on MGUS and 140 normal plasma cells are available under accession number GSE5900. 141 Statistical analyses of microarray results relied on GCOS1.1 software 142 (Affymetrix, Santa Clara, CA), and included log-rank tests for univariate 143 association with disease-related survival. 144 145 Human myeloma cell lines (HMCLs) and primary cells 146 All human MM cell lines were grown under mycoplasma-free conditions and 147 maintained in complete culture medium (RPMI1640 supplemented with 10%

148 FBS and 100 IU/mL penicillin) in tissue culture flasks, at 37°C in a 5% CO2 149 humidified incubator. HEK293T and MCF-7 cells were cultured in DMEM with 150 10% FBS and 100 IU/mL penicillin. All the cells were maintained in our lab and 151 shown to be mycoplasma-free via polymerase chain reaction (PCR); they were 152 used for experiments within 8 passages after thawing. Identities of the cell lines 153 were confirmed by STR testing. 154 155 Bone marrow mononuclear cells (BMMCs) were obtained from healthy donors 156 and MM patients who were newly diagnosed at the Department of Lymphoma 157 and Myeloma, Institute of Hematology and Blood Disease Hospital, Chinese 158 Academy of Medical Sciences, Peking Union Medical College. BMMC were 159 isolated from bone marrow (BM) via density gradient centrifugation using 160 ficoll-hypaque (GE Healthcare). MM cells and normal plasma cells were 161 purified from bone marrow aspirates, using anti-CD138 microbeads (Miltenyi 162 Biotech, Auburn, CA, USA). All studies with human samples were done under 163 the approval of the School Review Board, in accordance with the Declaration of 6

164 Helsinki. Good clinical practice guidelines were followed, and written informed 165 consents were obtained from healthy donors and MM patients to process 166 research on their samples. 167 168 Antibodies and reagents 169 The following primary antibodies were used for Western blotting: anti-PHF19 170 (#77271), anti-EZH2 (#5246), anti-Mcl-1(#94296), anti-Bcl-xL (#2764), 171 anti-phosphorylated PDK1 (S241) (#3061), anti-pan AKT, anti-phosphorylated 172 AKT (Ser473), anti-phosphorylated AKT (T308), anti-β actin, anti-GAPDH from 173 Cell Signaling Technology (Danvers, USA); anti-phospho-EZH2(Ser21) 174 (#IHC-00388) from Bethyl Laboratories Inc., and anti-HIF-1α(#36169) from 175 Abcam. Secondary antibodies, including horseradish peroxidase 176 (HRP)-conjugated anti-mouse and anti-rabbit antibodies were from Cell 177 Signaling Technology. 178 179 The following drugs (their sources) were used in this study: bortezomib 180 (Velcade, Janssen Pharmaceuticals, USA), melphalan (L-PAM), epirubicin 181 hydrochloride (Pharmorubicin®, Pfizer, UK); Doxycycline and LY294002 were 182 obtained from Sigma-Aldrich (Millipore Sigma, USA) and dissolved in water or 183 dimethyl sulfoxide, respectively, at appropriate concentrations. The final 184 concentration of dimethyl sulfoxide in the culture medium was <0.1%, and did 185 not affect drug effects and cell growth per se. 186 187 Primers or shRNA sequences 188 Primers for PHF19 cDNA clones (forward, 5’-GTG TCT AGA ATG CTG GTC 189 TTG GTA ATC CGT GG-3’; reverse, 5’-GTG GGA TCC TCA GTA AGG GGT 190 GGT CCC TTC CCA C-3’) were purchased from Beijing Genomics Institute 191 (BGI, China). PHF19 shRNAs were: sh-1, 5’-CCT CAA GTC CTC TAT CAC 192 CAA-3’; sh-2, 5’-GCG GCT GCC TCG TGA CTT TCG AAG ATA ATA GTG 193 AAG CCA CAG ATG TAT TAT CTT CGA AAG TCA CGA GGC AGC TGC-3’; 7

194 and sh-3, 5’-GCG GAA GGA CAT ACA GCA TGC CGG TGT TTA GTG AAG 195 CCA CAG ATG TAA ACA CCG GCA TGC TGT ATG TCC TTC TGC-3’. 196 197 Plasmids and transfection 198 The full-length coding sequence for PHF19 was inserted into the 199 pCDH-EF1-MCS-GFP vector. PHF19 was knocked down by shRNA were 200 designed with use of an RNA interference platform provided by the Broad 201 Institute, MA, USA. The oligomers were cloned into the pTRIPZ vector. The 202 PMIRH15a-PA-1 vector was purchased from System Biosciences (SBI) 203 (California, USA). Lentivirus was packaged in HEK293T cells, harvested after 204 48 hours and concentrated 10-fold by the Lenti-X™ Concentrator (Takara Bio 205 USA, Inc.). A total of 1×106 cells were plated in a 12-well plate. Lentivirus were 206 added to the cells along with 8 ug/ml polybrene, and stable cells were selected 207 after 72 hours according to the manufacturer’s instruction. The expression of 208 target proteins was examined by western blot. 209 210 Western blotting and co-immunoprecipitation (Co-IP) assays 211 Whole MM cells were lysed with RIPA lysis buffer; proteins (30ug) were 212 separated with SDS polyacrylamide gel electrophoresis, transferred onto a 213 polyvinylidene ﬂuoride (PVDF) membrane, blocked with 5% non-fat milk in 214 Tris-buffered saline solution containing 0.1% Tween 20 (TBS-T) at room 215 temperature for 1 h, and then incubated overnight at 4°C with primary antibody. 216 Protein bands were visualized with Super Signal West Pico Chemiluminescent 217 Substrate (Thermo Fisher Scientific, USA). Co-IPs were carried out with the 218 Pierce™ Direct Magnetic IP/Co-IP Kit (Thermo Fisher Scientific), using 219 antibodies as above. Normal mAb IgG from Cell Signaling Technology was 220 used as an isotype control. 221 222 Cell proliferation and apoptosis assay

