KMT2D mutations promoted tumor progression in diffuse large B-cell lymphoma through altering tumor-induced regulatory T cell trafficking via FBXW7-NOTCH-MYC/TGF-ß1 axis.

Histone methyltransferase KMT2D is one of the most frequently mutated genes in diffuse large B-cell lymphoma (DLBCL) and has been identified as an important pathogenic factor and prognostic marker. However, the biological relevance of KMT2D mutations on tumor microenvironment remains to be determined. KMT2D mutations were assessed by whole-genome/exome sequencing (WGS/WES) in 334 patients and by targeted sequencing in 427 patients with newly diagnosed DLBCL. Among all 761 DLBCL patients, somatic mutations in KMT2D were observed in 143 (18.79%) patients and significantly associated with advanced Ann Arbor stage and MYC expression ≥ 40%, as well as inferior progression-free survival and overall survival. In B-lymphoma cells, the mutation or knockdown of KMT2D inhibited methylation of lysine 4 on histone H3 (H3K4), downregulated FBXW7 expression, activated NOTCH signaling pathway and downstream MYC/TGF-ß1, resulting in alterations of tumor-induced regulatory T cell trafficking. In B-lymphoma murine models established with subcutaneous injection of SU-DHL-4 cells, xenografted tumors bearing KMT2D mutation presented lower H3K4 methylation, higher regulatory T cell recruitment, thereby provoking rapid tumor growth compared with wild-type KMT2D via FBXW7-NOTCH-MYC/TGF-ß1 axis.

Deletion of Fbxw7 in oocytes causes follicle loss and premature ovarian insufficiency in mice.

Premature ovarian insufficiency (POI) is one of the important causes of female infertility. Yet the aetiology for POI is still elusive. FBXW7 (F-box with 7 tandem WD) is one of the important components of the Skp1-Cullin1-F-box (SCF) E3 ubiquitin ligase. FBXW7 can regulate cell growth, survival and pluripotency through mediating ubiquitylation and degradation of target proteins via triggering the ubiquitin-proteasome system, and is associated with tumorigenesis, haematopoiesis and testis development. However, evidence establishing the function of FBXW7 in ovary is still lacking. Here, we showed that FBXW7 protein level was significantly decreased in the ovaries of the cisplatin-induced POI mouse model. We further showed that mice with oocyte-specific deletion of Fbxw7 demonstrated POI, characterized with folliculogenic defects, early depletion of follicle reserve, disordered hormonal secretion, ovarian dysfunction and female infertility. Impaired oocyte-GCs communication, manifested as down-regulation of connexin 37, may contribute to follicular development failure in the Fbxw7-mutant mice. Furthermore, single-cell RNA sequencing and in situ hybridization results indicated an accumulation of Clu and Ccl2 transcripts, which may alter follicle microenvironment deleterious to oocyte development and accelerate POI. Our results establish the important role of Fbxw7 in folliculogenesis and ovarian function, and might provide valuable information for understanding POI and female infertility.

Inhibition of KIF20A enhances the immunotherapeutic effect of hepatocellular carcinoma by enhancing c-Myc ubiquitination.

Immune therapy has significantly improved the prognosis of hepatocellular carcinoma (HCC) patients, yet its efficacy remains limited, underscoring the urgency to identify new therapeutic targets and biomarkers. Here, we investigated the pathological and physiological roles of KIF20A and assess its potential in enhancing HCC treatment efficacy when combined with PD-1 inhibitors. We initially assess KIF20A's oncogenic function using liver-specific KIF20A knockout (Kif20a CKO) mouse models and orthotopic xenografts. Subsequently, we establish a regulatory axis involving KIF20A, FBXW7, and c-Myc, validated through construction of c-Myc splicing mutants. Large-scale clinical immunohistochemistry (IHC) analyses confirm the pathological relevance of the KIF20A-FBXW7-c-Myc axis in HCC. We demonstrate that KIF20A overexpression correlates with poor prognosis in HCC by competitively inhibiting FBXW7-mediated degradation of c-Myc, thereby promoting glycolysis and enhancing tumor proliferation. Conversely, KIF20A downregulation suppresses these effects, impairing tumor growth through c-Myc downregulation. Notably, KIF20A inhibition attenuates c-Myc-induced MMR expression, associated with improved prognosis in HCC patients receiving PD-1 inhibitor therapy. Furthermore, in Kif20a CKO HCC mouse models, we observe synergistic effects between Kif20a knockout and anti-PD-1 antibodies, significantly enhancing immunotherapeutic efficacy against HCC. Our findings suggest that targeting the KIF20A-c-Myc axis could identify HCC patients likely to benefit from anti-PD-1 therapy. In conclusion, we propose that combining KIF20A inhibitors with anti-PD-1 treatment represents a promising therapeutic strategy for HCC, offering new avenues for clinical development and patient stratification.

