MYC Regulation Research Articles

EYA2 tyrosine phosphatase inhibition reduces MYC and prevents medulloblastoma progression.

Medulloblastoma is the most common pediatric brain malignancy. Patients with the Group 3 subtype of medulloblastoma (MB) often exhibit MYC amplification and/or overexpression and have the poorest prognosis. While Group 3 MB is known to be highly dependent on MYC, direct targeting of MYC remains elusive. Patient gene expression data were used to identify highly expressed EYA2 in Group 3 MB samples, assess the correlation between EYA2 and MYC, and examine patient survival. Genetic and pharmacological studies were performed on EYA2 in Group 3 derived MB cell models to assess MYC regulation and viability in vitro and in vivo. EYA2 is more highly expressed in Group 3 MB than other MB subgroups and is essential for Group 3 MB growth in vitro and in vivo. EYA2 regulates MYC expression and protein stability in Group 3 MB, resulting in global alterations of MYC transcription. Inhibition of EYA2 tyrosine phosphatase activity, using a novel small molecule inhibitor (NCGC00249987, or 9987), significantly decreases Group 3 MB MYC expression in both flank and intracranial growth in vivo. Human MB RNA-seq data show that EYA2 and MYC are significantly positively correlated, high EYA2 expression is significantly associated with a MYC transcriptional signature, and patients with high EYA2 and MYC expression have worse prognoses than those that do not express both genes at high levels. Our data demonstrate that EYA2 is a critical regulator of MYC in Group 3 MB and suggest a novel therapeutic avenue to target this highly lethal disease.

Cryo‐EM structure of the chain‐elongating E3 ubiquitin ligase UBR5

UBR5 is a nuclear E3 ligase that ubiquitinates a vast range of substrates for proteasomal degradation. This HECT domain‐containing ubiquitin ligase has recently been identified as an important regulator of oncogenes, e.g., MYC, but little is known about its structure or mechanisms of substrate engagement and ubiquitination. Here, we present the cryo‐EM structure of human UBR5, revealing an α‐solenoid scaffold with numerous protein–protein interacting motifs, assembled into an antiparallel dimer that adopts further oligomeric states. Using cryo‐EM processing tools, we observe the dynamic nature of the UBR5 catalytic domain, which we postulate is important for its enzymatic activity. We characterise the proteasomal nuclear import factor AKIRIN2 as an interacting protein and propose UBR5 as an efficient ubiquitin chain elongator. This preference for ubiquitinated substrates and several distinct domains for protein–protein interactions may explain how UBR5 is linked to several different signalling pathways and cancers. Together, our data expand on the limited knowledge of the structure and function of HECT E3 ligases.

Loss of PRDX6 Aborts Proliferative and Migratory Signaling in Hepatocarcinoma Cell Lines.

Peroxiredoxin 6 (PRDX6), the only mammalian 1-Cys member of the peroxiredoxin family, has peroxidase, phospholipase A2 (PLA2), and lysophosphatidylcholine (LPC) acyltransferase (LPCAT) activities. It has been associated with tumor progression and cancer metastasis, but the mechanisms involved are not clear. We constructed an SNU475 hepatocarcinoma cell line knockout for PRDX6 to study the processes of migration and invasiveness in these mesenchymal cells. They showed lipid peroxidation but inhibition of the NRF2 transcriptional regulator, mitochondrial dysfunction, metabolic reprogramming, an altered cytoskeleton, down-regulation of PCNA, and a diminished growth rate. LPC regulatory action was inhibited, indicating that loss of both the peroxidase and PLA2 activities of PRDX6 are involved. Upstream regulators MYC, ATF4, HNF4A, and HNF4G were activated. Despite AKT activation and GSK3β inhibition, the prosurvival pathway and the SNAI1-induced EMT program were aborted in the absence of PRDX6, as indicated by diminished migration and invasiveness, down-regulation of bottom-line markers of the EMT program, MMP2, cytoskeletal proteins, and triggering of the "cadherin switch". These changes point to a role for PRDX6 in tumor development and metastasis, so it can be considered a candidate for antitumoral therapies.

