Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

Expanded profiling of WD repeat domain 5 inhibitors reveals actionable strategies for the treatment of hematologic malignancies.

WD40 Repeat Domain 5 (WDR5) is a highly conserved nuclear protein that recruits MYC oncoprotein transcription factors to chromatin to stimulate ribosomal protein gene expression. WDR5 is tethered to chromatin via an arginine-binding cavity known as the "WIN" site. Multiple pharmacological inhibitors of the WDR5-interaction site of WDR5 (WINi) have been described, including those with picomolar affinity and oral bioavailability in mice. Thus far, however, WINi have only been shown to be effective against a number of rare cancer types retaining wild-type p53. To explore the full potential of WINi for cancer therapy, we systematically profiled WINi across a panel of cancer cells, alone and in combination with other agents. We report that WINi are unexpectedly active against cells derived from both solid and blood-borne cancers, including those with mutant p53. Among hematologic malignancies, we find that WINi are effective as a single agent against leukemia and diffuse large B cell lymphoma xenograft models, and can be combined with the approved drug venetoclax to suppress disseminated acute myeloid leukemia in vivo. These studies reveal actionable strategies for the application of WINi to treat blood-borne cancers and forecast expanded utility of WINi against other cancer types.

Structure-based discovery of potent WD repeat domain 5 inhibitors that demonstrate efficacy and safety in preclinical animal models.

WD repeat domain 5 (WDR5) is a core scaffolding component of many multiprotein complexes that perform a variety of critical chromatin-centric processes in the nucleus. WDR5 is a component of the mixed lineage leukemia MLL/SET complex and localizes MYC to chromatin at tumor-critical target genes. As a part of these complexes, WDR5 plays a role in sustaining oncogenesis in a variety of human cancers that are often associated with poor prognoses. Thus, WDR5 has been recognized as an attractive therapeutic target for treating both solid and hematological tumors. Previously, small-molecule inhibitors of the WDR5-interaction (WIN) site and WDR5 degraders have demonstrated robust in vitro cellular efficacy in cancer cell lines and established the therapeutic potential of WDR5. However, these agents have not demonstrated significant in vivo efficacy at pharmacologically relevant doses by oral administration in animal disease models. We have discovered WDR5 WIN-site inhibitors that feature bicyclic heteroaryl P7 units through structure-based design and address the limitations of our previous series of small-molecule inhibitors. Importantly, our lead compounds exhibit enhanced on-target potency, excellent oral pharmacokinetic (PK) profiles, and potent dose-dependent in vivo efficacy in a mouse MV4:11 subcutaneous xenograft model by oral dosing. Furthermore, these in vivo probes show excellent tolerability under a repeated high-dose regimen in rodents to demonstrate the safety of the WDR5 WIN-site inhibition mechanism. Collectively, our results provide strong support for WDR5 WIN-site inhibitors to be utilized as potential anticancer therapeutics.

Structural studies of WDR5 in complex with MBD3C WIN motif reveal a unique binding mode.

The nucleosome remodeling and deacetylase (NuRD) complex plays a pivotal role in chromatin regulation and transcriptional repression. In mice, methyl-CpG binding domain 3 isoform C (MBD3C) interacts specifically with the histone H3 binding protein WD repeat-containing protein 5 (WDR5) and forms the WDR5-MBD3C/Norde complex. Despite the functional significance of this interaction on embryonic stem cell gene regulation, the molecular mechanism underlying MBD3C recognition by WDR5 remains elusive. Here, we determined the crystal structure of WDR5 in complex with the peptide (residues 40-51) derived from the MBD3C protein at a resolution of 1.9 Å. Structural analysis revealed that MBD3C utilizes a unique binding mode to interact with WDR5, wherein MBD3C Arg43 and Phe47 are involved in recognizing the WDR5-interacting (WIN) site and Tyr191-related B site on the small surface of WDR5, respectively. Notably, the binding induces a ∼91° rotation of WDR5 Tyr191, generating the hydrophobic B site. Furthermore, mutation experiments combined with isothermal titration calorimetry (ITC) assays confirmed the importance of both Arg43 and Phe47 in mediating WDR5 binding affinity. By determining structures of various peptides bound to WDR5, we demonstrated that the WDR5 WIN site and B site can be concurrently recognized by WIN motif peptides containing ''Arg-Cies/Ser-Arg-Val-Phe'' consensus sequence. Overall, this study reveals the structural basis for the formation of the WDR5-MBD3C subcomplex and provides new insights into the recognition mode of WDR5 for the WIN motif. Moreover, these findings shed light on structural-based designs of WDR5-targeted anti-cancer small molecule inhibitors or peptide-mimic drugs.

