MYC Activity Research Articles

Radiation-induced morphea of the breast – characterization and treatment of fibroblast dysfunction with repurposed mesalazine

Radiation-induced morphea (RIM) is a rare complication of radiotherapy presenting as inflammatory fibrosis, most commonly reported in breast cancer patients. As underlying disease mechanisms are not well understood, targeted therapies are lacking. Since fibroblasts are the key mediators of all fibroproliferative diseases, this study aimed to characterize patient-derived fibroblasts to identify therapeutic targets. We studied primary human control and RIM-fibroblasts on a functional and molecular basis, analyzed peripheral blood and tissue samples and conducted, based on our findings, a treatment attempt in one patient. In RIM, we identified a distinct myofibroblast phenotype reflected by increased alpha-smooth-muscle-actin (αSMA) expression, reduced proliferation and migration rates, and overexpression of osteopontin (OPN). Our RNA sequencing identified aberrant Myc activation as a potential disease driver in RIM fibroblasts, similar to previous findings in systemic sclerosis. Treatment with the anti-inflammatory drug mesalazine reversed the myofibroblast phenotype by targeting Myc. Based on these findings, a patient with RIM was successfully treated with mesalazine, resulting in reduced inflammation and pain and tissue softening, while serum OPN was halved. The present study provides a comprehensive characterization of RIM fibroblasts, suggests a disease-driving role for Myc, demonstrates promising antifibrotic effects of mesalazine and proposes OPN as a biomarker for RIM.

Abstract C067: Tuft cells transdifferentiate to neural-like progenitor cells in the progression of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is partly initiated through the transdifferentiation of acinar cells to metaplastic ducts that act as precursors of neoplasia and cancer. Tuft cells are solitary chemosensory cells not found in the normal pancreas but arise in metaplasia and neoplasia, diminishing as neoplastic lesions progress to carcinoma. Metaplastic tuft cells (mTCs) function to suppress tumor progression through communication with the tumor microenvironment, but their fate during progression is unknown. To determine the fate of mTCs during PDA progression, we have created a lineage tracing model that uses a tamoxifen-inducible tuft-cell specific Pou2f3CreERT/+ driver to induce transgene expression, including the lineage tracer tdTomato or the oncogene Myc. mTC lineage trace models of pancreatic neoplasia and carcinoma were used to follow mTC fate. We found that mTCs, in the carcinoma model, transdifferentiate into neural-like progenitor cells (NRPs), a cell type associated with poor survival in PDA patients. Using conditional knock-out and overexpression systems, we found that Myc activity in mTCs is necessary and sufficient to induce this Tuft-to- Neuroendocrine-Transition (TNT). Citation Format: Daniel J. Salas-Escabillas, Megan T. Hoffman, Sydney M. Brender, Jacee S. Moore, Hui-Ju Wen, Simone Benitz, Erick T. Davis, Daniel Long, Allison Wombwell, Nina G. Steele, Brittany Allen-Petersen, Rosalie C. Sears, Ichiro Matsumoto, Kathleen E. DelGiorno, Howard C. Crawford. Tuft cells transdifferentiate to neural-like progenitor cells in the progression of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C067.

Abstract IA-05: Targeting KRAS for pancreatic cancer treatment

Abstract The approval of clinically effective inhibitors for KRAS G12C mutant non-small cell lung cancers in 2021 and 2022 marks a significant milestone in anti-KRAS oncogene drug discovery. This success has fueled intense efforts to develop inhibitors targeting additional KRAS mutations. However, primary and treatment-associated acquired resistance limit the depth and duration of efficacy of KRAS G12C selective and likely other KRAS inhibitors. Thus, a major challenge moving forward will be the elucidation of mechanisms of resistance to then guide development of effective combination anti-KRAS therapies. Recent studies have begun to identify mechanisms of acquired resistance, where most involve alterations in components in the RAS signaling network that then cause reactivation of KRAS effector signaling that bypass drug activity. Our studies have established the critical role of reactivation of the RAF-MEK-ERK mitogen activated protein kinase cascade, and the ERK target MYC, in driving resistance in KRAS-mutant pancreatic cancer. To fully understand how aberrant ERK and MYC activation overcomes KRAS inhibitor response, we have established a comprehensive molecular portrait of ERK- and MYC-dependent activities. We have also identified YAP/TAZ-TEAD activation as a mechanism of acquired resistance and have characterized KEAP1 loss as a mechanism of primary resistance. Together, these studies have identified drug combination strategies that enhance KRAS inhibitor activity. Progress in these and other studies will be presented. Citation Format: Channing J. Der. Targeting KRAS for pancreatic cancer treatment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr IA-05.

