MYC Target Research Articles

The tumor immune microenvironment and therapeutic efficacy of trastuzumab deruxtecan in gastric cancer.

Trastuzumab deruxtecan (T-DXd), an anti-HER2 antibody-drug conjugate with a topoisomerase I inhibitor connected by a cleavable linker, has been approved for patients with HER2-positive gastric or gastroesophageal junction tumors. This biomarker study assessed HER2 expression and immune cell infiltration in relation to the therapeutic response to T-DXd. This retrospective analysis included samples from patients treated with T-DXd in three clinical trials. We performed RNA sequencing and multiplex immunohistochemistry on archival tumor samples obtained at baseline, during treatment, and after treatment. Flow cytometry was performed on tumor infiltrating immune cells freshly isolated from tumor tissues. Samples from 28 patients were included in this study. ERBB2 mRNA levels and CD20+ cell infiltration in tumors were significantly higher at baseline in responders than in nonresponders. Patients were classified into three biological groups based on their baseline tumor/stroma-infiltrating immune cell densities. Two groups reported similar response rates, but a trend was observed toward a shorter progression-free-survival in the group with more immunosuppressive regulatory T cells and programmed death-ligand 1 (PD-L1) expression at baseline. T-DXd treatment tended to increase the levels of tumor-infiltrating CD8+ T cells and PD1+CD8+ T cells, particularly in responders. Gene expression signatures of CTL and helper T cells increased during treatment, whereas signatures related to hypoxia, MYC targets, collagen formation, and interleukin-10 were downregulated. Our data suggest that HER2 expression levels and baseline tumor microenvironment (TME) characteristics correlate with T-DXd efficacy. Furthermore, this treatment may modulate TME immune profiles. Further validation using a larger sample size is warranted.

Design, Screening and Development of Asymmetric siRNAs Targeting the MYC Oncogene in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks hormone receptor and Her2 (ERBB2) expression, leaving chemotherapy as the only treatment option. The urgent need for targeted therapy for TNBC patients has led to the investigation of small interfering RNAs (siRNAs), which can target genes in a sequence-specific manner, unlike other drugs. However, the clinical translation of siRNAs has been hindered by the lack of an effective delivery system, except in the case of liver diseases. The MYC oncogene is commonly overexpressed in TNBC compared to other breast cancer subtypes. In this study, we used siRNA to target MYC in MDA-MB-231, MDA-MB-157, MDA-MB-436 and Hs-578T cells. We designed various symmetric and asymmetric (asiRNAs), screened them for in vitro efficacy, modified them for enhanced nuclease resistance and reduced off-target effects, and conjugated them with cholesterol (ChoL) and docosanoic acid (DCA) as a delivery system. DCA was conjugated to the 3' end of asiRNA by a cleavable phosphodiester linker for in vivo delivery. Our findings demonstrated that asiRNA-VP and Mod_asiRNA10-6 efficiently downregulated MYC and its downstream targets, including RRM2, RAD51 and PARP1. Moreover, in a tumor xenograft model, asiRNA-VP-DCA effectively knocked down MYC mRNA and protein expression. Remarkably, durable knockdown persisted for at least 46 days postdosing in mouse tumor xenografts, with no visible signs of toxicity, underscoring the safety of DCA-conjugated asiRNAs. In conclusion, this study developed novel asiRNAs, design platforms, validated modification patterns, and in vivo delivery systems specifically targeting MYC in TNBC.

Extrajunctional CLDN10 cooperates with LAT1 and accelerates clear cell renal cell carcinoma progression