Unraveling MLL1-fusion leukemia: Epigenetic revelations from an iPS cell point mutation.

Our understanding of acute leukemia pathology is heavily dependent on 11q23 chromosomal translocations involving the mixed lineage leukemia-1 (MLL1) gene, a key player in histone H3 lysine 4 (H3K4) methylation. These translocations result in MLL1-fusion (MLL1F) proteins that are thought to drive leukemogenesis. However, the mechanism behind increased H3K4 trimethylation in MLL1F-leukemic stem cells (MLL1F-LSCs), following loss of the catalytic SET domain of MLL1 (known for H3K4 monomethylation and dimethylation) remains unclear. In our investigation, we introduced a homozygous loss-of-function point mutation in MLL1 within human-induced pluripotent stem cells. This mutation mimics the histone methylation, gene expression, and epithelial-mesenchymal transition phenotypes of MLL1F-LSCs-without requiring a translocation or functional WT MLL1. The mutation caused a genome-wide redistribution of the H3K4 trimethyl mark and upregulated LSC-maintenance genes like HoxA9-A13, Meis1, and the HOTTIP long noncoding RNA. Epithelial-mesenchymal transition markers such as ZEB1, SNAI2, and HIC-5 were also increased leading to enhanced cellular migration and invasiveness. These observations underscore the essential role of MLL1's enzymatic activity in restraining the cascade of epigenetic changes associated with the gene-activating H3K4 trimethylation mark, which we show may be catalyzed by mislocalized SETd1a H3K4 trimethyltransferase in the absence of MLL1's enzymatic activity. Challenging existing models, our findings imply that MLL1F-induced leukemias arise from a dominant-negative impact on MLL1's histone methyltransferase activity. We propose targeting SETd1a in precision medicine as a new therapeutic approach for MLL1-associated leukemias.

Host WD repeat-containing protein 5 inhibits protein kinase R-mediated integrated stress response during measles virus infection.

Some negative-sense RNA viruses, including measles virus (MeV), share the characteristic that during their infection cycle, cytoplasmic inclusion bodies (IBs) are formed where components of the viral replication machinery are concentrated. As a foci of viral replication, how IBs act to enhance the efficiency of infection by affecting virus-host interactions remains an important topic of investigation. We previously established that upon MeV infection, the epigenetic host protein, WD repeat-containing protein 5 (WDR5), translocates to cytoplasmic viral IBs and facilitates MeV replication. We now show that WDR5 is recruited to IBs by forming a complex with IB-associated MeV phosphoprotein via a conserved binding motif located on the surface of WDR5. Furthermore, we provide evidence that WDR5 promotes viral replication by suppressing a major innate immune response pathway, the double-stranded RNA-mediated activation of protein kinase R and integrated stress response. IMPORTANCE: MeV is a pathogen that remains a global concern, with an estimated 9 million measles cases and 128,000 measles deaths in 2022 according to the World Health Organization. A large population of the world still has inadequate access to the effective vaccine against the exceptionally transmissible MeV. Measles disease is characterized by a high morbidity in children and in immunocompromised individuals. An important area of research for negative-sense RNA viruses, including MeV, is the characterization of the complex interactome between virus and host occurring at cytoplasmic IBs where viral replication occurs. Despite the progress made in understanding IB structures, little is known regarding the virus-host interactions within IBs and the role of these interactions in promoting viral replication and antagonizing host innate immunity. Herein we provide evidence suggesting a model by which MeV IBs utilize the host protein WDR5 to suppress the protein kinase R-integrated stress response pathway.

