c-Myc Target Genes Research Articles

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.

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.

LncRNA CCAT2 Knockdown Alleviates Pressure Overload or Ang II-Induced Cardiac Hypertrophy Via Disruption of the Wnt/β-Catenin Signaling.

Sustained pathological cardiac hypertrophy (CH) is an independent risk factor for increased incidence and mortality of cardiovascular events. This research was designed to unravel the role of long non-coding RNA (LncRNA) CCAT2 in CH progression. Transverse aortic constriction (TAC) procedures were conducted to construct a pressure overload-induced in vivo CH model. Angiotensin II (Ang II) treatment was utilized to induce hypertrophic rat cardiomyocyte H9c2 cells. In vivo results showed that silencing of CCAT2 reduced cardiomyocyte surface area, alleviated cardiac fibrosis, and decreased β-MHC, ANP, and BNP levels in CH mouse models. In vitro results revealed that CCAT2 knockdown reduced cell surface area and attenuated β-MHC, ANP, and BNP levels in hypertrophic H9c2 cells. Besides, CCAT2 silencing decreased the levels of active β-catenin, phosphorylated-GSK-3β, and Wnt target genes (c-Myc, cyclinD1, and c-Jun) in CH mice and hypertrophic H9c2 cells. Importantly, treatment with the Wnt/β-catenin pathway activator LiCl reversed the suppression of CCAT2 knockdown on H9c2 cell surface area and MHC, ANP, and BNP levels. Collectively, CCAT2 silencing plays a protective role against CH through inactivating the Wnt/β-catenin signaling, which suggests that CCAT2 might become a promising therapeutic target for CH.

CXCR4 regulates macrophage M1 polarization by altering glycolysis to promote prostate fibrosis

BackgroundC-X-C receptor 4(CXCR4) is widely considered to be a highly conserved G protein-coupled receptor, widely involved in the pathophysiological processes in the human body, including fibrosis. However, its role in regulating macrophage-related inflammation in the fibrotic process of prostatitis has not been confirmed. Here, we aim to describe the role of CXCR4 in modulating macrophage M1 polarization through glycolysis in the development of prostatitis fibrosis.MethodsUse inducible experimental chronic prostatitis as a model of prostatic fibrosis. Reduce CXCR4 expression in immortalized bone marrow-derived macrophages using lentivirus. In the fibrotic mouse model, use adenovirus carrying CXCR4 agonists to detect the silencing of CXCR4 and assess the in vivo effects.ResultsIn this study, we demonstrated that reducing CXCR4 expression during LPS treatment of macrophages can alleviate M1 polarization. Silencing CXCR4 can inhibit glycolytic metabolism, enhance mitochondrial function, and promote macrophage transition from M1 to M2. Additionally, in vivo functional experiments using AAV carrying CXCR4 showed that blocking CXCR4 in experimental autoimmune prostatitis (EAP) can alleviate inflammation and experimental prostate fibrosis development. Mechanistically, CXCR4, a chemokine receptor, when silenced, weakens the PI3K/AKT/mTOR pathway as its downstream signal, reducing c-MYC expression. PFKFB3, a key enzyme involved in glucose metabolism, is a target gene of c-MYC, thus impacting macrophage polarization and glycolytic metabolism processes.

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.

Myct1 Alleviates Hypoxia-Induced Dysfunction by Regulating Pericyte Reprogramming

As one of the main causes of death from cardiovascular diseases, myocardial infarction has brought a heavy burden to society. However, its underlying mechanism has not been elucidated. Irreversible contraction of pericytes will cause capillary contraction, resulting in microcirculatory disorder, which finally lead to no-reflow after myocardial infarction. In the current study, we used hypoxia to simulate the environment of myocardial infarction in vitro, and found that under hypoxia conditions, the contractility of pericytes was significantly enhanced, the apoptosis rate and the content of angiogenic factors was increased. Besides, a target gene of c-Myc, Myct1, could regulate pericytes reprogramming into endothelial cells. After reprogramming of pericytes, the contractile ability was reduced, and the ability to promote angiogenesis was also inhibited. Moreover, pericyte reprogramming significantly reduced the expressions of myocardial enzymes CK-MB and LDH, troponin TnT and inflammatory cytokine IL-6. In conclusion, the reprogramming of pericytes regulated by Myct1 could alleviate the dysfunction of pericytes, thereby inhibiting the expression of myocardial infarction markers, which was conducive to improving the phenomenon of no-reflow after myocardial infarction.