223 Cell proliferation assays were carried out with use of a cell counting kit-8 (CCK8) 224 (Dojindo Laboratories, Japan) assay in 96-well plates and counted once a day 225 for 5 days. Equal number of multiple myeloma cells were seeded in 96-well 226 plates (5,000 cells/well). Cells were incubated in RPMI1640 medium at 37 °C in

227 a 5% CO2 humidified atmosphere. After incubation, 10 μL CCK8 was added to 228 a plate each day and plates were incubated at 37 °C for another 2-4 h. Finally, 229 absorbance was determined at 450 nm using a microplate reader SpectraMax 230 3 (Molecular Devices, Walpole, MA, USA). Apoptosis was quantified with use of 231 the Annexin V: PE and Annexin V: FITC Apoptosis Detection kits (BD 232 Bioscience, USA), following the manufacturer’s protocol. Fluorescence was 233 measured on an LSR II cytometer (BD Biosciences) and data analyzed using 234 FlowJo Software v10.0 (Tree Star, Ashland, OR, USA). 235 236 RNA-sequencing analysis 237 RNA was extracted using the Qiagen RNeasy Kit. Whole RNA (500ng) was 238 subjected to library preparation, with use of the Beijing Genomics Institute (BGI) 239 Library prep for Illumina kit. Paired-end reads were mapped to the human 240 genome (hg19) using Tophat, with only unique mapped reads with fewer than 2 241 mismatches used for downstream analysis. Genes were assembled using 242 Cufflinks. Normalized transcript abundance was computed using Cufflinks and 243 expressed as FPKM (Fragments Per Kilobase of transcripts per Million mapped 244 reads). Gene-level FPKM values were computed by summing the FPKM values 245 of their corresponding transcripts. A gene is expressed if the FPKM is greater or 246 equal to 1 in at least one sample. Edge R was adopted to calculate the p-values 247 of differential gene expression using the normalized gene level read counts. 248 For calling differentially expressed genes, we used the False Discovery Rate 249 (FDR) to correct the p-values, with a cutoff of 0.05 as significantly differentially 250 expressed genes. To evaluate the biological functional relevance of genes, we 251 employed the gene ontology enrichment analysis on groups of genes, using the

252 hyper-geometric test via the Gene Ontology. P-values were corrected through 253 BH test with a cutoff value of 0.05. 254 255 In vivo tumor xenograft model 256 A total of 1× 106 ARP1 cells expressing either normal (ARP1 EV) or high (ARP1 257 OE) levels of PHF19 were injected subcutaneously (SC) into the right flank of 258 nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice 259 (n=4/group). After 7 days, mice were treated with bortezomib (1mg/kg) or PBS, 260 twice weekly for 3 weeks. On day 28, mice were executed to harvest and 261 examine the tumors. Tumor volumes (measured by caliper) were calculated by 262 the formula: length × width2 × 0.52. And cells with PHF19-inducible knockdown 263 of ARP1 (PHF19 shRNA) were injected under the skin of the abdominal flank of 264 NOD/SCID mice (n=4). Depletion of PHF19 expression was induced with 265 doxycycline (2mg/ml). All procedures involving use of live animals were 266 approved by the Institutional Animal Care and Use Committee (IACUC) of the 267 Institute of Hematology, Chinese Academy of Medical Science. 268 269 RNA extraction and real-time quantitative RT-PCR 270 Total RNA yields were obtained with the RNeasy Mini Kit (QIAGEN, Germany) 271 according to manufacturer’s instructions, and first-strand cDNA synthesis 272 reactions were performed with the Transcriptor First Strand cDNA Synthesis Kit 273 (Roche, Switzerland). Real-time quantitative RT-PCR (RQ-PCR) reactions 274 were performed in triplicate on an ABI 7500 Real-Time PCR System (Applied 275 Biosystems, USA). Data were analyzed according to the ΔΔCt method. Primers 276 were purchased from Beijing Genomics Institute (BGI, China). 277 278 Dual luciferase reporter assay 279 To confirm that PHF19 is the target of miR-15a, we used a luciferase reporter 280 assay. PHF19 3’UTR wild type (wt) and mutated (mut) recombined with 281 SV40-Luc-MCS plasmids were obtained from Genechem corporation in China. 10

282 HEK293T cells were cultured and planted with 3 ×104 cells/well in96 well plates. 283 After 24 hours, plasmids were transfected to these cells, with use of 284 lipofectamine 2000 (Invitrogen, CA, USA). PHF19 3’UTR wt or mut plasmids 285 (1000ng) were co-transfected with miR-15a or empty vector. The phRL-CMV 286 plasmids (Promega WI, USA) that constitutively express Renilla luciferase was 287 included in all transfections. Forty-eight hours after transfection, cells were 288 analyzed by the Dual-Luciferase® Reporter (DLR™) Assay (Promega, WI, 289 USA), and the ratio of Firefly luciferase activity to Renilla luciferase activity was 290 calculated. 291 292 Statistical analysis 293 In accordance with PHF19 expression, survival curves were plotted using the 294 Kaplan-Meier method. The two-tailed Student’s t-test was used to compare two 295 groups. One-way analysis of variance (ANOVA) was used to assess more than 296 two groups. GraphPad Prism (version 6.01, GraphPad software Inc.) was 297 utilized, and p≤0.05 was considered statistically significant. Image J software 298 was utilized for the statistical analysis of these protein expression levels.

