MYC Upregulation Research Articles

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.

Neoadjuvant sunitinib plus exemestane in post-menopausal women with hormone receptor-positive/HER2-negative early-stage breast cancer (SUT_EXE-08): a phase I/II trial

Neoadjuvant endocrine therapy (NET) for hormone receptor-positive (HR+) breast cancer might be as effective as chemotherapy, with a better toxicity profile. Blocking a crucial process such as angiogenesis with sunitinib may have a synergistic effect with NET. We aimed to assess the efficacy and safety of neoadjuvant sunitinib plus exemestane in early-stage HR+/HER2-negative breast cancer. In this phase I/II study, postmenopausal women with HR+/HER2− stage II-III breast cancer received neoadjuvant exemestane at conventional dose of 25mg plus sunitinib in a 3 + 3 design at 25mg (3/1weeks scheme) or 37.5mg continuous dose, for 6 months. Coprimary endpoints were the recommended dose of sunitinib combined with exemestane and objective response. Secondary endpoints included safety and biomarkers of early response. For 15 months, 18 patients were enrolled, 15 at sunitinib 25mg and 3 at 37.5mg. Median age was 73, 77% of patients had T2 tumors and 67% node-positive disease. The most common grade 2 toxicity was asthenia (44%), as was hypertension (22%) for grade 3. No grade 4–5 were reported. Twelve patients (66%) achieved an objective response. VEGFR-2 levels significantly decreased after one month of treatment. Differential gene expression analysis showed downregulation of ESR1, PGR and NAT1 in post-treatment samples and upregulation of EGFR, MYC, SFRP1, and FOXC1. PAM50 analysis on 83% of patients showed a prevalence of luminal A subtype, both in pre-treatment (63.6%) and post-treatment tumors (54.5%). Sunitinib plus exemestane was associated with substantial yet reversible toxicities, providing safety, efficacy and biological impact insights of combining an antiangiogenic drug with hormone therapy in early-stage breast cancer.Trial registration: Registered with ClinicalTrials.gov, NCT00931450. 02/07/2009

MYC upstream region orchestrates resistance to PI3K inhibitors in cancer cells through FOXO3a-mediated autophagic adaptation.

The MYC oncogene is frequently overexpressed in tumors and inhibition of its translation is considered an attractive therapeutic opportunity. Despite numerous reports proposing an internal ribosome entry site (IRES) within the MYC Upstream Region (MYC UR) to sustain MYC translation during cellular stress or chemotherapy, conflicting evidence remains regarding the validity of such a mechanism. Through comprehensive investigations in MYC-driven Colorectal Cancer (CRC) and Burkitt Lymphoma (BL) cells, we demonstrate that MYC UR does not facilitate cap-independent translation, but instead orchestrates resistance to PI3K inhibitors. Genomic deletion of MYC UR neither impacts MYC protein levels nor viability in CRC cells, either untreated or exposed to cellular stress. However, in response to PI3K inhibitors, MYC UR drives a FOXO3a-dependent transcriptional upregulation of MYC, conferring drug resistance. This resistance is mediated by enhanced autophagic flux, governed by MYC, and blockade of autophagy sensitizes CRC cells to PI3K inhibition in vitro and in vivo. Remarkably, BL cells lacking the translocation of MYC UR exhibit sensitivity to PI3K inhibitors, whereas MYC UR-translocated cells respond to these drugs only when autophagy is inhibited. These findings challenge previous notions regarding IRES-mediated translation and highlight a promising strategy to overcome resistance to PI3K inhibitors in MYC-driven malignancies, offering potential clinical implications for CRC and BL treatment.

MYC Induces Oncogenic Stress through RNA Decay and Ribonucleotide Catabolism in Breast Cancer.

Upregulation of MYC is a hallmark of cancer, wherein MYC drives oncogenic gene expression and elevates total RNA synthesis across cancer cell transcriptomes. Although this transcriptional anabolism fuels cancer growth and survival, the consequences and metabolic stresses induced by excess cellular RNA are poorly understood. Herein, we discover that RNA degradation and downstream ribonucleotide catabolism is a novel mechanism of MYC-induced cancer cell death. Combining genetics and metabolomics, we find that MYC increases RNA decay through the cytoplasmic exosome, resulting in the accumulation of cytotoxic RNA catabolites and reactive oxygen species. Notably, tumor-derived exosome mutations abrogate MYC-induced cell death, suggesting excess RNA decay may be toxic to human cancers. In agreement, purine salvage acts as a compensatory pathway that mitigates MYC-induced ribonucleotide catabolism, and inhibitors of purine salvage impair MYC+ tumor progression. Together, these data suggest that MYC-induced RNA decay is an oncogenic stress that can be exploited therapeutically. Significance: MYC is the most common oncogenic driver of poor-prognosis cancers but has been recalcitrant to therapeutic inhibition. We discovered a new vulnerability in MYC+ cancer where MYC induces cell death through excess RNA decay. Therapeutics that exacerbate downstream ribonucleotide catabolism provide a therapeutically tractable approach to TNBC (Triple-negative Breast Cancer) and other MYC-driven cancers.