Background & aimsIn addition to their adhesive properties, cell adhesion molecules such as claudins (CLDNs) exhibit signaling ability to organize diverse cellular events. Although the CLDN-adhesion signaling stimulates or inhibits cancer progression, the underlying mechanism remains poorly established. Here, we verified whether and how CLDN10 promotes intracellular signals and malignant phenotypes in clear cell renal cell carcinoma (ccRCC).MethodsWe developed a novel monoclonal antibody that specifically recognizes CLDN10. By immunohistochemistry using this antibody, the clinicopathological significance of aberrant CLDN10 expression in 165 ccRCC patients was determined. We next generated the ccRCC cells (786-O, ACHN, and OS-RC-2) expressing CLDN10, and compared their phenotypes with those of control cells. Immunoprecipitation-mass spectrometry was used to identify a CLDN10-interacting protein, followed by evaluation of its association with CLDN10 and loss-of-functions in ccRCC cells.ResultsHigh CLDN10 expression predicted poor outcome in ccRCC patients and represented an independent prognostic marker for cancer-specific survival. Cell surface CLDN10 promoted cell viability, proliferation, and migration of ccRCC cells, as well as their tumor growth. CLDN10 also activated mTOR signaling and expression of downstream targets, including MYC target genes. Notably, we found that CLDN10 forms a complex with an amino acid transporter, LAT1, and that CLDN10–LAT1 signaling facilitates malignant phenotypes in ccRCC cells. Structural prediction and immunoprecipitation analysis results strongly suggest an interaction between CLDN10-TM1 (transmembrane domain 1) and LAT1-TM4.ConclusionsWe conclude that CLDN10–LAT1 signaling drives ccRCC progression. Taken together with our previous findings on CLDN–Src-family kinases signaling, CLDNs propagate distinct intracellular signals depending on their association with different binding partners.

An oncoprotein CREPT functions as a co-factor in MYC-driven transformation and tumor growth

Understanding the mechanisms behind MYC-driven oncogenic transformation could pave the way for identifying novel drug targets. This study explored the role of CREPT in MYC-induced malignancy by generating MYC-transformed mouse embryonic fibroblasts (MEFs) with conditional CREPT deletion. Our results demonstrated that the loss of CREPT significantly impaired MYC-induced colony formation and cell proliferation, indicating that CREPT is essential for the malignant transformation of MEFs. Reintroducing CREPT in CREPT-deficient cells restored malignant properties. Furthermore, CREPT overexpression alone enhanced colony formation upon MYC induction but was insufficient to induce transformation without MYC, suggesting a cooperative interaction between CREPT and MYC in malignant transformation. CREPT deletion resulted in delayed cell cycle progression during the G2/M and S phases. CREPT enhanced the expression of MYC target genes by directly interacting with MYC through the CID domain of CREPT and the PEST domain of MYC. Arginine 34 of CREPT was identified as a critical residue for the interaction with MYC, and its mutation lost the ability of CREPT to promote MYC-driven colony formation and tumor growth in colorectal cancer models. Additionally, CREPT facilitated the recruitment of RNA Polymerase II (RNAPII) to MYC-binding promoters, promoting transcriptional initiation of MYC-targeted genes. Our study also revealed a strong correlation between CREPT and MYC expression in various human cancers, particularly in colorectal cancer, where their interaction appears to play a significant role in tumorigenesis. These findings suggest that the CREPT-MYC interaction is crucial for the progression of MYC-driven cancers and presents a potential target for therapeutic intervention.

EZH2 inhibition sensitizes MYC-high medulloblastoma cancers to PARP inhibition by regulating NUPR1-mediated DNA repair.

MYC-driven medulloblastomas (MB) are highly aggressive pediatric brain tumors with poor outcomes, and effective therapies remain limited despite intensive multimodal treatments. Targeting MYC directly is challenging, but exploiting MYC-mediated synthetic lethality holds promise. In this study, we investigated the combined effects of EZH2 and PARP inhibitors in MYC-high medulloblastoma and demonstrated that EZH2 inhibition significantly increased the sensitivity of MYC-high MB tumor cells to PARP inhibitors. This effect occurs through the upregulation of NUPR1, which promotes error-prone non-homologous end-joining (NHEJ) DNA repair by facilitating the recruitment of the XRCC4-LIG4 complex to DNA damage sites. This amplification of error-prone NHEJ DNA repair leads to genetic instability and eventual cell death in cells treated with the PARP inhibitor. The synergistic effect of EZH2 and PARP inhibitors was further validated in both in vitro and in vivo MB models without observed toxicity. These findings reveal a novel therapeutic strategy for MYC-high MB by co-targeting EZH2 and PARP, suggesting that this combination could potentially overcome the clinical challenges associated with this aggressive tumor subtype and warrants further investigation in clinical trials.