Highly expressed B3GALT5-AS1 contributes to gastric cancer progression by recruiting WDR5 to mediate B3GALT5 and regulating ß-catenin/ZEB1 axis.

Long non-coding RNAs (lncRNAs) play an important role in the progression of gastric cancer (GC), but its specific regulatory mechanism remains to be further studied. We previously identified that lncRNA B3GALT5-AS1 was upregulated in GC serum. Here, we investigated the functions and molecular mechanisms of B3GALT5-AS1 in GC tumorigenesis. qRT-PCR was used to detect B3GALT5-AS1 expression in GC. EdU, CCK-8, and colony assays were utilized to assess the proliferation ability of B3GAL5-AS1, and transwell, tube formation assay were used to assess the invasion and metastasis ability. Mechanically, FISH and nuclear plasmolysis PCR identified the subcellular localization of B3GALT5-AS1. RIP and CHIP assays were used to analyse the regulation of B3GALT5-AS1 and B3GALT5. We observed that B3GALT5-AS1 was highly expressed in GC, and silencing B3GALT5-AS1 could inhibit the proliferation, invasion, and migratory capacities of GC. Additionally, B3GALT5-AS1 was bound to WDR5 and modulated the expression of B3GALT5 via regulating the ZEB1/ß-catenin pathway. High-expressed B3AGLT5-AS1 promoted GC tumorigenesis and regulated B3GALT5 expression via recruiting WDR5. Our study is expected to provide a new idea for clinical diagnosis and treatment.

Mitotic gene regulation by the N-MYC-WDR5-PDPK1 nexus.

BACKGROUND: During mitosis the cell depends on proper attachment and segregation of replicated chromosomes to generate two identical progeny. In cancers defined by overexpression or dysregulation of the MYC oncogene this process becomes impaired, leading to genomic instability and tumor evolution. Recently it was discovered that the chromatin regulator WDR5-a critical MYC cofactor-regulates expression of genes needed in mitosis through a direct interaction with the master kinase PDPK1. However, whether PDPK1 and WDR5 contribute to similar mitotic gene regulation in MYC-overexpressing cancers remains unclear. Therefore, to characterize the influence of WDR5 and PDPK1 on mitotic gene expression in cells with high MYC levels, we performed a comparative transcriptomic analysis in neuroblastoma cell lines defined by MYCN-amplification, which results in high cellular levels of the N-MYC protein. RESULTS: Using RNA-seq analysis, we identify the genes regulated by N-MYC and PDPK1 in multiple engineered CHP-134 neuroblastoma cell lines and compare them to previously published gene expression data collected in CHP-134 cells following inhibition of WDR5. We find that as expected N-MYC regulates a multitude of genes, including those related to mitosis, but that PDPK1 regulates specific sets of genes involved in development, signaling, and mitosis. Analysis of N-MYC- and PDPK1-regulated genes reveals a small group of commonly controlled genes associated with spindle pole formation and chromosome segregation, which overlap with genes that are also regulated by WDR5. We also find that N-MYC physically interacts with PDPK1 through the WDR5-PDPK1 interaction suggesting regulation of mitotic gene expression may be achieved through a N-MYC-WDR5-PDPK1 nexus. CONCLUSIONS: Overall, we identify a small group of genes highly enriched within functional gene categories related to mitotic processes that are commonly regulated by N-MYC, WDR5, and PDPK1 and suggest that a tripartite interaction between the three regulators may be responsible for setting the level of mitotic gene regulation in N-MYC amplified cell lines. This study provides a foundation for future studies to determine the exact mechanism by which N-MYC, WDR5, and PDPK1 converge on cell cycle related processes.

The Wdr5-H3K4me3 Epigenetic Axis Regulates Pancreatic Tumor Immunogenicity and Immune Suppression.