Targeting STAT3 potentiates CDK4/6 inhibitors therapy in head and neck squamous cell carcinoma

Anti-CDK4/6 therapy has been employed for the treatment for head and neck squamous cell carcinoma (HNSCC) with CDK4/6 hyperactivation, but the response rate is relatively low. In this study, we first showed that CDK4 and CDK6 was over-expressed and conferred poor prognosis in HNSCC. Moreover, in RB-positive HNSCC, STAT3 signaling was activated induced by CDK4/6 inhibition and STAT3 promotes RB deficiency by upregulation of MYC. Thirdly, the combination of Stattic and CDK4/6 inhibitor results in striking anti-tumor effect in vitro and in Cal27 derived animal models. Additionally, phospho-STAT3 level negatively correlates with RB expression and predicts poor prognosis in patients with HNSCC. Taken together, our findings suggest an unrecognized function of STAT3 confers to CDK4/6 inhibitors resistance and presenting a promising combination strategy for patients with HNSCC.

Tigecycline Opposes Bortezomib Effect on Myeloma Cells Decreasing Mitochondrial Reactive Oxygen Species Production.

Multiple myeloma is an incurable plasma cell malignancy. Most patients end up relapsing and developing resistance to antineoplastic drugs, like bortezomib. Antibiotic tigecycline has activity against myeloma. This study analyzed tigecycline and bortezomib combination on cell lines and plasma cells from myeloma patients. Apoptosis, autophagic vesicles, mitochondrial mass, mitochondrial superoxide, cell cycle, and hydrogen peroxide were studied by flow cytometry. In addition, mitochondrial antioxidants and electron transport chain complexes were quantified by reverse transcription real-time PCR (RT-qPCR) or western blot. Cell metabolism and mitochondrial activity were characterized by Seahorse and RT-qPCR. We found that the addition of tigecycline to bortezomib reduces apoptosis in proportion to tigecycline concentration. Supporting this, the combination of both drugs counteracts bortezomib in vitro individual effects on the cell cycle, reduces autophagy and mitophagy markers, and reverts bortezomib-induced increase in mitochondrial superoxide. Changes in mitochondrial homeostasis and MYC upregulation may account for some of these findings. These data not only advise to avoid considering tigecycline and bortezomib combination for treating myeloma, but caution on the potential adverse impact of treating infections with this antibiotic in myeloma patients under bortezomib treatment.

Synergy between BRD9- and IKZF3-Targeting as a Therapeutic Strategy for Multiple Myeloma.

Progress in the treatment of multiple myeloma (MM) has resulted in improvement in the survival rate. However, there is still a need for more efficacious and tolerated therapies. We and others have shown that bromodomain-containing protein 9 (BRD9), a member of the non-canonical SWI/SNF chromatin remodeling complex, plays a role in MM cell survival, and targeting BRD9 selectively blocks MM cell proliferation and synergizes with IMiDs. We found that synergy in vitro is associated with the downregulation of MYC and Ikaros proteins, including IKZF3, and overexpression of IKZF3 or MYC could partially reverse synergy. RNA-seq analysis revealed synergy to be associated with the suppression of pathways associated with MYC and E2F target genes and pathways, including cell cycle, cell division, and DNA replication. Stimulated pathways included cell adhesion and immune and inflammatory response. Importantly, combining IMiD treatment and BRD9 targeting, which leads to the downregulation of MYC protein and upregulation of CRBN protein, was able to override IMiD resistance of cells exposed to iberdomide in long-term culture. Taken together, our results support the notion that combination therapy based on agents targeting BRD9 and IKZF3, two established dependencies in MM, represents a promising novel therapeutic strategy for MM and IMiD-resistant disease.

Abstract 6994: MerTK drives proliferation and metastasis activity in triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is characterized by the absence of estrogen, progesterone, and HER2 receptors. It exhibits a higher level of aggressiveness compared to other types of breast cancer, with a greater likelihood of recurrence following standard treatments such as surgery, chemotherapy, and radiation. Despite limited FDA-approved targeted therapies for TNBC, there is an ongoing need for additional molecular targeting agents.Our investigation focused on the role of the receptor tyrosine kinase MerTK and its impact on proliferation and invasion/metastatic potential. Immunohistochemistry analysis revealed that approximately 86% (128/148) expressed varying levels of MerTK in human TNBC samples, whereas approximately 50% (23/47) of TNBC patient-derived xenografts (PDX) expressed MerTK. Additionally, three established TNBC cell lines (BT549, MDAMB231, and MDAMB436) exhibited elevated MerTK expression levels.To further explore the role of MerTK in TNBC, we stably overexpressed MerTK in human TNBC cell lines SUM102 and SUM159, which naturally have low MerTK levels. The results demonstrated that MerTK overexpression led to increases in proliferative potential, robust in vivo tumor growth, heightened migration/invasion potential, and increased lung metastasis. NanoString nCounter analysis of MerTK-overexpressing SUM102 cells (SUM102-MerTK) revealed upregulation of several signaling pathways, including PI3k-Akt, PDGF, and Myc, ultimately driving cell cycle progression, reducing apoptosis, and enhancing cell survival in TNBC.Further investigation using a cytokine array showed increased endoglin production in SUM102-MerTK cells compared to vector control cells. This suggested that MerTK might be creating a conducive environment for increased proliferative and metastatic activity via elevated endoglin expression in TNBC. To ascertain the role of endoglin in increase proliferation and/or invasion potential, we knocked out endoglin in SUM102-MerTK cells. While endoglin knockout SUM102-MerTK cells exhibited similar growth to SUM102-MerTK cells, the number of lung metastases was significantly impacted, indicating that MerTK regulates invasion and metastasis through endoglin in TNBC. Collectively, our data suggests that MerTK regulates a unique proliferative signature allowing for robust tumor growth and increased metastatic potential through endoglin regulation. This suggests that targeting MerTK and endoglin simultaneously may be a viable approach for TNBC patients. Citation Format: Christine Glitchev, Mari Iida, Bridget E. Crossman, Kourtney L. Kostecki, Carlene A. Kranjac, Jillian M. Adams, Peng Liu, Irene Ong, David T. Yang, Irene Kang, Ravi Salgia, Deric L. Wheeler. MerTK drives proliferation and metastasis activity in triple negative breast cancer [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 6994.