299 Results 300 PHF19 is a high-risk gene that predicts poor survival and drug resistance 301 in multiple myeloma patients 302 We compared the expression of PHF19 through gene expression profiling 303 (GEP) in plasma cells from healthy donors (NPC, n=22), individuals with 304 monoclonal gammopathy of undetermined significance (MGUS, n=44), 305 individuals with smoldering multiple myeloma (SMM, n=12), and newly 306 diagnosed MM patients (n=351) from the Total Therapy 2 (TT2) dataset. When 307 an Affymetrix signal of 1,500 for PHF19 was set at as the cutoff, plasma cells in 308 96 out of 351 (27.3%) MM patients exhibited an obviously high level of PHF19 309 expression compared to normal plasma cells; plasma cells from individuals in 310 the MGUS and SMM groups did not show high PHF19 expression levels 311 (Figure 1A). Expression of PHF19 was significantly higher in relapsed and 312 high-risk myeloma subgroups (Figure 1B and 1C). In sum, these results 313 strongly suggest that high levels of PHF19 is strongly correlated with an 314 aggressive MM phenotype. 315 316 Subsequently, we correlated PHF19 expression with MM patient outcomes, 317 determined by the P-value and hazard ratio (HR) at the best expression signal 318 cut-off, using the R package. The 21% (74/351) of MM patients with elevated 319 PHF19 expression had inferior event-free survival (EFS) (Figure 1D) and 320 overall survival (OS) (Figure 1E) in the TT2 clinical trial. Similar results were 321 obtained in an independent IFM cohort that included 119 MM patients 322 (Supplementary Figure 1A). These findings strongly suggest that PHF19 is a 323 high-risk gene and that PHF19 expression positively correlates with drug 324 resistance and poor prognosis in MM. 325 326 Enhanced PHF19 expression promotes cell growth and induces drug 327 resistance in MM cells

328 The association between the level of PHF19 expression and the high risk and 329 poor survival of MM patients described above strongly suggests that PHF19 is 330 a gene related to myeloma progression. However, the biological roles of PHF19 331 in myeloma cell formation and development, have not been fully characterized. 332 To address this gap, we overexpressed full-length PHF19 in ARP1 and 333 OCI-My5 MM cell lines via lentivirus-mediated transfection and observed the 334 alterations in cancer-related behavior in these two cell lines. Western blots 335 verified that PHF19 was significantly increased in these cells (Figure 2A). 336 PHF19 OE cells demonstrated significantly enhanced cell proliferation 337 compared to those transfected with an empty vector (EV) (Figure 2B). PHF19 338 OE also induced resistance to anti-MM treatments. Twenty-four hours after 339 treatment with bortezomib (BTZ), epirubicin (EPI), or melphalan (MEL) in 340 designated concentration, flow cytometry revealed a decrease in MM cell 341 apoptosis in PHF19 OE groups compared to the EV groups (Figure 2C and 342 2D). 343 344 Next, we examined the growth inhibitory effect of the drugs in PHF19 OE cells. 345 OCI-My5 cells were cultured for 48 hours in the presence of a series of diluted 346 concentrations of BTZ, EPI or MEL. Enhanced PHF19 expression decreased 347 cell sensitivity significantly after 48 hours in a dose-dependent manner, with an 348 increased the half maximal inhibitory concentration (IC50) in PHF19 OE cells 349 (Supplementary Fig.2A-2C). These results suggest that ectopic expression of 350 PHF19 promotes multiple drug resistance in MM cells. We then tested the 351 effects of PHF19 OE in a xenograft mouse model. ARP1 EV or PHF19 OE cells 352 were injected into the right flank of NOD/SCID mice. After 28 days, the mice 353 were euthanized to measure tumor burden. We found that mice engrafted with 354 ARP1 PHF19 OE cells had significantly larger tumor volumes than the control 355 mice which were engrafted with ARP1 EV cells (P=0.0079) (Figure 2E and 2F). 356 Both in vitro and in vivo data demonstrated that overexpression of PHF19 357 enhances the tumorigenic capacity of MM. 13

358 359 Knockdown of PHF19 inhibits MM cell growth and sensitizes MM cells to 360 chemotherapeutic drugs 361 To determine whether MM cell survival relies on PHF19, we also knocked down 362 PHF19 in ARP1 and OCI-My5 MM cell lines, via a doxycycline-inducible shRNA 363 lentivirus delivery system. Upon induction with 2μg/mL doxycycline, PHF19 364 protein expression was remarkably downregulated (Figure 3A). Cell 365 proliferation assays indicated that, compared to controls, PHF19 depletion 366 significantly suppressed cell growth in ARP1 (day 6, p<0.001) as well as 367 OCI-My5 (day 6, p<0.001) cell lines (Figure 3B). We next evaluated whether 368 PHF19 knockdown promotes drug sensitivity in MM cells. PHF19-knockdown 369 cells were treated for 24 hours with BTZ, EPI or MEL at indicated doses. As 370 expected, the knockdown of PHF19 increased cell apoptosis in both ARP1 and 371 OCI-My5 cell lines in the presence of doxycycline (Figure 3C and 3D). We then 372 used CCK8 assays to examine the growth inhibitory effect of the drugs in 373 PHF19- knockdown cells. OCI-My5 cells were cultured for 48 hours in the 374 presence of a series of diluted concentrations of BTZ, EPI or MEL. The 375 knockdown of PHF19 expression increased cell sensitivity significantly after 48 376 hours, in a dose-dependent manner, with a reduced IC50 in PHF19-knockdown 377 cells (Supplementary Fig.2A-2C). The in vivo study also showed that tumor 378 volumes in the PHF19 shRNA knockdown group induced by doxycycline was 379 significantly smaller than those in the control mice (P=0.04) (Figure 3E and 3F). 380 In sum, these results further support that PHF19 promotes myeloma growth 381 and induces resistance to chemotherapy. 382 383 PHF19 inactivates EZH2 through phosphorylation of Ser21 in MM 384 PHF19 is a Tudor domain-containing protein that plays an important role in 385 recruiting the PRC2 complex to CpG islands (21). EZH2 is the enzymatic 386 subunit of PRC2 that methylates lysine 27 of histone H3 (H3K27), to promote 387 transcriptional silencing (10-12). Our co-IP analysis indicated that PHF19 binds 14

