Background Emerging evidence has revealed that PKM2 has oncogenic functions independent of its canonical pyruvate kinase activity, serving as a protein kinase that regulates gene expression. However, the mechanism by which PKM2, as a histone kinase, regulates the transcription of genes involved in triple-negative breast cancer (TNBC) metastasis remains poorly understood. Methods We integrated cellular analysis, including cell viability, proliferation, colony formation, and migration assays; biochemical assays, including protein interaction studies and ChIP; clinical sample analysis; RNA-Seq and CUT&Tag data; and xenograft or mammary-specific gene knockout mouse models, to investigate the epigenetic modulation of TNBC metastasis via NONO-dependent interactions with nuclear PKM2. Results We report that the transcription factor NONO directly interacts with nuclear PKM2 and directs PKM2-mediated phosphorylation of histone H3 at threonine 11 (H3T11ph) to promote TNBC metastasis. We show that H3T11ph cooperates with TIP60-mediated acetylation of histone H3 at lysine 27 (H3K27ac) to activate SERPINE1 expression and to increase the proliferative, migratory, and invasive abilities of TNBC cells in a NONO-dependent manner. Conditional mammary loss of NONO or PKM2 markedly suppressed SERPINE1 expression and attenuated the malignant progression of spontaneous mammary tumors in mice. Importantly, elevated expression of NONO or PKM2 in TNBC patients is positively correlated with SERPINE1 expression, enhanced invasiveness, and poor clinical outcomes. Conclusion These findings revealed that the NONO-dependent interaction with nuclear PKM2 is key for the epigenetic modulation of TNBC metastasis, suggesting a novel intervention strategy for treating TNBC.

Renal cell carcinoma (RCC) is a frequent malignancy of the urinary system with high mortality and morbidity. However, the molecular mechanisms underlying RCC progression are still largely unknown. In this study, we identified FOXA2, a pioneer transcription factor, as a driver oncogene for RCC. We show that FOXA2 was commonly upregulated in human RCC samples and promoted RCC proliferation, as evidenced by assays of cell viability, colony formation, migratory and invasive capabilities, and stemness properties. Mechanistically, we found that FOXA2 promoted RCC cell proliferation by transcriptionally activating HIF2 alpha expression in vitro and in vivo. Furthermore, we found that FOXA2 could interact with VHL (von Hippel-Lindau), which ubiquitinated FOXA2 and controlled its protein stability in RCC cells. We showed that mutation of lysine at position 264 to arginine in FOXA2 could mostly abrogate its ubiquitination, augment its activation effect on HIF2 alpha expression, and promote RCC proliferation in vitro and RCC progression in vivo. Importantly, elevated expression of FOXA2 in patients with RCC positively correlated with the expression of HIF2 alpha and was associated with shorter overall and disease-free survival. Together, these fi ndings reveal a novel role of FOXA2 in RCC development and provide insights into the underlying molecular mechanisms of FOXA2-driven pathological processes in RCC.

Decoding the molecular mechanisms underlying human fetal (γ) globin gene silencing impacts therapeutic strategies for β-thalassemia and sickle cell disease. Here, we identified a nucleolar protein, fibrillarin (FBL), which mediates the methylation of glutamine104 in histone H2A and functions as a repressor of the γ-globin gene in cultured erythroid cells, including those from β-thalassemia patients. Conditional Fbl depletion in adult β-YAC transgenic mice or in βIVS-2-654-thalassemic mice reactivated the human γ-globin gene or murine embryonic globin expression, respectively, which corrects hematologic and pathologic defects in β-thalassemic mice. We showed that FBL plays a dual role in activating BCL11A expression and repressing γ-globin gene expression, which is dependent on its histone methyltransferase activity. Our study may provide an alternative strategy for therapeutic targeted treatment of β-hemoglobinopathies. ### Competing Interest Statement The authors have declared no competing interest.

Genomic studies have demonstrated a high frequency of genetic alterations in components of the SWI/SNF complex including the core subunit SMARCA4. However, the mechanisms of tumorigenesis driven by SMARCA4 mutations, particularly in colorectal cancer (CRC), remain largely unknown. In this study, we identified a specific, hotspot mutation in SMARCA4 (c. 3721C>T) which results in a conversion from arginine to tryptophan at residue 1157 (R1157W) in human CRC tissues associated with higher-grade tumors and controls CRC progression. Mechanistically, we found that the SMARCA4 R1157W mutation facilitated its recruitment to PRMT1-mediated H4R3me2a (asymmetric dimethylation of Arg 3 in histone H4) and enhanced the ATPase activity of SWI/SNF complex to remodel chromatin in CRC cells. We further showed that the SMARCA4 R1157W mutant reinforced the transcriptional expression of EGFR and TNS4 to promote the proliferation of CRC cells and patient-derived tumor organoids. Importantly, we demonstrated that SMARCA4 R1157W CRC cells and mutant cell-derived xenografts were more sensitive to the combined inhibition of PRMT1 and SMARCA4 which act synergistically to suppress cell proliferation. Together, our findings show that SMARCA4-R1157W is a critical activating mutation, which accelerates CRC progression through facilitating chromatin recruitment and remodeling. Our results suggest a potential precision therapeutic strategy for the treatment of CRC patients carrying the SMARCA4 R1157W mutation.