Abstract 1101: A patient-derived xenograft in vitro platform identifies panobinostat as the most efficient drug to treat high-risk hepatoblastoma patients

Abstract Hepatoblastoma is the most common pediatric liver tumor, and the treatment of patients with preoperative chemotherapy and surgical resection of the tumor has led to survival rates of up to 83%. However, resistance of the primary tumor towards chemotherapy and the persistence of metastases still represents major challenges to achieve complete remission in high-risk patients, which is associated with poor prognosis. In order to identify new treatment options, we have used a newly developed in vitro drug-testing platform comprised of conventional cell lines and cultures from dissociated patient-derived xenografts of pediatric liver cancers to test standard-of-care medications used in the current PHITT trial as well as compounds targeting a wide range of different cellular pathways. We identified histone deacetylase (HDAC) inhibitors as a promising drug family to reduce tumor cell growth in a dose-dependent manner, with panobinostat being the most effective drug tested. Subsequent in vitro assays showed that panobinostat reduced short- and long-term proliferation, retarded three-dimensional spheroid growth and ultimately induced apoptosis of hepatoblastoma cells. RNA sequencing indicated that panobinostat results in downregulation of MYC target genes and upregulation of the dual specificity phosphatase 1. Of clinical relevance, the combination of panobinostat with doxorubicin revealed a strong synergistic effect, even stronger than the one of cisplatin and doxorubicin, which is currently given to high-risk hepatoblastoma patients. Moreover, increased expression of HDAC4 and HDAC11 in primary tumor tissues was significantly associated with metastatic disease. Our results strongly suggest panobinostat to be used in future therapeutic trials for high-risk hepatoblastoma patients, especially when combined with doxorubicin. Citation Format: Salih Demir, Emilie Indersie, Sophie Branchereau, Eiso Hiyama, Stefano Cairo, Roland Kappler. A patient-derived xenograft in vitro platform identifies panobinostat as the most efficient drug to treat high-risk hepatoblastoma patients [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 1101.

Abstract 6601: Menin drives oncogenesis in Ewing sarcoma cells by activating transcription of key metastatic factors

Abstract Ewing sarcoma (EwS) is a bone and soft tissue tumor that is driven by the fusion of FET and ETS genes, most commonly resulting in creation of the EWS::FLI1 chimeric oncoprotein. The scaffolding protein Menin regulates transcription through interactions with transcription factors and chromatin modifying enzymes and has both oncogenic and tumor suppressive roles, depending on the cellular context. The half-life of Menin is prolonged in EwS cells leading to high levels of the protein that is dependent on EWS::FLI1 expression. To investigate tumorigenic functions of Menin in EwS, we used CRISPR/Cas9 to generate clonal A673 and TC32 Menin knockout cells. Consistent with our earlier studies using shRNA-mediated knockdown, loss of Menin had little effect on proliferation in 2-D culture but resulted in reduced anchorage-independent growth in soft agar assays. When MEN1-KO cells were injected into NSG mice, either subcutaneously or by tail vein, tumor formation was delayed. Injection of MEN1-KO cells into the renal subcapsule of NSG mice resulted in locally invasive tumors but their capacity to metastasize to the liver was reduced compared to control cells. To elucidate how loss of Menin led to diminished tumorigenic and metastatic potential, we performed RNA-seq of control and MEN1-KO cells. This analysis revealed significant and reproducible down-regulation of MYC target genes and up-regulation of the epithelial to mesenchymal transition pathway and extracellular matrix protein-encoding genes in MEN1-KO cells. To determine if genes altered in MEN1-KO cells were dependent on the role of Menin in Menin/MLL methyltransferase complexes, we analyzed transcriptomes of EwS cells that had been exposed to the Menin/MLL interaction inhibitor, VTP-50469. Only a subset of genes altered by the MEN1-KO were also altered by the Menin/MLL inhibitor, suggesting that Menin regulates gene expression in EwS cells by both MLL-dependent and MLL-independent mechanisms. To test this, we performed CUT&RUN to define Menin binding sites and discovered that Menin binds not only at H3K4me3-marked gene promoters but also at intragenic enhancers marked by H3K27Ac. Importantly, comparison of RNA-seq data revealed substantial overlap between Menin-regulated and established EWS::FLI1 target genes and we identified that a subset of EWS::FLI1-bound intragenic enhancers are also bound by Menin. Significantly, these co-bound, co-regulated loci encode for pro-metastatic genes, including IL1RAP, STEAP1, and CAV1. Finally, we found that Menin and EWS::FLI1 co-immunoprecipitate in EwS nuclear extracts suggesting that they may exist in complex with one another at sites of shared gene regulation. Taken together, these data indicate that Menin promotes EwS metastasis and that this is achieved by its function as a scaffolding protein that augments the transcriptional activity of EWS::FLI1 at intragenic enhancers of pro-metastasis genes. Citation Format: Katherine A. Braun, Aya Miyaki, Nicolas M. Garcia, Darleen Tu, Feinan Wu, Jay F. Sarthy, Elizabeth R. Lawlor. Menin drives oncogenesis in Ewing sarcoma cells by activating transcription of key metastatic factors [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 6601.