The WDR5/MLL1-H3K4me3 epigenetic axis is often activated in both tumor cells and tumor-infiltrating immune cells to drive various cellular responses in the tumor microenvironment and has been extensively studied in hematopoietic cancer, but its respective functions in tumor cells and immune cells in the context of tumor growth regulation of solid tumor is still incompletely understood. We report here that WDR5 exhibits a higher expression level in human pancreatic tumor tissues compared with adjacent normal pancreas. Moreover, WDR5 expression is negatively correlated with patients' response to chemotherapy or immunotherapy in human colon cancer and melanoma. However, WDR5 expression is positively correlated with the HLA level in human cancer cells, and H3K4me3 enrichment is observed at the promoter region of the HLA-A, HLA-B, and HLA-C genes in pancreatic cancer cells. Using mouse tumor cell lines and in vivo tumor models, we determined that WDR5 deficiency or inhibition significantly represses MHC I expression in vitro and in vivo in pancreatic tumor cells. Mechanistically, we determine that WDR5 deficiency inhibits H3K4me3 deposition at the MHC I (H2K) promoter region to repress MHC I (H2K) transcription. On the other hand, WDR5 depletion leads to the effective downregulation of immune checkpoints and immunosuppressive cytokines, including TGFß and IL6, in the pancreatic tumor microenvironments. Our data determine that WDR5 not only regulates tumor cell immunogenicity to suppress tumor growth but also activates immune suppressive pathways to promote tumor immune evasion. Selective activation of the WDR5-MHC I pathway and/or selective inhibition of the WDR5-immune checkpoint and WDR5-cytokine pathways should be considered in WDR5-based epigenetic cancer immunotherapy.

The SCF-FBXW7 E3 ubiquitin ligase triggers degradation of histone 3 lysine 4 methyltransferase complex component WDR5 to prevent mitotic slippage.

During prolonged mitotic arrest induced by antimicrotubule drugs, cell fate decision is determined by two alternative pathways, one leading to cell death and the other inducing premature escape from mitosis by mitotic slippage. FBWX7, a member of the F-box family of proteins and substrate-targeting subunit of the SKP1-CUL1-F-Box E3 ubiquitin ligase complex, promotes mitotic cell death and prevents mitotic slippage, but molecular details underlying these roles for FBWX7 are unclear. In this study, we report that WDR5 (WD-repeat containing protein 5), a component of the mixed lineage leukemia complex of histone 3 lysine 4 methyltransferases, is a substrate of FBXW7. We determined by coimmunoprecipitation experiments and in vitro binding assays that WDR5 interacts with FBXW7 in vivo and in vitro. SKP1-CUL1-F-Box-FBXW7 mediates ubiquitination of WDR5 and targets it for proteasomal degradation. Furthermore, we find that WDR5 depletion counteracts FBXW7 loss of function by reducing mitotic slippage and polyploidization. In conclusion, our data elucidate a new mechanism in mitotic cell fate regulation, which might contribute to prevent chemotherapy resistance in patients after antimicrotubule drug treatment.

LncRNA AC142119.1 facilitates the progression of neuroblastoma by epigenetically initiating the transcription of MYCN.