Lipid metabolism-related gene signature predicts prognosis and unveils novel anti-tumor drugs in specific type of diffuse large B cell lymphoma

BackgroundDiffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma which possess highly aggressive and heterogeneous. Despite advances in understanding heterogeneity and development of novel targeted agents, the prognosis of DLBCL patients remains unsatisfied. Lipids are crucial components of biological membranes and signal transduction while accumulating evidence has supported the vital roles of abnormal lipid metabolism in tumorigenesis. Furthermore, some related pathways could serve as prognostic biomarkers and potential therapeutic targets. However, the clinical significance of abnormal lipid metabolism reprogramming in DLBCL has not been investigated. In the current study, we developed a prognostic risk model for DLBCL based on the abnormal expressed lipid metabolism genes and moreover based on our risk model we classified patients with DLBCL into novel subtypes and identified potential drugs for DLBCL patients with certain lipid metabolism profiles.MethodsWe utilized univariate Cox regression analysis to identify the prognosis-related lipid metabolism genes, and then performed LASSO Cox regression to identify prognostic related lipid metabolism related genes. Multivariate cox regression was used to establish the prognostic model. Patients were divided in to high and low risk groups based on the median risk score. Immune cell infiltration and GSEA were used to identify the pathways between high and low risk groups. Oncopredict algorithm was utilized to identify potential drug for high-risk patients. In vitro cell apoptosis and viability analysis were employed to verify the specific tumor inhibition effects of AZD5153.ResultsNineteen survival related lipid metabolism genes TMEM176B, LAYN, RAB6B, MMP9, ATAD3B, SLC2A11, CD3E, SLIT2, SLC2A13, SLC43A3, CD6, SIRPG, NEK6, LCP2, CTTN, CXCL2, SNX22, BCL6 and FABP4 were identified and subjected to build the prognostic model which was further verified in four external microarray cohorts and one RNA seq cohorts. Tumor immune microenvironment analysis and GSEA results showed that the activation of MYC targets genes rather than immunosuppression contribute to the poor survival outcome of patients in the high-risk group. AZD5153, a novel bivalent BET bromodomain inhibitor which could inhibit the transcription of MYC and E2F exhibited specific antitumor function for cells with high-risk score.ConclusionsOur results provide the first lipid metabolism-based gene signature for predicting the survival of patients with DLBCL. Furthermore, by determining novel subtypes with our lipid metabolism prognostic model we illustrated that drugs that compromising MYC target genes rather than immune checkpoint inhibitors may be beneficial to DLBCL patients with certain lipid metabolism profiles.

Myc 9aaTAD activation domain binds to mediator of transcription with superior high affinity

The overexpression of MYC genes is frequently found in many human cancers, including adult and pediatric malignant brain tumors. Targeting MYC genes continues to be challenging due to their undruggable nature. Using our prediction algorithm, the nine-amino-acid activation domain (9aaTAD) has been identified in all four Yamanaka factors, including c-Myc. The predicted activation function was experimentally demonstrated for all these short peptides in transactivation assay. We generated a set of c-Myc constructs (1–108, 69–108 and 98–108) in the N-terminal regions and tested their ability to initiate transcription in one hybrid assay. The presence and absence of 9aaTAD (region 100–108) in the constructs strongly correlated with their activation functions (5-, 3- and 67-times respectively). Surprisingly, we observed co-activation function of the myc region 69–103, called here acetyl-TAD, previously described by Faiola et al. (Mol Cell Biol 25:10220–10234, 2005) and characterized in this study as a new domain collaborating with the 9aaTAD. We discovered strong interactions on a nanomolar scale between the Myc-9aaTAD activation domains and the KIX domain of CBP coactivator. We showed conservation of the 9aaTADs in the MYC family. In summary for the c-Myc oncogene, the acetyl-TAD and the 9aaTAD domains jointly mediated activation function. The c-Myc protein is largely intrinsically disordered and therefore difficult to target with small-molecule inhibitors. For the c-Myc driven tumors, the strong c-Myc interaction with the KIX domain represents a promising druggable target.

