c-Myc Target Research Articles

CSIG-18. YAP/TAZ TARGET GENES MAY DETERMINE THE SELF-RENEWAL AND SURVIVAL OF GLIOBLASTOMA STEM CELLS

Abstract Glioblastoma, a neoplasm arising from neuro-glial stem/progenitor cells, is the most aggressive and lethal form of brain cancer, with a median survival of 14 months. Despite intensive treatments like surgery, radiation, and chemotherapy, recurrence is inevitable, underscoring the urgent need for new treatment strategies. Progress is limited by an incomplete understanding of the biological mechanisms that drive glioblastoma formation and progression, hindering the development of effective therapies. Key molecular pathways, particularly the receptor tyrosine kinases, such as EGFR, and their effector pathways play a critical role in the initiation and maintenance of glioblastoma. Our lab’s research using patient-derived glioblastoma neurospheres and mouse models has shown that the transcriptional co-activators YAP and TAZ are overexpressed downstream of EGFR activity in EGFR-amplified or mutant glioblastoma tumors. YAP/TAZ proteins interact with the TEAD family DNA-binding proteins to promote expression of transcriptional target genes, including SOX2, C-MYC, and EGFR itself in glioblastoma, creating a positive feedback loop that enhances tumor stem cell renewal, proliferation, and survival. Furthermore, our research has demonstrated that inhibiting YAP/TAZ activity with the drug Verteporfin induces apoptosis and promotes growth arrest in patient-derived EGFR-amplified/mutant glioblastoma models. Our findings highlight YAP/TAZ-TEAD as key mediators of glioblastoma stem cell (GSC) self-renewal and survival. Ongoing experiments aim to identify tumor cell-specific YAP/TAZ-TEAD transcriptional target genes predictive of response to YAP/TAZ inhibition in GSCs within EGFR-amplified glioblastomas. Our research seeks to uncover the epigenetic basis of YAP/TAZ dependencies and to determine which genes and cellular processes are critical for tumor stem cell survival, tumor progression, and sensitivity to YAP/TAZ inhibition. These target genes could serve as biomarkers for predicting therapeutic response to YAP/TAZ inhibitors in glioblastoma.

Inhibition of UBE2N in regulatory T-cells boosts immunity against cancer.

Regulatory T (Treg) cells prevent autoimmunity and facilitate cancer immune evasion. Depletion of Tregs is a promising cancer therapy, but risks of autoimmune reactions hamper its clinical translation. Here, we demonstrate that temporally induced deletion of Ube2n in Tregs (Ube2n Treg-KO ) of adult mice results in a robust expansion and activation of cytotoxic CD8 + T-cells in response to cancer cell challenges, producing a long-lasting survival benefit without autoimmune complications. The anti-tumor effect persists following adoptive T-cell transfer to T-cell-deficient Rag1-knockout mice. Single-cell transcriptomic analysis revealed that UBE2N deletion shifted immunosuppressive Tregs to effector-like T-cells. This shift is characterized by the downregulation of c-Myc target genes, resembling that observed in tumor-infiltrating Tregs of melanoma patients. Further analyses confirm that UBE2N maintains c-Myc protein stability via suppression of K48-Ubiquitin-mediated proteasomal degradation. Taken together, our studies uncover a hitherto unexplored and potentially druggable UBE2N/c-Myc signaling axis to eradicate Treg-enabled cancer immune escape.

Discovery of a Potent, selective and orally bioavailable CDK9 degrader for targeting transcription regulation in Triple-Negative breast cancer

Triple-negative breast cancer (TNBC) is a highly aggressive, heterogeneous and invasive subtype of breast cancer with very limited effective modalities of treatment. Degrading the critical transcription regulator cyclin-dependent kinase 9 (CDK9) by proteolysis targeting chimeras (PROTACs) has shown promising potential for treating TNBC. However, to date, CDK9-targeting PROTACs for oral administration in treatment of cancers have not been reported. We herein present the design, synthesis, and extensive biological evaluation of a series of novel PROTACs as orally bioavailable, potent and selective degraders of CDK9 for targeting transcription regulation in triple-negative breast cancer. The developed compound 29 exhibited a desired potency (DC50 = 3.94 nM) with high efficacy (Dmax = 96 %) on CDK9 degradation, and effectively inhibited the proliferation of TNBC MDA-MB-231 cells. Mechanistic investigations revealed that compound 29 is a bona fide CDK9 degrader and can substantially downregulate the downstream targets c-Myc and MCL-1. Furthermore, compound 29 displayed favorable oral bioavailability in mice, and oral administration of degrader 29 significantly depleted CDK9 protein in TNBC tumor tissues and exhibited tumor growth inhibition in TNBC xenograft mice models. Collectively, our work established that degrader 29 is a highly potent and selective degraders of CDK9 with satisfactory oral bioavailability, which holds promising potential for the treatment of TNBC.