Targeted panel sequencing of pharmacogenes and oncodrivers in colorectal cancer patients reveals genes with prognostic significance.

BACKGROUND: Colorectal cancer is still the second leading cause of cancer-related deaths and thus biomarkers allowing prediction of the resistance of patients to therapy and estimating their prognosis are needed. We designed a panel of 558 genes with pharmacogenomics records related to 5-fluorouracil resistance, genes important for sensitivity to other frequently used drugs, major oncodrivers, and actionable genes. We performed a target enrichment sequencing of DNA from tumors and matched blood samples of patients, and compared the results with patient prognosis stratified by systemic adjuvant chemotherapy. RESULTS: The median number of detected variants per tumor sample was 18.5 with 4 classified as having a high predicted functional effect and 14.5 moderate effect. APC, TP53, and KRAS were the most frequent mutated genes (64%, 59%, and 42% of mutated samples, respectively) followed by FAT4 (23%), FBXW7, and PIK3CA (16% for both). Patients with advanced stage III had more frequently APC, TP53, or KRAS mutations than those in stages I or II. KRAS mutation counts followed an increasing trend with grade (G1 < G2 < G3). The response to adjuvant therapy was worse in carriers of frameshift mutations in APC or 12D variant in KRAS, but none of these oncodrivers had prognostic value. Carriage of somatic mutations in any of the genes ABCA13, ANK2, COL7A1, NAV3, or UNC80 had prognostic relevance for worse overall survival (OS) of all patients. In contrast, mutations in FLG, GLI3, or UNC80 were prognostic in the same direction for patients untreated, and mutations in COL6A3, LRP1B, NAV3, RYR1, RYR3, TCHH, or TENM4 for patients treated with adjuvant therapy. The first association was externally validated. From all germline variants with high or moderate predicted functional effects (median 326 per patient), > 5% frequency and positive Manhattan plot based on 3-year RFS, rs72753407 in NFACS, rs34621071 in ERBB4, and rs2444274 in RIF1 were significantly associated with RFS, OS or both. CONCLUSIONS: The present study identified several putative somatic and germline genetic events with prognostic potential for colorectal cancer that should undergo functional characterization.

CYR61 Acts as an Intracellular Microtubule-Associated Protein and Coordinates Mitotic Progression via PLK1-FBW7 Pathway.

Cysteine-rich angiogenic inducer 61 (CYR61), also called CCN1, has long been characterized as a secretory protein. Nevertheless, the intracellular function of CYR61 remains unclear. Here, we found that CYR61 is important for proper cell cycle progression. Specifically, CYR61 interacts with microtubules and promotes microtubule polymerization to ensure mitotic entry. Moreover, CYR61 interacts with PLK1 and accumulates during the mitotic process, followed by degradation as mitosis concludes. The proteolysis of CYR61 requires the PLK1 kinase activity, which directly phosphorylates two conserved motifs on CYR61, enhancing its interaction with the SCF E3 complex subunit FBW7 and mediating its degradation by the proteasome. Mutations of phosphorylation sites of Ser167 and Ser188 greatly increase CYR61's stability, while deletion of CYR61 extends prophase and metaphase and delays anaphase onset. In summary, our findings highlight the precise control of the intracellular CYR61 by the PLK1-FBW7 pathway, accentuating its significance as a microtubule-associated protein during mitotic progression.