388 directly to EZH2 in ARP1, KMS11, and HEK293T cell lines (Figure 4A and 389 Supplementary Fig.3). Further study demonstrated that high levels of PHF19 390 suppressed bi- and tri-methylation of H3K27 (Supplementary Fig.4). 391 Correspondingly, the knockdown of PHF19 by shRNA decreased EZH2 392 phosphorylation and restored H3K27 methylation (Figure 4B and 4C, 393 Supplementary Fig.4). However, according to our RNA-seq analysis, 394 expression of three lysine demethylases (KDM7A, KDM6A, and KDM6B), 395 which induces the demethylation of H3K27, was not elevated in PHF19 OE 396 cells (data not shown). The level of EZH2 phosphorylation at Ser21 was 397 enhanced significantly in PHF19 OE cells (Figure 4B). When EZH2 is 398 phosphorylated (Ser21), its trans-methylase function is inactivated, causing 399 H3K27 demethylation, which then upregulates the expression of downstream 400 genes. Furthermore, high levels of PHF19 were associated with increased 401 pho-Ser21 EZH2 in primary MM cells (Supplementary Fig.4). Since 402 phosphorylation-mediated inactivation of EZH2 leads to drug resistance in MM 403 (22), these data strongly indicate that PHF19 directly affects the enzymatic 404 activity of EZH2 methyltransferase and exerts a drug-resistance function 405 through the phosphorylation of EZH2. We also found that the drug-resistance 406 genes of MM, (Bcl-xL, Mcl-1, and HIF-1α) were significantly upregulated by 407 PHF19 (Figure 4D), while they were significantly downregulated in cells that 408 were depleted of PHF19 (Figure 4E). 409 410 AKT mediates EZH2 phosphorylation by PHF19

411 To establish which pathways are involved in PHF19-mediated MM cell survival 412 and drug resistance, we performed RNA sequencing on ARP1 cells with 413 aberrant PHF19 expression. Distinct clusters of ARP1 PHF19 OE samples 414 were distinguished from the EV samples through a principal component 415 analysis (Supplementary Fig.5A). The heatmap revealed that 1,426 genes 416 were significantly differentially expressed between ARP1 PHF19 OE and EV 417 cells (FDR<0.05). MM pathogenesis genes (CCL3, FOS, JUN, KLF and RELB) 15

418 were significantly upregulated by PHF19 overexpression (Supplementary 419 Fig.5B). The UBB, UBA52, UBE2L6, and UBXN6 genes, which control 420 unfolded protein degradation through ubiquitination and the proteasomes 421 system, were also upregulated in PHF19 OE cells (Supplementary Fig.5C). 422 Gene set enrichment analysis (GSEA) showed that differential expression of 423 genes in PHF19 OE cells was enriched in signal pathways, such as the NF-κB, 424 hypoxia, and p53 pathways, which play pivotal roles in MM cell proliferation. 425 We also used RNA-seq to profile gene expression in shRNA-PHF19 and 426 control ARP1 cells, and we identified 1,505 differentially expressed genes 427 (Supplementary Fig. 5D). GSEA also indicated that the MYC, NF-κB, and 428 KRAS signaling pathways were enriched in shRNA-PHF19 MM cells 429 (Supplementary Fig. 5E). Further analysis indicated that 224 genes were 430 overlapped between the 2 RNA-seq studies (Figure 5A and 5B), and the 431 GSEA hallmark gene analysis indicated that these genes were involved in 432 multiple signaling pathways (such as the NF-κB, EMT transition, hypoxia and 433 PI3K/AKT/mTOR pathways) that were implicated in cell growth and 434 tumorigenesis (Figure 5C). These data further support our hypothesis that 435 PHF19 promotes myeloma cell survival through multiple signaling pathways. 436 These RNA-seq data have been submitted to the Gene Expression Omnibus 437 (GEO) database (GSE128406) of the national Center for Biotechnology 438 Information (NCBI). 439 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE128406). 440 441 Cha et al. reported that AKT-mediated phosphorylation of EZH2 suppresses 442 methylation of H3K27 (23), and our RNA-seq data strongly indicated that the 443 PI3K/AKT pathway was activated in PHF19 OE cells (Figure 5D). We therefore 444 investigated the relationship between AKT activation and phosphorylation of 445 EZH2, and we found significantly increased levels of phosphorylation of AKT at 446 Ser-473, phosphorylation of PDK1 at Ser-241, and phosphorylation of EZH2 at 447 Ser-21 in PHF19 OE cells. These findings showed a correlation between the 16

448 activation of AKT pathways and EZH2 phosphorylation (Figure 6A). To further 449 verify whether EZH2 phosphorylation is regulated by AKT in this context, we 450 treated the PHF19 OE ARP1 and OCI-My5 cells with LY294002, a commonly 451 used broad-spectrum inhibitor of PI3K/AKT. Western blot showed that 452 pharmacological inhibition of the PI3K/AKT pathway significantly reduced 453 EZH2 phosphorylation at Ser21 in both ARP1 and OCI-My5 cells (Figure 6B), 454 similar results were also found in PHF19 OE JJN3 cells (Supplementary Fig.6) 455 These results showed that PHF19 induced EZH2 phosphorylation by PI3K/AKT 456 pathway. 457 458 PHF19 is a direct target of miR-15a, which is downregulated in MM 459 Complex genomic events underlie MM development. Gains of 9,15 or 19 are 460 common clonal events observed in MM (24). The PHF19 gene is located on 461 chromosome 9q33.2, and MM cells commonly display DNA amplification in this 462 region. To determine the mechanism that underlies the increase of PHF19 in 463 myeloma cells, we first investigated whether PHF19 is caused by the DNA 464 amplification of chromosome 9q; however, we found a negative correlation 465 between those two (Figure 7A), indicating that high levels of PHF19 are not 466 induced by DNA amplification. We then hypothesized that a miRNA-based 467 post-transcriptional mechanism was likely involved in PHF19 regulation. Based 468 on our report that downregulated miR-15a in MM cells is correlated with drug 469 resistance and poor survival of MM patients (25-27), we examined the 470 expression of miR-15a and PHF19 in newly diagnosed MM patients (n=27). 471 Our data revealed that miR-15a expression was negatively correlated with 472 PHF19 (r=-0.651, P=0.0005, Figure 7B and 7C). We also hypothesized that 473 miR-15a was directly correlated with PHF19, which may explain the 474 overexpression of PHF19 in MM. Our results confirmed that overexpression of 475 miR-15a via transient transfection of a mimic oligo (PMIRH 15a-PA-1) 476 significantly downregulated PHF19 expression at the mRNA level as well as in 477 protein levels (Figure 7D and 7E, Supplementary Fig.7). To establish a direct 17