Abstract Background: Genomic studies have demonstrated a high frequency of genetic alterations in components of the SWI/SNF complex including the core subunit SMARCA4. However, the mechanisms of tumorigenesis driven by SMARCA4 mutations, particularly in colorectal cancer (CRC), remain largely unknown. Methods: To reveal the role of the SMARCA4R1157W mutation in CRC cells and its consequences on CRC progression, we used multiple approaches and techniques. These include DNA sequencing, the CRISPR/Cas9 genome editing system, biochemical assays including protein interaction studies, chromatin immunoprecipitation (ChIP) and chromatin remodeling assays, cellular analysis including cell proliferation, colony formation, migration assays and culture of CRC patient-derived organoids, clinical sample analysis and treatment of cell line-derived xenograft (CDX) mouse models. Results: We identified a specific, hotspot mutation in SMARCA4 (c. 3721 C>T) which results in conversion from arginine to tryptophan at residue 1157 (R1157W) in human CRC tissues associated with higher grade tumors and controls CRC progression. Mechanistically, we found that the SMARCA4R1157W mutation facilitated its recruitment to PRMT1-mediated H4R3me2a (asymmetric dimethylation of Arg 3 in histone H4) and enhanced the ATPase activity of SWI/SNF complex to remodel chromatin in CRC cells. We further showed that the SMARCA4R1157W mutant reinforced the transcriptional expression of EGFR and TNS4 to promote proliferation of CRC cells and patient-derived tumor organoids. Importantly, we demonstrated that SMARCA4R1157W CRC cells and mutant cell-derived xenografts were more sensitive to combined inhibition of PRMT1 and SMARCA4 which act synergistically to suppress cell proliferation. Conclusions: Together, our findings show that SMARCA4-R1157W is a novel gain-of-function mutation, which accelerates CRC progression through epigenetic reprograming of EGFR signaling. Our results suggest a novel therapeutic strategy for the treatment of CRC patients carrying the SMARCA4R1157W mutation.

Background Aberrant changes in epigenetic mechanisms such as histone modifications play an important role in cancer progression. PRMT1 which triggers asymmetric dimethylation of histone H4 on arginine 3 (H4R3me2a) is upregulated in human colorectal cancer (CRC) and is essential for cell proliferation. However, how this dysregulated modification might contribute to malignant transitions of CRC remains poorly understood. Methods In this study, we integrated biochemical assays including protein interaction studies and chromatin immunoprecipitation (ChIP), cellular analysis including cell viability, proliferation, colony formation, and migration assays, clinical sample analysis, microarray experiments, and ChIP-Seq data to investigate the potential genomic recognition pattern of H4R3me2s in CRC cells and its effect on CRC progression. Results We show that PRMT1 and SMARCA4, an ATPase subunit of the SWI/SNF chromatin remodeling complex, act cooperatively to promote colorectal cancer (CRC) progression. We find that SMARCA4 is a novel effector molecule of PRMT1-mediated H4R3me2a. Mechanistically, we show that H4R3me2a directly recruited SMARCA4 to promote the proliferative, colony-formative, and migratory abilities of CRC cells by enhancing EGFR signaling. We found that EGFR and TNS4 were major direct downstream transcriptional targets of PRMT1 and SMARCA4 in colon cells, and acted in a PRMT1 methyltransferase activity-dependent manner to promote CRC cell proliferation. In vivo, knockdown or inhibition of PRMT1 profoundly attenuated the growth of CRC cells in the C57BL/6 J-Apc Min/+ CRC mice model. Importantly, elevated expression of PRMT1 or SMARCA4 in CRC patients were positively correlated with expression of EGFR and TNS4, and CRC patients had shorter overall survival. These findings reveal a critical interplay between epigenetic and transcriptional control during CRC progression, suggesting that SMARCA4 is a novel key epigenetic modulator of CRC. Our findings thus highlight PRMT1/SMARCA4 inhibition as a potential therapeutic intervention strategy for CRC. Conclusion PRMT1-mediated H4R3me2a recruits SMARCA4, which promotes colorectal cancer progression by enhancing EGFR signaling.