RGS19 activates the MYH9/β-catenin/c-Myc positive feedback loop in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common fatal cancers worldwide, and the identification of novel treatment targets and prognostic biomarkers is urgently needed because of its unsatisfactory prognosis. Regulator of G-protein signaling 19 (RGS19) is a multifunctional protein that regulates the progression of various cancers. However, the specific function of RGS19 in HCC remains unclear. The expression of RGS19 was determined in clinical HCC samples. Functional and molecular biology experiments involving RGS19 were performed to explore the potential mechanisms of RGS19 in HCC. The results showed that the expression of RGS19 is upregulated in HCC tissues and is significantly associated with poor prognosis in HCC patients. RGS19 promotes the proliferation and metastasis of HCC cells in vitro and in vivo. Mechanistically, RGS19, via its RGS domain, stabilizes the MYH9 protein by directly inhibiting the interaction of MYH9 with STUB1, which has been identified as an E3 ligase of MYH9. Moreover, RGS19 activates β-catenin/c-Myc signaling via MYH9, and RGS19 is also a transcriptional target gene of c-Myc. A positive feedback loop formed by RGS19, MYH9, and the β-catenin/c-Myc axis was found in HCC. In conclusion, our research revealed that competition between RGS19 and STUB1 is a critical mechanism of MYH9 regulation and that the RGS19/MYH9/β-catenin/c-Myc feedback loop may represent a promising strategy for HCC therapy.

ARMC10 regulates mitochondrial dynamics and affects mitochondrial function via the Wnt/β-catenin signalling pathway involved in ischaemic stroke.

Mitochondrial dynamics has emerged as an important target for neuronal protection after cerebral ischaemia/reperfusion. Therefore, the aim of this study was to investigate the mechanism by which ARMC10 regulation of mitochondrial dynamics affects mitochondrial function involved in ischaemic stroke (IS). Mitochondrial morphology was detected by laser scanning confocal microscopy (LSCM), and mitochondrial ultrastructural alterations were detected by electron microscopy. The expression of mitochondrial dynamics-related genes Drp1, Mfn1, Mfn2, Fis1, OPA1 and ARMC10 and downstream target genes c-Myc, CyclinD1 and AXIN2 was detected by RT-qPCR. Western blot was used to detect the protein expression of β-catenin, GSK-3β, p-GSK-3β, Bcl-2 and Bax. DCFH-DA fluorescent probe was to detect the effect of ARMC10 on mitochondrial ROS level, Annexin V-FITC fluorescent probe was to detect the effect of ARMC10 on apoptosis, and ATP assay kit was to detect the effect of ARMC10 on ATP production. Mitochondrial dynamics was dysregulated in clinical IS samples and in the OGD/R cell model, and the relative expression of ARMC10 gene was significantly decreased in IS group (p < 0.05). Knockdown and overexpression of ARMC10 could affect mitochondrial dynamics, mitochondrial function and neuronal apoptosis. Agonist and inhibitor affected mitochondrial function and neuronal apoptosis by targeting Wnt/β-Catenin signal pathway. In the OGD/R model, ARMC10 affected mitochondrial function and neuronal apoptosis through the mechanism that regulates Wnt/β-catenin signalling pathway. ARMC10 regulates mitochondrial dynamics and protects mitochondrial function by activating Wnt/β-catenin signalling pathway, to exert neuroprotective effects.

Staphylococcal enterotoxin B exposed to pregnant rats inhibits the hedgehog signaling pathway in thymic T lymphocytes of the offspring

The Hedgehog (Hh) signaling pathway is involved in T cell differentiation and development and plays a major regulatory part in different stages of T cell development. A previous study by us suggested that prenatal exposure to staphylococcal enterotoxin B (SEB) changed the percentages of T cell subpopulation in the offspring thymus. However, it is unclear whether prenatal SEB exposure impacts the Hh signaling pathway in thymic T cells. In the present study, pregnant rats at gestational day 16 were intravenously injected once with 15 μg SEB, and the thymi of both neonatal and adult offspring rats were aseptically acquired to scrutinize the effects of SEB on the Hh signaling pathway. It firstly found that prenatal SEB exposure clearly caused the increased expression of Shh and Dhh ligands of the Hh signaling pathway in thymus tissue of both neonatal and adult offspring rats, but significantly decreased the expression levels of membrane receptors of Ptch1 and Smo, transcription factor Gli1, as well as target genes of CyclinD1, C-myc, and N-myc in Hh signaling pathway of thymic T cells. These data suggest that prenatal SEB exposure inhibits the Hh signaling pathway in thymic T lymphocytes of the neonatal offspring, and this effect can be maintained in adult offspring via the imprinting effect.