Regular exercise suppresses steatosis-associated liver cancer development by degrading E2F1 and c-Myc via circadian gene upregulation.

Regular exercise is believed to suppress cancer progression. However, the precise molecular mechanisms by which exercise prevents cancer development remain unclear. In this study, using a steatosis-associated liver cancer mouse model, we found that regular exercise at a speed of 18 m/min for 20 min daily suppressed liver cancer development. To explore the underlying mechanisms, we examined the gene expression profiles in the livers of the exercise and non-exercise groups. The expressions of circadian genes, such as Per1 and Cry2, were upregulated in the exercise group. As circadian rhythm disruption is known to cause various diseases, including cancer, improving circadian rhythm through exercise could contribute to cancer prevention. We further found that the expression of a series of E2F1 and c-Myc target genes that directly affect the proliferation of cancer cells was downregulated in the exercise group. However, the expression of E2F1 and c-Myc was transcriptionally unchanged but degraded at the post-translational level by exercise. Cry2, which is regulated by the Skp1-Cul1-FBXL3 (SCFFBXL3) ubiquitin ligase complex by binding to FBXL3, can form a complex with E2F1 and c-Myc, which we think is the mechanism to degrade them. Our study revealed a previously unknown mechanism by which exercise prevents cancer development.

Deferasirox, an iron chelator, impacts myeloid differentiation by modulating NF-kB activity via mitochondrial ROS.

The iron chelator deferasirox (DFX) is effective in the treatment of iron overload. In certain patients with myelodysplastic syndrome, DFX can also provide a dramatic therapeutic benefit, improving red blood cell production and decreasing transfusion requirements. Nuclear Factor-kappa B (NF-kB) signalling has been implicated as a potential mechanism behind this phenomenon, with studies focusing on the effect of DFX on haematopoietic progenitors. Here, we examine the phenotypic and transcriptional effects of DFX throughout myeloid cell maturation in both murine and human model systems. The effect of DFX depends on the stage of differentiation, with effects on mitochondrial reactive oxygen species (ROS) production and NF-kB pathway regulation that vary between progenitors and neutrophils. DFX triggers a greater increase in mitochondrial ROS production in neutrophils and this phenomenon is mitigated when cells are cultured in hypoxic conditions. Single-cell transcriptomic profiling revealed that DFX decreases the expression of NF-kB and MYC (c-Myc) targets in progenitors and decreases the expression of PU.1 (SPI1) gene targets in neutrophils. Together, these data suggest a role of DFX in impairing terminal maturation of band neutrophils.

Targeting Molecular Signaling Pathways and Cytokine Responses to Modulate c-MYC in Acute Myeloid Leukemia.

Overexpression of the MYC oncogene, encoding c-MYC protein, contributes to the pathogenesis and drug resistance of acute myeloid leukemia (AML) and many other hematopoietic malignancies. Although standard chemotherapy has predominated in AML therapy over the past five decades, the clinical outcomes and patient response to treatment remain suboptimal. Deeper insight into the molecular basis of this disease should facilitate the development of novel therapeutics targeting specific molecules and pathways that are dysregulated in AML, including fms-like tyrosine kinase 3 (FLT3) gene mutation and cluster of differentiation 33 (CD33) protein expression. Elevated expression of c-MYC is one of the molecular features of AML that determines the clinical prognosis in patients. Increased expression of c-MYC is also one of the cytogenetic characteristics of drug resistance in AML. However, direct targeting of c-MYC has been challenging due to its lack of binding sites for small molecules. In this review, we focused on the mechanisms involving the bromodomain and extra-terminal (BET) and cyclin-dependent kinase 9 (CDK9) proteins, phosphoinositide-Akt-mammalian target of rapamycin (PI3K/AKT/mTOR) and Janus kinase-signal transduction and activation of transcription (JAK/STAT) pathways, as well as various inflammatory cytokines, as an indirect means of regulating MYC overexpression in AML. Furthermore, we highlight Food and Drug Administration (FDA)-approved drugs for AML, and the results of preclinical and clinical studies on novel agents that have been or are currently being tested for efficacy and tolerability in AML therapy. Overall, this review summarizes our current knowledge of the molecular processes that promote leukemogenesis, as well as the various agents that intervene in specific pathways and directly or indirectly modulate c-MYC to disrupt AML pathogenesis and drug resistance.