N-methyl-D-aspartate receptor regulates the circadian clock in megakaryocytic cells and impacts cell proliferation through BMAL1

Peripheral circadian clocks control cell proliferation and survival, but little is known about their role and regulation in megakaryocytic cells. N-methyl-D-aspartate receptor (NMDAR) regulates the central clock in the brain. The purpose of this study was to determine whether NMDAR regulates the megakaryocytic cell clock and whether the megakaryocytic clock regulates cell proliferation and cell death. We found that both the Meg-01 megakaryocytic cell line and native murine megakaryocytes expressed circadian clock genes. Megakaryocyte-directed deletion of Grin1 in mice caused significant disruption of the circadian rhythm pathway at the transcriptional level and increased expression of BMAL1 at the protein level. Similarly, both pharmacological (MK-801) and genetic (GRIN–/–) inhibition of NMDAR in Meg-01 cells in vitro resulted in widespread changes in clock gene expression including increased expression of BMAL1, the core clock transcription factor. BMAL1 overexpression reduced Meg-01 cell proliferation and altered the time-dependent expression of the cell cycle regulators MYC and WEE1, whereas BMAL1 knockdown led to increased cell death in Meg-01-GRIN1–/– cells. Our results demonstrate that NMDAR regulates the circadian clock in megakaryocytic cells and that the circadian clock component BMAL1 contributes to the control of Meg-01 cell proliferation and survival.

The proliferation of human mucosal-associated invariant T cells requires a MYC-SLC7A5-glycolysis metabolic axis

Mucosal-associated invariant T (MAIT) cells are an abundant population of innate T cells that recognize bacterial ligands and play a key role in host protection against bacterial and viral pathogens. Upon activation, MAIT cells undergo proliferative expansion and increase their production of effector molecules such as cytokines. In this study, we found that both mRNA and protein abundance of the key metabolism regulator and transcription factor MYC was increased in stimulated MAIT cells. Using quantitative mass spectrometry, we identified the activation of two MYC-controlled metabolic pathways, amino acid transport and glycolysis, both of which were necessary for MAIT cell proliferation. Last, we showed that MAIT cells isolated from people with obesity showed decreased MYC mRNA abundance upon activation, which was associated with defective MAIT cell proliferation and functional responses. Collectively, our data uncover the importance of MYC-regulated metabolism for MAIT cell proliferation and provide additional insight into the molecular basis for the functional defects of MAIT cells in obesity.

Abstract 2756: Custom G4 DNA microarray can determine large-scale binding selectivity of small molecules and proteins to oncogene G-quadruplexes

Abstract G-quadruplexes are novel DNA secondary structures that are formed in guanine-rich genomic regions with functional importance in cancers, such as human telomeres and oncogene promoters. G-quadruplexes adopt globular structures distinct from duplex B-DNA and their structural diversity suggests specific recognition. G-quadruplexes have become attractive drug targets because they provide a means of addressing otherwise undruggable targets. As one example, MYC is an important oncogene that is difficult to target by traditional drug design; however, its regulation involves a promoter G-quadruplex (MycG4) that is a potential drug target. 3,6-bis(1-Methyl-4-vinylpyridinium) carbazole diiodide (BMVC) is the first fluorescent probe developed for detecting G-quadruplex structures in cells and has been tested for cancer cell diagnosis. Although it was designed to detect the G-quadruplexes formed in human telomeres, we found BMVC binds to the MYC promoter G-quadruplex (MycG4) with higher affinity and specificity. We determined the high-resolution NMR K+ solution structure of the 2:1 BMVC-MycG4 complex showing a novel conformational adjustment of BMVC for an optimal binding to G-quadruplexes. Intriguingly, BMVC binds the 5’-end of MycG4 with higher affinity, but it binds the 3’-end with greater sequence selectivity. To determine ligand-binding selectivity on a large-scale, we designed a new custom G-quadruplex microarray platform with more than 25,000 potential oncogene G4 sequences and mutants. The new microarray was able to systematically assess binding affinity and selectivity of drugs targeted to G4s like the G-quadruplex involved in MYC regulation. The microarray results show that BMVC preferentially binds to the parallel type of G-quadruplexes, especially MycG4. Importantly, a strong sequence selectivity of BMVC for a 3’ flanking T was observed, consistent with the NMR data. The new microarray was also able to systematically assess binding of proteins and antibodies in a high-throughput and unbiased manner, and we tested the G-quadruplex binding proteins FANCJ, PIF1, BLM, DHX36, WRN, IGF2, CNBP, nucleolin, as well as the G-quadruplex-specific antibody BG4. In conclusion, we establish the custom G4 DNA microarray which can be used to determine binding selectivity of small molecules and proteins to oncogene G-quadruplexes in large-scale and high-throughput. Citation Format: Guanhui Wu, Luying Chen, Kristen Huseman, Desiree Tillo, Sreejana Ray, Ta-Chau Chang, John S. Schneekloth, Charles Vinson, Danzhou Yang. Custom G4 DNA microarray can determine large-scale binding selectivity of small molecules and proteins to oncogene G-quadruplexes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2756.