Smooth muscle NF90 deficiency ameliorates diabetic atherosclerotic calcification in male mice via FBXW7-AGER1-AGEs axis.

Hyperglycemia accelerates calcification of atherosclerotic plaques in diabetic patients, and the accumulation of advanced glycation end products (AGEs) is closely related to the atherosclerotic calcification. Here, we show that hyperglycemia-mediated AGEs markedly increase vascular smooth muscle cells (VSMCs) NF90/110 activation in male diabetic patients with atherosclerotic calcified samples. VSMC-specific NF90/110 knockout in male mice decreases obviously AGEs-induced atherosclerotic calcification, along with the inhibitions of VSMC phenotypic changes to osteoblast-like cells, apoptosis, and matrix vesicle release. Mechanistically, AGEs increase the activity of NF90, which then enhances ubiquitination and degradation of AGE receptor 1 (AGER1) by stabilizing the mRNA of E3 ubiquitin ligase FBXW7, thus causing the accumulation of more AGEs and atherosclerotic calcification. Collectively, our study demonstrates the effects of VSMC NF90 in mediating the metabolic imbalance of AGEs to accelerate diabetic atherosclerotic calcification. Therefore, inhibition of VSMC NF90 may be a potential therapeutic target for diabetic atherosclerotic calcification.

The HTLV-I oncoprotein Tax inactivates the tumor suppressor FBXW7.

Human T-cell leukemia virus type 1 (HTLV-I) is the etiological agent of adult T-cell leukemia (ATL). Mutational analysis has demonstrated that the tumor suppressor, F-box and WD repeat domain containing 7 (FBXW7/FBW7/CDC4), is mutated in primary ATL patients. However, even in the absence of genetic mutations, FBXW7 substrates are stabilized in ATL cells, suggesting additional mechanisms can prevent FBXW7 functions. Here, we report that the viral oncoprotein Tax represses FBXW7 activity, resulting in the stabilization of activated Notch intracellular domain, c-MYC, Cyclin E, and myeloid cell leukemia sequence 1 (BCL2-related) (Mcl-1). Mechanistically, we demonstrate that Tax directly binds to FBXW7 in the nucleus, effectively outcompeting other targets for binding to FBXW7, resulting in decreased ubiquitination and degradation of FBXW7 substrates. In support of the nuclear role of Tax, a non-degradable form of the nuclear factor kappa B subunit 2 (NFκB2/p100) was found to delocalize Tax to the cytoplasm, thereby preventing Tax interactions with FBXW7 and Tax-mediated inhibition of FBXW7. Finally, we characterize a Tax mutant that is unable to interact with FBXW7, unable to block FBXW7 tumor suppressor functions, and unable to effectively transform fibroblasts. These results demonstrate that HTLV-I Tax can inhibit FBXW7 functions without genetic mutations to promote an oncogenic state. These results suggest that Tax-mediated inhibition of FBXW7 is likely critical during the early stages of the cellular transformation process. IMPORTANCE: F-box and WD repeat domain containing 7 (FBXW7), a critical tumor suppressor of human cancers, is frequently mutated or epigenetically suppressed. Loss of FBXW7 functions is associated with stabilization and increased expression of oncogenic factors such as Cyclin E, c-Myc, Mcl-1, mTOR, Jun, and Notch. In this study, we demonstrate that the human retrovirus human T-cell leukemia virus type 1 oncoprotein Tax directly interacts with FBXW7, effectively outcompeting other targets for binding to FBXW7, resulting in decreased ubiquitination and degradation of FBXW7 cellular substrates. We further demonstrate that a Tax mutant unable to interact with and inactivate FBXW7 loses its ability to transform primary fibroblasts. Collectively, our results describe a novel mechanism used by a human tumor virus to promote cellular transformation.

Transcriptional Up-Regulation of FBXW7 by KCa1.1 K+ Channel Inhibition through the Nrf2 Signaling Pathway in Human Prostate Cancer LNCaP Cell Spheroid Model.