Screening and identification of miRNAs negatively regulating FAM83A/Wnt/β-catenin signaling pathway in non-small cell lung cancer

The prevalence of non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers, with the Wnt/β-catenin signaling pathway exhibiting robust activation in this particular subtype. The expression of FAM83A (family with sequence similarity 83, member A) has been found to be significantly upregulated in lung cancer, leading to the stabilization of β-catenin and activation of the Wnt signaling pathway. In this study, we conducted a screening of down-regulated miRNAs in lung cancer with FAM83A as the target. Ultimately, we identified miR-1 as a negative regulator of FAM83A and confirmed that FAM83A is a direct target gene of miR-1 through dual luciferase reporter assays. The overexpression of miR-1 significantly attenuated the expression level of FAM83A and suppressed the Wnt signaling pathway, leading to a reduction in the expression levels of downstream target genes AXIN2, CyclinD1, and C-MYC. Additionally, it decreased the nuclear translocation of β-catenin. In addition, overexpression of miR-1 accelerated the degradation of β-catenin by inhibiting FAM83A, promoted the assembly of β-catenin degradation complex, and inhibited the proliferation, migration and invasion of NSCLC cells. In summary, miR-1 may be a potential candidate miRNA for the treatment of NSCLC.

USP11 promotes prostate cancer progression by up-regulating AR and c-Myc activity

Androgen receptor (AR) is a main driver for castration-resistant prostate cancer (CRPC). c-Myc is an oncogene underlying prostate tumorigenesis. Here, we find that the deubiquitinase USP11 targets both AR and c-Myc in prostate cancer (PCa). USP11 expression was up-regulated in metastatic PCa and CRPC. USP11 knockdown (KD) significantly inhibited PCa cell growth. Our RNA-seq studies revealed AR and c-Myc as the top transcription factors altered after USP11 KD. ChIP-seq analysis showed that either USP11 KD or replacement of endogenous USP11 with a catalytic-inactive USP11 mutant significantly decreased chromatin binding by AR and c-Myc. We find that USP11 employs two mechanisms to up-regulate AR and c-Myc levels: namely, deubiquitination of AR and c-Myc proteins to increase their stability and deubiquitination of H2A-K119Ub, a repressive histone mark, on promoters of AR and c-Myc genes to increase their transcription. AR and c-Myc reexpression in USP11-KD PCa cells partly rescued cell growth defects. Thus, our studies reveal a tumor-promoting role for USP11 in aggressive PCa through upregulation of AR and c-Myc activities and support USP11 as a potential target against PCa.

Effect of vitamin E δ-tocotrienol and aspirin on Wnt signaling in human colon cancer stem cells and in adenoma development in APCmin/+ mice.

In this study, we evaluated the effects of vitamin E δ-tocotrienol (DT3) and aspirin on Wnt signaling in human colon cancer stem cells (CCSCs) and in the prevention of adenoma formation in APCmin/+ mice. We found that knockdown of the adenomatous polyposis coli (APC) gene led to subsequent activation of Wnt signaling in colon epithelial cells (NCM460-APCsiRNA) and induction of β-catenin and its downstream target proteins c-MYC, cyclin D1, and survivin. When aspirin and DT3 were combined, cell growth and survival were inhibited and apoptosis was induced in colon epithelial cells and in CCSCs. However, DT3 and/or aspirin had little or no effect on control normal colon epithelial cells (NCM460-NCsiRNA). The induction of apoptosis was directly related to activation of caspase 8 and cleavage of BID to truncated BID. In addition, DT3 and/or aspirin-induced apoptosis was associated with cleaved PARP, elevated levels of cytosolic cytochrome c and BAX, and depletion of anti-apoptotic protein BCl-2 in CCSCs. The combination of aspirin and DT3 inhibited the self-renewal capacity, Wnt/β-catenin receptor activity, and expression of β-catenin and its downstream targets c-MYC, cyclin D1 and survivin in CCSCs. We also found that treatment with DT3 alone or combined with aspirin significantly inhibited intestinal adenoma formation and Wnt/ β-catenin signaling and induced apoptosis, compared to vehicle, in APCmin/+ mice. Our study demonstrated a rationale for further investigation of the combination of DT3 and aspirin for colorectal cancer prevention and therapy.