Drug prioritization identifies panobinostat as a tailored treatment element for patients with metastatic hepatoblastoma

BackgroundPatients with metastatic hepatoblastoma are treated with severely toxic first-line chemotherapies in combination with surgery. Yet, inadequate response of lung metastases to neo-adjuvant chemotherapy still compromises patient outcomes making new treatment strategies, tailored to more efficient lung clearance, mandatory.MethodsWe harnessed a comprehensive patient-derived xenograft platform and a variety of in vitro and in vivo assays to establish the preclinical and biological rationale for a new drug for patients with metastatic hepatoblastoma.ResultsThe testing of a library of established drugs on patient-derived xenografts identified histone deacetylase inhibitors, most notably panobinostat, to be highly efficacious on hepatoblastoma cells, as compared to non-cancerous cells. Molecularly, the anti-tumor effect of panobinostat is mediated by posttranslational obstruction of the MYC oncoprotein as a result of dual specificity phosphatase 1 upregulation, thereby leading to growth inhibition and programmed cell death. Of clinical importance, upregulation of the MYC target gene nucleophosmin 1 is indicative of response to panobinostat and associated with metastatic disease in patients with hepatoblastoma. The combination of panobinostat with the current SIOPEL 4 induction protocol, consisting of cisplatin and doxorubicin, revealed high synergies already at low nanomolar levels. The simulation of a clinical trial, with this combination therapy, in patient-derived xenograft models, and ultimately heterotypic lung metastasis mimics clearly underscored the potency of this approach.ConclusionIntegrated studies define MYC inhibition by panobinostat as a novel treatment element to be introduced into the therapeutic strategy for patients with metastatic hepatoblastoma.

EPCO-57. INTEGRATED ANALYSIS OF BULK AND SINGLE-CELL RNA SEQUENCING DATA OF PRIMARY AND METASTATIC PEDIATRIC MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is a highly aggressive and the most common brain tumor in childhood. MB presents a high intertumoral heterogeneity, with at least four molecular subgroups identified (SHH, WNT, Group 3, and Group 4). Metastatic MB, or Leptomeningeal Disease (LMD), is predominantly found in the MB Group 3 type. Although LMD represents a main clinical challenge, its molecular mechanisms remain poorly characterized. Recent research has shown that primary and MB metastasis diverge dramatically. Our work has focused on establishing therapy naïve Group 3 Patient-Derived Xenografts models of primary and metastatic Medulloblastoma to conduct transcriptomic profiling at the bulk and single-nuclei RNAseq levels to identify genetic drivers/pathways that sustain leptomeningeal disease compartment. Our results show various signaling pathways enriched across LMD models, such as MYC targets, unfolded protein response, and fatty acid metabolism. Using single-sample GSEA analysis (ssGSEA) and deconvolution approaches, we have also identified that our PDXes models retain neoplastic subpopulations previously identified in MB single-cell sequencing studies. Similarly, we have identified slight differences in cell subpopulation proportions between primary and leptomeningeal compartments. Our single-nuclei studies have confirmed these results and differentially expressed genes previously found in bulk RNAseq analyses. These results suggest the presence of cell populations enriched in the metastatic compartment with an aberrant transcription phenotype and adaptations in fatty acid metabolism to survive the leptomeningeal space. In conclusion, our work supports the notion that primary and LMD retain subpopulation clusters present in MB Group 3 tumors with different proportions in the metastatic compartment shown by bulkRNAseq deconvolution and single nuclei RNA analysis. We also show that primary and LMD are transcriptionally different, with various enriched pathways in the metastatic compartment and shared among LMD Group 3 PDX models. Through these approaches, we aim to elucidate the genetic dependencies of metastatic Medulloblastoma that will help for targeted therapies.

Delineating MYC-Mediated Escape Mechanisms from Conventional and T Cell-Redirecting Therapeutic Antibodies.