BACKGROUND: Oncogene MYCN is closely related with malignant progression and poor prognosis of neuroblastoma (NB). Recently, long non-coding RNAs (lncRNAs) have been recognized as crucial regulators in various cancers. However, whether lncRNAs contribute to the overexpression of MYCN in NB is unclear. METHODS: Microarray analysis were applied to analyze the differentially expressed lncRNAs between MYCN-amplified and MYCN-non-amplified NB cell lines. Bioinformatic analyses were utilized to identify lncRNAs nearby MYCN locus. qRT-PCR was used to detect the expression level of lncRNA AC142119.1 in NB cell lines and tissues. Gain- and loss-of-function assays were conducted to investigate the biological effect of AC142119.1 in NB. Fluorescence in situ hybridization, RNA pull-down, RNA immunoprecipitation, mass spectrometry, RNA electrophoretic mobility shift, chromatin immunoprecipitation and chromatin isolation by RNA purification assays were performed to validate the interaction between AC142119.1 and WDR5 protein as well as MYCN promoter. RESULTS: AC142119.1 was significantly elevated in NB tissues with MYCN amplification, advanced INSS stage and high risk, and associated with poor survival of NB patients. Moreover, enforced expression of AC142119.1 reinforced the proliferation of NB cells in vitro and in vivo. Additionally, AC142119.1 specifically recruited WDR5 protein to interact with MYCN promoter, further initiating the transcription of MYCN and accelerating NB progression. CONCLUSIONS: We identified a novel lncRNA AC142119.1, which promoted the progression of NB through epigenetically initiating the transcription of MYCN via interacting with both WDR5 protein and the promoter of MYCN, indicating that AC142119.1 might be a potential diagnostic biomarker and therapeutic target for NB.

Lnc-17Rik promotes the immunosuppressive function of Myeloid-Derived suppressive cells in esophageal cancer.

Myeloid-derived suppressor cells (MDSCs) are a population of immature bone marrow cells that accumulate in large numbers in the spleen, peripheral blood, bone marrow, lymph nodes, and local and metastatic foci of tumors. C/EBP homologous protein (CHOP) and CCAAT/enhancer binding protein ß (C/EBPß) play key roles in regulating the immunosuppressive function and differentiation of MDSCs. Our study revealed that the long noncoding RNA Lnc-17Rik was able to promote immunosuppression in tumors by facilitating the activation and expression of key genes involved in MDSC differentiation. Lnc-17Rik was shown to directly interact with CHOP and C/EBPß LIP to facilitate their dissociation from the transcriptional repressor complex involving C/EBP LAP/LIP/CHOP. Moreover, Lnc-17Rik increased the association of WD repeat-containing protein 5 (WDR5) with C/EBP LAP, promoting H3K4me3 enrichment in the promoter regions of arginase 1 (Arg-1), cyclooxygenase 2 (COX2), nitric oxide synthase 2 (NOS2) and NADPH oxidase 2 (NOX2) to enhance the expression of these genes. Furthermore, using a CD45 chimeric model we confirmed that Lnc-17Rik promoted the differentiation of monocytic (M)-MDSCs in vivo with the introduction of Lnc-17Rik-overexpressing MDSCs shown to promote tumor growth as a result of enhancing their immunosuppressive function. Notably, human Lnc-17Rik is highly homologous to mouse Lnc-17Rik and fulfills similar functions in human MDSC-like cells. In addition, we also found a high level of Lnc-17Rik in peripheral blood MDSC of patients with esophageal cancer. These findings suggest that Lnc-17Rik plays an important role in controlling the immunosuppressive function of MDSCs in the tumor environment and may further serve as a potential therapeutic target for patients with esophageal cancer.

Discovery, evaluation and mechanism study of WDR5-targeted small molecular inhibitors for neuroblastoma.

Neuroblastoma is the most common and deadliest tumor in infancy. WDR5 (WD Repeat Domain 5), a critical factor supporting an N-myc transcriptional complex via its WBM site and interacting with chromosome via its WIN site, promotes the progression of neuroblastoma, thus making it a potential anti-neuroblastoma drug target. So far, a few WIN site inhibitors have been reported, and the WBM site disruptors are rare to see. In this study we conducted virtual screening to identify candidate hit compounds targeting the WBM site of WDR5. As a result, 60 compounds were selected as candidate WBM site inhibitors. Cell proliferation assay demonstrated 6 structurally distinct WBM site inhibitors, numbering as compounds 4, 7, 11, 13, 19 and 22, which potently suppressed 3 neuroblastoma cell lines (MYCN-amplified IMR32 and LAN5 cell lines, and MYCN-unamplified SK-N-AS cell line). Among them, compound 19 suppressed the proliferation of IMR32 and LAN5 cells with EC50 values of 12.34 and 14.89 µM, respectively, and exerted a moderate inhibition on SK-N-AS cells, without affecting HEK293T cells at 20 µM. Analysis of high-resolution crystal complex structure of compound 19 against WDR5 revealed that it competitively occupied the hydrophobic pocket where V264 was located, which might disrupt the interaction of MYC with WDR5 and further MYC-medicated gene transcription. By performing RNA-seq analysis we demonstrated the differences in molecular action mechanisms of the compound 19 and a WIN site inhibitor OICR-9429. Most interestingly, we established the particularly high synergy rate by combining WBM site inhibitor 19 and the WIN site inhibitor OICR-9429, providing a novel therapeutic avenue for neuroblastoma.