HOXD12 defines an age-related aggressive subtype of oligodendroglioma

Oligodendroglioma, IDH-mutant and 1p/19q-codeleted has highly variable outcomes that are strongly influenced by patient age. The distribution of oligodendroglioma age is non-Gaussian and reportedly bimodal, which motivated our investigation of age-associated molecular alterations that may drive poorer outcomes. We found that elevated HOXD12 expression was associated with both older patient age and shorter survival in the TCGA (FDR < 0.01, FDR = 1e−5) and the CGGA (p = 0.03, p < 1e−3). HOXD12 gene body hypermethylation was associated with older age, higher WHO grade, and shorter survival in the TCGA (p < 1e−6, p < 0.001, p < 1e−3) and with older age and higher WHO grade in Capper et al. (p < 0.002, p = 0.014). In the TCGA, HOXD12 gene body hypermethylation and elevated expression were independently prognostic of NOTCH1 and PIK3CA mutations, loss of 15q, MYC activation, and standard histopathological features. Single-nucleus RNA and ATAC sequencing data showed that HOXD12 activity was elevated in neoplastic tissue, particularly within cycling and OPC-like cells, and was associated with a stem-like phenotype. A pan-HOX DNA methylation analysis revealed an age and survival-associated HOX-high signature that was tightly associated with HOXD12 gene body methylation. Overall, HOXD12 expression and gene body hypermethylation were associated with an older, atypically aggressive subtype of oligodendroglioma.

Bioinformatic prediction of miR-320a as a potential negative regulator of CDGSH iron-sulfur domain 2 (CISD2), involved in lung adenocarcinoma bone metastasis via MYC activation, and associated with tumor immune infiltration.

Ferroptosis, a form of regulated cell death associated with iron-dependent lipid peroxidation, plays a role in cancer progression. However, the specific mechanisms of ferroptosis in lung adenocarcinoma (LUAD) bone metastasis (BM) remain unclear. Using bioinformatics analysis, this study sought to identify the ferroptosis-associated genes involved in BM in LUAD, thus providing potential novel targets for the treatment of BM in LUAD. The RNA expression dataset GSE10799 was acquired from the Gene Expression Omnibus (GEO) database, and intersected with the ferroptosis dataset to identify ferroptosis-related differentially expressed genes (DEGs). The expression of candidate genes and their correlation with the prognosis of LUAD patients were validated in The Cancer Genome Atlas (TCGA) database. A protein gene interaction network was constructed using GeneMania and Retrieval of Interacting Genes/Proteins (STRING) databases. The association between the candidate genes and immune cells was assessed via TCGA and Tumor IMmune Estimation Resource (TIMER) databases. The potential mechanisms were elucidated by a gene set enrichment analysis (GSEA). The relevant microRNAs (miRNAs or miRs) that bind to the 3'untranslated region (3'UTR) end of candidate genes' mRNA was explored using the TargetScan database. The expression of these candidate miRNAs in LUAD was validated and the correlation between candidate miRNAs and candidate mRNAs was tested using the TCGA database. Finally, the clinical data of 40 LUAD patients were retrospectively analyzed to evaluate the clinical value of candidate gene expression for LUAD BM patients. In this research, 15 ferroptosis-related DEGs in LUAD BM were identified. TCGA database analysis indicated that patients with low levels of CDGSH iron-sulfur domain 2 (CISD2) in LUAD had better disease-specific survival (DSS), overall survival (OS), and a better progression-free interval (PFI) than those with high levels of CISD2. The TIMER database results show that the expression of CISD2 is correlated with the infiltration levels of various immune cells. The GSEA indicated that CISD2 might influence biological activity in LUAD by participating in cell-cycle regulation, mitochondrial translation, DNA damage repair, c-Myc (MYC) activation, and the P53 signaling pathway. Through the combined analysis of the TargetScan and TCGA databases, hsa-miR-320a was identified as the optimal upstream regulatory miRNA. The immunohistochemistry data indicated that the positive CISD2 expression rates and immunohistochemistry scores of the patients with BM were significantly higher than those of the patients without BM (P<0.05). The high expression of CISD2 is a significant risk factor for BM in LUAD. The downregulation of CISD2 expression may extend DSS, OS, and the PFI of LUAD patients. Thus, CISD2 could serve as a novel predictive biomarker for LUAD patients. Further, miR-320a might negatively regulate CISD2 and participate in LUAD BM by activating MYC. These data provide a potential perspective for developing anticancer therapies for LUAD-BM patients.