In B-cell malignancies, the overexpression of MYC is associated with poor prognosis, but its mechanism underlying resistance to immunochemotherapy remains less clear. In further investigations of this issue, we show here that the pharmacological inhibition of MYC in various lymphoma and multiple myeloma cell lines, as well as patient-derived primary tumor cells, enhances their susceptibility to NK cell-mediated cytotoxicity induced by conventional antibodies targeting CD20 (rituximab) and CD38 (daratumumab), as well as T cell-mediated cytotoxicity induced by the CD19-targeting bispecific T-cell engager blinatumomab. This was associated with upregulation of the target antigen only for rituximab, suggesting additional escape mechanisms. To investigate these mechanisms, we targeted the MYC gene in OCI-LY18 cells using CRISPR-Cas9 gene-editing technology. CRISPR-Cas9-mediated MYC targeting not only upregulated CD20 but also triggered broader apoptotic pathways, upregulating pro-apoptotic PUMA and downregulating anti-apoptotic proteins BCL-2, XIAP, survivin and MCL-1, thereby rendering tumor cells more prone to apoptosis, a key tumor-lysis mechanism employed by T-cells and NK-cells. Moreover, MYC downregulation boosted T-cell activation and cytokine release in response to blinatumomab, revealing a MYC-mediated T-cell suppression mechanism. In conclusion, MYC overexpressing tumor cells mitigated the efficacy of therapeutic antibodies through several non-overlapping mechanisms. Given the challenges associated with direct MYC inhibition due to toxicity, successful modulation of MYC-mediated immune evasion mechanisms may improve the outcome of immunotherapeutic approaches in B-cell malignancies.

Development of DuoMYC: a synthetic cell penetrant miniprotein that efficiently inhibits the oncogenic transcription factor MYC.

The master regulator transcription factor MYC is implicated in numerous human cancers, and its targeting is a long-standing challenge in drug development. MYC is a typical 'undruggable' target, with no binding pockets on its DNA binding domain and extensive intrinsically disordered regions. Rather than trying to target MYC directly with classical modalities, here we engineer synthetic miniproteins that can bind to MYC's target DNA, the enhancer box (E-Box), and potently inhibit MYC-driven transcription. We crafted the miniproteins via structure-based design and a combination of solid phase peptide synthesis and site-specific crosslinking. Our lead variant, DuoMYC, binds to E-Box DNA with high affinity (KD ~0.1 μM) and is able to enter cells and inhibit MYC-driven transcription with submicromolar potency (IC50=464 nM) as shown by reporter gene assay and confirmed by RNA sequencing. Notably, DuoMYC surpasses the efficacy of several other recently developed MYC inhibitors. Our results highlight the potential of engineered synthetic protein therapeutics for addressing challenging intracellular targets.

Development of DuoMYC: a synthetic cell penetrant miniprotein that efficiently inhibits the oncogenic transcription factor MYC

AbstractThe master regulator transcription factor MYC is implicated in numerous human cancers, and its targeting is a long‐standing challenge in drug development. MYC is a typical ‘undruggable’ target, with no binding pockets on its DNA binding domain and extensive intrinsically disordered regions. Rather than trying to target MYC directly with classical modalities, here we engineer synthetic miniproteins that can bind to MYC's target DNA, the enhancer box (E‐Box), and potently inhibit MYC‐driven transcription. We crafted the miniproteins via structure‐based design and a combination of solid phase peptide synthesis and site‐specific crosslinking. Our lead variant, DuoMYC, binds to E‐Box DNA with high affinity (KD ~0.1 μM) and is able to enter cells and inhibit MYC‐driven transcription with submicromolar potency (IC50=464 nM) as shown by reporter gene assay and confirmed by RNA sequencing. Notably, DuoMYC surpasses the efficacy of several other recently developed MYC inhibitors. Our results highlight the potential of engineered synthetic protein therapeutics for addressing challenging intracellular targets.

Inhibition of p300/CBP Overcomes BTK Inhibitor Resistance in MCL By Counteracting IL-6/JAK/STAT3 Signaling