Photoactivated Controlled Dnazyme Platform for on-Demand Activation Sensitive Electrochemiluminescence mRNA Analysis.

Programming ultrasensitive and stimuli-responsive DNAzyme-based probes holds great potential for on-demand biomarker detection. Here, an optically triggered DNAzyme platform was reported for on-demand activation-sensitive electrochemiluminescence (ECL) c-myc mRNA analysis. In this design, the sensing and recognition function of the split DNAzyme (SDz) probe was silent by engineering a blocking sequence containing a photocleavable linker (PC-linker) group at a defined site that could be indirectly cleaved by 302 nm ultraviolet (UV) light. When the SDz probes were assembled on the Au nanoparticles and potassium (K) element doped graphitic carbon nitride nanosheet (K-doped g-C3N4) covered electrode, UV light activation induces the configurational switching and consequently the formation of an active DNAzyme probe with the help of target c-myc mRNA, allowing the cleavage of the substrate strand by magnesium ions (Mg2+). Thus, the release of a ferrocene (Fc)-labeled DNAzyme 2 strand contributed to an extreme ECL signal recovery. In the meantime, the released target c-myc mRNA combined another inactive SDz motif to form active DNAzyme and repeat the cyclic cleavage reaction, resulting in the signal amplification. Furthermore, according to the responses toward two other designed nPC-SDz and m-SDz probes, we demonstrated that controlled UV light mediated photoactivation of the DNAzyme biosensor "on demand" effectively constrained the ECL signal to the mRNA of interest. Moreover, false positive signals could also be avoided due to such a photoactivation design with UV light. Therefore, this study provided a simple methodology that may be broadly applicable for investigating the mRNA-associated physiological events that were difficult to access using traditional DNAzyme probes.

Liver cancer development driven by the AP-1/c-Jun~Fra-2 dimer through c-Myc

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death. HCC incidence is on the rise, while treatment options remain limited. Thus, a better understanding of the molecular pathways involved in HCC development has become a priority to guide future therapies. While previous studies implicated the Activator Protein-1 (AP-1) (Fos/Jun) transcription factor family members c-Fos and c-Jun in HCC formation, the contribution of Fos-related antigens (Fra-) 1 and 2 is unknown. Here, we show that hepatocyte-restricted expression of a single chain c-Jun~Fra-2 protein, which functionally mimics the c-Jun/Fra-2 AP-1 dimer, results in spontaneous HCC formation in c-Jun~Fra-2hep mice. Several hallmarks of human HCC, such as cell cycle dysregulation and the expression of HCC markers are observed in liver tumors arising in c-Jun~Fra-2hep mice. Tumorigenesis occurs in the context of mild inflammation, low-grade fibrosis, and Pparγ-driven dyslipidemia. Subsequent analyses revealed increased expression of c-Myc, evidently under direct regulation by AP-1 through a conserved distal 3' enhancer. Importantly, c-Jun~Fra-2-induced tumors revert upon switching off transgene expression, suggesting oncogene addiction to the c-Jun~Fra-2 transgene. Tumors escaping reversion maintained c-Myc and c-Myc target gene expression, likely due to increased c-Fos. Interfering with c-Myc in established tumors using the Bromodomain and Extra-Terminal motif inhibitor JQ-1 diminished liver tumor growth in c-Jun~Fra-2 mutant mice. Thus, our data establish c-Jun~Fra-2hep mice as a model to study liver tumorigenesis and identify the c-Jun/Fra-2-Myc interaction as a potential target to improve HCC patient stratification and/or therapy.