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.

Hyperoside Inhibits RNF8-mediated Nuclear Translocation of β-catenin to Repress PD-L1 Expression and Prostate Cancer.

Hyperoside is a flavonol glycoside isolated from Hypericum perforatum L. that has inhibitory effects on cancer cells; however, its effects on prostate cancer (PCa) remain unclear. Therefore, we studied the anti-PCa effects of hyperoside and its underlying mechanisms in vitro and in vivo. This study aimed to explore the mechanism of hyperoside in anti-PCa. 3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl Tetrazolium Bromide (MTT), transwell, and flow cytometry assays were used to detect PCa cell growth, invasion, and cell apoptosis. Immunoblot analysis, immunofluorescence, immunoprecipitation, and quantitative real-time PCR (qRT-PCR) were used to analyze the antitumor mechanism of hyperoside. Hyperoside inhibited PCa cell growth, invasion, and cell cycle and induced cell apoptosis. Furthermore, RING finger protein 8 (RNF8), an E3 ligase that assembles K63 polyubiquitination chains, was predicted to be a direct target of hyperoside and was downregulated by hyperoside. Downregulation of RNF8 by hyperoside impeded the nuclear translocation of β-catenin and disrupted the Wnt/β-catenin pathway, which reduced the expression of the target genes c-myc, cyclin D1, and programmed death ligand 1 (PD-L1). Decreased PD-L1 levels contributed to induced immunity in Jurkat cells in vitro. Finally, in vivo studies demonstrated that hyperoside significantly reduced tumor size, inhibited PD-L1 and RNF8 expression, and induced apoptosis in tumor tissues of a subcutaneous mouse model. Hyperoside exerts its anti-PCa effect by reducing RNF8 protein, inhibiting nuclear translocation of β-catenin, and disrupting the Wnt/β-catenin pathway, in turn reducing the expression of PD-L1 and improving Jurkat cell immunity.

Enhanced bacterial cancer therapy delivering therapeutic RNA interference of c-Myc

BackgroundBacterial cancer therapy was first trialled in patients at the end of the nineteenth century. More recently, tumour-targeting bacteria have been harnessed to deliver plasmid-expressed therapeutic interfering RNA to a range of solid tumours. A major limitation to clinical translation of this is the short-term nature of RNA interference in vivo due to plasmid instability. To overcome this, we sought to develop tumour-targeting attenuated bacteria that stably express shRNA by virtue of integration of an expression cassette within the bacterial chromosome and demonstrate therapeutic efficacy in vitro and in vivo.ResultsThe attenuated tumour targeting Salmonella typhimurium SL7207 strain was modified to carry chromosomally integrated shRNA expression cassettes at the xylA locus. The colorectal cancer cell lines SW480, HCT116 and breast cancer cell line MCF7 were used to demonstrate the ability of these modified strains to perform intracellular infection and deliver effective RNA and protein knockdown of the target gene c-Myc. In vivo therapeutic efficacy was demonstrated using the Lgr5creERT2Apcflx/flx and BlgCreBrca2flx/flp53flx/flx orthotopic immunocompetent mouse models of colorectal and breast cancer, respectively. In vitro co-cultures of breast and colorectal cancer cell lines with modified SL7207 demonstrated a significant 50–95% (P < 0.01) reduction in RNA and protein expression with SL7207/c-Myc targeted strains. In vivo, following establishment of tumour tissue, a single intra-peritoneal administration of 1 × 106 CFU of SL7207/c-Myc was sufficient to permit tumour colonisation and significantly extend survival with no overt toxicity in control animals.ConclusionsIn summary we have demonstrated that tumour tropic bacteria can be modified to safely deliver therapeutic levels of gene knockdown. This technology has the potential to specifically target primary and secondary solid tumours with personalised therapeutic payloads, providing new multi-cancer detection and treatment options with minimal off-target effects. Further understanding of the tropism mechanisms and impact on host immunity and microbiome is required to progress to clinical translation.

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.