478 connection between miR-15a and PHF19, we undertook a bioinformatics 479 analysis to test the possibility that the seed sequence of miR-15a binds with the 480 3’UTR of PHF19, as shown in Figure 7F,followed by a dual luciferase reporter 481 assay. We also found that overexpression of miR-15a reduced the activity of 482 the luciferase reporter gene fused to the wild type PHF19 3’UTR by a significant 483 52.5%, compared with the control group in HEK293T cells, while a deletion of 6 484 nucleotides in the miR-15a binding site in the 3’UTR of PHF19 induced no such 485 change (Figure 7G). These data underscore that PHF19 is one of the direct 486 targets of miR-15a. Based on these findings, we have developed a working 487 model, shown in Supplementary Fig.8. 488 489 Discussion 490 Wang et al. first described a human homologue of the Drosophila polycomb-like 491 protein PHF19 (PCL3) in 2004 (28). Since then, PHF19 has been ascribed 492 putative roles in the memory of cellular identity based on establishing and 493 faithfully maintaining transcription states at the chromatin level. PHF19 is 494 markedly overexpressed in many types of cancers and is highly correlated with 495 tumor progression (14-18). Recently, new evidence has emerged that PHF19, 496 a gene downstream of miR-155, participates in CD8+T cell biology (29). Here 497 we have demonstrated through GEP analysis that PHF19 is significantly 498 upregulated in samples from MM patients, especially in the relapsed and 499 high-risk myeloma samples. Survival analysis clearly indicates that MM 500 patients with elevated PHF19 have shorter survival and inferior prognoses in 2 501 different clinical cohorts: TT2 and IFM. Collectively, these clinical data suggest 502 that the PHF19 gene plays a pivotal role in drug resistance and disease 503 progression in MM. 504 505 Drug resistance is a major obstacle limiting the effectiveness of MM treatment 506 and significantly contributing to the cause of relapse in patients with the disease. 507 Elucidating the mechanism that underlies drug resistance and, of equal 18

508 importance, developing a corresponding treatment, is critical for the clinical 509 management of MM. Here, we have identified the high-risk gene PHF19, which 510 induces drug resistance and shorter survival of patients, our findings are 511 consistent with the report of Zhou et al (30,31), which found that PHF19 is one 512 of 56 drug resistance genes. We have also used in vitro and in vivo approaches 513 to further demonstrate that high levels of PHF19 promote proliferation and 514 multidrug resistance in MM cells. Our RNA-seq results have uncovered 515 that many transcription factors and oncogenes are downstream targets of 516 PHF19 in promoting the cell growth and drug resistance of MM cells 517 (Supplementary Fig.5).We document that JUN, JUNB, JUND, FOSB, KLF and 518 RELB, which play pivotal roles in the pathogenesis and progression of MM 519 (32,33), are significantly upregulated in PHF19 OE cells. In addition, we report 520 that ubiquitin genes-including UBB, UBA52, UBE2L6, and UBXN6-are 521 upregulated in PHF19 OE cells: given that high levels of ubiquitin genes 522 facilitate protein degradation via the ubiquitin-proteasome pathway, this 523 upregulation likely contributes to the cell growth and drug resistance induced by 524 PHF19 overexpression. Our data also indicate that CCL3, which is critical for 525 the development of MM bone disease, is significantly increased in MM cells that 526 express high levels of PHF19, indicating that PHF19 overexpression likely 527 plays a role in bone disease related to MM. Finally, the GSEA analysis shows 528 that critical signaling pathways - such as NF-κB, hypoxia, MYC-known to be 529 involved in the pathogenesis of MM, are dysregulated by PHF19. 530 531 PHF19 is a PRC2-associated factor that forms sub-complexes with PRC2 core 532 component EZH2, which is a histone methyltransferase (10) that participates in 533 the proliferation and differentiation of cells. Our data reveal that PHF19 534 promotes the phosphorylation-related inactivation of EZH2 and subsequently 535 decreases histone H3K27 methylation, leading to upregulation of HIF-1α, 536 Bcl-xL, and Mcl-1, and promoting the proliferation and drug resistance of MM

537 cells. These data are consistent with previous reports by other groups that 538 phosphorylation-mediated EZH2 inactivation promotes CAM-DR in MM (22). 539 PHF19 has no kinase enzyme domain, and it promotes EZH2 phosphorylation. 540 The underlying mechanism is still unclear. We hypothesize that upstream 541 proteins of the AKT signaling pathway are directly linked to PHF19 (34). We 542 performed immuno-precipitation for PHF19, followed by mass spectrometry, 543 and detected the insulin receptor substrate 4 (IRS4), which is a poorly studied 544 member of the IRS family. IRS4 activates the PI3K/AKT pathway in breast 545 cancer and induces mammary tumorigenesis and confers resistance to 546 HER2-targeted therapy (35). Another interesting molecule listed was receptor 547 for activated protein C kinase 1 (RACK1), shown to be a multifaceted 548 scaffolding protein involved in multiple biological events via interaction with 549 different partners, including cell migration, and angiogenesis. RACK1 is an 550 EphB3-binding protein and mediates the assembly of a ternary signal complex 551 that comprises protein phosphatase 2A, AKT and itself in response to EphB3 552 activation, thereby leading to reduced AKT phosphorylation and subsequent 553 inhibition of cell migration in non-small-cell lung cancer (36). Further 554 experiments are needed to verify our hypotheses. Based on our RNA-seq and 555 western blot analysis, the PI3K/AKT pathway is significantly activated in PHF19 556 OE MM cells. Depression of AKT activation by the inhibitor LY294002 notably 557 decreases the phosphorylation of EZH2 and phosphorylation-related 558 inactivation of EZH2 down-regulates of the level of methylation of H3K27 (a 559 transcriptionally repressive marker), which in turn activates expression of 560 downstream targets. Our results thus establish that the phosphorylation-related 561 inactivation of EZH2 plays a pivotal role in mediating PHF19-induced MM cell 562 proliferation and drug resistance. 563 564 Recent reports show that, PHF19 is highly expressed in various forms of cancer, 565 including MM (16-19); however, the molecular mechanism involved in the 566 upregulation of PHF19 is poorly elucidated. Because the human PHF19 gene is 20