The tumor suppressor gene F-box and WD repeat domain-containing (FBXW) 7 reduces cancer stemness properties by promoting the protein degradation of pluripotent stem cell markers. We recently demonstrated the transcriptional repression of FBXW7 by the three-dimensional (3D) spheroid formation of several cancer cells. In the present study, we found that the transcriptional activity of FBXW7 was promoted by the inhibition of the Ca2+-activated K+ channel, KCa1.1, in a 3D spheroid model of human prostate cancer LNCaP cells through the Akt-Nrf2 signaling pathway. The transcriptional activity of FBXW7 was reduced by the siRNA-mediated inhibition of the CCAAT-enhancer-binding protein C/EBP δ (CEBPD) after the transfection of miR223 mimics in the LNCaP spheroid model, suggesting the transcriptional regulation of FBXW7 through the Akt-Nrf2-CEBPD-miR223 transcriptional axis in the LNCaP spheroid model. Furthermore, the KCa1.1 inhibition-induced activation of FBXW7 reduced (1) KCa1.1 activity and protein levels in the plasma membrane and (2) the protein level of the cancer stem cell (CSC) markers, c-Myc, which is a molecule degraded by FBXW7, in the LNCaP spheroid model, indicating that KCa1.1 inhibition-induced FBXW7 activation suppressed CSC conversion in KCa1.1-positive cancer cells.

CRL2KLHDC3 and CRL1Fbxw7 cooperatively mediate c-Myc degradation.

c-Myc is a proto-oncoprotein that regulates various cellular processes and whose abnormal expression leads to tumorigenesis. c-Myc protein stability has been shown to be predominantly controlled by the ubiquitin ligase (E3) CRL1Fbxw7 in a manner dependent on glycogen synthase kinase 3 (GSK3)-mediated phosphorylation. Here we show that, in some types of cancer cells, c-Myc degradation is largely insensitive to the GSK3 inhibitor (GSK3i) CHIR99021, suggesting the existence of an E3 other than CRL1Fbxw7 for c-Myc degradation. Mass spectrometry identified CRL2KLHDC3 as such an E3. In GSK3i-insensitive cancer cells, combined depletion of Fbxw7 and KLHDC3 resulted in marked stabilization of c-Myc, suggestive of a cooperative action of Fbxw7 and KLHDC3. Furthermore, transplantation of such cells deficient in both Fbxw7 and KLHDC3 into immunodeficient mice gave rise to larger tumors compared with those formed by cells lacking only Fbxw7. GSK3i-insensitive pancreatic cancer cells expressed lower levels of SHISA2, a negative regulator of the Wnt signaling pathway, than did GSK3i-sensitive cells. KLHDC3 mRNA abundance was associated with prognosis in pancreatic cancer patients with a low level of SHISA2 gene expression. These results suggest that KLHDC3 cooperates with Fbxw7 to promote c-Myc degradation in a subset of cancer cells with low GSK3 activity.

Increased H19/miR-675 Expression in Adult T-Cell Leukemia Is Associated with a Unique Notch Signature Pathway.

The Notch pathway is a key cancer driver and is important in tumor progression. Early research suggested that Notch activity was highly dependent on the expression of the intracellular cleaved domain of Notch-1 (NICD). However, recent insights into Notch signaling reveal the presence of Notch pathway signatures, which may vary depending on different cancer types and tumor microenvironments. Herein, we perform a comprehensive investigation of the Notch signaling pathway in adult T-cell leukemia (ATL) primary patient samples. Using gene arrays, we demonstrate that the Notch pathway is constitutively activated in ATL patient samples. Furthermore, the activation of Notch in ATL cells remains elevated irrespective of the presence of activating mutations in Notch itself or its repressor, FBXW7, and that ATL cells are dependent upon Notch-1 expression for proliferation and survival. We demonstrate that ATL cells exhibit the expression of pivotal Notch-related genes, including notch-1, hes1, c-myc, H19, and hes4, thereby defining a critical Notch signature associated with ATL disease. Finally, we demonstrate that lncRNA H19 is highly expressed in ATL patient samples and ATL cells and contributes to Notch signaling activation. Collectively, our results shed further light on the Notch pathway in ATL leukemia and reveal new therapeutic approaches to inhibit Notch activation in ATL cells.