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

Abstract NG14: Dissecting immune microenvironment of T-cell acute lymphoblastic leukemia

Abstract 6713: Myc-regulated miR17, 20a modulate RANK expression in osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. Approximately 25-30% of these tumors carry amplification of chromosome 8q24, which harbors the oncogene c-Myc, and correlates with a poor prognosis in patients with OS. To understand the mechanisms that underlie the ability of Myc to alter both the tumor and its surrounding tumor immune microenvironment (TiME), we generated and molecularly characterized an osteoblast-specific Cre-Lox-Stop-Lox;(LSL)-c-MycT58A;p53f/+ knockin genetically engineered mouse model (GEMM). Phenotypically, the Myc knockin-GEMM had rapid tumor development with a high incidence of metastasis. Myc-dependent gene signature in our murine model demonstrated significant homology to the human Myc-amplified OS.Interestingly, we noticed a significant reduction in the osteoclast (OCL) cell population in the Myc knockin OS tumor compared to the p53-driven. We found the expression of RANK was significantly downregulated in the Myc knockin tumor compared to the Non-knockin p53 heterozygous tumors. The RANK/RANKL pathway is vital in OCL maturation and bone modeling/remodeling. To understand the involvement of Myc in RANK regulation, we used murine-derived OS cell lines and transiently knocked down of Myc expression using siRNA. We observed a significant upregulation in RANK expression after Myc knockdown. To decipher the molecular mechanism behind the Myc-dependent regulation of RANK expression in OS, we looked into the Myc-mediated microRNAs. Myc regulates the expression of several microRNAs, including the polycistronic miR-17-92 cluster. The expression of miR17-5p and Mir20a-5p was significantly higher in the GEMM tumor tissue samples isolated from the Myc knockin compared to the p53-driven. Further, we validated the Myc-dependent regulation of miR-17-5p/20a-5p expression using transient knockdown of Myc in mouse Myc knockin-derived cell lines. To examine the role of miR17-5p/20a-5p on the RANK regulation, we performed both gain and loss-of-function studies using microRNA-17/20a mimics and inhibitors. After the treatment with miR-17-5p/20a-5p inhibitors, the expression of RANK was significantly upregulated whereas in the case of miR17/20a mimics reversed these effects and led to a downregulation of RANK expression. We established that miR-17-5p/20a-5p is causally responsible for at least part of the mechanism by which Myc regulates the RANK expression in OS.We concluded that the Myc-regulated miR17/20a modulates the RANK expression that is involved in the OCL cell population regulation and function in the OS. Citation Format: Bikesh Kumar Nirala, Lyazat Kurenbekova, Tajhal Patel, Ryan Lane Shuck, Atreyi Dasgupta, Nino Carlo Rainusso, Jason T. Yustein. Myc-regulated miR17, 20a modulate RANK expression in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6713.

Abstract 2475: Bromodomain inhibitor: MT1 and its potential role in modulation of prostate cancer progression

Abstract Bromodomains (BD) are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation of protooncogene cellular myelocytomatosis (c- Myc) and other genes. Because c-Myc cannot be directly targeted by small molecular inhibitors due to disordered alpha helical structure, epigenetic targeting of c-Myc by BD inhibitors is an attractive therapeutic strategy for diseases such as prostate cancer (PC) associated with increased c-Myc upregulation with advancing disease. We studied the efficacy of MT1, a novel bivalent BD inhibitor that is 100-fold more potent than the first in class BD inhibitor JQ1, at inhibiting PC growth. We tested the effect on viability by MT-1 on PC cell lines, 3D spheroids derived from clinically annotated drug resistant patient derived xenografts (PDX), mice PDX models and corroborated the molecular mechanism of MT1 down regulation of Myc leading to downstream Myc-dependent up regulation of Protein Kinase D1 (PrKD) substrate phosphorylation by western blot. MT-1 inhibited growth of PC in castration sensitive (LNCaP) and resistant PC cells (PC-3). MT-1 treatment upregulated PrKD expression and phosphorylation of known PrKD substrates: threonine 120 (Thr-120) residues in beta-catenin and the serine 216 in Cell Division Cycle 25 (CDC25C) in PC-3 cells. Moreover, MT-1 was effective in inhibition of 3D spheroids growth at IC 50 between 0.27-0.92µM in Abiraterone, Enzalutamide, Docetaxel, Cabazitaxel metastatic castrate resistant PCa patient-derived tumor 3D spheroids. Additionally, MT1 was effective in inhibiting the tumor growth in PDX mice model. A combined intra-peritoneal administration of MT-1 with another c-Myc inhibitor (3JC48-3), an obligate c-Myc and MYC-associated protein X (MAX) heterodimerization inhibitor, increased the efficacy of inhibiting the PDX growth in mice. This study provides strong pre-clinical in vitro and in vivo evidence for advancing MT- 1 as a novel c-Myc targeting drug in PC. The MT-1 drug development will likely be highly impactful as c-Myc is dysregulated in three fourths of men with advanced PC. Citation Format: Sanjeev Shukla, Mohammed Al-Toubat, Allison H. Feibus, Ahmed Elshafei, Carlos Riveros, Nathalie Meurice, Justyna Gleba, Jonathan Chardon-Robles, Adam M. Kase, John A. Copland III, Joachim L. Petit, Teruko Osumi, K.C. Balaji. Bromodomain inhibitor: MT1 and its potential role in modulation of prostate cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2475.