567 located on chromosome 9q33.2, which is commonly amplified in MM cells, we 568 initially looked for a correlation between the PHF19 gene and chromosome 569 9q33 amplification; however, we did not find that the level of expression of 570 PHF19 is correlated with 9q amplification in primary patient samples. In 571 keeping with this, results from use of cancer profiling arrays reported in the 572 literature, PHF19 is upregulated in other kinds of tumors without amplification of 573 chromosome 9q (28,37). Therefore, we concluded that other mechanisms 574 underlie PHF19 regulation in MM. Of note, no activation mutations, gene 575 amplifications, or a hypomethylated promoter of PHF19 has been reported in 576 tumor cells, suggesting that increased accumulation of PHF19 results from 577 changes in post-transcriptional regulation. Our previous studies demonstrate 578 that aberrant expression of miRNAs serves a pivotal role in the pathogenesis of 579 MM (38,39). For example, miR-15a is significantly downregulated in MM cells, 580 promoting cell proliferation and drug resistance (25-27). Here we used 581 bioinformatics analysis and a luciferase report assay to identify PHF19 as a 582 novel target of miR-15a. Our data validate that the seed sequence of miR-15a 583 directly binds to the 3’UTR of PHF19 and suppresses PHF19 mRNA 584 transcription and expression. 585 586 In sum, this study reveals that the gene for the PRC2 complex member 587 polycomb-like protein PHF19 is overexpressed in high-risk myeloma patients, 588 and that this overexpression correlates with drug resistance and inferior 589 outcome of MM patients. PHF19 promotes the phosphorylation-related 590 inactivation of EZH2 by activating PI3K/AKT pathways that mediate 591 PHF19-induced proliferation and drug resistance of MM cells. PHF19 causes 592 demethylation of histone H3K27, and promotes expression of HIF-1a, Bcl-xL 593 and Mcl-1, thereby inducing MM cell proliferation and conferring drug 594 resistance. We also show that PHF19-induced inactivation of EZH2 by 595 phosphorylation is a novel epigenetic mechanism involved in the promotion of 596 MM cell proliferation and drug resistance. Our findings implicate the 21

597 miR-15a/PHF19/pho-EZH2 epigenetic axis as a critical participant in MM 598 tumorigenesis and suggest that it is a promising target for MM treatment. 599 Authors’ Contributions 600 Conception and design: T. Yu, C. Du, L. Qiu, F. Zhan, M. Hao 601 Development of methodology: T. Yu, C. Du L. Qiu, F. Zhan, M. Hao 602 Acquisition of data (provided animals, acquired and managed patients, 603 provided facilities, etc.): T. Yu, C. Du, Z. Yu, L. Liu, L. Zhao, Z. Li, J. Xu, X. Wei, 604 S. Deng, D. Zou. G. An, W, Sui, W, Zhou 605 Analysis and interpretation of data (e.g., statistical analysis, biostatistics, 606 computational analysis): T. Yu, C.X. Du, G. Tricot, G. An, X. Ma, M. Hao 607 Writing, review, and/or revision of the manuscript: T. Yu, C.X. Du, G. Tricot, 608 Y-T. Tai, K.C. Anderson, L. Qiu, F. Zhan, M. Hao 609 Administrative, technical, or material support (i.e., reporting or organizing data, 610 constructing databases): L. Liu, G. An, Z. Yu, L. Qiu, M. Hao 611 Study supervision: L. Qiu, F. Zhan, M. Hao

612

613 Acknowledgments 614 The authors acknowledge the patients and physicians who participated in 615 sample collections. We thank Dr. Sonal Jhaveri-Schneider (Postdoc and 616 Graduate Student Affairs Office, Dana-Farber Cancer Institute) and Dr. Jie Ni 617 (South Eastern Sydney Local Health District) for editing some drafts of the 618 manuscript. We thank Dr. Teru Hideshima, Mr. Kenneth Wen and Dr. Wenjuan 619 Yang (Dana-Farber Cancer Institute) for their assistance and constructive 620 advice on this project. 621

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742

743 FIGURE LEGENDS 744 Figure 1. High PHF19 levels are related to MM cell myeloma drug 745 resistance and relapse. (A) A bar graph depicting the range of PHF19 mRNA 746 level in normal bone marrow (BM) plasma cells (NPC), “premalignant” BM 747 plasma cells from individuals with monoclonal gammopathy of undetermined 748 significance (MGUS) and malignant plasma cells from patients with newly 749 diagnosed MM from the University of Arkansas Total Therapy 2 (TT2) cohort 750 (GSE5900 and GSE2658). (B) GSE31161 expression data from primary 751 multiple myeloma plasma cells from patients treated by Total Therapy 2, 3, 752 and other protocols at baseline and relapse. The data were available from the 753 National Institutes of Health (NIH) GEO database under accession number 754 GSE31161. (C)The data were available from the NIH GEO database under 755 accession number GSE2658. (D and E) Elevated PHF19 expression predicts 756 poor survival in patients with newly diagnosed MM. Kaplan-Meier analyses 757 show event-free survival (EFS) and overall survival (OS) of MM patients 758 enrolled in the TT2 cohorts. Each line represents different subgroups with high 759 or low PHF19 expression. 760 761 Figure 2. Overexpression of PHF19 promotes MM cell growth and drug 762 resistance. (A) Western blots were performed to measure expression of the 763 gene for the PHF19 protein in ARP1 and OCI-My5 cell lines transfected with 764 pCDH-PHF19 or a control vector. (B) ARP1 and OCI-My5 cells with or without 765 PHF19 transfection were counted daily for 6 days. All results were expressed 766 as means ± standard error of the mean (SEM) of 3 independent experiments. *, 767 p<0.05; **, p<0.01. (C) Cell apoptosis was evaluated in ARP1 or OCI-My5 EV 768 and PHF19 OE cells treated with bortezomib, epirubicin, and melphalan; this 769 was followed by Annexin V staining flow cytometry. The right peak indicated 770 cells undergoing apoptosis. (D) The statistical analysis showed the drug 771 resistance of PHF19 OE cells. *, p<0.05. (E) ARP1 EV and PHF19 OE cells 772 were injected subcutaneously into the right axilla of NOD/SCID mice. After 28