Introduction: Although Bruton's tyrosine kinase inhibitors (BTKi) are effective against relapsed refractory mantle cell lymphoma (MCL), their limited efficacy and resistance remain clinical issues. Previously, we conducted CRISPR/Cas9 screening and found that p300/CBP, lysine acetyltransferases, are the genes responsible for one mechanism of BTKi resistance. A p300/CBP inhibitor A-485 with ibrutinib synergistically induced apoptosis and cell cycle arrest in ibrutinib-resistant MCL cells. Methods: To elucidate the synergistic mechanism, we conducted RNA sequencing using an ibrutinib-resistant MCL cell line, Granta519, and compared the transcriptomic profiles of Granta519, treated with ibrutinib, A-485, or both. Results: Genes related to the G2M checkpoint were upregulated when treated with the combination therapy. Pathway enrichment analysis revealed that the combination therapy downregulated pathways including Myc targets, TNF-α signaling via NF-κB, E2F targets, mTORC1 signaling, and IL-2/STAT5 signaling activation. Moreover, we observed that addition of A-485 restored the efficiency of ibrutinib to suppress downstream BCR signaling in Granta519 cells. We also detected IL6/JAK/STAT3 signaling activation by ibrutinib monotherapy, and the addition of A-485 significantly counteracted IL6/JAK/STAT3 signaling activation, suggesting that IL6/JAK/STAT3 signaling contributes to ibrutinib resistance. Pathway analysis showed that Myc was the most suppressed target by the combination therapy, reducing Myc mRNA levels by 90%. Using the MTS assay, we confirmed that Myc overexpression impaired the therapeutic effect of A-485 and significantly reduced the synergistic therapeutic effect of the combination therapy. This impairment suggests that the combination therapy achieves therapeutic effects by attenuating Myc function. We next focused on IL6/JAK/STAT3 signaling and tested ruxolitinib to inhibit JAK1/2. Interestingly, the IC50 value of ruxolitinib was lower in ibrutinib-resistant cells than ibrutinib-sensitive cells. To confirm that A-485 suppresses IL6/JAK/STAT3 signaling, we also confirmed that A-485 inhibited the nuclear translocation of STAT3 and the acetylation of STAT3 by Western blotting. Conclusion: p300/CBP inhibition overcomes BTKi resistance in MCL through IL-6/JAK/STAT3 signaling inhibition and attenuation of Myc transcription. Thus, p300/CBP inhibition could be a novel strategy for treating BTKi-resistant MCL.

Study of PC-002 (Sepantronium Bromide), a First-in-Class Inhibitor of Deubiquitinases (DUBs) Targeting Myc Degradation in Relapsed/Refractory c-Myc Rearranged High-Grade B Cell Lymphoma (HGBCL): Updated Phase 2 Results

Study of PC-002 (Sepantronium Bromide), a First-in-Class Inhibitor of Deubiquitinases (DUBs) Targeting Myc Degradation in Relapsed/Refractory c-Myc Rearranged High-Grade B Cell Lymphoma (HGBCL): Updated Phase 2 Results

Epigenetic features support the diagnosis of B-cell prolymphocytic leukemia and identify 2 clinicobiological subtypes

AbstractThe recognition of B-cell prolymphocytic leukemia (B-PLL) as a separate entity is controversial based on the current classification systems. Here, we analyzed the DNA methylome of a cohort of 20 B-PLL cases diagnosed according to the International Consensus Classification/World Health Organization HAEM4R guidelines, and compared them with chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), splenic marginal zone lymphoma (SMZL), and normal B-cell subpopulations. Unsupervised principal component analyses suggest that B-PLL is epigenetically distinct from CLL, MCL, and SMZL, which is further supported by robust differential methylation signatures in B-PLL. We also observe that B-PLL can be segregated into 2 epitypes with differential clinicobiological characteristics. B-PLL epitype 1 carries lower immunoglobulin heavy variable somatic hypermutation and a less profound germinal center–related DNA methylation imprint than epitype 2. Furthermore, epitype 1 is significantly enriched in mutations affecting MYC and SF3B1, and displays DNA hypomethylation and gene upregulation signatures enriched in MYC targets. Despite the low sample size, patients from epitype 1 have an inferior overall survival than those of epitype 2. This study provides relevant insights into the biology and differential diagnosis of B-PLL, and potentially identifies 2 subgroups with distinct biological and clinical features.