Targeting of mutant-p53 and MYC as a novel strategy to inhibit oncogenic SPAG5 activity in triple negative breast cancer

Triple negative breast cancer (TNBC) is an aggressive disease which currently has no effective therapeutic targets and prominent biomarkers. The Sperm Associated antigen 5 (SPAG5) is a mitotic spindle associated protein with oncogenic function in several human cancers. In TNBC, increased SPAG5 expression has been associated with tumor progression, chemoresistance, relapse, and poor clinical outcome. Here we show that high SPAG5 expression in TNBC is regulated by coordinated activity of YAP, mutant p53 and MYC. Depletion of YAP or mutant p53 proteins reduced SPAG5 expression and the recruitment of MYC onto SPAG5 promoter. Targeting of MYC also reduced SPAG5 expression and concomitantly tumorigenicity of TNBC cells. These effects of MYC targeting were synergized with cytotoxic chemotherapy and markedly reduced TNBC oncogenicity in SPAG5-expression dependent manner. These results suggest that mutant p53-MYC-SPAG5 expression can be considered as bona fide predictors of patient’s outcome, and reliable biomarkers for effective anticancer therapies.

Transcription factor 4 is a key mediator of oncogenesis in neuroblastoma by promoting MYC activity.

Super-enhancer-associated transcription factor networks define cell identity in neuroblastoma (NB). Dysregulation of these transcription factors contributes to the initiation and maintenance of NB by enforcing early developmental identity states. We report that the class I basic helix-loop-helix (bHLH) transcription factor 4 (TCF4; also known as E2-2) is a critical NB dependency gene that significantly contributes to these identity states through heterodimerization with cell-identity-specific bHLH transcription factors. Knockdown of TCF4 significantly induces apoptosis in vitro and inhibits tumorigenicity in vivo. We used genome-wide expression profiling, TCF4 chromatin immunoprecipitation sequencing (ChIP-seq) and TCF4 immunoprecipitation-mass spectrometry to determine the role of TCF4 in NB cells. Our results, along with recent findings in NB for the transcription factors T-box transcription factor TBX2, heart- and neural crest derivatives-expressed protein 2 (HAND2) and twist-related protein 1 (TWIST1), propose a role for TCF4 in regulating forkhead box protein M1 (FOXM1)/transcription factor E2F-driven gene regulatory networks that control cell cycle progression in cooperation with N-myc proto-oncogene protein (MYCN), TBX2, and the TCF4 dimerization partners HAND2 and TWIST1. Collectively, we showed that TCF4 promotes cell proliferation through direct transcriptional regulation of the c-MYC/MYCN oncogenic program that drives high-risk NB. Mechanistically, our data suggest the novel finding that TCF4 acts to support MYC activity by recruiting multiple factors known to regulate MYC function to sites of colocalization between critical NB transcription factors, TCF4 and MYC oncoproteins. Many of the TCF4-recruited factors are druggable, giving insight into potential therapies for high-risk NB. This study identifies a new function for class I bHLH transcription factors (e.g., TCF3, TCF4, and TCF12) that are important in cancer and development.

Loss of multi-level 3D genome organization during breast cancer progression.

Breast cancer entails intricate alterations in genome organization and expression. However, how three-dimensional (3D) chromatin structure changes in the progression from a normal to a breast cancer malignant state remains unknown. To address this, we conducted an analysis combining Hi-C data with lamina-associated domains (LADs), epigenomic marks, and gene expression in an in vitro model of breast cancer progression. Our results reveal that while the fundamental properties of topologically associating domains (TADs) are overall maintained, significant changes occur in the organization of compartments and subcompartments. These changes are closely correlated with alterations in the expression of oncogenic genes. We also observe a restructuring of TAD-TAD interactions, coinciding with a loss of spatial compartmentalization and radial positioning of the 3D genome. Notably, we identify a previously unrecognized interchromosomal insertion event, wherein a locus on chromosome 8 housing the MYC oncogene is inserted into a highly active subcompartment on chromosome 10. This insertion is accompanied by the formation of de novo enhancer contacts and activation of MYC , illustrating how structural genomic variants can alter the 3D genome to drive oncogenic states. In summary, our findings provide evidence for the loss of genome organization at multiple scales during breast cancer progression revealing novel relationships between genome 3D structure and oncogenic processes.

Medaka liver developed Human NAFLD-NASH transcriptional signatures in response to ancestral bisphenol A exposure.