773 days, the mice were euthanized to obtain the tumors. (F) Tumor volume 774 assessment revealed that the mice injected with PHF19 OE cells had larger 775 tumors than the EV group. n=4, bars represent the means ± SEM of each 776 group. *, p<0.05; **, p<0.01. 777 778 Figure 3. PHF19 knockdown inhibits tumor growth and enhances 779 sensitivity to bortezomib. (A) Western blots confirmed PHF19 expression in 780 ARP1 and OCI-My5 cells transfected with pTRIPZ-shRNA-PHF19 and the 781 control group. (B) Cell proliferation was measured in ARP1 and OCI-My5 cells 782 transfected with shRNA-PHF19 and controls by counting the number of cells. 783 All results are expressed as means ± SEM of 3 independent experiments. **, 784 p<0.01; ***, p<0.001. (C and D) Drug sensitivity was determined by measuring 785 cell apoptosis in ARP1 and OCI-My5 cells expressing shRNA-PHF19 and 786 controls with or without treatment with bortezomib, epirubicin, or melphalan; 787 this was followed by Annexin V staining flow cytometry. (E) Silencing PHF19 788 inhibits MM growth in vivo. ARP1 shPHF19 cells were inoculated into 789 NOD/SCID mice. shRNA expression was induced by doxycycline (DOX), 790 which was given on the day after the injection of tumor cells and was 791 administered every other day. (F) Tumor volume assessment revealed that the 792 mice in Dox-induced shRNA PHF19 group had smaller tumors than the control 793 group. n=4, bars represent the means ± SEM each group. *, p<0.05. 794 795 Figure 4. PHF19 induces EZH2 phosphorylation.(A) ARP1 cells were lysed, 796 and PHF19 was immunoprecipitated using PHF19 antibodies. Western blots 797 were probed using PHF19 and EZH2 antibodies. (B and C) Western blots 798 showed the expression of p-EZH2 (Ser21) in ARP1 and OCI-My5 cells with 799 over-expression or knockdown of PHF19. (B) in PHF19 OE cells; (C) in 800 shRNA-PHF19 cells. (D and E) Western blots showed the pro-survival proteins 801 Bcl-xL, Mcl-1, and HIF-1a in ARP1 and OCI-My5 MM cells with

802 over-expression or knockdown of PHF19. (D) in PHF19 OE cells; (E) in 803 shRNA-PHF19 cells. 804 Figure 5. RNA-seq indicates that PHF19 activates MM cell 805 proliferation-related signaling pathways. (A) PCA (principal component 806 analysis) showed the distinct clusters of each group separately and a heatmap 807 of genes that were significantly differentially regulated upon PHF19 aberrant 808 expression. (B) The Venn diagram of differential gene expression analysis 809 combined with PHF19 OE and shRNA-PHF19. A total of 224 genes were 810 overlapped in both groups. The red circle indicates the differential genes 811 between the ARP1 PHF19 OE and EV cells. The blue circle indicates the 812 differential genes in the shRNA-PHF19 ARP1 cells. (C) The GSEA based on 813 the 224 differential genes combined in PHF19 OE and shRNA revealed

814 significantly enriched signaling pathways, including the NF-κB, EMT, hypoxia

815 and PI3K-AKT-mTOR pathways. (D) GSEA analysis revealed that the 816 AKT/mTOR pathway was enriched in the PHF19 OE group. 817 818 Figure 6. AKT mediates EZH2 phosphorylation induced by PHF19. (A) 819 Western blots revealed the expression levels of p-PDK1-(Ser241), 820 p-AKT-(Ser473) and p-EZH2-(Ser21) in ARP1 and OCI-My5 cells with PHF19 821 OE. (B) Western blots showed the expression of PHF19, p-EZH2(S21), EZH2 822 in EV and PHF19 OE ARP1 and OCI-My5 cells with or without LY294002 823 treatment for 6 hours. 824 825 Figure 7. PHF19 is a direct target of miR-15a, which is downregulated in 826 MM cells. (A) Box plots present PHF19 copy number variation (CNV) (X-axis) 827 by exome sequencing and mRNA expression (Y-axis) by RNA sequencing 828 from 718 primary MM samples. (B) RT-qPCR analysis revealed the expression 829 of miR-15a in CD138+ plasma cells derived from newly diagnosed multiple 830 myeloma (NDMM) patients and from the healthy donors. (C) This scatter plot

831 shows that PHF19 expression was negatively correlated with miR-15a level in 832 27 samples collected from NDMM patients (r=-0.651, p=0.0005). (D and E) 833 ARP1 cells were transiently transfected with miR-15a expressing plasmids for 834 72 hours; PHF19 mRNA (D) and protein (E) were significantly decreased, as 835 revealed by qRT-PCR and western blot analysis, respectively. (F) Illustration 836 of the 3’ UTR of PHF19 mRNA in a CMV-driven luciferase construct used for a 837 luciferase reporter assay. It showed the predicted pairing with the target sites 838 and their respective mutant (Mut) sequences. (G) Dual luciferase report 839 assays confirmed that PHF19 is a direct target of miR-15a.HEK 293T cells 840 were co-transfected with luciferase fused with wild type or mutant PHF19 841 3’UTR and an empty vector or a miR-15a-expressing construct. The bar-view 842 showed the luciferase signals in HEK293T cells containing wild type or mutant 843 PHF19 3’UTR. 844

Figure 1 A B C 2500 ) 2500

1) *** PHF19 *** 58 16 2000 2000 31 SE 1500 1500 l (GSE26 l (G a a n g

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F 500 500 F H PH 0 P 0 ine e k asel D B E Relaps low-risk high-ris