Prostaglandin E2-EP2/EP4 signaling induces immunosuppression in human cancer by impairing bioenergetics and ribosome biogenesis in immune cells

While prostaglandin E2 (PGE2) is produced in human tumor microenvironment (TME), its role therein remains poorly understood. Here, we examine this issue by comparative single-cell RNA sequencing of immune cells infiltrating human cancers and syngeneic tumors in female mice. PGE receptors EP4 and EP2 are expressed in lymphocytes and myeloid cells, and their expression is associated with the downregulation of oxidative phosphorylation (OXPHOS) and MYC targets, glycolysis and ribosomal proteins (RPs). Mechanistically, CD8+ T cells express EP4 and EP2 upon TCR activation, and PGE2 blocks IL-2-STAT5 signaling by downregulating Il2ra, which downregulates c-Myc and PGC-1 to decrease OXPHOS, glycolysis, and RPs, impairing migration, expansion, survival, and antitumor activity. Similarly, EP4 and EP2 are induced upon macrophage activation, and PGE2 downregulates c-Myc and OXPHOS in M1-like macrophages. These results suggest that PGE2-EP4/EP2 signaling impairs both adaptive and innate immunity in TME by hampering bioenergetics and ribosome biogenesis of tumor-infiltrating immune cells.

Caerin 1.1 and 1.9 peptides halt B16 melanoma metastatic tumours via expanding cDC1 and reprogramming tumour macrophages

BackgroundCancer immunotherapy, particularly immune checkpoint inhibitors (ICBs) such as anti-PD-1 antibodies, has revolutionised cancer treatment, although response rates vary among patients. Previous studies have demonstrated that caerin 1.1 and 1.9, host-defence peptides from the Australian tree frog, enhance the effectiveness of anti-PD-1 and therapeutic vaccines in a murine TC-1 model by activating tumour-associated macrophages intratumorally.MethodsWe employed a murine B16 melanoma model to investigate the therapeutic potential of caerin 1.1 and 1.9 in combination with anti-CD47 and a therapeutic vaccine (triple therapy, TT). Tumour growth of caerin-injected primary tumours and distant metastatic tumours was assessed, and survival analysis conducted. Single-cell RNA sequencing (scRNAseq) of CD45+ cells isolated from distant tumours was performed to elucidate changes in the tumour microenvironment induced by TT.ResultsThe TT treatment significantly reduced tumour volumes on the treated side compared to untreated and control groups, with notable effects observed by Day 21. Survival analysis indicated extended survival in mice receiving TT, both on the treated and distant sides. scRNAseq revealed a notable expansion of conventional type 1 dendritic cells (cDC1s) and CD4+CD8+ T cells in the TT group. Tumour-associated macrophages in the TT group shifted toward a more immune-responsive M1 phenotype, with enhanced communication observed between cDC1s and CD8+ and CD4+CD25+ T cells. Additionally, TT downregulated M2-like macrophage marker genes, particularly in MHCIIhi and tissue-resident macrophages, suppressing Cd68 and Arg1 expression across all macrophage types. Differential gene expression analysis highlighted pathway alterations, including upregulation of oxidative phosphorylation and MYC target V1 in Arg1hi macrophages, and activation of pro-inflammatory pathways in MHCIIhi and tissue-resident macrophages.ConclusionOur findings suggest that caerin 1.1 and 1.9, combined with immunotherapy, effectively modulate the tumour microenvironment in primary and secondary tumours, leading to reduced tumour growth and enhanced systemic immunity. Further investigation into these mechanisms could pave the way for improved combination therapies in advanced melanoma treatment.

RHOF promotes Snail1 lactylation by enhancing PKM2-mediated glycolysis to induce pancreatic cancer cell endothelial–mesenchymal transition

BackgroundThe influence of the small Rho GTPase Rif (RHOF) on tumor growth, glycolysis, endothelial–mesenchymal transition (EMT), and the potential mechanism of RHOF in pancreatic cancer (PC) were explored.MethodsRHOF expression in PC tissues and cells was assessed by qRT-PCR and western blotting. The viability, proliferation, apoptosis, migration, and invasion of PC cells were assessed using CCK-8, colony formation, EdU, flow cytometry, scratch, and Transwell assays. The expression of EMT- and glycolysis-related proteins was determined using western blotting. The potential mechanisms of action of RHOF in PC were identified using bioinformatic analysis. The effects of RHOF were assessed in vivo using a xenograft mouse model.ResultsPC cell proliferation, migration, and invasion are accelerated by RHOF overexpression, which inhibited apoptosis. RHOF overexpression promoted EMT and glycolysis as evidenced by a decrease in E-cadherin expression and an increase in N-cadherin, Vimentin, HK2, PKM2, and LDHA expression. Bioinformatic analysis indicated that RHOF activated EMT, glycolysis, and Myc targets and that c-Myc could bind to the PKM2 promoter. RHOF overexpression promotes the lactylation and nuclear translocation of Snail1. Silencing Snail1 reversed the promoting effects of RHOF and lactate on cell migration, invasion, and EMT. Moreover, in vivo tumor growth and EMT were inhibited by RHOF silencing.ConclusionRHOF plays an oncogenic role in PC. c-Myc is upregulated by RHOF and promotes PKM2 transcription. PKM2 further induces glycolysis, and the lactate produced by glycolysis causes the lactylation of Snail1, ultimately promoting EMT.