Abstract 4450: MYC upregulates MCT4 expression and promotes metabolic reprogramming and enhanced lactate utilization in LKB1-deficient NSCLC

Abstract STK11, which encodes the serine/threonine kinase LKB1, is the second most commonly altered tumor suppressor in non-small cell lung cancer (NSCLC). LKB1 loss is frequently co-mutated with KRAS and associated with primary resistance to immunotherapy, which is partially due to MCT4-dependent lactate secretion. However, the mechanisms driving MCT4 elevation and metabolic reprogramming remains unclear. We hypothesized that increased MYC expression, which is associated with LKB1 loss, plays a pivotal role in metabolic alterations, promoting lactate incorporation and maintaining redox homeostasis. To explore the impact of lactate on tumor cells with LKB1 loss, we performed metabolic profiling on KRAS mutant (K) murine lung cancer cells with or without LKB1 knockout (KL) treated with either glucose or lactate as the carbon source. PLS analysis indicated that there was no notable difference between K and KL cells after glucose treatment. However, we observed significant changes in metabolites between K and KL cells when treated with lactate. Differentially altered metabolites included TCA-related components such as acetyl-CoA and glutamate, indicating enhanced lactate utilization in KL tumor cells. We also noticed that KL cells showed increased oxidative phosphorylation (OXPHOS) with the addition of lactate, with increased GSH/GSSH and NADPH/NADP+ ratios, suggesting that, as compared to LKB1-intact cells, LKB1-deficient cells had an enhanced ability to utilize OXPHOS and maintain redox homeostasis with lower levels of ROS generation when utilizing lactate as an energy source. However, in KL cells, MYC knockout (KO) or MCT4 KO partially abolished the lactate-induced OXPHOS and increased ROS. Moreover, [U-13C]lactate tracing revealed that isotopologues were significantly enriched in TCA components in KL cells, and this was reversed by either MYC KO or MCT4 KO, suggesting that MYC and MCT4 were critical for lactate reutilization in KL tumor cells. We observed that MYC could directly bind to the promoter region and transcriptionally activate MCT4 expression, and restoring MCT4 in MYC KO cells enhanced lactate incorporation and decreased ROS levels. Finally, we injected KL murine tumor cells with or without MYC/MCT4 KO into mice and infused [U-13C]lactate to detect the isotopologues distribution in vivo. KL tumors showed significantly enhanced lactate incorporation as compared to K tumors, while MYC KO or MCT4 KO reversed these phenotypes. Collectively, our data indicates that in LKB1-deficient tumors, upregulation of MYC promotes MCT4 expression and increases lactate incorporation and utilization, and that can be inhibited by targeting MYC or MCT4. These findings provide insight into the mechanisms driving the metabolic reprogramming and aggressive phenotype of KRAS-mutant LKB1-deficient tumors and identify a novel therapeutic strategy for this recalcitrant subgroup. Citation Format: Yu Qian, David Molkentine, Yifan Kong, Qian Huang, Chendong Yang, Alissa Poteete, Alvaro P. Guimaraes, Peixin Jiang, Ferdinandos Skoulidis, Alexandre Reuben, John D. Minna, Ralph J. DeBerardinis, John V. Heymach. MYC upregulates MCT4 expression and promotes metabolic reprogramming and enhanced lactate utilization in LKB1-deficient NSCLC [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 4450.

Functional characterization of cooperating MGA mutations in RUNX1::RUNX1T1 acute myeloid leukemia

MGA (Max-gene associated) is a dual-specificity transcription factor that negatively regulates MYC-target genes to inhibit proliferation and promote differentiation. Loss-of-function mutations in MGA have been commonly identified in several hematological neoplasms, including acute myeloid leukemia (AML) with RUNX1::RUNX1T1, however, very little is known about the impact of these MGA alterations on normal hematopoiesis or disease progression. We show that representative MGA mutations identified in patient samples abolish protein-protein interactions and transcriptional activity. Using a series of human and mouse model systems, including a newly developed conditional knock-out mouse strain, we demonstrate that loss of MGA results in upregulation of MYC and E2F targets, cell cycle genes, mTOR signaling, and oxidative phosphorylation in normal hematopoietic cells, leading to enhanced proliferation. The loss of MGA induces an open chromatin state at promoters of genes involved in cell cycle and proliferation. RUNX1::RUNX1T1 expression in Mga-deficient murine hematopoietic cells leads to a more aggressive AML with a significantly shortened latency. These data show that MGA regulates multiple pro-proliferative pathways in hematopoietic cells and cooperates with the RUNX1::RUNX1T1 fusion oncoprotein to enhance leukemogenesis.