Rspo2-LGR4 exacerbates hepatocellular carcinoma progression via activation of Wnt/β-catenin signaling pathway

BackgroundThe leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) plays an important role in stem cell differentiation, organ development and cancer. Whether LGR4 affects the progression of hepatocellular carcinoma (HCC) remains unknown. This study aimed to reveal the role of LGR4 in HCC. MethodsClinical samples of HCC were collected to assess the expression of LGR4 and its correlation with patients’ clinical characteristics. The expression level of LGR4 in HCC cells was altered by pharmacological and genetic methods, and the role of LGR4 in HCC progression was analyzed by in vivo and in vitro assays. HCC was induced by diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) in wild-type and LGR4 deficient mice, the effect of LGR4 on HCC was examined by histopathological evaluation and biochemical assays. ResultsLGR4 expression was up-regulated in HCC samples, and its expression level was positively correlated with tumor size, microvascular invasion (MVI), TNM stage and pathological differentiation grade of HCC patients. In the mouse HCC model induced by DEN+CCl4, knockdown of LGR4 effectively inhibited the progression of HCC. Silencing of LGR4 inhibited the proliferation, migration, invasion, stem cell-like properties and Warburg effect of HCC cells. These phenotypes were promoted by R-spondin2 (Rspo2), an endogenous ligand for LGR4. Rspo2 markedly increased the nuclear translocation of β-catenin, whereas IWR-1, an inhibitor of Wnt/β-catenin signaling, reversed its effect. Deficiency of LGR4 significantly reduced the nuclear translocation of β-catenin and the expression of its downstream target genes cyclinD1 and c-Myc. ConclusionsLGR4 promotes HCC progression via Wnt/β-catenin signaling pathway.

Abstract 3039: Pan cancer therapeutic inhibition of oncogenic c-MYC by the engineered destabilized 3’UTR of c-MYC

Abstract c-MYC is a master transcription factor that is over expressed in more than 70% of human cancers. There is no clinically approved direct inhibitor of c-MYC. Recently, we reported the development of the engineered destabilized 3’UTR of MYC constructs which is a novel mRNA overwriting technology through which we directly destabilized and degraded c-MYC. Here we report that we have extended this technology in directly targeting and degrading c-MYC in multiple ethnically diverse triple negative breast cancer cell lines, ovarian cancers, neuroendocrine prostate cancers and pancreatic cancers. The constructs are specific, titratable, and directly engaged the c-MYC target validated by RNA transcript technologies. We have performed head-to-head IC50 dose dependent titration comparison with the engineered destabilized 3’UTR of c-MYC constructs and chemotherapies (DNA damaging agents, microtubules inhibiting drugs, alkylating agents) as well as immune checkpoint inhibitor. We found evidence that suggests that the engineered destabilized 3’UTR of c-MYC is likely to be superior to these therapies in pan cancers driven by c-MYC. Citation Format: Chidiebere U. Awah. Pan cancer therapeutic inhibition of oncogenic c-MYC by the engineered destabilized 3’UTR of c-MYC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3039.

Abstract 3044: Novel agents with efficacy against germline mutant RUNX1 familial platelet disorder with myeloid malignancy (FPD-MM)

Abstract RUNX1 is a master-transcriptional regulator involved in normal and malignant hematopoiesis. Germline, mono-allelic, missense mutations in RUNX1 cause large deletions or truncations that are mostly loss-of-function mutations. They cause familial platelet disorder (RUNX1-FPD), which evolves into myeloid malignancy (FPD-MM), e.g., MDS or AML. Although curative in some patients, allogeneic stem cell transplantation from matched, un-related donors carries the risk of GVHD and relapse in FPD-MM. Thus, there is a need for novel therapies to revert FPD-MM back to FPD. LINCS1000-CMap analysis, of the RUNX1 knockdown RNA-Seq signature revealed homoharringtonine (HHT or omacetaxine) and the anthelmintic fenbendazole (analog of mebendazole (MB)) as the top expression mimickers. In our current studies, we demonstrate that treatment with HHT or MB induced significantly greater loss of viability in seven patient-derived samples of FPD-MM vs RUNX1-FPD. Analyses conducted on BMA cells harvested longitudinally from the same patient at the RUNX1-FPD versus FPD-MM stage demonstrated greater chromatin accessibility in FPD-MM cells along with increased mRNA expressions of MYB, MECOM and BCL2. Following HHT treatment, scRNA-Seq analysis demonstrated a marked reduction in the HSC population, but concomitant increase in the macrophage population in FPD-MM. This was associated with positive enrichment of apoptosis signaling and TP53 targets gene-sets, while negatively enriching those of c-Myc targets. CyTOF analysis demonstrated that HHT treatment reduced c-Myc, EVI1, MCL1, BFL1 and CDK6 expression in phenotypically characterized FPD-MM stem-progenitor cells. Utilizing an FPD-MM sample harboring Runx1 K194N, also detected in other pedigree members with FPD or FPD-MM, we established the first ever cell line (GMR-AML1) expressing germline mtRUNX1. GMR-AML1 cells exhibited high surface expression of CD117 (c-KIT), CD123 (IL-3R) and CD33. GMR-AML1 cells in which Runx1 was knocked-out by CRISPR-gRNA were outcompeted in vitro and in vivo, by cells that retained Runx1. Treatment with HHT or MB induced loss of viability in GMR-AML1 cells (LD50: 40 or 330 nM, respectively). HHT also caused concordant decline in ATAC-Seq and RNA-Seq peaks in ribosomal genes involved in protein translation. Treatment with MB inhibited cell cycle and protein translation. CyTOF analysis revealed that MB treatment also depleted CDK6, RUNX1, c-Myc and EVI1 in phenotypically defined FPD-MM stem cells. Finally, in the GMR-AML1 cell xenograft, treatment with omacetaxine or MB significantly reduced AML burden and improved overall survival of NSG mice. These preclinical findings highlight the molecular features associated with progression of RUNX1-FPD to FPD-MM and highlight HHT or MB as effective against cellular models of FPD-MM. Citation Format: Christopher P. Mill, Warren C. Fiskus, Courtney D. DiNardo, Patrick K. Reville, John A. Davis, Christine E. Birdwell, Kaberi Das, Hanxi Hou, Koichi Takahashi, Sanam Loghavi, Joseph D. Khoury, Kapil Bhalla. Novel agents with efficacy against germline mutant RUNX1 familial platelet disorder with myeloid malignancy (FPD-MM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3044.