Copy number variants at 4q31.3 affecting the regulatory region of FBXW7 associated with neurodevelopmental delay.

Emerging research has demonstrated that genomic alterations disrupting topologically associated domains (TADs) and chromatin interactions underlie the pathogenic mechanisms of specific copy number variants (CNVs) in neurodevelopmental disorders. We report two patients with a de novo deletion and a duplication in chromosome 4q31, potentially causing FBX-related neurodevelopmental syndrome by affecting the regulatory region of FBXW7. High-throughput chromosome conformation capture (Hi-C) analysis using available capture data in neural progenitor cells revealed the rewiring of the TAD boundary close to FBXW7. Both patients exhibited facial dysmorphisms, cardiac and limb abnormalities, and neurodevelopmental delays, showing significant clinical overlap with previously reported FBXW7-related features. We also included an additional 10 patients with CNVs in the 4q31 region from the literature and the DECIPHER database for Hi-C analysis, which confirmed that disruption of the regulatory region of FBXW7 likely contributes to the developmental defects observed in these patients.

Recurrent mutations in tumor suppressor FBXW7 bypass Wnt/ß-catenin addiction in cancer.

Pathologic Wnt/ß-catenin signaling drives various cancers, leading to multiple approaches to drug this pathway. Appropriate patient selection can maximize success of these interventions. Wnt ligand addiction is a druggable vulnerability in RNF43-mutant/RSPO-fusion cancers. However, pharmacologically targeting the biogenesis of Wnt ligands, e.g., with PORCN inhibitors, has shown mixed therapeutic responses, possibly due to tumor heterogeneity. Here, we show that the tumor suppressor FBXW7 is frequently mutated in RNF43-mutant/RSPO-fusion tumors, and FBXW7 mutations cause intrinsic resistance to anti-Wnt therapies. Mechanistically, FBXW7 inactivation stabilizes multiple oncoproteins including Cyclin E and MYC and antagonizes the cytostatic effect of Wnt inhibitors. Moreover, although FBXW7 mutations do not mitigate ß-catenin degradation upon Wnt inhibition, FBXW7-mutant RNF43-mutant/RSPO-fusion cancers instead lose dependence on ß-catenin signaling, accompanied by dedifferentiation and loss of lineage specificity. These FBXW7-mutant Wnt/ß-catenin-independent tumors are susceptible to multi-cyclin-dependent kinase inhibition. An in-depth understanding of primary resistance to anti-Wnt/ß-catenin therapies allows for more appropriate patient selection and use of alternative mechanism-based therapies.

Overcoming BRAF and CDK4/6 inhibitor resistance by inhibiting MAP3K3-dependent protection against YAP lysosomal degradation.

Transcriptional programs governed by YAP play key roles in conferring resistance to various molecular-targeted anticancer agents. Strategies aimed at inhibiting YAP activity have garnered substantial interest as a means to overcome drug resistance. However, despite extensive research into the canonical Hippo-YAP pathway, few clinical agents are currently available to counteract YAP-associated drug resistance. Here, we present a novel mechanism of YAP stability regulation by MAP3K3 that is independent of Hippo kinases. Furthermore, we identified MAP3K3 as a target for overcoming anticancer drug resistance. Depletion of MAP3K3 led to a substantial reduction in the YAP protein level in melanoma and breast cancer cells. Mass spectrometry analysis revealed that MAP3K3 phosphorylates YAP at serine 405. This MAP3K3-mediated phosphorylation event hindered the binding of the E3 ubiquitin ligase FBXW7 to YAP, thereby preventing its p62-mediated lysosomal degradation. Robust YAP activation was observed in CDK4/6 inhibitor-resistant luminal breast cancer cells. Knockdown or pharmacological inhibition of MAP3K3 effectively suppressed YAP activity and restored CDK4/6 inhibitor sensitivity. Similarly, elevated MAP3K3 expression supported the prosurvival activity of YAP in BRAF inhibitor-resistant melanoma cells. Inhibition of MAP3K3 decreased YAP-dependent cell proliferation and successfully restored BRAF inhibitor sensitivity. In conclusion, our study reveals a previously unrecognized mechanism for the regulation of YAP stability, suggesting MAP3K3 inhibition as a promising strategy for overcoming resistance to CDK4/6 and BRAF inhibitors in cancer treatment.