Abstract 1570: Novel Myc-dependent compound DL78 selectively induces mitotic catastrophe and cell death in high grade serous ovarian cancer

Abstract While the rates of new cases and deaths for high grade serous ovarian cancer (HGSC) have declined steadily in the past decade, HGSC remains the fifth leading cause of cancer death in women. As many patients succumb to chemoresistance, there is a need to uncover novel targeted drugs. One way to do so is by drug repurposing. A computational screen, DrugPredict, led to the discovery that amiodarone, an antiarrhythmic, is a potent apoptosis-inducer and Myc-degrader in numerous patient-derived ovarian cancer cell lines. In search of a more selective, but less toxic compound, structure-activity relationship was applied to identify DL78. Due to its structural differences, DL78 is considered to be independent of amiodarone. DL78 lacks hERG activity, retains Myc regulation, and gains selective anticancer properties. This compound produces significant cell death selectively in patient-derived HGSC cells, including isogenic cisplatin-resistant lines, but does not harm non-transformed fallopian tube cells. Cancer cells treated with DL78 arrest prior to anaphase and showcase microtubule abnormalities and upregulation of mitotic regulators. The prolonged arrest leads to loss of Myc, mitotic catastrophe, and apoptosis selectively in the HGSC cells, while fallopian tube cells can cycle normally. DL78’s activity is confirmed to be dependent on Myc expression because targeted inhibition of Myc decreases its potency and prevents the DL78-induced cell cycle arrest. Finally, pharmacokinetic analysis shows that DL78 saturates the tumor following intraperitoneal injection, yet drug concentrations in the blood and liver present at normal levels. Overall, these phenotypes do not show with amiodarone treatment, suggesting that structurally different DL78 performs through a different mechanism. Further classification of this tumor-selective compound may reveal targeted ovarian cancer vulnerabilities. Citation Format: Jessica Teitel, Michele Cusato, Margaret Farah, Agharnan Gandhi, Pil Lee, Andrew White, John Takyi-Williams, Bo Wen, Andre Monterio Da Rocha, Analisa DiFeo. Novel Myc-dependent compound DL78 selectively induces mitotic catastrophe and cell death in high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1570.