The progression of fatty liver disease to non-alcoholic steatohepatitis (NASH) is a leading cause of death in humans. Lifestyles and environmental chemical exposures can increase the susceptibility of humans to NASH. In humans, the presence of bisphenol A (BPA) in urine is associated with fatty liver disease, but whether ancestral BPA exposure leads to the activation of human NAFLD-NASH-associated genes in the unexposed descendants is unclear. In this study, using medaka fish as an animal model for human NAFLD, we investigated the transcriptional signatures of human NAFLD-NASH and their associated roles in the pathogenesis of the liver of fish that were not directly exposed, but their ancestors were exposed to BPA during embryonic and perinatal development three generations prior. Comparison of bulk RNA-Seq data of the liver in BPA lineage male and female medaka with publicly available human NAFLD-NASH patient data revealed transgenerational alterations in the transcriptional signature of human NAFLD-NASH in medaka liver. Twenty percent of differentially expressed genes (DEGs) were upregulated in both human NAFLD patients and medaka. Specifically in females, among the total shared DEGs in the liver of BPA lineage fish and NAFLD patient groups, 27.69% were downregulated, and 20% were upregulated. Of all DEGs, 52.31% of DEGs were found in ancestral BPA-lineage females, suggesting that NAFLD in females shared the majority of human NAFLD gene networks. Pathway analysis revealed beta-oxidation, lipoprotein metabolism, and HDL/LDL-mediated transport processes linked to downregulated DEGs in BPA lineage males and females. In contrast, the expression of genes encoding lipogenesis-related proteins was significantly elevated in the liver of BPA lineage females only. BPA lineage females exhibiting activation of myc, atf4, xbp1, stat4, and cancerous pathways, as well as inactivation of igf1, suggest their possible association with an advanced NAFLD phenotype. The present results suggest that gene networks involved in the progression of human NAFLD and the transgenerational NAFLD in medaka are conserved and that medaka can be an excellent animal model to understand the development and progression of liver disease and environmental influences in the liver.

Body-Wide Inactivation of the Myc-Like Mlx Transcription Factor Network Accelerates Aging and Increases the Lifetime Cancer Incidence.

The "Mlx" and "Myc" transcription factor networks cross-communicate and share many common gene targets. Myc's activity depends upon its heterodimerization with Max, whereas the Mlx Network requires that the Max-like factor Mlx associate with the Myc-like factors MondoA or ChREBP. The current work demonstrates that body-wide Mlx inactivation, like that of Myc, accelerates numerous aging-related phenotypes pertaining to body habitus and metabolism. The deregulation of numerous aging-related Myc target gene sets is also accelerated. Among other functions, these gene sets often regulate ribosomal and mitochondrial structure and function, genomic stability, and aging. Whereas "MycKO" mice have an extended lifespan because of a lower cancer incidence, "MlxKO" mice have normal lifespans and a higher cancer incidence. Like Myc, the expression of Mlx, MondoA, and ChREBP and their control over their target genes deteriorate with age in both mice and humans. Collectively, these findings underscore the importance of lifelong and balanced cross-talk between the two networks to maintain proper function and regulation of the many factors that can affect normal aging.

WNT Oncogenic Transcription Requires MYC Suppression of Lysosomal Activity and EPCAM Stabilization in Gastric Tumors

Background and aimsWNT signaling is central to spatial tissue arrangement, regulating stem cell activity, and represents the hallmark of gastrointestinal cancers. While its role in driving intestinal tumors is well characterized, WNT's role in gastric tumorigenesis remains elusive. MethodsWe have developed mouse models to control the specific expression of an oncogenic form of B-CATENIN in combination with MYC activation in Lgr5+ cells of the gastric antrum. We used multi-omics approaches applied in vivo and in organoid models to characterize their cooperation in driving gastric tumorigenesis. ResultsWe report that constitutive B-CATENIN stabilization in the stomach has negligible oncogenic effects and requires MYC activation to induce gastric tumour formation. While physiologically low MYC levels in gastric glands limit B-CATENIN transcriptional activity, increased MYC expression unleashes the WNT oncogenic transcriptional program, promoting B-CATENIN enhancer invasion without a direct transcriptional cooperation. MYC activation induces a metabolic rewiring that suppresses lysosomal biogenesis through mTOR and ERK activation and MiT/TFE inhibition. This prevents EPCAM degradation by macropinocytosis, promoting B-CATENIN chromatin accumulation and activation of WNT oncogenic transcription. ConclusionOur results uncovered a new signaling framework with important implications for the control of gastric epithelial architecture and WNT-dependent oncogenic transformation.