Selective deletion of E3 ubiquitin ligase FBW7 in VE-cadherin-positive cells instigates diffuse large B-cell lymphoma in mice in vivo.

During the maturation of hematopoietic stem/progenitor cells (HSPCs) to fully differentiated mature B lymphocytes, developing lymphocytes may undergo malignant transformation and produce B-cell lymphomas. Emerging evidence shows that through the endothelial-hematopoietic transition, specialized endothelial cells called the hemogenic endothelium can differentiate into HSPCs. However, the contribution of genetic defects in hemogenic endothelial cells to B-cell lymphomagenesis has not yet been investigated. Here, we report that mice with endothelial cell-specific deletion of Fbw7 spontaneously developed diffuse large B-cell lymphoma (DLBCL) following Bcl6 accumulation. Using lineage tracing, we showed that B-cell lymphomas in Fbw7 knockout mice were hemogenic endothelium-derived. Mechanistically, we found that FBW7 directly interacted with Bcl6 and promoted its proteasomal degradation. FBW7 expression levels are inversely correlated with BCL6 expression. Additionally, pharmacological disruption of Bcl6 abolished Fbw7 deletion-induced B-cell lymphomagenesis. We conclude that selective deletion of E3 ubiquitin ligase FBW7 in VE-cadherin positive endothelial cells instigates diffuse large B-cell lymphoma via upregulation of BCL6 stability. In addition, the mice with endothelial cell-specific deletion of Fbw7 provide a valuable preclinical platform for in vivo development and evaluation of novel therapeutic interventions for the treatment of DLBCL.

Epidermal growth factor receptor (EGFR) is a target of the tumor-suppressor E3 ligase FBXW7.

FBXW7 is an E3 ubiquitin ligase that targets proteins for proteasome-mediated degradation and is mutated in various cancer types. Here, we use CRISPR base editors to introduce different FBXW7 hotspot mutations in human colon organoids. Functionally, FBXW7 mutation reduces EGF dependency of organoid growth by ~10,000-fold. Combined transcriptomic and proteomic analyses revealed increased EGFR protein stability in FBXW7 mutants. Two distinct phosphodegron motifs reside in the cytoplasmic tail of EGFR. Mutations in these phosphodegron motifs occur in human cancer. CRISPR-mediated disruption of the phosphodegron motif at T693 reduced EGFR degradation and EGF growth factor dependency. FBXW7 mutant organoids showed reduced sensitivity to EGFR-MAPK inhibitors. These observations were further strengthened in CRC-derived organoid lines and validated in a cohort of patients treated with panitumumab. Our data imply that FBXW7 mutations reduce EGF dependency by disabling EGFR turnover.

The FBXW7-binding sites on FAM83D are potential targets for cancer therapy.

Increasing evidence shows the oncogenic function of FAM83D in human cancer, but how FAM83D exerts its oncogenic function remains largely unclear. Here, we investigated the importance of FAM83D/FBXW7 interaction in breast cancer (BC). We systematically mapped the FBXW7-binding sites on FAM83D through a comprehensive mutational analysis together with co-immunoprecipitation assay. Mutations at the FBXW7-binding sites on FAM83D led to that FAM83D lost its capability to promote the ubiquitination and proteasomal degradation of FBXW7; cell proliferation, migration, and invasion in vitro; and tumor growth and metastasis in vivo, indicating that the FBXW7-binding sites on FAM83D are essential for its oncogenic functions. A meta-evaluation of FAM83D revealed that the prognostic impact of FAM83D was independent on molecular subtypes. The higher expression of FAM83D has poorer prognosis. Moreover, high expression of FAM83D confers resistance to chemotherapy in BCs, which is experimentally validated in vitro. We conclude that identification of FBXW7-binding sites on FAM83D not only reveals the importance for FAM83D oncogenic function, but also provides valuable insights for drug target.