Ubiquitin‐dependent regulation of MYC: A promising therapeutic target

Ubiquitin‐dependent regulation of MYC: A promising therapeutic target

PD04-07 THE BIVALENT BROMODOMAIN INHIBITOR; MT-1 INHIBITS PROSTATE CANCER GROWTH

You have accessJournal of UrologyCME1 Apr 2023PD04-07 THE BIVALENT BROMODOMAIN INHIBITOR; MT-1 INHIBITS PROSTATE CANCER GROWTH Sanjeev Shukla, Mohammed Al-Toubat, Carlos Riveros, Allison H. Feibus, Ahmed El-shafie, Adam M. Kase, John A. Copland, Joachim L. Petit, Nathalie Meurice, Justyna Gleba, Jonathan Chardon-Robles, Teruko Osumi, and K. C. Balaji Sanjeev ShuklaSanjeev Shukla More articles by this author , Mohammed Al-ToubatMohammed Al-Toubat More articles by this author , Carlos RiverosCarlos Riveros More articles by this author , Allison H. FeibusAllison H. Feibus More articles by this author , Ahmed El-shafieAhmed El-shafie More articles by this author , Adam M. KaseAdam M. Kase More articles by this author , John A. CoplandJohn A. Copland More articles by this author , Joachim L. PetitJoachim L. Petit More articles by this author , Nathalie MeuriceNathalie Meurice More articles by this author , Justyna GlebaJustyna Gleba More articles by this author , Jonathan Chardon-RoblesJonathan Chardon-Robles More articles by this author , Teruko OsumiTeruko Osumi More articles by this author , and K. C. BalajiK. C. Balaji More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003228.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Bromodomains (BD) are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation of protooncogene cellular myelocytomatosis (c-Myc) and other genes. Because c-Myc cannot be directly targeted by small molecular inhibitors due to disordered alpha helical structure, epigenetic targeting of c-Myc by BD inhibitors is an attractive therapeutic strategy for advanced prostate cancer (PC) associated with increased c-Myc upregulation . We studied the efficacy of MT1, a novel bivalent BD inhibitor that is 100-fold more potent than the first in class BD inhibitor JQ1, at inhibiting PC growth METHODS: We tested the effect on viability by MT-1 on PC cell lines, 3D spheroids derived from clinically annotated drug resistant patient derived xenografts (PDX), mice PDX models and corroborated the molecular mechanism of MT1 down regulation of Myc leading to downstream Myc-dependent up regulation of Protein Kinase D1 (PrKD) substrate phosphorylation by western blot. RESULTS: MT-1 inhibited growth of PC in castration sensitive (LNCaP) and resistant PC cells (PC-3). MT-1 treatment upregulated PrKD expression and phosphorylation of known PrKD substrates: threonine 120 (Thr-120) residues in beta-catenin and the serine 216 in Cell Division Cycle 25 (CDC25C) in PC-3 cells. Moreover, MT-1 was effective in inhibition of 3D spheroids growth at IC50 between 0.27-0.92µM in Abiraterone, Enzalutamide, Docetaxel, Cabazitaxel metastatic castrate resistant PCa patient-derived tumor 3D spheroids. Additionally, MT1 was effective in inhibiting the tumor growth in PDX mice model. A combined intra-peritoneal administration of MT-1 with another c-Myc inhibitor (3JC48-3), an obligate c-Myc and MYC-associated protein X (MAX) heterodimerization inhibitor, increased the efficacy of inhibiting the PDX growth in mice. CONCLUSIONS: This study provides strong pre-clinical in vitro and in vivo evidence for advancing MT-1 as a novel c-Myc targeting drug in PC. The MT-1 drug development will likely be highly impactful as c-Myc is dysregulated in three fourths of men with advanced PC. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e144 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Sanjeev Shukla More articles by this author Mohammed Al-Toubat More articles by this author Carlos Riveros More articles by this author Allison H. Feibus More articles by this author Ahmed El-shafie More articles by this author Adam M. Kase More articles by this author John A. Copland More articles by this author Joachim L. Petit More articles by this author Nathalie Meurice More articles by this author Justyna Gleba More articles by this author Jonathan Chardon-Robles More articles by this author Teruko Osumi More articles by this author K. C. Balaji More articles by this author Expand All Advertisement PDF downloadLoading ...

Obesity defined molecular endotypes in the synovium of patients with osteoarthritis provides a rationale for therapeutic targeting of fibroblast subsets.

Osteoarthritis (OA), a multifaceted condition, poses a significant challenge for the successful clinical development of therapeutics due to heterogeneity. However, classifying molecular endotypes of OA pathogenesis could provide invaluable phenotype-directed routes for stratifying subgroups of patients for targeted therapeutics, leading to greater chances of success in trials. This study establishes endotypes in OA soft joint tissue driven by obesity in both load-bearing and non-load bearing joints. Hand, hip, knee and foot joint synovial tissue was obtained from OA patients (n=32) classified as obese (BMI>30) or normal weight (BMI 18.5-24.9). Isolated fibroblasts (OA SF) were assayed by Olink proteomic panel, seahorse metabolic flux assay, Illumina's NextSeq 500 bulk and Chromium 10X single cell RNA-sequencing, validated by Luminex and immunofluorescence. Targeted proteomic, metabolic and transcriptomic analysis found the inflammatory landscape of OA SFs are independently impacted by obesity, joint loading and anatomical site with significant heterogeneity between obese and normal weight patients, confirmed by bulk RNAseq. Further investigation by single cell RNAseq identified four functional molecular endotypes including obesity specific subsets defined by an inflammatory endotype related to immune cell regulation, fibroblast activation and inflammatory signaling, with up-regulated CXCL12, CFD and CHI3L1 expression. Luminex confirmed elevated chitase3-like-1(229.5vs. 49.5ng/ml, p<.05) and inhibin (20.6vs. 63.8 pg/ml, p<.05) in obese and normal weight OA SFs, respectively. Lastly, we find SF subsets in obese patients spatially localise in sublining and lining layers of OA synovium and can be distinguished by differential expression of the transcriptional regulators MYC and FOS. These findings demonstrate the significance of obesity in changing the inflammatory landscape of synovial fibroblasts in both load bearing and non-load bearing joints. Describing multiple heterogeneous OA SF populations characterised by specific molecular endotypes, which drive heterogeneity in OA disease pathogenesis. These molecular endotypes may provide a route for the stratification of patients in clinical trials, providing a rational for the therapeutic targeting of specific SF subsets in specific patient populations with arthritic conditions.