Abstract PO-037: Machine learning-enabled transomics identifies three therapeutic targets for MYC-driven diffuse large B cell lymphoma

Abstract MYC activation and dysregulation is a powerful oncogenic driver in multiple cancers, including diffuse large B cell lymphoma (DLBCL). There is a known correlation between MYC expression and poor prognosis. While the 5-year overall survival for DLBCL patients on first-line therapy, R-CHOP, is approximately 75%, the 5-year overall survival drops to a staggering 30% for patients co-expressing MYC and BCL2 (double expressor lymphoma). However, it is challenging to target MYC directly. Therefore, we identified a novel approach to circumvent targeting MYC directly by leveraging Pepper’s proprietary machine learning (ML) transomic analysis platform, COMPASS, to identify novel targets associated with high MYC activity that, when inhibited, are predicted to recapitulate the effect of MYC inactivation. Specifically, COMPASS unlocks functional drivers of disease to identify novel drug targets. To study the role of MYC in lymphoma biology, we utilize a MYC-conditional T cell acute lymphoblastic leukemia/lymphoma (T-ALL) cell line (4188) with tunable MYC expression. We collect genomic, transcriptomic, proteomic, and phosphoproteomic data from samples with MYC transgene expression “on” and “off” and compare these biological cell states via COMPASS to identify novel targets that reproduce MYC inactivation. This provides new hope for patients suffering from MYC driven DLBCL. The MYC-conditional cell line allows the regulation of MYC expression via the tetracycline regulatory (Tet-Off) system. Four omic datasets were collected from each sample: genomics (next-generation sequencing, NGS), transcriptomics (NGS), proteomics (mass spectrometry), and phosphoproteomics (mass spectrometry). We employed the COMPASS target prioritization algorithm to identify and rank novel targets that mimic “turning MYC off”. Targets were then filtered to select those with an available pharmacological tool compound (PTC). The PTCs were used to evaluate the targets in the SU-DHL-06 xenograft model of DLBCL. The PTCs inhibited tumor growth from 8-91%. A total of 20 targets were tested of which 13/20 (65%) were validated as evidenced by significant inhibition of tumor growth (p&amp;lt;0.05, two-way ANOVA). All targets were kinases or related to kinase activity and pathways. Three of the PTCs resulted in maximum tumor growth inhibition of 86%, 89% and 91% at end of treatment, resulting in stasis of tumor growth and increased survival compared with control vehicle-treated mice. We have previously shown how the COMPASS transomics analysis approach can identify novel drug targets for drug-resistant EGFRm NSCLC and for MYC-driven hepatocellular carcinoma (HCC). The success rate of target validation across several indications including EGFRm NSCLC, HCC and lymphoma is 73%. The data presented here on MYC-driven DLBCL further validates this approach. Further validation studies will investigate novel targets using gene silencing, as PTCs were unavailable for many of the novel high-ranked targets identified in this study. Citation Format: Simon P Fricker, Christopher J Nicholson, Samuel J Roth, Arudhir Singh, Caitlin Brown, Jon Hu, Petronela Buiga, Vishnu P Kanakaveti, Anja Deutzmann, Dean Felsher, Samantha D Strasser. Machine learning-enabled transomics identifies three therapeutic targets for MYC-driven diffuse large B cell lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-037.

MDB-109. CHARACTERIZING DISTINCT SIGNATURES OF RNA TRANSLATIONAL REGULATION UPON MYC OVEREXPRESSION IN H9 NEURAL STEM CELLS

Abstract BACKGROUND Medulloblastoma (MB), a childhood brain tumor, emerges typically between 6-8 years, originating from cerebellar neuronal stem cells. Recurrent genetic alterations, notably MYC amplifications, are associated with poor therapy response. Subgrouped as WNT-MB, SHH-MB, Group 3 MB, and Group 4 MB, Group 3 MB is characterized by high-level MYC amplification in approximately 17% of cases. Patients with Group 3 MB and MYC activation present distinct transcriptional and proteomic signatures, including elevated levels of ribosomal proteins and ribosome assembly proteins. METHODS We conducted a comprehensive analysis of RNA-seq and Ribo-seq in human neural stem cells (H9 NSCs) with amplified MYC expression. H9-NSCs were cultured in tumor stem media and transduced with lentivirus for either GFP control or MYC cDNA. RNA and ribosome-protected fragments were isolated from cell lysates for RNA-seq and Ribo-seq in biological triplicates. Ribo-seq data was analyzed using ORFquant for coding sequence quantification and deltaTE for translational efficiency. RESULTS Overexpressing MYC in H9-NSCs led to significant differential expression of 4194 genes (p adj &amp;lt; 0.05) using RNA-seq and 2860 genes (p adj &amp;lt; 0.05) using Ribo-seq. While RNA-seq and Ribo-seq exhibited high correlation for most genes, we identified 364 differentially translated genes (p adj &amp;lt; 0.05) upon MYC overexpression. Genes regulated solely at the translational level were enriched for functions in cytoplasmic translation initiation, viral translation, and response to interleukin-4. Key genes highly regulated at the Ribo-seq level included CCND3, YBX1, and EEF2. Validating with proteomics data from the CPTAC medulloblastoma tissue set confirmed protein-specific upregulation of these targets in MYC-driven Group 3 medulloblastomas. CONCLUSIONS MYC overexpression revealed distinct behaviors in differentially expressed genes, either regulated at the level of translation alone, correlated with transcription, or independent of transcription. We anticipate that our findings will contribute to a refined understanding of the molecular landscape of MB, particularly in the context of Group 3 MB.