WWOX promotes osteosarcoma development via upregulation of Myc

Osteosarcoma is an aggressive bone tumor that primarily affects children and adolescents. This malignancy is highly aggressive, associated with poor clinical outcomes, and primarily metastasizes to the lungs. Due to its rarity and biological heterogeneity, limited studies on its molecular basis exist, hindering the development of effective therapies. The WW domain-containing oxidoreductase (WWOX) is frequently altered in human osteosarcoma. Combined deletion of Wwox and Trp53 using Osterix1-Cre transgenic mice has been shown to accelerate osteosarcoma development. In this study, we generated a traceable osteosarcoma mouse model harboring the deletion of Trp53 alone (single-knockout) or combined deletion of Wwox/Trp53 (double-knockout) and expressing a tdTomato reporter. By tracking Tomato expression at different time points, we detected the early presence of tdTomato-positive cells in the bone marrow mesenchymal stem cells of non-osteosarcoma-bearing mice (young BM). We found that double-knockout young BM cells, but not single-knockout young BM cells, exhibited tumorigenic traits both in vitro and in vivo. Molecular and cellular characterization of these double-knockout young BM cells revealed their resemblance to osteosarcoma tumor cells. Interestingly, one of the observed significant transcriptomic changes in double-knockout young BM cells was the upregulation of Myc and its target genes compared to single-knockout young BM cells. Intriguingly, Myc-chromatin immunoprecipitation sequencing revealed its increased enrichment on Myc targets, which were upregulated in double-knockout young BM cells. Restoration of WWOX in double-knockout young BM cells reduced Myc protein levels. As a prototype target, we demonstrated the upregulation of MCM7, a known Myc target, in double-knockout young BM relative to single-knockout young BM cells. Inhibition of MCM7 expression using simvastatin resulted in reduced proliferation and tumor cell growth of double-knockout young BM cells. Our findings reveal BM mesenchymal stem cells as a platform to study osteosarcoma and Myc and its targets as WWOX effectors and early molecular events during osteosarcomagenesis.

SNAP25-induced MYC upregulation promotes high-grade neuroendocrine lung carcinoma progression.

This study investigated the expression and role of Synaptosome associated protein 25 (SNAP25) in high-grade neuroendocrine carcinoma (HGNEC). We used differentially expressed analysis and weighted gene co-expression network analysis (WGCNA) to identify key genes and modules in HGNEC. KEGG and GO analyses helped understand these genes' roles, and ROC curves assessed their diagnostic value. We also studied SNAP25's relation to immune infiltration and confirmed findings with in vitro and vivo experiments and datasets. WGCNA identified 595 key genes related to pathways like MAPK signaling, GABAergic synapse, and cancer-related transcriptional misregulation. Top genes included SNAP25, MYC, NRXN1, GAD2, and SYT1. SNAP25 was notably associated with M2 macrophage infiltration. Dataset GSE40275 confirmed SNAP25's high expression and poor prognosis in HGNEC. qRT-PCR and WB analyses showed increased SNAP25 and c-MYC levels in HGNEC, promoting MEK/ERK pathway activity. Reducing SNAP25 decreased H1299 cell proliferation, migration, invasion, and levels of c-MYC, MEK, and ERK. Finally, in vivo experiments further confirmed that SNAP25 knockout can inhibit tumor growth. SNAP25 regulates c-MYC activation by stimulating the MEK/ERK pathway, ultimately influencing the development of HGNEC.

Collaborative effect of Csnk1a1 haploinsufficiency and mutant p53 in Myc induction can promote leukemic transformation

AbstractIt is still not fully understood how genetic haploinsufficiency in del(5q) myelodysplastic syndrome (MDS) contributes to malignant transformation of hematopoietic stem cells. We asked how compound haploinsufficiency for Csnk1a1 and Egr1 in the common deleted region on chromosome 5 affects hematopoietic stem cells. Additionally, Trp53 was disrupted as the most frequently comutated gene in del(5q) MDS using CRISPR/Cas9 editing in hematopoietic progenitors of wild-type (WT), Csnk1a1–/+, Egr1–/+, Csnk1a1/Egr1–/+ mice. A transplantable acute leukemia only developed in the Csnk1a1–/+Trp53–edited recipient. Isolated blasts were indefinitely cultured ex vivo and gave rise to leukemia after transplantation, providing a tool to study disease mechanisms or perform drug screenings. In a small-scale drug screening, the collaborative effect of Csnk1a1 haploinsufficiency and Trp53 sensitized blasts to the CSNK1 inhibitor A51 relative to WT or Csnk1a1 haploinsufficient cells. In vivo, A51 treatment significantly reduced blast counts in Csnk1a1 haploinsufficient/Trp53 acute leukemias and restored hematopoiesis in the bone marrow. Transcriptomics on blasts and their normal counterparts showed that the derived leukemia was driven by MAPK and Myc upregulation downstream of Csnk1a1 haploinsufficiency cooperating with a downregulated p53 axis. A collaborative effect of Csnk1a1 haploinsufficiency and p53 loss on MAPK and Myc upregulation was confirmed on the protein level. Downregulation of Myc protein expression correlated with efficient elimination of blasts in A51 treatment. The “Myc signature” closely resembled the transcriptional profile of patients with del(5q) MDS with TP53 mutation.

A Critical Interpretive Synthesis of the Role of Arecoline in Oral Carcinogenesis: Is the Local Cholinergic Axis a Missing Link in Disease Pathophysiology?