Intermittent hypoxia BMSCs-derived exosomal miR-31-5p promotes lung adenocarcinoma development via WDR5-induced epithelial mesenchymal transition.

BACKGROUND: Intermittent hypoxia (IH) is a factor involved in the incidence and progression of lung adenocarcinoma (LUAD). Bone marrow-derived bone mesenchymal stem cells (BMSCs)-derived exosomes are related to the promotion of tumor development. The objective of this experiment was to clarify the mechanism of exosomes from BMSCs in promoting the progression of LUAD induced by IH. METHODS: This study examined if IH BMSCS-derived exosomes affect the malignancy of LUAD cells in vitro. Dual-luciferase assays were conducted to confirm the target of miR-31-5p with WD repeat domain 5 (WDR5). We further investigated whether or not  exosomal miR-31-5p or WDR5 could regulate epithelial-mesenchymal transition (EMT). We determined the effect of IH exosomes using a tumorigenesis model in vivo. RESULTS: miR-31-5p entered into LUAD cells via exosomes. MiR-31-5p was greatly upregulated in IH BMSCs-derived exosomes compared with RA exosomes. Increased expression of exosomal miR-31-5p induced by IH was discovered to target WDR5 directly, increased activation of WDR5, and significantly facilitated EMT, thereby promoting LUAD progression. CONCLUSIONS: The promoting effect of IH on LUAD is achieved partly through BMSCs-derived exosomal miR-31-5p triggering WDR5 and promoting EMT.

TM4SF19-AS1 facilitates the proliferation of lung squamous cell carcinoma by recruiting WDR5 to mediate TM4SF19.

BACKGROUND: As reported, long non-coding RNAs are a pivotal player in lung squamous cell carcinoma (LSCC) progression. We noticed the remarkably upregulated transmembrane-4-l-six-family-19 antisense RNA 1 (TM4SF19-AS1) in LSCC and further demonstrated the function it played in LSCC and the possible molecular mechanism. METHODS: Via bioinformatics approach, we evaluated TM4SF19-AS1 and TM4SF19 levels in LSCC tissue, and real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot revealed their mRNA and protein levels in LSCC cells. Cell Counting Kit-8 and colony formation assays analyzed the proliferation ability of LSCC cells, and cell adhesion ability was detected via cell adhesion assay. RNA immunoprecipitation and chromatin immunoprecipitation analyzed the underlying mechanism of TM4SF19-AS1 regulating its target, while methylation-specific PCR indicated the methylation level of TM4SF19-AS1. RESULTS: TM4SF19-AS1 was markedly upregulated in LSCC. Functional assays revealed that TM4SF19-AS1 could facilitate the proliferation and adhesion of LSCC. Besides, we revealed the mechanism of TM4SF19-AS1 regulation that it directly bound to WD repeat-containing protein 5 (WDR5), and was then recruited to TM4SF19 promoter region, which activated DNA demethylation, thereby suppressing malignant LSCC progression. CONCLUSION: Our research demonstrated that TM4SF19-AS1 affected LSCC cell proliferation by recruiting WDR5 to manipulate transmembrane-4-lsix-family-member-19 (TM4SF19), which offers a new observation on LSCC pathogenesis, indicating that TM4SF19-AS1 is able to be a promising target for LSCC treatment.