Abstract B004: An isogenic CRISPR screen identifies novel MYC-driven vulnerabilities

Abstract c-MYC (MYC) is a central regulatory protein that is dysregulated in &amp;gt;50% of all human cancers and is linked to aggressive disease. Developing MYC inhibitors would revolutionize cancer treatment; however, developing small molecules that directly target MYC is challenging. An alternative approach is to identify and inhibit critical MYC partner proteins to inactivate MYC and trigger cancer cell death. Inhibiting these targets therapeutically can result in synthetic lethality (MYC-SL), which can be exploited in MYC-dysregulated cancers. To identify MYC-SL targets, we performed a genome-wide CRISPR knock-out screen using an isogenic pair of normal and MYC-driven breast cancer cells. In contrast to other screens, this model is dependent on MYC and recapitulates human disease at both pathological and molecular levels in vivo. We identified high-priority hits to validate from the screen using two independent approaches: 1) a traditional gene-set enrichment analysis to identify highly represented biological pathways; and 2) analyzing the Cancer Dependency Map (DEPMAP) to select hits that are likely to be robust beyond the context of our screening conditions. Using a traditional gene-set enrichment analysis approach, we identified topoisomerase 1 (TOP1) as an actionable vulnerability that can be targeted with clinically approved inhibitors. Genetic and pharmacological inhibition of TOP1 resulted in MYC-driven cell death compared to that in control cells. Finally, drug response to TOP1 inhibitors significantly correlated with MYC levels and activity across panels of breast cancer cell lines and patient-derived organoids, highlighting TOP1 as a promising target for MYC-driven cancers. As a secondary approach to interpreting our CRISPR screen hits, we analyzed DEPMAP to identify MYC-SLs that are differentially essential in MYC-dependent cancer cells. Specifically, data from RNA interference screens in hundreds of cancer cell lines were used to describe the response of these cells to MYC knockdown. These data were used to define MYC-dependent and MYC-independent cell lines within the context of this analysis. These two groups were then investigated for their in silico response to the knockdown of each of our MYC-SL hits. MYC-SLs, which were also differentially essential in MYC-dependent cancer cells from DEPMAP, were prioritized for further investigation. Critical MYC cofactors that have been validated by us and others (e.g., CDK9) were identified, providing confidence in this approach, and rationalizing ongoing investigations. Together, this work features two successful strategies to prioritize hits from hundreds of synthetic-lethal genome-wide CRISPR screens to identify novel MYC-driven vulnerabilities in cancer. Citation Format: Peter Lin, Linda Penn. An isogenic CRISPR screen identifies novel MYC-driven vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B004.

Dose-dependent effects of Dnmt3a in an inducible murine model of KrasG12D-driven leukemia

DNMT3A mutations are frequently found in clonal hematopoiesis and a variety of hematologic malignancies, including acute myeloid leukemia. An assortment of mouse models have been engineered to explore the tumorigenic potential and malignant lineage bias due to loss of function of DNMT3A in consort with commonly comutated genes in myeloid malignancies, such as Flt3, Nras, Kras, and c-Kit. We employed several tamoxifen-inducible Cre-ERT2 murine model systems to study the effects of constitutively active KrasG12D-driven myeloid leukemia (Kras) development together with heterozygous (3aHet) or homozygous Dnmt3a deletion (3aKO). Due to the rapid generation of diverse nonhematologic tumors appearing after tamoxifen induction, we employed a transplantation model. With pretransplant tamoxifen induction, most Kras mice died quickly of T-cell malignancies regardless of Dnmt3a status. Using posttransplant induction, we observed a dose-dependent effect of DNMT3A depletion that skewed the leukemic phenotype toward a myeloid lineage. Specifically, 64% of 3aKO/Kras mice had exclusively myeloid disease compared with 36% of 3aHet/Kras and only 13% of Kras mice. Here, 3aKO combined with Kras led to increased disease burden, multiorgan infiltration, and faster disease progression. DOT1L inhibition exerted profound antileukemic effects in malignant 3aKO/Kras cells, but not malignant cells with Kras mutation alone, consistent with the known sensitivity of DNMT3A-mutant leukemia to DOT1L inhibition. RNAseq from malignant myeloid cells revealed that biallelic Dnmt3a deletion was associated with loss of cell-cycle regulation, MYC activation, and TNF⍺ signaling. Overall, we developed a robust model system for mechanistic and preclinical investigations of acute myeloid leukemia with DNMT3A and Ras-pathway lesions.

