Mechanistic basis and efficacy of targeting the ß-catenin-TCF7L2-JMJD6-c-Myc axis to overcome resistance to BET inhibitors.

The promising activity of BET protein inhibitors (BETi's) is compromised by adaptive or innate resistance in acute myeloid leukemia (AML). Here, modeling of BETi-persister/resistance (BETi-P/R) in human postmyeloproliferative neoplasm (post-MPN) secondary AML (sAML) cells demonstrated accessible and active chromatin in specific superenhancers/enhancers, which was associated with increased levels of nuclear ß-catenin, TCF7L2, JMJD6, and c-Myc in BETi-P/R sAML cells. Following BETi treatment, c-Myc levels were rapidly restored in BETi-P/R sAML cells. CRISPR/Cas9-mediated knockout of TCF7L2 or JMJD6 reversed BETi-P/R, whereas ectopic overexpression conferred BETi-P/R in sAML cells, confirming the mechanistic role of the ß-catenin-TCF7L2-JMJD6-c-Myc axis in BETi resistance. Patient-derived, post-MPN, CD34+ sAML blasts exhibiting relative resistance to BETi, as compared with sensitive sAML blasts, displayed higher messenger RNA and protein expression of TCF7L2, JMJD6, and c-Myc and following BETi washout exhibited rapid restoration of c-Myc and JMJD6. CRISPR/Cas9 knockout of TCF7L2 and JMJD6 depleted their levels, inducing loss of viability of the sAML blasts. Disruption of colocalization of nuclear ß-catenin with TBL1 and TCF7L2 by the small-molecule inhibitor BC2059 combined with depletion of BRD4 by BET proteolysis-targeting chimera reduced c-Myc levels and exerted synergistic lethality in BETi-P/R sAML cells. This combination also reduced leukemia burden and improved survival of mice engrafted with BETi-P/R sAML cells or patient-derived AML blasts innately resistant to BETi. Therefore, multitargeted disruption of the ß-catenin-TCF7L2-JMJD6-c-Myc axis overcomes adaptive and innate BETi resistance, exhibiting preclinical efficacy against human post-MPN sAML cells.

Protein arginine methyltransferase 5 represses tumor suppressor miRNAs that down-regulate CYCLIN D1 and c-MYC expression in aggressive B-cell lymphoma.

Protein arginine methyltransferase-5 (PRMT5) is overexpressed in aggressive B-cell non-Hodgkin's lymphomas, including mantle cell lymphoma and diffuse large B-cell lymphoma, and supports constitutive expression of CYCLIN D1 and c-MYC. Here, we combined ChIP analysis with next-generation sequencing to identify microRNA (miRNA) genes that are targeted by PRMT5 in aggressive lymphoma cell lines. We identified enrichment of histone 3 dimethylation at Arg-8 (H3(Me2)R8) in the promoter regions of miR33b, miR96, and miR503. PRMT5 knockdown de-repressed transcription of all three miRNAs, accompanied by loss of recruitment of epigenetic repressor complexes containing PRMT5 and either histone deacetylase 2 (HDAC2) or HDAC3, enhanced binding of co-activator complexes containing p300 or CREB-binding protein (CBP), and increased acetylation of specific histones, including H2BK12, H3K9, H3K14, and H4K8 at the miRNA promoters. Re-expression of individual miRNAs in B-cell lymphoma cells down-regulated expression of PRMT5, CYCLIN D1, and c-MYC, which are all predicted targets of these miRNAs, and reduced lymphoma cell survival. Luciferase reporter assays with WT and mutant 3'UTRs of CYCLIN D1 and c-MYC mRNAs revealed that binding sites for miR33b, miR96, and miR503 are critical for translational regulation of the transcripts of these two genes. Our findings link altered PRMT5 expression to transcriptional silencing of tumor-suppressing miRNAs in lymphoma cells and reinforce PRMT5's relevance for promoting lymphoma cell growth and survival.