Arecoline is the primary active carcinogen found in areca nut and has been implicated in the pathogenesis of oral squamous cell carcinoma (OSCC) and oral submucous fibrosis (OSF). For this study, we conducted a stepwise review process by combining iterative scoping reviews with a post hoc search, with the aim of identifying the specific mechanisms by which arecoline initiates and promotes oral carcinogenesis. Our initial search allowed us to define the current trends and patterns in the pathophysiology of arecoline-induced OSF and OSCC, which include the induction of cell proliferation, facilitation of invasion, adhesion, and migration, increased collagen deposition and fibrosis, imbalance in immune and inflammatory mechanisms, and genotoxicity. Key molecular pathways comprise the activation of NOTCH1, MYC, PRDX2, WNT, CYR61, EGFR/Pl3K, DDR1 signaling, and cytokine upregulation. Despite providing a comprehensive overview of potential pathogenic mechanisms of OSF, the involvement of molecules functioning as areca alkaloid receptors, namely, the muscarinic and nicotinic acetylcholine receptors (AChRs), was not elucidated with this approach. Accordingly, our search strategy was refined to reflect these evidence gaps. The results of the second round of reviews with the post hoc search highlighted that arecoline binds preferentially to muscarinic AChRs, which have been implicated in cancer. Consistently, AChRs activate the signaling pathways that partially overlap with those described in the context of arecoline-induced carcinogenesis. In summary, we used a theory-driven interpretive review methodology to inform, extend, and supplement the conventional systematic literature assessment workflow. On the one hand, the results of this critical interpretive synthesis highlighted the prevailing trends and enabled the consolidation of data pertaining to the molecular mechanisms involved in arecoline-induced carcinogenesis, and, on the other, brought up knowledge gaps related to the role of the local cholinergic axis in oral carcinogenesis, thus suggesting areas for further investigation.

The Universal Antitumor Activity of TAS0612, a Triple Inhibitor for RSK, AKT, and S6K, in B-Cell Lymphomas Regardless of Disease Subtype

B-cell lymphomas (BCLs) are the most common disease entity among hematological malignancies and have various genetically and molecularly distinct subtypes. Although the prognosis of most BCL subtypes has been greatly improved by the advancements of disease subtype-oriented molecular-targeted therapeutics, there remain patients who are incurable with currently available therapeutics. Therefore, further development of new therapies that are effective regardless of disease subtypes is still awaited. 3-phosphoinositide dependent protein kinase 1 (PDPK1) is the master serine-threonine kinase of >20 AGC kinases, including RSK family proteins, AKT, S6K, and several non-AGC kinases. By regulating a series of downstream molecules and mediating the RAS/ERK/RSK2 and PI3K/AKT pathways, PDPK1 plays a pivotal role in disease development and progression in various malignancies. We have also shown that PDPK1/RSK2 axis plays pivotal roles in the pathophysiology of multiple myeloma and mantle cell lymphoma (MCL) (Chinen Y, Cancer Res 2014; Shimura Y, Mol Cancer Ther 2012; Matsumura-Kimoto Y, Cancer Med 2020). In the search for novel molecular targeted therapy for BCLs, these provoked us to investigate the role of PDPK1 and its effector molecules as the therapeutic targets in a broad range of BCLs. We first revealed that the blockade of phosphoinositide-dependent kinase-1 (PDPK1) using its specific inhibitor BX-912 induces a growth inhibition via G2/M cell cycle arrest and the induction of apoptosis in all 8 BCL-derived cell lines examined, including two from activated B-cell-like diffuse large B cell lymphoma (DLBCL), two from double expressor DLBCL, two from Burkitt lymphoma, and two from follicular lymphoma (FL). By using specific inhibitors for the downstream kinases of PDPK1, we also demonstrated that RSK2, AKT, and S6K, but not other PDPK1 substrates, such as PLK1, SGK, or PKC, plays critical roles for cell survival and proliferation as target molecules of PDPK1 in these cell lines examined. In addition, RSK2 plays a central role and AKT and S6K play subsidiary functional roles as the downstream effectors of PDPK1. Following these results, we next investigated the anti-BCL efficacy of TAS0612 which is the triple inhibitor for total RSK, including RSK2, AKT, and S6K, by inhibiting the binding of its target RSK, AKT, and S6K with their substrate molecules (Shibata T, Mol Cancer Ther 2020). As the results, TAS0612 showed the antiproliferative effect in all 8 BCL-derived cell lines examined with IC80s ranging from 1.18 to 8.46 µM, leading to G1 or G2/M cell cycle blockade, and induced apoptosis. At the protein level, TAS0612 caused the accumulation of its direct substrates p-total RSK Ser380, p-AKT Ser473, and p-S6K Thr389 in most cell lines examined as the results of the inhibition of binding to their respective substrates, and the subsequent inactivation through dephosphorylation of their respective target substrates in all cell lines examined. The 72-hour treatment with TAS0612 at 2.5 and 5.0 µM also showed the ex vivo antiproliferative effect in 25 patients-derived lymphoma cells of various disease subtypes, including nine DLBCLs, seven FLs, three MCLs, one mucosa-associated lymphoid tissue lymphoma, one splenic marginal zone B cell lymphoma, one chronic lymphocytic leukemia, and three B-lymphoblastic lymphomas. We finally examined the global molecular effects of TAS0612 using gene expression profiling. Among several tumor suppressor genes upregulated by TAS0612, we found the increased expression of tumor protein 53 inducible nuclear protein 1 (TP53INP1) in cell lines regardless of disease subtypes. Moreover, gene set enrichment analysis identified that TAS0612 significantly downregulated the gene sets driven by the upregulation of MYC and mTORC1, reactive oxygen species, and the unfolded protein response. Thus, this study revealed that the concomitant blockade of RSK2, AKT, and S6K, which are the pivotal downstream substrates of PDPK1, is a novel universally active therapeutic target for the various disease subtypes of BCLs, and line up TAS0612 as an attractive candidate agent for BCLs for future clinical development.