Dynamic Transcriptional Programs During Single NK Cell Killing: Connecting Form to Function in Cellular Immunotherapy.

Natural killer (NK) cell-based therapies are a promising new method for treating indolent cancer, however engineering new therapies is complex and progress towards therapy for solid tumors is slow. New methods for determining the underlying intracellular signaling driving the killing phenotype would significantly improve this progress. We combined single-cell RNA sequencing with live cell imaging of a model system of NK cell killing to correlate transcriptomic data with functional output. A model of NK cell activity, the NK-92 cell line killing of HeLa cervical cancer cells, was used for these studies. NK cell killing activity was observed by microscopy during co-culture with target HeLa cells and killing activity subsequently manually mapped based on NK cell location and Annexin V expression. NK cells from this culture system were profiled by single-cell RNA sequencing using the 10× Genomics platform, and transcription factor activity inferred using the Viper and DoRothEA R packages. Luminescent microscopy of reporter constructs in the NK cells was then used to correlate activity of inferred transcriptional activity with killing activity. NK cells had heterogeneous killing activity during 10h of culture with target HeLa cells. Analysis of the single cell sequencing data identified Nuclear Factor Kappa B (NF-κB), Signal Transducer and Activator of Transcription 1 (STAT1) and MYC activity as potential drivers of NK cell functional phenotype in our model system. Live cell imaging of the transcription factor activity found NF-κB activity was significantly correlated with past killing activity. No correlation was observed between STAT1 or MYC activity and NK cell killing. Combining luminescent microscopy of transcription factor activity with single-cell RNA sequencing is an effective means of assigning functional phenotypes to inferred transcriptomics data. The online version contains supplementary material available at 10.1007/s12195-024-00812-3.

Pharmacokinetic analysis of OMO-103 in patient tissues with advanced solid tumors.

e15168 Background: MYC is a pleiotropic transcription factor aberrantly expressed in up to 70% of human cancers. OMO-103 (Omomyc) is a 91-amino-acids-long polypeptide that derives from the B-HLH-LZ domain of human MYC, containing four amino acid substitutions. It is capable of forming homos and heterodimers with MYC and with MAX and blunt MYC activity by preventing its access to target DNA sequences (E-boxes) and replacing it by inactive complexes. Previous in vivo preclinical experiments showed that Omomyc blocks MYC-driven transcription and proliferation, induces apoptosis and recruitment of intratumoral immune infiltrates, significantly abrogating tumor progression and reducing tumor grading. Here, we present PK results from the first-in-human trial (NCT04808362) of OMO-103 in patients with advanced solid tumors. Methods: Patient eligibility The study included histologically or cytologically proven advanced solid tumors for which there was no curative therapy. Patients had to demonstrate measurable disease according to RECIST v.1.1 criteria as demonstrated by CT/magnetic resonance imaging and documented progression on or following the last line of therapy. Description and dose selection OMO-103 was administered by weekly I.V. infusion over 30–45 min, one treatment cycle corresponding to three infusions. Patients were treated until progression. The starting dose of 0.48 mg kg−1 was determined based on nonclinical toxicology and efficacy studies (anticipated pharmacologically active dose) and subsequent conversion to a human equivalent dose. Pharmacokinetic assays A targeted mass spectrometry (MS) assay for the absolute quantification of 4 Omomyc proteotypic peptides was developed to quantify functional OMO-103 in tumor tissue. LLOQ and linear range of the panel was determined by spiking in dilution series of recombinant OMO-103 protein into tumor tissue lysates. Finally, in the phase I trial, we used the panel for the quantification of OMO-103 in FFPE tumor biopsies from 12 patients who received i.v. injection of OMO-103. Results: MS Data show that 2 h after i.v. administration, higher concentrations of the drug are reached in the tumors compared with serum and persist there with at least one order of magnitude higher concentration than in serum 72 h after administration in preclinical models. Functional Omomyc protein could be quantified in all 12 clinical tumor biopsies from treated patients and PK results confirmed previous observations in preclinical models. where Omomyc exhibits higher tissue concentration and prolonged residency compared to plasma. Conclusions: We demonstrate the utility of targeted MS assay for precise quantification of therapeutic (mini)proteins in clinical tissue samples. Thanks to its multiplexing capability, up to 60 peptides (epitopes) can be monitored simultaneously which enables high sequence coverage and profiling of additional PD or other supporting biomarkers. Clinical trial information: NCT04808362 .