SLC26A9 promotes colorectal tumorigenesis by modulating Wnt/β-catenin signaling

Solute carrier family 26 member 9 (SLC26A9) is a member of the Slc26a family of multifunctional anion transporters that functions as a Cl- channel in parietal cells during acid secretion. We explored the role of SLC26A9 in colorectal cancer (CRC) and its related mechanisms through clinical samples from CRC patients, CRC cell lines and mouse models. We observed that SLC26A9 was expressed at low levels in the cytoplasm of adjacent tissues, polyps and adenomas but was significantly increased in colorectal adenocarcinoma. Moreover, increased levels of SLC26A9 were associated with a high risk of disease and poor prognosis. In addition, downregulation of SLC26A9 in CRC cells induced cell cycle arrest and apoptosis but inhibited cell proliferation and xenograft tumor growth both in vitro and in vivo. Mechanistic analysis revealed that SLC26A9 was colocalized with β-catenin in the nucleus of CRC cells. The translocation of these two proteins from the cytoplasm to the nucleus reflected the activation of Wnt/β-catenin signaling, and promoted the transcription of downstream target proteins, including CyclinD1, c-Myc and Snail, but inhibited the expression of cytochrome C (Cyt-c), cleaved Caspase9, cleaved Caspase3 and apoptosis-inducing factor (AIF). CRC is accompanied by alteration of epithelial mesenchymal transition (EMT) markers. Meanwhile, further studies showed that in SW48 cells, overexpressing SLC26A9 was cocultured with the β-catenin inhibitor XAV-939, β-catenin was downregulated, and EMT was reversed. Our study demonstrated SLC26A9 may be responsible for alterations in the proliferative ability and aggressive potential of CRC by regulating the Wnt/β-catenin signaling pathway.

Cellular nucleic acid binding protein facilitates cardiac repair after myocardial infarction by activating β-catenin signaling

The regenerative capacity of the adult mammalian heart is limited, while the neonatal heart is an organ with regenerative and proliferative ability. Activating adult cardiomyocytes (CMs) to re-enter the cell cycle is an effective therapeutic method for ischemic heart disease such as myocardial infarction (MI) and heart failure. Here, we aimed to reveal the role and potential mechanisms of cellular nucleic acid binding protein (CNBP) in cardiac regeneration and repair after heart injury. CNBP is highly expressed within 7 days post-birth while decreases significantly with the loss of regenerative ability. In vitro, overexpression of CNBP promoted CM proliferation and survival, whereas knockdown of CNBP inhibited these processes. In vivo, knockdown of CNBP in CMs robustly hindered myocardial regeneration after apical resection in neonatal mice. In adult MI mice, CM-specific CNBP overexpression in the infarct border zone ameliorated myocardial injury in acute stage and facilitated CM proliferation and functional recovery in the long term. Quantitative proteomic analysis with TMT labeling showed that CNBP overexpression promoted the DNA replication, cell cycle progression, and cell division. Mechanically, CNBP overexpression increased the expression of β-catenin and its downstream target genes CCND1 and c-myc; Furthermore, Luciferase reporter and Chromatin immunoprecipitation (ChIP) assays showed that CNBP could directly bind to the β-catenin promoter and promote its transcription. CNBP also upregulated the expression of G1/S-related cell cycle genes CCNE1, CDK2, and CDK4. Collectively, our study reveals the positive role of CNBP in promoting cardiac repair after injury, providing a new therapeutic option for the treatment of MI.