Transducin ß-like protein 1 controls multiple oncogenic networks in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common Non-Hodgkin's lymphoma and is characterized by a remarkable heterogeneity with diverse variants that can be identified histologically and molecularly. Large-scale gene expression profiling studies have identified the germinal center B-cell (GCB-) and activated B-cell (ABC-) subtypes. Standard chemo-immunotherapy remains standard front line therapy, curing approximately two thirds of patients. Patients with refractory disease or those who relapse after salvage treatment have an overall poor prognosis highlighting the need for novel therapeutic strategies. Transducin ß-like protein 1 (TBL1) is an exchange adaptor protein encoded by the TBL1X gene and known to function as a master regulator of the Wnt signalling pathway by binding to ß-CATENIN and promoting its downstream transcriptional program. Here, we show that, unlike normal B-cells, DLBCL cells express abundant levels of TBL1 and its overexpression correlates with poor clinical outcome regardless of DLBCL molecular subtype. Genetic deletion of TBL1 and pharmacological approach using tegavivint, a first-in-class small molecule targeting TBL1 (Iterion Therapeutics), promotes DLBCL cell death in vitro and in vivo. Through an integrated genomic, biochemical, and pharmacologic analyses, we characterized a novel, ß-CATENIN independent, post-transcriptional oncogenic function of TBL1 in DLBCL where TBL1 modulates the stability of key oncogenic proteins such as PLK1, MYC, and the autophagy regulatory protein BECLIN-1 through its interaction with a SKP1-CUL1-F-box (SCF) protein supercomplex. Collectively, our data provide the rationale for targeting TBL1 as a novel therapeutic strategy in DLBCL.

Protein arginine methyltransferase 5 (PRMT5) promotes survival of lymphoma cells via activation of WNT/ß-catenin and AKT/GSK3ß proliferative signaling.

Epigenetic regulation by the type II protein arginine methyltransferase, PRMT5, plays an essential role in the control of cancer cell proliferation and tumorigenesis. In this report, we investigate the relationship between PRMT5 and WNT/ß-CATENIN as well as AKT/GSK3ß proliferative signaling in three different types of non-Hodgkin's lymphoma cell lines, clinical samples, and mouse primary lymphoma cells. We show that PRMT5 stimulates WNT/ß-CATENIN signaling through direct epigenetic silencing of pathway antagonists, AXIN2 and WIF1, and indirect activation of AKT/GSK3ß signaling. PRMT5 inhibition with either shRNA-mediated knockdown or a specific small molecule PRMT5 inhibitor, CMP-5, not only leads to derepression of WNT antagonists and decreased levels of active phospho-AKT (Thr-450 and Ser-473) and inactive phospho-GSK3ß (Ser-9) but also results in decreased transcription of WNT/ß-CATENIN target genes, CYCLIN D1, c-MYC, and SURVIVIN, and enhanced lymphoma cell death. Furthermore, PRMT5 inhibition leads to reduced recruitment of co-activators CBP, p300, and MLL1, as well as enhanced recruitment of co-repressors HDAC2 and LSD1 to the WNT/ß-CATENIN target gene promoters. These results indicate that PRMT5 governs expression of prosurvival genes by promoting WNT/ß-CATENIN and AKT/GSK3ß proliferative signaling and that its inhibition induces lymphoma cell death, which warrants further clinical evaluation.

PRMT5-Selective Inhibitors Suppress Inflammatory T Cell Responses and Experimental Autoimmune Encephalomyelitis.

In the autoimmune disease multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), expansion of pathogenic, myelin-specific Th1 cell populations drives active disease; selectively targeting this process may be the basis for a new therapeutic approach. Previous studies have hinted at a role for protein arginine methylation in immune responses, including T cell-mediated autoimmunity and EAE. However, a conclusive role for the protein arginine methyltransferase (PRMT) enzymes that catalyze these reactions has been lacking. PRMT5 is the main PRMT responsible for symmetric dimethylation of arginine residues of histones and other proteins. PRMT5 drives embryonic development and cancer, but its role in T cells, if any, has not been investigated. In this article, we show that PRMT5 is an important modulator of CD4+ T cell expansion. PRMT5 was transiently upregulated during maximal proliferation of mouse and human memory Th cells. PRMT5 expression was regulated upstream by the NF-κB pathway, and it promoted IL-2 production and proliferation. Blocking PRMT5 with novel, highly selective small molecule PRMT5 inhibitors severely blunted memory Th expansion, with preferential suppression of Th1 cells over Th2 cells. In vivo, PRMT5 blockade efficiently suppressed recall T cell responses and reduced inflammation in delayed-type hypersensitivity and clinical disease in EAE mouse models. These data implicate PRMT5 in the regulation of adaptive memory Th cell responses and suggest that PRMT5 inhibitors may be a novel therapeutic approach for T cell-mediated inflammatory disease.

Selective inhibition of protein arginine methyltransferase 5 blocks initiation and maintenance of B-cell transformation.