CDK12-inactivation-induced MYC signaling causes dependency on the splicing kinase SRPK1.

Inactivation of cyclin-dependent kinase 12 (CDK12) characterizes an aggressive sub-group of castration-resistant prostate cancer (CRPC). Hyper-activation of MYC transcription factor is sufficient to confer the CRPC phenotype. Here, we show that loss of CDK12 promotes MYC activity, which renders the cells dependent on the otherwise non-essential splicing regulatory kinase SRSF protein kinase 1 (SRPK1). High MYC expression is associated with increased levels of SRPK1 in patient samples, and overexpression of MYC sensitizes prostate cancer cells to SRPK1 inhibition using pharmacological and genetic strategies. We show that Endovion (SCO-101), a compound currently in clinical trials against pancreatic cancer, phenocopies the effects of the well-characterized SRPK1 inhibitor SRPIN340 on nascent transcription. This is the first study to show that Endovion is an SRPK1 inhibitor. Inhibition of SRPK1 with either of the compounds promotes transcription elongation, and transcriptionally activates the unfolded protein response. In brief, here we discover that CDK12 inactivation promotes MYC signaling in an SRPK1-dependent manner, and show that the clinical grade compound Endovion selectively targets the cells with CDK12 inactivation.

Sex-specific expression of the human NAFLD-NASH transcriptional signatures in the liver of medaka with a history of ancestral bisphenol A exposure.

The progression of fatty liver disease to non-alcoholic steatohepatitis (NASH) is a leading cause of death in humans. Lifestyles and environmental chemical exposures can increase the susceptibility of humans to NASH. In humans, the presence of bisphenol A (BPA) in urine is associated with fatty liver disease, but whether ancestral BPA exposure leads to the activation of human NAFLD-NASH-associated genes in the unexposed descendants is unclear. In this study, using medaka fish as an animal model for human NAFLD, we investigated the transcriptional signatures of human NAFLD-NASH and their associated roles in the pathogenesis of the liver of fish who were not directly exposed but their ancestors were exposed to BPA during embryonic and perinatal development three generations prior. Comparison of bulk RNA-Seq data of the liver in BPA lineage male and female medaka with publicly available human NAFLD-NASH patient data revealed transgenerational alterations in the transcriptional signature of human NAFLD-NASH in medaka liver. Twenty percent of differentially expressed genes (DEGs) were upregulated in both human NAFLD patients and medaka. Specifically in females, among the total shared DEGs in the liver of BPA lineage fish and NAFLD patient groups, 27.69% DEGs were downregulated and 20% DEGs were upregulated. Off all DEGs, 52.31% DEGs were found in ancestral BPA-lineage females, suggesting that NAFLD in females shared majority of human NAFLD gene networks. Pathway analysis revealed beta-oxidation, lipoprotein metabolism, and HDL/LDL-mediated transport processes linked to downregulated DEGs in BPA lineage males and females. In contrast, the expression of genes encoding lipogenesis-related proteins was significantly elevated in the liver of BPA lineage females only. BPA lineage females exhibiting activation of myc, atf4, xbp1, stat4, and cancerous pathways, as well as inactivation of igf1, suggest their possible association with an advanced NAFLD phenotype. The present results suggest that gene networks involved in the progression of human NAFLD and the transgenerational NAFLD in medaka are conserved and that medaka can be an excellent animal model to understand the development and progression of liver disease and environmental influences in the liver.

Allele-level visualization of transcription and chromatin by high-throughput imaging

The spatial arrangement of the genome within the nucleus is a pivotal aspect of cellular organization and function with implications for gene expression and regulation. While all genome organization features, such as loops, domains, and radial positioning, are nonrandom, they are characterized by a high degree of single-cell variability. Imaging approaches are ideally suited to visualize, measure, and study single-cell heterogeneity in genome organization. Here, we describe two methods for the detection of DNA and RNA of individual gene alleles by fluorescence in situ hybridization (FISH) in a high-throughput format. We have optimized combined DNA/RNA FISH approaches either using simultaneous or sequential detection of DNA and nascent RNA. These optimized DNA and RNA FISH protocols were implemented in a 384-well plate format alongside automated image and data analysis and enable accurate detection of individual gene alleles and their gene expression status across a large cell population. We successfully visualized MYC and EGFR DNA and nascent RNA with allele-level resolution in multiple cell types, and we determined the radial position of active and inactive MYC and EGFR alleles. These optimized DNA/RNA detection approaches are versatile and sensitive tools for mapping of chromatin features and gene activity at the single-allele level and at high throughput.