MYC Pathway Upregulation Identified in Non-Responders to Hypomethylating Agents and Presents a Therapeutic Target in Acute Myeloid Leukemia

Background: In the current era of acute myeloid leukemia (AML) management, hypomethylating agents (HMAs) remain as the backbone of combination regimens for front-line treatment of elderly or unfit patients (pts) and/or as salvage therapy in relapsed/refractory pts. Data on biological predictors of HMA response are limited. Our previous work explored cytidine deaminase (CDA), known to inactivate HMAs, and nucleophosmin 1 (NPM1), determined by core pathway analysis to indirectly influence CDA expression. No clear correlation was identified in pt samples between CDA protein expression and NPM1 status or response, and pharmacogenomic analysis showed no CDA single nucleotide polymorphisms were predictive of response to HMAs. We aimed to expand prior findings using RNA sequencing (RNA seq) and gene set enrichment analysis (GSEA) to identify gene signatures predictive of HMA response and propose potential therapeutic targets. Methods: AML pts with banked samples who received frontline, bridging, or salvage HMA-based therapy between January 2014 to December 2018 were reviewed. Responses following at least 2 cycles of HMA were categorized as complete response (CR), CRi (CR with incomplete hematologic recovery), morphologic leukemia-free state (MLFS), complete hematologic response (CHR), or refractory. Pts were categorized as responders (CR, CRi, MLFS, CHR) or non-responders (refractory). Tumor cells were purified using immunomagnetic selection from bone marrow aspirates collected at diagnosis (dx). RNA seq was performed on 20 available pt tumor samples. Unsupervised clustering was performed and GSEA was completed to compare identified clusters using a false discovery rate (FDR) cut-off of less than 25%. GSEA-derived gene pathway scores were assessed by Wilcoxon rank-sum test. To evaluate AML cell lines to explore the non-responder phenotype, viability of AML cell lines was assessed following treatment with HMA azacitidine (Aza) and MS177 (a proteolysis targeting chimeric EZH2 degrader, also known to degrade MYC). Immunoblotting was used to assay proteolysis targeting chimeric (PROTAC)-induced MYC degradation. Results: Unsupervised clustering of RNA seq from 20 tumor samples at dx, labeled for response and NPM1 status, revealed 3 distinct groups: a non-responder group, a responder group, and a mixed response group ( Figure 1A). GSEA comparing the non-responder group to the responder group revealed several enriched genes in the non-responder group, including MYC targets (FDR <0.001), E2F targets (FDR <0.001), and G2M checkpoint (FDR=0.244). Upregulation of MYC targets was confirmed by enrichment profiling. Application of enrichment pathway scores revealed significant differences between non-responders and responders for E2F targets (p=0.026), G2M checkpoint (p=0.026), and MYC targets (p=0.0022). To explore MYC as a therapeutic target, we assayed MYC degradation in leukemia cells treated with two PROTAC degraders. The PROTAC MS177 induced marked MYC degradation in HL-60 cells ( Figure 1B). Cytotoxicity assays showed that in Kasumi-1 cells, the 50% inhibitory concentrations (IC50) for Aza and MS177 were 2.9 µM (responder phenotype) and 1.1 µM, respectively ( Figure 1C); in HL-60 cells the IC50 for Aza and MS177 were 10.9 µM (non-responder phenotype) and 0.06 µM, respectively ( Figure 1D). MS177 also showed efficacy in cytotoxicity assays using pt derived tumor samples. Conclusions: Using tumor samples from AML pts receiving HMAs, we identified a non-responder gene phenotype enriched with MYC upregulation. GSEA-derived pathway scoring differentiated non-responders from responders in our dataset. To further evaluate whether the enrichment pathway score is predictive for response across other AML cohorts, we are applying enrichment scores to publicly available AML datasets. With MYC upregulation identified as a potential driver of HMA failure, MYC degradation or inhibitory targets are recognized as potential therapeutic targets for pts with a non-responder gene signature. We established the HL-60 AML cell line, found to be less sensitive to Aza than the Kasumi-1 cell line, demonstrated greater sensitivity to MYC degradation compared to Kasumi-1 cells and may reflect the non-responder phenotype. Further in vitro efforts are ongoing to evaluate MYC targets for the HMA non-responder phenotype.

Lesser prevalence of polyps/WNT-dysregulation and concomitant upregulation of gamma-catenin/MYC point to alternate pathways in colorectal cancer in India.