CDK7 controls E2F- and MYC-driven proliferative and metabolic vulnerabilities in multiple myeloma

AbstractTherapeutic targeting of CDK7 has proven beneficial in preclinical studies, yet the off-target effects of currently available CDK7 inhibitors make it difficult to pinpoint the exact mechanisms behind MM cell death mediated by CDK7 inhibition. Here, we show that CDK7 expression positively correlates with E2F and MYC transcriptional programs in cells from patients with multiple myeloma (MM); its selective targeting counteracts E2F activity via perturbation of the cyclin-dependent kinases/Rb axis and impairs MYC-regulated metabolic gene signatures translating into defects in glycolysis and reduced levels of lactate production in MM cells. CDK7 inhibition using the covalent small-molecule inhibitor YKL-5-124 elicits a strong therapeutic response with minimal effects on normal cells, and causes in vivo tumor regression, increasing survival in several mouse models of MM including a genetically engineered mouse model of MYC-dependent MM. Through its role as a critical cofactor and regulator of MYC and E2F activity, CDK7 is therefore a master regulator of oncogenic cellular programs supporting MM growth and survival, and a valuable therapeutic target providing rationale for development of YKL-5-124 for clinical use.

MYC determines lineage commitment in KRAS-driven primary liver cancer development

Primary liver cancer (PLC) comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), two frequent and lethal tumour types that differ regarding their tumour biology and responses to cancer therapies. Liver cells harbour a high degree of cellular plasticity and can give rise to either HCC or iCCA. However, little is known about the cell-intrinsic mechanisms directing an oncogenically transformed liver cell to either HCC or iCCA. The scope of this study was to identify cell-intrinsic factors determining lineage commitment in PLC. Cross-species transcriptomic and epigenetic profiling was applied to murine HCCs and iCCAs and to two human PLC cohorts. Integrative data analysis comprised epigenetic Landscape In Silico deletion Analysis (LISA) of transcriptomic data and Hypergeometric Optimization of Motif EnRichment (HOMER) analysis of chromatin accessibility data. Identified candidate genes were subjected to functional genetic testing in non-germline genetically engineered PLC mouse models (shRNAmir knockdown or overexpression of full-length cDNAs). Integrative bioinformatic analyses of transcriptomic and epigenetic data pinpointed the Forkhead-family transcription factors FOXA1 and FOXA2 as MYC-dependent determination factors of the HCC lineage. Conversely, the ETS family transcription factor ETS1 was identified as a determinant of the iCCA lineage, which was found to be suppressed by MYC during HCC development. Strikingly, shRNA-mediated suppression of FOXA1 and FOXA2 with concomitant ETS1 expression fully switched HCC to iCCA development in PLC mouse models. The herein reported data establish MYC as a key determinant of lineage commitment in PLC and provide a molecular explanation why common liver-damaging risk factors such as alcoholic or non-alcoholic steatohepatitis can lead to either HCC or iCCA. Liver cancer is a major health problem and comprises hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), two frequent and lethal tumour types that differ regarding their morphology, tumour biology, and responses to cancer therapies. We identified the transcription factor and oncogenic master regulator MYC as a switch between HCC and iCCA development. When MYC levels are high at the time point when a hepatocyte becomes a tumour cell, an HCC is growing out. Conversely, if MYC levels are low at this time point, the result is the outgrowth of an iCCA. Our study provides a molecular explanation why common liver-damaging risk factors such as alcoholic or non-alcoholic steatohepatitis can lead to either HCC or iCCA. Furthermore, our data harbour potential for the development of better PLC therapies.

