Roles And Mechanistic Studies Of The HRP2 In Multiple Myeloma Drug Resistance

Objective 1.To elucidate the significance of hepatoma-derived growth factor 2(HRP2)expression level in the clinical treatment of MM patients and the role in bortezomib(BTZ)induced drug resistance in multiple myeloma(MM).2.To explore the epigenetic transcriptional regulation mechanism of HRP2 as a histone H3K36me2 recognition protein promoting MM sensitivity to proteasome inhibitor(PIs)drugs.3.To evaluate the effect of PIs combined with targeting H3K27me3 on reversing MM resistance in vitro and in vivo.Methods and results1.The low expression of HRP2 indicates poor prognosis.We collected the bone marrow image samples of patients with MM and detected the protein expression level of HRP2 by immunohistochemistry.We found that the expression level of HRP2 was positively correlated with the clinical treatment effect.Compared with the control group,the expression of HRP2 was lower in relapsed / refractory multiple myeloma patients with poor prognosis and severe bone destruction.Combined with the analysis of patients’ genotypes,the expression of HRP2 was negatively correlated with the occurrence of chromosomal t(4;14)abnormalities.2.The expression level of HRP2 was decreased in bortezomib resistant cells of multiple myeloma.Compared with wild-type cells,the m RNA and protein levels of HRP2 in bortezomib resistant cells were decreased by Western blot and cell viability assay.Knockdown of HRP2 in multiple myeloma cells by gene editing technology significantly reduced the sensitivity of cells to bortezomib.Combined with cytogenetic background analysis,it was found that in multiple myeloma cells with heterotopic mutation on chromosome 4 and 14,the tolerance to bortezomib was more obvious after knockdown of HRP2.The same results were obtained by flow cytometry and Western blot.3.Multiple myeloma cells with low expression of HRP2 have stronger drug resistance and bone destruction ability in xenograft models.The subcutaneous tumor volume was measured and the bone destruction was observed by microtomography.It was found that in the bortezomib treated group,the subcutaneous tumor volume was larger and the bone destruction was more serious than that in the control group.4.HRP2 down regulates apoptosis related genes in response to external stress.By RNA sequencing analysis,compared with the control group,HRP2 knockdown in MM cells in response to external stimuli,regulation of reactive oxygen species metabolism process and regulation of endoplasmic reticulum unfolded protein response related gene expression were significantly reduced.5.HRP2 directly regulates the transcription of endoplasmic reticulum stress and apoptosis related genes.Chromatin immunoprecipitation technique was used to analyze the genes directly regulated by HRP2 in MM cells.Combined with the results of RNA seq analysis,the genes down regulated after HRP2 knockdown were analyzed.In vivo enrichment analysis found that some of the genes were enriched in endoplasmic reticulum stress and apoptosis related pathways.6.HRP2 was negatively correlated with the trimethylation of lysine at position 27 of histone H3(H3K27me3).Western blotting results showed that H3K27me3 was significantly increased in HRP2 low expression cells.The level of H3K27me3 was detected by immunofluorescence assay in a pair of cell lines simulating the heterotopic mutation of chromosome 4 and 14 in multiple myeloma.In the cells with translocation of chromosome 4 and 14,the level of H3K27me3 increased significantly after HRP2 knockdown.At the same time,the distribution of H3K27me3 on the target gene was significantly increased after HRP2 knockdown,which was consistent with the change of chromatin immunoprecipitation assay peak.7.HRP2 regulates H3K27me3 level by interacting with MINA.HRP2 interacting protein MINA was identified by immunoprecipitation and mass spectrometry.The drug resistance of HEK293 T cells to bortezomib was significantly increased after MINA knockdown.The results of chromatin immunoprecipitation assay showed that the distribution of MINA on target genes was significantly decreased after HRP2 knockdown,the binding of H3K27me3 was significantly increased.8.Tazemetostat increases the sensitivity of MM cells with low HRP2 expression to bortezomib.The cytotoxicity and apoptosis of MM cells co treated with tazemetostat and bortezomib were detected by cell viability and apoptosis assay.It was found that tazemetostat and bortezomib had synergistic killing effect on MM cells with low expression of HRP2.This result was also verified in animal models.Conclusion 1.The expression level of HRP2 in patients with MM is positively correlated with the clinical treatment effect.The overall survival rate of patients with high expression of HRP2 is higher.2.After HRP2 knockdown,the sensitivity of multiple myeloma cells to bortezomib decreased,the transcriptome changed,the response of cells to external stress decreased significantly,and the drug resistance was more obvious in the cells with chromosome 4 and 14 translocation positive.3.HRP2 can regulate the level of H3K27me3 by directly binding to MINA.Small molecule inhibitors that interfere with the level of H3K27me3 can enhance the killing effect of bortezomib on drug-resistant multiple myeloma cells with low expression of HRP2.