Endometrial cancer (EC) is the most common gynecological malignancy worldwide. However, the molecular mechanisms underlying EC progression are still largely unknown, and chemotherapeutic options for EC patients are currently very limited. In this study, we found that histone methyltransferase EZH2 and DNA methyltransferase DNMT3B were upregulated in EC samples from patients, and promoted EC cell proliferation as evidenced by assays of cell viability, cell cycle, colony formation. Mechanistically, we found that EZH2 promoted EC cell proliferation by epigenetically repressing TCF3, a direct transcriptional activator of CCKN1A (p21WAF1/Cip1), in vitro and in vivo. In addition, we found that DNMT3B specifically methylated the TCF3 promoter, repressing TCF3 expression and accelerating EC cell proliferation independently of EZH2. Importantly, elevated expression of EZH2 or DNMT3B in EC patients inversely correlated with expression of TCF3 and p21, and was associated with shorter overall survival. We show that combined treatment with GSK126 and 5-Aza-2d treatment wit synergistically inhibited methyltransferase activity of EZH2 and DNMT3B, resulting in a profound block of EC cell proliferation as well as EC tumor progression in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models. These findings reveal that TCF3 functions as a tumor suppressor epigenetically silenced by EZH2 and DNMT3B in EC, and support the notion that targeting the EZH2/DNMT3B/TCF3/p21 axis may be a novel and effective therapeutic strategy for treatment of EC.

The proto-oncogene c-Myc regulates multiple biological processes mainly through selectively activating gene expression. However, the mechanisms underlying c-Myc-mediated gene repression in the context of cancer remain less clear. This study aimed to clarify the role of PRMT5 in the transcriptional repression of c-Myc target genes in gastric cancer. Methods: Immunohistochemistry was used to evaluate the expression of PRMT5, c-Myc and target genes in gastric cancer patients. PRMT5 and c-Myc interaction was assessed by immunofluorescence, co-immunoprecipitation and GST pull-down assays. Bioinformatics analysis, immunoblotting, real-time PCR, chromatin immunoprecipitation, and rescue experiments were used to evaluate the mechanism. Results: We found that c-Myc directly interacts with protein arginine methyltransferase 5 (PRMT5) to transcriptionally repress the expression of a cohort of genes, including PTEN, CDKN2C (p18INK4C), CDKN1A (p21CIP1/WAF1), CDKN1C (p57KIP2) and p63, to promote gastric cancer cell growth. Specifically, we found that PRMT5 was required to promote gastric cancer cell growth in vitro and in vivo, and for transcriptional repression of this cohort of genes, which was dependent on its methyltransferase activity. Consistently, the promoters of this gene cohort were enriched for both PRMT5-mediated symmetric di-methylation of histone H4 on Arg 3 (H4R3me2s) and c-Myc, and c-Myc depletion also upregulated their expression. H4R3me2s also colocalized with the c-Myc-binding E-box motif (CANNTG) on these genes. We show that PRMT5 directly binds to c-Myc, and this binding is required for transcriptional repression of the target genes. Both c-Myc and PRMT5 expression levels were upregulated in primary human gastric cancer tissues, and their expression levels inversely correlated with clinical outcomes. Conclusions: Taken together, our study reveals a novel mechanism by which PRMT5-dependent transcriptional repression of c-Myc target genes is required for gastric cancer progression, and provides a potential new strategy for therapeutic targeting of gastric cancer.

Aurora kinase B (AURKB) triggers the phosphorylation of serine 10 on histone H3 (H3S10ph), which is important for chromosome condensation and cytokinesis during mitosis in mammals. However, how exactly AURKB controls cell cycle and contributes to tumorigenesis as an oncoprotein under pathological conditions remains largely unknown. Here, we report that AURKB promotes gastric cancer cell proliferation in vitro and in vivo. Silencing AURKB expression inhibits gastric cell proliferation and arrests the cell cycle in G2/M phase. We demonstrate that cyclin D1 (CCND1) is a direct downstream target of AURKB that plays a key role in gastric cancer cell proliferation. AURKB is able to activate the expression of CCND1 through mediating H3S10ph in the promoter of the CCND1 gene. Furthermore, we show that AZD1152, a specific inhibitor of AURKB, can suppress the expression of CCND1 in the gastric cancer cells and inhibit cell proliferation in vitro and in vivo. Importantly, we found that high AURKB and CCND1 expression levels are correlated with shorter overall survival of gastric cancer patients. This study demonstrates that AURKB promotes gastric tumorigenesis potentially through epigenetically activating CCND1 expression, suggesting AURKB as a promising therapeutic target in gastric cancer.