O Knockdown de LncRNA CCAT2 Alivia a Sobrecarga de Pressão ou a Hipertrofia Cardíaca Induzida por Ang II por Meio da Interrupção da Sinalização Wnt/β-catenina

Abstract Background Sustained pathological cardiac hypertrophy (CH) is an independent risk factor for increased incidence and mortality of cardiovascular events. Objectives This research was designed to unravel the role of long non-coding RNA (LncRNA) CCAT2 in CH progression. Methods Transverse aortic constriction (TAC) procedures were conducted to construct a pressure overload-induced in vivo CH model. Angiotensin II (Ang II) treatment was utilized to induce hypertrophic rat cardiomyocyte H9c2 cells. Results In vivo results showed that silencing of CCAT2 reduced cardiomyocyte surface area, alleviated cardiac fibrosis, and decreased β-MHC, ANP, and BNP levels in CH mouse models. In vitro results revealed that CCAT2 knockdown reduced cell surface area and attenuated β-MHC, ANP, and BNP levels in hypertrophic H9c2 cells. Besides, CCAT2 silencing decreased the levels of active β-catenin, phosphorylated-GSK-3β, and Wnt target genes (c-Myc, cyclinD1, and c-Jun) in CH mice and hypertrophic H9c2 cells. Importantly, treatment with the Wnt/β-catenin pathway activator LiCl reversed the suppression of CCAT2 knockdown on H9c2 cell surface area and MHC, ANP, and BNP levels. Conclusions Collectively, CCAT2 silencing plays a protective role against CH through inactivating the Wnt/β-catenin signaling, which suggests that CCAT2 might become a promising therapeutic target for CH.

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 &amp;gt;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&amp;lt;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.

Novel Protac-Based EZH2 Degraders Effectively Target EZH2-Driven Lymphoid Malignancies By Abolishing Both Methylation-Dependent and -Independent Functions

Across various cancers, overexpression or mutation of the epigenetic silencer EZH2 correlates with invasive cancer growth, resistance and poor survival outcomes. While the tumorigenic role of EZH2 is largely attributed to the H3K27me3-mediated repression of tumor suppressor genes, reports have also established a methylation-independent role for EZH2 in the transcriptional activation of oncogenes, particularly in aggressive cancers like Natural Killer T-cell Lymphoma (NKTL). Here, traditional EZH2 inhibitors have been ineffective. We hypothesized that targeted depletion of the EZH2 protein will more effectively disrupt oncogenic signals arising from enzyme-independent activities of EZH2. Thus, we embarked on the development of EZH2 Proteolysis Targeting Chimeras (PROTACs) to trigger targeted ubiquitination and proteasome-mediated degradation of EZH2. No clinical EZH2 PROTAC has been reported yet. Our initial efforts focused on the iterative synthesis of a series of PROTAC candidates considering factors such as permeability, solubility and stable ternary complex formation. We developed a library of 53 compounds with various permutations of EZH2- and E3 ligase-binding ligands as well as linker lengths. Through extensive screening and testing of the EZH2 degradation activity via the Nano-Glo HiBiT lytic detection system and immunoblotting, we identified three lead Protac candidates. Compared to the clinical EZH2 inhibitor Tazemetostat (Taz), EZH2 PROTACs, C2911 and C1311 demonstrated higher cytotoxic activity in diffuse large B-cell lymphoma (DLBCL), a cancer in which Taz was evaluated during clinical trial. Importantly, C2911 and C1311 also successfully triggered EZH2 degradation in a dose-dependent manner thereby qualifying for further evaluation in NKTL. Previous work has highlighted a critical role for JAK3-mediated phosphorylation of EZH2 in promoting NKTL proliferation. Treatment of NKTL with C2911 and C1311 leads to a dose and time-dependent EZH2 degradation, loss of cell viability and increased apoptosis. These effects were absent with Taz and Pomalidomide treatments. The concomitant loss of H3K27me3 levels as well as PRC2 complex subunits, SUZ12 and EED suggest effective degradation of the EZH2 protein. We also observed that induction of NKTL cell death required a longer treatment period as compared to DLBCL. This may be potentially ascribed to the longer half-life of the EZH2 protein in NKTL as determined through cycloheximide treatment. In order to validate the mechanism of action of the EZH2 PROTACs, cells were either pretreated with pomalidomide or siRNA targeting cereblon. Indeed, this mitigated PROTAC-induced degradation of the EZH2 protein and rescued cancer cell death. Similarly, inhibiting the activity of the 26S proteasome through bortezomib also prevented EZH2 degradation. Characterization of EZH2 PROTAC-induced protein expression changes through immunoblotting highlighted that there was specific loss of H3K27me3 and H3K27me2 but not H3K27me1. Of note, we detected a concomitant decrease of Jak3 protein expression and STAT5 phosphorylation levels, more pronounced than PRC2 complex subunits. This suggests that the PROTAC may act by disrupting the non-canonical binding of EZH2 to Jak3 in NKTL. Furthermore, gene expression analyses revealed that EZH2 PROTAC treatment more effectively reactivated canonical EZH2 target genes such as MYT1 and ANPEP as compared to Taz. As the JAK-STAT-MYC axis is dysregulated in NK leukemia, we also evaluated the gene expression of c-Myc and c-Myc target gene, E2F1. EZH2 PROTAC treatment reduces the transcription levels of these oncogenes corroborating with loss of c-Myc protein expression. Studying the pharmacokinetics of the lead PROTACs in a mouse model revealed good plasma stability, half-life (&amp;gt; 4 h), and low clearance. Collectively, our data supports a critical role for EZH2 PROTACs in abolishing both the enzyme-dependent and independent activities of EZH2 through protein degradation, resulting in enhanced anti-cancer activity against DLBCL and NKTL. This may potentially benefit other EZH2-driven cancers. Further evaluation of the EZH2 PROTACs for clinical application is in progress. EZH2 PROTACs will next be tested in vivo. An in-depth investigation of the mechanisms stabilizing EZH2 protein levels and underlying PROTAC induced-cell death will be made to identify potential mechanisms of resistance.