Stratification of Colorectal Patients Based on Survival Analysis Shows the Value of Consensus Molecular Subtypes and Reveals the CBLL1 Gene as a Biomarker of CMS2 Tumours.

The consensus molecular subtypes (CMSs) classification of colorectal cancer (CRC) is a system for patient stratification that can be potentially applied to therapeutic decisions. Hakai (CBLL1) is an E3 ubiquitin-ligase that induces the ubiquitination and degradation of E-cadherin, inducing epithelial-to-mesenchymal transition (EMT), tumour progression and metastasis. Using bioinformatic methods, we have analysed CBLL1 expression on a large integrated cohort of primary tumour samples from CRC patients. The cohort included survival data and was divided into consensus molecular subtypes. Colon cancer tumourspheres were used to analyse the expression of stem cancer cells markers via RT-PCR and Western blotting. We show that CBLL1 gene expression is specifically associated with canonical subtype CMS2. WNT target genes LGR5 and c-MYC show a similar association with CMS2 as CBLL1. These mRNA levels are highly upregulated in cancer tumourspheres, while CBLL1 silencing shows a clear reduction in tumoursphere size and in stem cell biomarkers. Importantly, CMS2 patients with high CBLL1 expression displayed worse overall survival (OS), which is similar to that associated with CMS4 tumours. Our findings reveal CBLL1 as a specific biomarker for CMS2 and the potential of using CMS2 with high CBLL1 expression to stratify patients with poor OS.

Expression of microRNA-155 in thalassemic erythropoiesis.

Ineffective erythropoiesis (IE) is the primary cause of anemia and associated pathologies in β-thalassemia. The characterization of IE is imbalance of erythroid proliferation and differentiation, resulting in increased erythroblast proliferation that fails to differentiate and gives rise to enucleate RBCs. MicroRNAs (miRs) are known to play important roles in hematopoiesis. miR-155 is a multifunctional molecule involved in both normal and pathological hematopoiesis, and its upregulation is observed in patients with β-thalassemia/HbE. However, the expression and function of miR-155, especially in β-thalassemia, have not yet been explored. To study miR-155 expression in thalassemia, erythroblast subpopulations, CD45-CD71+Ter-119+ and CD45-CD71-Ter-119+ were collected from β IVSII-654 thalassemic bone marrow. Additionally, a two-phase culture of mouse bone marrow erythroid progenitor cells was performed. Expression of miR-155 and predicted mRNA target genes, c-myc, bach-1 and pu-1, were determined by quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and normalized to small nucleolar RNA (snoRNA) 202 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. To investigate the effect of miR-155 expression, erythroblasts were transfected with miR-inhibitor and -mimic in order to elevate and eliminate miR-155 expression, respectively. Erythroid cell differentiation was evaluated by Wright-Giemsa staining and flow cytometry. miR-155 was upregulated, both in vivo and in vitro, during erythropoiesis in β-thalassemic mice. Our study revealed that gain- and loss of function of miR-155 were involved in erythroid proliferation and differentiation, and augmented proliferation and differentiation of thalassemic mouse erythroblasts may be associated with miR-155 upregulation. miR-155 upregulation in β-thalassemic mice significantly increased the percentage of basophilic and polychromatic erythroblasts. Conversely, a significant decrease in percentage of basophilic and polychromatic erythroblasts was observed in β-thalassemic mice transfected with anti-miR-155 inhibitor. We also examined the mRNA targets (c-myc, bach-1 and pu-1) of miR-155, which indicated that c-myc is a valid target gene of miR-155 that regulates erythroid differentiation. miR-155 regulates IE in β-thalassemia via c-myc expression controlling erythroblast proliferation and differentiation.