BRD9 Degradation Disrupts Ribosome Biogenesis in Multiple Myeloma.

BRD9 is a defining component of the noncanonical SWI/SNF complex, which regulates gene expression by controlling chromatin dynamics. Although recent studies have found an oncogenic role for BRD9 in multiple cancer types including multiple myeloma, its clinical significance and oncogenic mechanism have not yet been elucidated. Here, we sought to identify the clinical and biological impact of BRD9 in multiple myeloma, which may contribute to the development of novel therapeutic strategies. We performed integrated analyses of BRD9 in vitro and in vivo using multiple myeloma cell lines and primary multiple myeloma cells in established preclinical models, which identified the molecular functions of BRD9 contributing to multiple myeloma cell survival. We found that high BRD9 expression was a poor prognostic factor in multiple myeloma. Depleting BRD9 by genetic (shRNA) and pharmacologic (dBRD9-A; proteolysis-targeting chimera; BRD9 degrader) approaches downregulated ribosome biogenesis genes, decreased the expression of the master regulator MYC, and disrupted the protein-synthesis maintenance machinery, thereby inhibiting multiple myeloma cell growth in vitro and in vivo in preclinical models. Importantly, we identified that the expression of ribosome biogenesis genes was associated with the disease progression and prognosis of patients with multiple myeloma. Our results suggest that BRD9 promotes gene expression by predominantly occupying the promoter regions of ribosome biogenesis genes and cooperating with BRD4 to enhance the transcriptional function of MYC. Our study identifies and validates BRD9 as a novel therapeutic target in preclinical models of multiple myeloma, which provides the framework for the clinical evaluation of BRD9 degraders to improve patient outcome.

Effective oxygen metabolism-based prognostic signature for colorectal cancer.

Oxygen metabolism is an important factor affecting the development of tumors, but its roles and clinical value in Colorectal cancer are not clear. We developed an oxygen metabolism (OM) based prognostic risk model for colorectal cancer and explored the role of OM genes in cancer. Gene expression and clinical data obtained from The Cancer Genome Atlas, Clinical Proteomic Tumor Analysis Consortium databases were consider as discovery and validation cohort, respectively. The prognostic model based on differently expressed OM genes between tumor and GTEx normal colorectal tissues were constructed in discovery cohort and validated in validation cohort. The Cox proportional hazards analysis was used to test clinical independent. Upstream and downstream regulatory relationships and interaction molecules are used to clarify the roles of prognostic OM genes in colorectal cancer. A total of 72 common differently expressed OM genes were detected in the discovery and validation set. A five-OM gene prognostic model including LRT2, ATP6V0E2, ODC1, SEL1L3 and VDR was established and validated. Risk score determined by the model was an independent prognostic according to routine clinical factors. Besides, the role of prognostic OM genes involves transcriptional regulation of MYC and STAT3, and downstream cell stress and inflammatory response pathways. We developed a five-OM gene prognostic model and study the unique roles of oxygen metabolism in of colorectal cancer.

Intricate network of nuclear morphology, chromatin dynamics and macromolecular crowding facilitates efficient nuclear functioning.

The highly crowded nuclear interior impacts molecular motion and assembly, and therefore influence sub-cellular organization and function. However, we have limited understanding of nuclear crowding at the mesoscale (10-100 nanometers), which corresponds to the size of regulatory molecules such as RNA polymerase II. Here, we leveraged nuclear Genetically Encoded Multimeric nanoparticles (nucGEMs) to characterize physical properties of nucleus and to study its impact on transcription. nucGEMs are self-assembling fluorescent tracer particles of 40-nm diameter whose motion can be tracked to infer biophysical properties. By tracking GEMs, we found that nucleus is more crowded than the cytoplasm. Within the nucleus, nucGEMs majorly populated euchromatic compartments, excluded from heterochromatin and the nucleolus. Changing nuclear volume by growing cells to confluency or by exposing them to hypo and hyper osmotic shock analogously altered nuclear crowding. Increasing heterochromatin using HDAC inhibitors or inhibiting transcription using actinomycin D led to an immediate decrease in nucGEM diffusion followed by slow and gradual recovery. Interestingly, transcriptional activity, as monitored by 5EU incorporation, also changed in-correlation with nuclear crowding changes. These findings indicate that nuclear morphology, chromatin dynamics and nuclear processes altogether shape the molecular crowding of the nucleus, which in turn affects transcription. Furthermore, through an osmotic-stress resistance screen, we discovered that MYC confers resistance to increased molecular crowding. We found that overexpression of key transcriptional regulator MYC, increased crowding and elevated transcription rate. We propose that MYC enables transcription even in a hyper-crowded molecular environment ensuring cell proliferation, shedding a new light on the function of this molecule. Our findings indicate a direct link between crowding and transcription suggesting that nuclear crowding may play a crucial role in several biological processes such as stem cell differentiation and oncogenic transformation of normal cells.