Dysregulated Proteostasis Restrains the Developmental HSC Expansion in Fanconi Anemia

Hematopoietic stem cells (HSCs) require precise regulation of protein synthesis, processing, and degradation. Protein homeostasis (Proteostasis) is particularly critical during development, relying on chaperone proteins to maintain folding capacity in a highly proliferative HSC pool. Even modest changes in synthesis or degradation lead to the accumulation of unfolded proteins and compromise fetal liver HSC pool formation. Fanconi anemia (FA) is a multisystem disorder that manifests with bone marrow failure (BMF) early in life. Previous reports from our lab and others suggest that pre-natal HSC depletion in FA is a critical factor for the early onset of clinical BMF. To identify the cellular and molecular mechanisms underlying prenatal HSC defects in FA, we performed single-cell transcriptome profiling in both Fancd2 +/+and Fancd2 -/- FL samples. Gene set enrichment analysis (GSEA) of differentially expressed genes revealed significantly increased expression of c-Myc target genes in Fancd2 -/ - HSCs (normalized enrichment score, NES: 2.04, p&amp;lt;10 -5 FDR&amp;lt;10 -5), matched by an increase in c-Myc in Fancd2 -/- LT-HSC (CD150 + CD48 - Lineage - Sca-1 + c-KIT +) at the protein level (1.7-fold (p&amp;lt;0.05). Our sc-RNA Seq data further showed an increase in ribosome biogenesis (NES: 1.8, p&amp;lt;10 -5, FDR&amp;lt;10 -3) and translation (NES: 2.2, p&amp;lt;10 -5, FDR&amp;lt;10 -5) in Fancd2 -/- HSC, functionally corroborated by increased protein synthesis rates across Fancd2 -/- FL HSPC subpopulations (1.3-fold in LT- HSCs, p&amp;lt;0.01) using the in vivo O-Propargyl-puromycin (OPP) assay. Not surprisingly, the increased protein synthesis rates exceed the physiologic protein folding capacity, leading to elevated levels of unfolded proteins in Fancd2 -/- FL HSPCs by Tetraphenylethene maleimide (TPE-MI) stain. The aggregated misfolded proteins (aggresomes) in Fancd2 -/- FL HSPCs were also observed by immunofluorescence microscopy. Revisiting our scRNA Seq data, the observed proteotoxic stress was also reflected in the GSEA enrichment for unfolded protein response (UPR; NES: 1.30, p=0.01, FDR=0.01) as well as heat shock stress response (NES:1.7,p&amp;lt;0.0001, FDR=0.09), and coincided with upregulation of response to heat shock factor-1 ( Hsf-1; NES:1.86,p&amp;lt;0.0001, FDR=0.02) in Fancd2 -/- FL HSCs. Because chaperone supplementation has been shown to enhance HSC protein folding capacity, we serially injected the bile acid TUDCA (a chemical chaperone) into the pregnant dam. This not only significantly reduced unfolded protein levels, but indeed rescued FL HSC numbers in Fancd2 -/- littermates. Our observations in aggregate indicate that dysregulated proteostasis is a previously unrecognized pathogenic process constraining fetal Fancd2 -/- hematopoiesis. Protection of the HSPC proteome appears to be a new, non-canonical function of FANCD2, without immediate connection to the FA pathway and DNA crosslink repair. Importantly, we demonstrate the successful pharmacological rescue of the fetal Fancd2 -/- HSPC pool.