A recurrent de novo MAX p.Arg60Gln variant causes a syndromic overgrowth disorder through differential expression of c-Myc target genes

Cyclin D2 (CCND2) stabilization underpins a range of macrocephaly-associated disorders through mutation of CCND2 or activating mutations in upstream genes encoding PI3K-AKT pathway components. Here, we describe three individuals with overlapping macrocephaly-associated phenotypes who carry the same recurrent de novo c.179G>A (p.Arg60Gln) variant in Myc-associated factor X (MAX). The mutation, located in the b-HLH-LZ domain, causes increased intracellular CCND2 through increased transcription but it does not cause stabilization of CCND2. We show that the purified b-HLH-LZ domain of MAXArg60Gln (Max∗Arg60Gln) binds its target E-box sequence with a lower apparent affinity. This leads to a more efficient heterodimerization with c-Myc resulting in an increase in transcriptional activity of c-Myc in individuals carrying this mutation. The recent development of Omomyc-CPP, a cell-penetrating b-HLH-LZ-domain c-Myc inhibitor, provides a possible therapeutic option for MAXArg60Gln individuals, and others carrying similar germline mutations resulting in dysregulated transcriptional c-Myc activity.

Abstract B149: Anti-tumor and immunostimulatory properties of ST316, a peptide antagonist of beta-catenin for treatment of cancers with aberrant Wnt pathway activity

Abstract Beta-catenin represents a significant challenge as a therapeutic target due to its multifaceted role in tumor development as well as normal cell homeostasis. Dysregulation of the Wnt/beta-catenin signaling pathway is implicated in various cancers, including colorectal, hepatocellular, breast, ovarian, and pancreatic cancers. We developed ST316 as a peptide antagonist of the interaction of beta-catenin with BCL9, a co-activator implicated in oncogenic beta-catenin signaling. ST316 is currently being evaluated in a Phase 1-2 study (NCT05848739) that is currently enrolling patients with selected advanced solid tumors likely to harbor abnormalities of the Wnt/beta-catenin signaling pathway. In vitro studies indicate that ST316 selectively attenuates Wnt transcriptional activity in beta-catenin-dependent but not -independent cell lines. Gene expression profiling using RNA sequencing in beta-catenin mutant HCT116 cells and APC mutant Colo320DM cells demonstrates a significant downregulation of Wnt pathway target genes, oncogenic signature genes and pro-tumor immunologic gene sets within 24 hours of ST316 treatment. Consequently, ST316 induced a dose-dependent decrease in viability of beta-catenin-dependent cells while beta-catenin-independent cells were resistant, indicating that the anticancer effects of ST316 are mediated by inhibition of the Wnt/beta-catenin pathway. To evaluate the impact of ST316 in vivo, a 4T1 triple negative breast cancer orthotopic tumor model was employed. Once weekly injection of ST316 (5mg/kg SC) over eight weeks suppressed expression of Wnt target genes CDK4 and cMyc and resulted in a significant 84.3% inhibition of tumor growth compared to the control group. In addition to direct anti-tumor activity, ST316 triggers a pro-inflammatory immune microenvironment. ST316 induces polarization of human macrophages derived from peripheral blood mononuclear cells, resulting in >100-fold shift from immunosuppressive M2 macrophages towards the anti-tumor M1 phenotype. Co-cultures of M2 macrophages with T cells treated with ST316 demonstrate a significant three-fold increase in T-cell activation, as indicated by intracellular IFN-γ staining. Finally, ST316 enhances the anti-tumor activity of anti-PD-1 therapy in a syngeneic orthotopic 4T1 mouse model, where combination ST316 and anti-PD-1 antibody results in enhanced tumor growth inhibition compared to either single agent alone (p<0.01). These findings demonstrate the antitumor and immunostimulatory effects of ST316 and highlight its potential as a therapeutic agent for targeting cancers with aberrantly activated Wnt signaling pathways. Citation Format: Lila Ghamsari, Claudio Scuoppo, Erin Gallagher, Siok Leong, Mark Koester, Rick Ramirez, Zachary Mattes, Jerel Gonzales, Gene Merutka, Barry Kappel, Abi Vainstein, Jim Rotolo. Anti-tumor and immunostimulatory properties of ST316, a peptide antagonist of beta-catenin for treatment of cancers with aberrant Wnt pathway activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B149.