Identify potential driver genes for PAX-FOXO1 fusion-negative rhabdomyosarcoma through frequent gene co-expression network mining.

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma usually originated from skeletal muscle. Currently, RMS classification based on PAX-FOXO1 fusion is widely adopted. However, compared to relatively clear understanding of the tumorigenesis in the fusion-positive RMS, little is known for that in fusion-negative RMS (FN-RMS). We explored the molecular mechanisms and the driver genes of FN-RMS through frequent gene co-expression network mining (fGCN), differential copy number (CN) and differential expression analyses on multiple RMS transcriptomic datasets. We obtained 50 fGCN modules, among which five are differentially expressed between different fusion status. A closer look showed 23% of Module 2 genes are concentrated on several cytobands of chromosome 8. Upstream regulators such as MYC, YAP1, TWIST1 were identified for the fGCN modules. Using in a separate dataset we confirmed that, comparing to FP-RMS, 59 Module 2 genes show consistent CN amplification and mRNA overexpression, among which 28 are on the identified chr8 cytobands. Such CN amplification and nearby MYC (also resides on one of the above cytobands) and other upstream regulators (YAP1, TWIST1) may work together to drive FN-RMS tumorigenesis and progression. Up to 43.1% downstream targets of Yap1 and 45.8% of the targets of Myc are differentially expressed in FN-RMS vs. normal comparisons, which also confirmed the driving force of these regulators. We discovered that copy number amplification of specific cytobands on chr8 and the upstream regulators MYC, YAP1 and TWIST1 work together to affect the downstream gene co-expression and promote FN-RMS tumorigenesis and progression. Our findings provide new insights for FN-RMS tumorigenesis and offer promising targets for precision therapy. Experimental investigation about the functions of identified potential drivers in FN-RMS are in progress.

Synergistic Targeting of DNA-PK and KIT Signaling Pathways in KIT Mutant Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common and aggressive form of acute leukemia, with a 5-year survival rate of just 24%. Over a third of all AML patients harbor activating mutations in kinases, such as the receptor tyrosine kinases FLT3 (receptor-type tyrosine-protein kinase FLT3) and KIT (mast/stem cell growth factor receptor kit). FLT3 and KIT mutations are associated with poor clinical outcomes and lower remission rates in response to standard-of-care chemotherapy. We have recently identified that the core kinase of the non-homologous end joining DNA repair pathway, DNA-PK (DNA-dependent protein kinase), is activated downstream of FLT3; and targeting DNA-PK sensitized FLT3-mutant AML cells to standard-of-care therapies. Herein, we investigated DNA-PK as a possible therapeutic vulnerability in KIT mutant AML, using isogenic FDC-P1 mouse myeloid progenitor cell lines transduced with oncogenic mutant KIT (V560G and D816V) or vector control. Targeted quantitative phosphoproteomic profiling identified phosphorylation of DNA-PK in the T2599/T2605/S2608/S2610 cluster in KIT mutant cells, indicative of DNA-PK activation. Accordingly, proliferation assays revealed that KIT mutant FDC-P1 cells were more sensitive to the DNA-PK inhibitors M3814 or NU7441, compared with empty vector controls. DNA-PK inhibition combined with inhibition of KIT signaling using the kinase inhibitors dasatinib or ibrutinib, or the protein phosphatase 2A activators FTY720 or AAL(S), led to synergistic cell death. Global phosphoproteomic analysis of KIT-D816V cells revealed that dasatinib and M3814 single-agent treatments inhibited extracellular signal–regulated kinase and AKT (RAC-alpha serine/threonine-protein kinase)/MTOR (serine/threonine-protein kinase mTOR) activity, with greater inhibition of both pathways when used in combination. Combined dasatinib and M3814 treatment also synergistically inhibited phosphorylation of the transcriptional regulators MYC and MYB. This study provides insight into the oncogenic pathways regulated by DNA-PK beyond its canonical role in DNA repair and demonstrates that DNA-PK is a promising therapeutic target for KIT mutant cancers.