Reversible role of MIR654/3P and MIR9/3P in pathogenesis of Epstein-Barr Virus negative, but not positive, Burkitt's lymphoma.

The role of MIR654 in Burkitt's lymphoma (BL) and whether it impacts expression of MYC, and its downstream activated MIR9 is not known. Expression of MYC, MYCN, MYCL, MIR9/3P, MIR654/5P, and MIR654/3P were assessed by qRT-PCR in biopsy samples from Epstein-Barr virus (EBV)- and EBV+ BL patients and BL cell lines. Effects of modulation of MIR9/3P and MIR654/3P on cell proliferation, apoptosis and chemosensitivity were evaluated. Luciferase reporter assay was performed to validate putative target of MIR654/5P. Effects of MIR9/3P and MIR654/3P on tumor burden and disease outcome were evaluated using xenograft model of BL. Expression of MYC, MYCN, and MIR9/3P was higher in all BL patient samples and cell lines. Expression of MIR654/3P was downregulated in EBV- BL patient samples and cell lines compared to either noncancer lymphoid reactive hyperplasia (LRH) or EBV+ samples and cell lines. Additionally, MIR654/3P overexpression inhibited cell proliferation, induced apoptosis, and increased chemosensitivity in EBV- BL cell lines. Luciferase reporter assay confirmed that MYC is a target of MIR654/3P in both EBV- and EBV+ BL cell lines; however, the effect of MIR654/3P-mediated targeting of MYC is overridden in EBV+ cells. Administration of MIR654/3P mimic or MIR9/3P antagomir in the xenograft model decreased tumor burden and increased survival. Combined intervention with MIR654/3P mimic and MIR9/3P antagomir had synergistic action on decreasing tumor burden and improving disease outcome. MIR654/3P, as a putative tumor suppressor in EBV- BL, collaborated MIR9/3P might serve as a therapeutic agent to treat EBV- BL patients in combination with existing chemotherapy and immunotherapy regimes.

Modeling NK-cell lymphoma in mice reveals its cell-of-origin and microenvironmental changes and identifies therapeutic targets

Extranodal NK/T-cell lymphoma (ENKTCL) is an Epstein-Barr virus (EBV)-related neoplasm preferentially involving the upper aerodigestive tract. Here we show that NK-cell-specific Trp53 disruption in mice leads to the development of NK-cell lymphomas after long latency, which involve not only the hematopoietic system but also the salivary glands. Before tumor onset, Trp53 knockout causes extensive gene expression changes, resulting in immature NK-cell expansion, exclusively in the salivary glands. Both human and murine NK-cell lymphomas express tissue-resident markers, suggesting tissue-resident NK cells as their cell-of-origin. Murine NK-cell lymphomas show recurrent Myc amplifications and upregulation of MYC target gene signatures. EBV-encoded latent membrane protein 1 expression accelerates NK-cell lymphomagenesis and causes diverse microenvironmental changes, particularly myeloid propagation, through interferon-γ signaling. In turn, myeloid cells support tumor cells via CXCL16-CXCR6 signaling and its inhibition is effective against NK-cell tumors in vivo. Remarkably, KLRG1-expressing cells expand in the tumor and are capable of repopulating tumors in secondary recipients. Furthermore, targeting KLRG1 alone or combined with MYC inhibition using an eIF4 inhibitor is effective against NK-cell tumors. Therefore, our observations provide insights into the pathogenesis and highlight potential therapeutic targets, including CXCL16, KLRG1, and MYC, in ENKTCL, which can help improve its diagnostic and therapeutic strategies.