Events/N Deaths/N Low PHF19 115/227 Low PHF19 65/227 High PHF19 45/74 High PHF19 32/74 Proportion of Cases Proportion of Cases p < 0.001 p < 0.001

Downloaded from mcr.aacrjournals.orgMonths on September from 29, 2021. Start © 2020 American of Therapy Association for Cancer Months from Start of Therapy Research. Author Manuscript Published OnlineFirst on April 20, 2020; DOI: 10.1158/1541-7786.MCR-19-0852 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 2 A B C D DMSO BTZ EPI MEL 4 ARP1 ARP1 EV 100 ARP1 EV nm) ARP1 PHF19 OE 0 ARP1 PHF19 OE EV OE 3 80 cells

* + PHF19 2 60

xin V **** ****

* e GAPDH 40 1 ARP1

ative OD (45 20 l * % Ann re

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OCI-My5 OCI-My5 EV 100 OCI-My5 EV s l OCI-My5 PHF19 OE 4 OCI-My5 PHF19OE 80 cel

EV OE nm) + 0 * **** 3 60 ***

PHF19 in V

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Figure 3

A B C DMSO BTZ EPI MEL D shRNA-PHF19 shRNA PHF19 ARP1-Dox 2.0 100 shRNA PHF19 ARP1-DOX ARP1 shRNA PHF19 ARP1+Dox * 8.85% 41.7% 12.7% 34.2% s shRNA PHF19 ARP1 +DOX - + 1.5 80 cell +

* V PHF19 * 60 **** **** 1.0 n 40 RP1 tive OD (450nm) 10.1% GAPDH a 0.5 46.7% 17.2% 50.4% l ** * A e 20 % Annexi r *

0.0 0 0 1 2 3 4 5 +DOX -DOX DMSO BTZ EPI MEL Time (day) shRNA-PHF19

3.55% 46.3% 28% shRNA PHF19 OCI-My5-DOX shRNA-PHF19 shRNA PHF19 OCI-My5-Dox 23.5% 100 X OCI-My5 2.5 shRNA PHF19 OCI-My5+Dox s shRNA PHF19 OCI-My5+DOX

* O 80 - + 2.0 cell *** + D -DOX - 60 PHF19 1.5 n V ** xi ** 1.0 * e 40

4.20% 56.4% 37.2% 40.6% nn CI-My5

GAPDH A * 20

0.5 O % relative OD (450nm) DOX 0 0.0 +DOX + 0 1 2 3 4 5 shRNA-PHF19 DMSO BTZ EPI MEL Time (day)

400 )

E F 3

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shRNA PHF19 (m 300 me

ARP1-DOX lu 200 Vo

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Figure 4

A B ARP1 OCI-My5 C shRNA-PHF19 ARP1 OCI-My5 ARP1 PHF19 PHF19 EV OE DOX - + - + Input IgG PHF19 EZH2 EV OE PHF19 PHF19 PHF19 IP p-EZH2(S21) p-EZH2(S21) EZH2 EZH2 EZH2

GAPDH GAPDH

ARP1 OCI-My5 shRNA-PHF19 D PHF19 PHF19 E ARP1 OCI-My5 EV OE EV OE DOX - + - +

PHF19 PHF19 1.0 4.3 1.0 2.5 1.0 0.4 1.0 0.4 BCL-xL BCL-xL 1.0 1.5 1.0 1.2 1.0 0.6 1.0 0.7 MCL-1 MCL-1 1.0 1.2 1.0 1.5 1.0 0.5 1.0 0.5 HIF-1α HIF-1α 1.0 2.0 1.0 1.2 1.0 0.7 1.0 0.6 GAPDH GAPDH Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 20, 2020; DOI: 10.1158/1541-7786.MCR-19-0852 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Figure 5 A B ARP1 ARP1 shRNA-PHF19 PHF19 OE EV DOX - + Differential expression gene in PHF19 OE and shRNA-PHF19 ARP1 cells

PHF19 OE

1202 224 1277

PHF19 KD

C D AKT signaling PHF19 OE combined with shRNA

HALLMARK_TNFA_SIGNALING_VIA_NFKB[200] HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION HALLMARK_ALLOGRAFT_REJECTION[200] HALLMARK_MTORC1_SIGNALING[200] NES:1.445

HALLMARK_CHOLESTEROL_HOMEOSTASIS[74] ES HALLMARK_ESTROGEN_RESPONSE_EARLY[200] FDR q value: 0.090 HALLMARK_ESTROGEN_RESPONSE_LATE[200] HALLMARK_HYPOXIA[200] HALLMARK_MYOGENESIS[200] HALLMARK_PI3K_AKT_MTOR_SIGNALING[105] 0 2 4 6 8 PHF19 OE EV -log10(p-value)

Figure 6 A B

ARP1 OCI-My5 ARP1 OCI-My5 PHF19 PHF19 EV OE EV OE EV PHF19 OE EV PHF19 OE

PHF19 LY294002 - + - + - + - + p-PDK1 (S241) PHF19 p-EZH2(S21) p-AKT(S473) EZH2 AKT β-actin p-EZH2(S21)

GAPDH

Figure 7 A B C D

PHF19 (TRP) EV miR-15a 140 1.2 *** 120 p = 7.76 x 10-6 PHF19: Amp 384, on i 100 (a negative correlation) Nor 595 0.8 80

60 PHF19 40 0.4

20 Relative express 0 0.0 ARP1

E F G

ARP1 EV miR-15a ty miR-15a - + i 1.5 ** tiv c a e PHF19 s 1.0 ra e f i uc l

e 0.5 GAPDH v i t a

Rel 0.0 wild type mutant PHF19 3'UTR Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 20, 2020; DOI: 10.1158/1541-7786.MCR-19-0852 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Polycomb-like Protein 3 induces proliferation and drug resistance in multiple myeloma and is regulated by miRNA-15a

Tengteng Yu, Chenxing Du, Xiaoke Ma, et al.

Mol Cancer Res Published OnlineFirst April 20, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-19-0852

Supplementary Access the most recent supplemental material at: Material http://mcr.aacrjournals.org/content/suppl/2020/04/18/1541-7786.MCR-19-0852.DC1

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