Colorectal cancer (CRC) is known to follow adenoma carcinoma sequence (ACS) in majority of the tumors and the driver variants and associated pathways are well delineated. However, most of the published data are from the west and information in other ethnicities is sparse. We therefore comprehensively evaluated the CRC tumors from Indian ethnicity for the prevalence of ACS. In this cohort study, clinical data of 100,497 patients who attended hospital between 2013 and 2018 were accessed. Tumors from patients (n = 130) with CRC who were treated primarily by surgery were included. DNA and RNA were isolated to assess variants (direct sequencing) and WNT-pathway dysregulation in genes related to ACS. Global gene expression was generated and analyzed on microarrays (Affymetrix; N = 10) and next generation sequencing platforms (Illumina; N = 25). Gene expression at mRNA (qRT-PCR) and protein level (IHC) of JUP/CTNNB1/MYC were assessed. Correlation between expression of JUP and MYC was evaluated by Karl Pearson's correlation coefficient. The prevalence of polyps was 16.75%, while 18.26% variants in APC/CTNNB1, 20.00% in KRAS, and 18.33% WNT dysregulation were noted. Interestingly, 29/60 (48.33%) tumors showed only MYC upregulation with normal APC/CTNNB1 expression. Global gene expression and validation in an independent tumor cohort confirmed concomitant upregulation of JUP (gamma-catenin) & MYC (r = 0.71; p = 0.001) at mRNA and protein in sizeable number of tumors (45/96; 46.88%). Our study provides evidence for limited prevalence of ACSin the Indian ethnicity. Preventive colonoscopies for early identification and management of CRC may not be an effective strategy in this ethnicity.

Glycerolipid Metabolism Via the MGA-MYC-NME1-PGP Axis As a Key Regulator of Oxidative Phosphorylation in Richter's Transformation

Despite advances in targeted therapies for chronic lymphocytic leukemia (CLL), Richter's transformation (RT) remains a clinical challenge. RT is an aggressive shift from CLL to lymphoma, associated with metabolic changes and aberrations in the MYC transcriptional network. Genomic analysis of paired CLL and RT cases revealed recurrent mutations or deletions in the MYC suppressor Max-gene-associated (MGA) increase from 5% (CLL) to 30% (RT) cases, highlighting its critical yet unexplored role as a driver event. We recently developed a murine RT model using B cell-restricted in vitro editing of LSK progenitors from Cd19Cre-Cas9-del(13q)-Sf3b1 ( Sf3b1-K700E) with Mga-targeting sgRNAs. This murine model demonstrated upregulation of Mga targets Myc and Nme1 (nucleoside diphosphate kinase), a distinct OXPHOS-based transcription signature, increased oxygen consumption rate, elevated ROS levels, suggesting a significant link between the Mga-Myc-Nme1 axis and OXPHOS dysregulation in RT. Given the crucial role of metabolic reprogramming in cancer, we seek to understand the molecular mechanisms of metabolites driving OXPHOS and accelerating CLL transformation. Unraveling these processes could revolutionize therapeutic approaches for managing RT in clinical settings. To identify metabolites driving dysregulated OXPHOS in RT, we first conducted paired RNA-seq and unlabeled metabolomic profiling on normal and RT mouse splenic B cells (n=3/group). To translate our discovery from the murine RT model to human disease, we performed metabolomic profiling using MEC1 and Nalm6 cell lines with MGA deletion. Our metabolomic analysis of murine RT cells revealed 44 upregulated metabolites, including TCA cycle substrates (glutamine fold change (FC)=9.6, p=0.001;asparagine FC= 4.0, p=0.006), fatty-acid intermediates (glycerol-3P FC=11.7, p=0.003; carnitine FC=4.3, p=0.02; palmitate FC= 4.1, p= 0.08), and glycolysis intermediates (3-phosphoglycerate FC= 4.0, p=0.03; UDP-glucose FC=12.7, p=0.02; glycine FC=4.7, p=0.03). Moreover, integrated metabolite analysis (MetaboAnalyst v5.0) identified 63 common upregulated metabolites in MGA KO (knockout) human cell lines and murine RT, enriching glycerolipid, and fatty acid oxidation, associated pathways, indicating a potential role for MGA deletion in driving dysregulated fatty acid metabolism. Pathway enrichment analysis using integrated RNA-seq and metabolite analyses showed that the glycerolipid pathway was one of the most enriched pathways among mouse RT and B-cell lines (Nalm6 and MEC1) with MGA KO. Additionally, two key fatty-acid enzymes, fatty acid synthetase (FASN) and phosphoglycerate phosphatase (PGP), displayed significant mRNA and protein changes, as validated by qPCR and immunoblotting. Furthermore, elevated lipid accumulation and increased free glycerol were confirmed based on lipi-green flow cytometry and glycerol-bioluminescent assays, suggesting an important role of glycerolipid metabolism in MGA KO-associated OXPHOS. PGP, a glycerol-3-phosphatase, regulates glycerol-3-P levels, a key intermediate in glucose, lipid, and energy metabolism. Overexpression of MYC and NME1 increases PGP levels in B-cell lines (MEC1, Nalm6, and Jeko), confirming PGP as a direct target of the MGA-MYC-NME1 axis. To determine the role of PGP in MGA KO (knock-out) cells, we used CRISPR/Cas9 to KO PGP in MEC1, Nalm6, and JeKo cell lines. KO of PGP decreased cell growth and reduced OXPHOS compared to controls based on CCK8 absorbance and Seahorse Mitostress assays respectively. Moreover, PGP KO downregulated the mTOR (p4E-BP1) pathway, emphasizing the significance of dysregulated glycerol and fatty acid metabolism in CLL to RT-driven OXPHOS. Our study reveals key metabolites driving RT and highlights the crucial role of the MGA-MYC-NME1-PGP axis in regulating OXPHOS in Mga KO RT cells. Our results thus suggest targeting glycerolipid metabolism and related pathways may offer effective strategies to conquer RT