The SCF-FBW7ß E3 ligase mediates ubiquitination and degradation of the serine/threonine protein kinase PINK1.

Understanding the mechanisms that govern the stability of functionally crucial proteins is essential for various cellular processes, development, and overall cell viability. Disturbances in protein homeostasis are linked to the pathogenesis of neurodegenerative diseases. PTEN-induced kinase 1 (PINK1), a protein kinase, plays a significant role in mitochondrial quality control and cellular stress response, and its mutated forms lead to early-onset Parkinson's disease. Despite its importance, the specific mechanisms regulating PINK1 protein stability have remained unclear. This study reveals a cytoplasmic interaction between PINK1 and F-box and WD repeat domain-containing 7ß (FBW7ß) in mammalian cells. FBW7ß, a component of the Skp1-Cullin-1-F-box protein complex-type ubiquitin ligase, is instrumental in recognizing substrates. Our findings demonstrate that FBW7ß regulates PINK1 stability through the Skp1-Cullin-1-F-box protein complex and the proteasome pathway. It facilitates the K48-linked polyubiquitination of PINK1, marking it for degradation. When FBW7 is absent, PINK1 accumulates, leading to heightened mitophagy triggered by carbonyl cyanide 3-chlorophenylhydrazone treatment. Moreover, exposure to the toxic compound staurosporine accelerates PINK1 degradation via FBW7ß, correlating with increased cell death. This study unravels the intricate mechanisms controlling PINK1 protein stability and sheds light on the novel role of FBW7ß. These findings deepen our understanding of PINK1-related pathologies and potentially pave the way for therapeutic interventions.

Syngeneic murine models with distinct immune microenvironments represent subsets of human intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a poorly immunogenic malignancy associated with limited survival. Syngeneic immunocompetent mouse models of CCA are an essential tool to elucidate the tumor immune microenvironment (TIME), understand mechanisms of tumor immune evasion, and test novel immunotherapeutic strategies. The scope of this study was to develop and characterize immunocompetent CCA models with distinct genetic drivers, and correlate tumor genomics, immunobiology, and therapeutic response. METHODS: A multifaceted approach including scRNA-seq, CITE-seq, whole exome and bulk RNA sequencing was employed. FDA-approved PD-1/PD-L1 antibodies were tested in humanized PD-1/PD-L1 mice (HuPD-H1). RESULTS: A genetic mouse model of intrahepatic CCA (iCCA) driven by intrabiliary transduction of Fbxw7ΔF/Akt that mimics human iCCA was generated. From the Fbxw7ΔF/Akt tumors, a murine cell line (FAC) and syngeneic model with genetic and phenotypic characteristics of human iCCA were developed. Established SB1 (YAPS127A/Akt) and KPPC (KrasG12Dp53L/L) models were compared to the FAC model. Although the models had transcriptomic similarities, they had substantial differences as well. Mutation patterns of FAC, SB1, and KPPC cells matched different mutational signatures in Western and Japanese CCA patient cohorts. KPPC tumors had a high tumor mutation burden. FAC tumors had a T cell-infiltrated TIME, while SB1 tumors had a preponderance of suppressive myeloid cells. FAC, SB1, and KPPC tumors matched different immune signatures in human iCCA cohorts. Moreover, FAC, SB1, and KPPC tumor-bearing HuPD-H1 mice displayed differential responses to nivolumab or durvalumab. CONCLUSIONS: Syngeneic iCCA models display a correlation between tumor genotype and TIME phenotype, with differential responses to FDA-approved immunotherapies. This study underscores the importance of leveraging multiple preclinical models to understand responses to immunotherapy in different genetic subsets of human CCA. IMPACT AND IMPLICATIONS: Understanding the relationship between tumor genotype and the phenotype of the immune microenvironment is an unmet need in cholangiocarcinoma (CCA). Herein, we use syngeneic murine models of intrahepatic CCA with different genetic drivers to demonstrate a correlation between tumor genotype and immune microenvironment phenotype in murine models, which is associated with differential responses to FDA-approved immunotherapies. This information will help guide other preclinical studies. Additionally, it emphasizes that immune checkpoint inhibition in patients with CCA is not a "one-size-fits-all" approach. Our observations suggest that, as for targeted therapies, patients should be stratified and selected for treatment according to their tumor genetics.