Epigenetic events that are essential drivers of lymphocyte transformation remain incompletely characterized. We used models of Epstein-Barr virus (EBV)-induced B-cell transformation to document the relevance of protein arginine methyltransferase 5 (PRMT5) to regulation of epigenetic-repressive marks during lymphomagenesis. EBV(+) lymphomas and transformed cell lines exhibited abundant expression of PRMT5, a type II PRMT enzyme that promotes transcriptional silencing of target genes by methylating arginine residues on histone tails. PRMT5 expression was limited to EBV-transformed cells, not resting or activated B lymphocytes, validating it as an ideal therapeutic target. We developed a first-in-class, small-molecule PRMT5 inhibitor that blocked EBV-driven B-lymphocyte transformation and survival while leaving normal B cells unaffected. Inhibition of PRMT5 led to lost recruitment of a PRMT5/p65/HDAC3-repressive complex on the miR96 promoter, restored miR96 expression, and PRMT5 downregulation. RNA-sequencing and chromatin immunoprecipitation experiments identified several tumor suppressor genes, including the protein tyrosine phosphatase gene PTPROt, which became silenced during EBV-driven B-cell transformation. Enhanced PTPROt expression following PRMT5 inhibition led to dephosphorylation of kinases that regulate B-cell receptor signaling. We conclude that PRMT5 is critical to EBV-driven B-cell transformation and maintenance of the malignant phenotype, and that PRMT5 inhibition shows promise as a novel therapeutic approach for B-cell lymphomas.

Versatility of PRMT5-induced methylation in growth control and development.

Arginine methylation governs important cellular processes that impact growth and proliferation, as well as differentiation and development. Through their ability to catalyze symmetric or asymmetric methylation of histone and non-histone proteins, members of the protein arginine methyltransferase (PRMT) family regulate chromatin structure and expression of a wide spectrum of target genes. Unlike other PRMTs, PRMT5 works in concert with a variety of cellular proteins including ATP-dependent chromatin remodelers and co-repressors to induce epigenetic silencing. Recent work also implicates PRMT5 in the control of growth-promoting and pro-survival pathways, which demonstrates its versatility as an enzyme involved in both epigenetic regulation of anti-cancer target genes and organelle biogenesis. These studies not only provide insight into the molecular mechanisms by which PRMT5 contributes to growth control, but also justify therapeutic targeting of PRMT5.

Protein arginine methyltransferase 5 (PRMT5) inhibition induces lymphoma cell death through reactivation of the retinoblastoma tumor suppressor pathway and polycomb repressor complex 2 (PRC2) silencing.

Epigenetic regulation mediated by lysine- and arginine-specific enzymes plays an essential role in tumorigenesis, and enhanced expression of the type II protein arginine methyltransferase PRMT5 as well as the polycomb repressor complex PRC2 has been associated with increased cell proliferation and survival. Here, we show that PRMT5 is overexpressed in three different types of non-Hodgkin lymphoma cell lines and clinical samples as well as in mouse primary lymphoma cells and that it up-regulates PRC2 expression through inactivation of the retinoblastoma proteins RB1 and RBL2. Although PRMT5 epigenetically controls RBL2 expression, it indirectly promotes RB1 phosphorylation through enhanced cyclin D1 expression. Furthermore, we demonstrate that PRMT5 knockdown in non-Hodgkin lymphoma cell lines and mouse primary lymphoma cells leads to RBL2 derepression and RB1 reactivation, which in turn inhibit PRC2 expression and trigger derepression of its CASP10, DAP1, HOXA5, and HRK pro-apoptotic target genes. We also show that reduced PRMT5 expression leads to cyclin D1 transcriptional repression via loss of TP53K372 methylation, which results in decreased BCL3 expression and enhanced recruitment of NF-κB p52-HDAC1 repressor complexes to the cyclin D1 promoter. These findings indicate that PRMT5 is a master epigenetic regulator that governs expression of its own target genes and those regulated by PRC2 and that its inhibition could offer a promising therapeutic strategy for lymphoma patients.

Protein arginine methyltransferase 7 regulates cellular response to DNA damage by methylating promoter histones H2A and H4 of the polymerase δ catalytic subunit gene, POLD1.