WDR5 facilitates EMT and metastasis of CCA by increasing HIF-1α accumulation in Myc-dependent and independent pathways.

Cholangiocarcinoma (CCA) is a highly aggressive malignancy with extremely poor prognoses. The oncogenic role and prognostic value of c-Myc in CCA is not well elucidated. WD repeat domain 5 (WDR5) is a critical regulatory factor directly interacting with c-Myc to regulate c-Myc recruitment at chromosomal locations, but the interaction of WDR5 and c-Myc in CCA was uncovered. In our study, we detected WDR5 and c-Myc expression in all CCA types, including intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA) CCA, and evaluated their prognostic significance. Consequently, we demonstrated that WDR5 was significantly correlated with poor prognosis of CCA and that WDR5 and c-Myc co-expression was a more sensitive prognostic factor. With in vitro and in vivo experiments and bioinformatics, we showed that WDR5 interacted with the Myc box IIIb (MBIIIb) motif of c-Myc and facilitated Myc-induced HIF1A transcription, thereby promoting the epithelial-mesenchymal transition (EMT), invasion, and metastasis of CCA. Moreover, WDR5 enhanced hypoxia-inducible factor 1 subunit α (HIF-1α) accumulation by binding with histone deacetylase 2 (HDAC2) and increasing histone 3 lysine 4 acetylation (H3K4ac) deacetylation of the prolyl hydroxylase domain protein 2 (PHD2) promoter, resulting in the attenuation of chromatin opening and PHD2 expression, and eventually leading to HIF-1α stabilization and accumulation. In conclusion, WDR5 facilitated EMT and metastasis of CCA by increasing HIF-1α accumulation in a Myc-dependent pathway to promote HIF-1α transcription and a Myc-independent pathway to stabilize HIF-1α.

USP44 accelerates the growth of T-cell acute lymphoblastic leukemia through interacting with WDR5 and repressing its ubiquitination.

T-cell acute lymphoblastic leukemia (T-ALL) is a common hematologic malignancy. Based on the data from GSE66638 and GSE141140, T-ALL patients depicted a higher USP44 level. However, its role in T-ALL is still unclear. In the present study, we investigated the role of USP44 in T-ALL growth. USP44 overexpression elevated the proliferation of CCRF-CEM cells, while USP44 knockdown suppressed the proliferation of Jurkat and MOLT-4 cells. In addition, USP44 accelerated the cell cycle progression, with boosted cyclinD and PCNA levels. However, USP44 knockdown induced apoptosis in Jurkat and MOLT-4 cells, with an upheaval among cleaved caspase-3 and PARP levels. Mechanistically, USP44 co-localized and interacted with WDR5, leading to the repression of its ubiquitination and degradation. Interestingly, WDR5 overexpression abolished the apoptosis induced by USP44 knockdown. Consistently, the in vivo study revealed that USP44 knockdown restricted the leukemic engraftments in the bone marrow and spleens and reduced the infiltration of T-ALL cells in the livers and lungs. In conclusion, this study indicated that USP44 enhanced the growth of T-ALL through interacting with WDR5 and repressing its ubiquitination. This study highlights the potential use of USP44 as a therapeutic target of T-ALL.

USP7 sustains an active epigenetic program via stabilizing MLL2 and WDR5 in diffuse large B-cell lymphoma.