Targeted and Shallow Whole-Genome Sequencing Identifies Therapeutic Opportunities in p53abn Endometrial Cancers.

PURPOSE: Shallow whole-genome sequencing (sWGS) can detect copy-number (CN) aberrations. In high-grade serous ovarian cancer (HGSOC) sWGS identified CN signatures such as homologous recombination deficiency (HRD) to direct therapy. We applied sWGS with targeted sequencing to p53abn endometrial cancers to identify additional prognostic stratification and therapeutic opportunities. EXPERIMENTAL DESIGN: sWGS and targeted panel sequencing was performed on formalin-fixed, paraffin-embedded p53abn endometrial cancers. CN alterations, mutational data and CN signatures were derived, and associations to clinicopathologic and outcomes data were assessed. RESULTS: In 187 p53abn endometrial cancers, 5 distinct CN signatures were identified. Signature 5 was associated with BRCA1/2 CN loss with features similar to HGSOC HRD signature. Twenty-two percent of potential HRD cases were identified, 35 patients with signature 5, and 8 patients with BRCA1/2 somatic mutations. Signatures 3 and 4 were associated with a high ploidy state, and CCNE1, ERBB2, and MYC amplifications, with mutations in PIK3CA enriched in signature 3. We observed improved overall survival (OS) for patients with signature 2 and worse OS for signatures 1 and 3. Twenty-eight percent of patients had CCNE1 amplification and this subset was enriched with carcinosarcoma histotype. Thirty-four percent of patients, across all histotypes, had ERBB2 amplification and/or HER2 overexpression on IHC, which was associated with worse outcomes. Mutations in PPP2R1A (29%) and FBXW7 (16%) were among the top 5 most common mutations. CONCLUSIONS: sWGS and targeted sequencing identified therapeutic opportunities in 75% of patients with p53abn endometrial cancer. Further research is needed to determine the efficacy of treatments targeting these identified pathways within p53abn endometrial cancers.

eEF1A2 promotes PTEN-GSK3ß-SCF complex-dependent degradation of Aurora kinase A and is inactivated in breast cancer.

The translation elongation factor eEF1A promotes protein synthesis. Its methylation by METTL13 increases its activity, supporting tumor growth. However, in some cancers, a high abundance of eEF1A isoforms is associated with a good prognosis. Here, we found that eEF1A2 exhibited oncogenic or tumor-suppressor functions depending on its interaction with METTL13 or the phosphatase PTEN, respectively. METTL13 and PTEN competed for interaction with eEF1A2 in the same structural domain. PTEN-bound eEF1A2 promoted the ubiquitination and degradation of the mitosis-promoting Aurora kinase A in the S and G2 phases of the cell cycle. eEF1A2 bridged the interactions between the SKP1-CUL1-FBXW7 (SCF) ubiquitin ligase complex, the kinase GSK3ß, and Aurora-A, thereby facilitating the phosphorylation of Aurora-A in a degron site that was recognized by FBXW7. Genetic ablation of Eef1a2 or Pten in mice resulted in a greater abundance of Aurora-A and increased cell cycling in mammary tumors, which was corroborated in breast cancer tissues from patients. Reactivating this pathway using fimepinostat, which relieves inhibitory signaling directed at PTEN and increases FBXW7 expression, combined with inhibiting Aurora-A with alisertib, suppressed breast cancer cell proliferation in culture and tumor growth in vivo. The findings demonstrate a therapeutically exploitable, tumor-suppressive role for eEF1A2 in breast cancer.