Covalent modification of histones by protein arginine methyltransferases (PRMTs) impacts genome organization and gene expression. In this report, we show that PRMT7 interacts with the BRG1-based hSWI/SNF chromatin remodeling complex and specifically methylates histone H2A Arg-3 (H2AR3) and histone H4 Arg-3 (H4R3). To elucidate the biological function of PRMT7, we knocked down its expression in NIH 3T3 cells and analyzed global gene expression. Our findings show that PRMT7 negatively regulates expression of genes involved in DNA repair, including ALKBH5, APEX2, POLD1, and POLD2. Chromatin immunoprecipitation (ChIP) revealed that PRMT7 and dimethylated H2AR3 and H4R3 are enriched at target DNA repair genes in parental cells, whereas PRMT7 knockdown caused a significant decrease in PRMT7 recruitment and H2AR3/H4R3 methylation. Decreased PRMT7 expression also resulted in derepression of target DNA repair genes and enhanced cell resistance to DNA-damaging agents. Furthermore, we show that BRG1 co-localizes with PRMT7 on target promoters and that expression of a catalytically inactive form of BRG1 results in derepression of PRMT7 target DNA repair genes. Remarkably, reducing expression of individual PRMT7 target DNA repair genes showed that only the catalytic subunit of DNA polymerase, POLD1, was able to resensitize PRMT7 knock-down cells to DNA-damaging agents. These results provide evidence for the important role played by PRMT7 in epigenetic regulation of DNA repair genes and cellular response to DNA damage.

Bromodomain protein 7 interacts with PRMT5 and PRC2, and is involved in transcriptional repression of their target genes.

Histone modification regulates gene expression, and one major regulatory step in this process is the ability of proteins that recognize epigenetic marks to recruit enzymes required to specify transcriptional outcome. Here we show that BRD7 is a component of hSWI-SNF complexes that interacts with PRMT5 and PRC2. Recruitment studies revealed that BRD7 co-localizes with PRMT5 and PRC2 on 'suppressor of tumorigenecity 7' (ST7) and retinoblastoma-like protein 2 (RBL2) promoters in patient-derived B cell lines, and that its association with these target genes correlates with hypermethylation of H3R8, H4R3 and H3K27. Furthermore, inhibition of BRD7 expression reduces PRMT5 and PRC2 recruitment to ST7 and RBL2 promoters; however, only ST7 becomes transcriptionally derepressed. Evaluation of the PRMT5- and PRC2-induced epigenetic marks revealed that while H3(Me(2))R8, H4(Me(2))R3 and H3(Me(3))K27 marks are erased from the ST7 promoter, demethylation of RBL2 promoter histones is incomplete. We also show that the arginine demethylase (RDM) JMJD6, which can erase PRMT5-induced H4R3 methylation, and the H3K27-lysine-specific demethylases, KDM6A/UTX and KDM6B/JMJD3, are differentially recruited to ST7 and RBL2. These findings highlight the role played by BRD7 in PRMT5- and PRC2-induced transcriptional silencing, and indicate that recruitment of specific RDMs and KDMs is required for efficient transcriptional derepression.

Amino acid toxicities of Escherichia coli that are prevented by leucyl-tRNA synthetase amino acid editing.

Leucyl-tRNA synthetase (LeuRS) has evolved an editing function to clear misactivated amino acids. An Escherichia coli-based assay was established to identify amino acids that compromise the fidelity of LeuRS and translation. Multiple nonstandard as well as standard amino acids were toxic to the cell when LeuRS editing was inactivated.

A viable amino acid editing activity in the leucyl-tRNA synthetase CP1-splicing domain is not required in the yeast mitochondria.

Aminoacyl-tRNA synthetases are a family of enzymes that are responsible for translating the genetic code in the first step of protein synthesis. Some aminoacyl-tRNA synthetases have editing activities to clear their mistakes and enhance fidelity. Leucyl-tRNA synthetases have a hydrolytic active site that resides in a discrete amino acid editing domain called CP1. Mutational analysis within yeast mitochondrial leucyl-tRNA synthetase showed that the enzyme has maintained an editing active site that is competent for post-transfer editing of mischarged tRNA similar to other leucyl-tRNA synthetases. These mutations that altered or abolished leucyl-tRNA synthetase editing were introduced into complementation assays. Cell viability and mitochondrial function were largely unaffected in the presence of high levels of non-leucine amino acids. In contrast, these editing-defective mutations limited cell viability in Escherichia coli. It is possible that the yeast mitochondria have evolved to tolerate lower levels of fidelity in protein synthesis or have developed alternate mechanisms to enhance discrimination of leucine from non-cognate amino acids that can be misactivated by leucyl-tRNA synthetase.