Activated B-cell-like (ABC)-diffuse large B-cell lymphoma (ABC-DLBCL) is a common subtype of non-Hodgkin's lymphoma with poor prognosis. The survival of ABC-DLBCL relies on constitutive activation of BCR signaling, but the underlying molecular mechanism is not fully addressed. By mining The Cancer Genome Atlas database, we found that the expression of ubiquitin-specific protease 7 (USP7) is significantly elevated in three cancer types including DLBCL. Interestingly, unlike germinal center B-cell-like (GCB)-DLBCL, ABC-DLBCL shows upregulated expression of USP7. Inhibiting the enzymatic activity of USP7 (P22077) has a drastic effect on ABC-DLBCL, but not GCB-DLBCL cells. Compared to GCB-DLBCL, ABC-DLBCL cells show transcriptional upregulation of multiple components of BCR-signaling. USP7 inhibition significantly reduces the expression of upregulated components of BCR signaling. Mechanistically, USP7 inhibition greatly reduces the methylation of histone 3 on lysine 4 (H3K4me2), which is an epigenetic marker for active enhancers. USP7 inhibition greatly reduces the protein level of WDR5 and MLL2, key components of lysine-specific methyltransferase complex (complex of proteins associated with Set1 [COMPASS]). In ABC-DLBCL cells, USP7 stabilizes WDR5 and MLL2. In patients, the expression of USP7 is significantly associated with components of BCR signaling (LYN, SYK, BTK, PLCG2, PRKCB, MALT1, BCL10, and CARD11) and targets of BCR signaling (MYC and IRF4). In summary, we demonstrated an essential role of USP7 in ABC-DLBCL by organizing an oncogenic epigenetic program via stabilization of WDR5 and MLL2. Targeting USP7 might be a novel and efficient approach to treat patients with ABC-DLBCL and it might be better than targeting individual components such as BTK in BCR signaling.

WDR5 promotes the tumorigenesis of oral squamous cell carcinoma via CARM1/ß-catenin axis.

Oral squamous cell carcinoma (OSCC) is a malignancy all over the world. WD repeat domain 5 (WDR5) is involved in cancer progression. In addition, it was reported that WDR5 is upregulated in head and neck cancer, while its role in OSCC is unknown. First, the expression of WDR5 in oral cancer tissues and cells was examined by qRT-PCR, IHF and western blot. CCK-8 assay was performed to test the cell viability. Cell migration was assessed by transwell assay. Knocking down WDR5 or CARM1 in oral cancer cells to detect its function on cancer growth, WDR5 and CARM1 were significantly upregulated in OSCC. Silencing WDR5 suppressed OSCC cell viability and migration. CARM1 level in OSCC cells was significantly inhibited by WDR5 downregulation, and CARM1 elevation could rescue the effect of WDR5 knockdown on tumorigenesis of OSCC. Moreover, silencing of WDR5 notably inactivated ß-catenin signaling pathway, while this phenomenon was restored by CARM1 overexpression. Silencing of WDR5 attenuated the tumorigenesis of OSCC via CARM1/ß-catenin axis. Thus, WDR5 might be a target for OSCC treatment.

MKL1 regulates hepatocellular carcinoma cell proliferation, migration and apoptosis via the COMPASS complex and NF-κB signaling.

BACKGROUND: Histone modification plays essential roles in hepatocellular carcinoma (HCC) pathogenesis, but the regulatory mechanisms remain poorly understood. In this study, we aimed to analyze the roles of Megakaryoblastic leukemia 1 (MKL1) and its regulation of COMPASS (complex of proteins associated with Set1) in HCC cells. METHODS: MKL1 expression in clinical tissues and cell lines were detected by bioinformatics, qRT-PCR and western blot. MKL1 expression in HCC cells were silenced with siRNA, followed by cell proliferation evaluation via Edu staining and colony formation, migration and invasion using the Transwell system, and apoptosis by Hoechst staining. HCC cell tumorigenesis was assessed by cancer cell line-based xenograft model, combined with H&E staining and IHC assays. RESULTS: MKL1 expression was elevated in HCC cells and clinical tissues which was correlated with poor prognosis. MKL1 silencing significantly repressed proliferation, migration, invasion and colony formation but enhanced apoptosis in HepG2 and Huh-7 cells. MKL1 silencing also inhibited COMPASS components and p65 protein expression in HepG2 and Huh-7 cells. HepG2 cell tumorigenesis in nude mice was severely impaired by MKL1 knockdown, resulted into suppressed Ki67 expression and cell proliferation. CONCLUSION: MKL1 promotes HCC pathogenesis by regulating hepatic cell proliferation, migration and apoptosis via the COMPASS complex and NF-κB signaling.