Putative Oncogene Research Articles

Abstract PR-15: Identifying synergistic combinations with KRAS inhibition in PDAC

Abstract Background: To date, targeted therapies have largely failed in eliciting sustained responses in advanced pancreatic ductal adenocarcinoma (PDAC). KRAS-targeted therapies have the potential to drastically transform clinical management. Emerging data indicate that only a subset of patients respond, and that acquired resistance is common. Here, we used a combination of in vivo CRISPR activation (CRISPRa) screening and in vitro/preclinical models to identify synergistic combinations with KRAS inhibition (KRASi) in pancreatic and lung cancer. Material and Methods: We used our CRISPRa-competent PPKS (P53(F/F), Kras(LSL-G12D/+), R26(LSL-SAM)) model of adenocarcinoma to conduct targeted screening for drivers of resistance to MRTX133. Our library consisted of ∼450 frequently mutated and amplified putative oncogenes across human adenocarcinoma based on TCGA data. Mice were treated with 1e8 TU of lentiviral library through nasal instillation. After 6 weeks, treatment was initiated with 3 mg/kg MRTX1133 daily, 5 days/week (i.p.). Mice were sacrificed after 4 weeks of treatment and the guide distribution determined through bioinformatic analysis. Genes statistically significantly enriched (FDR<0.05) in the treatment group were determined. Cell lines with acquired resistance to MRTX1133 derived from the KPC PDAC model and the KPP lung adenocarcinoma (LUAD) model were generated using long-term, low-dose treatment. Resistance was confirmed with MTT drug response assays. In vitro combinatorial drug response was determined using a 72h, 96-well MTT assay, and clonogenic response using a 2 week, 384-well FACS assay. Mice were treated with 20 mg/kg MRTX1133 and/or 20mg/kg Temuterkib once daily, 7 days/week (i.p.) for 2 weeks. Results: We performed targeted in vivo CRISPRa screening for drivers of resistance to KRASi in an autochthons model of lung adenocarcinoma (LUAD) using a library targeting 452 genes commonly amplified or mutated in cancer. We identified a set of genes that were specifically enriched in at least 50% of tumors treated with MRTX1133, including MAPK1/ERK2. We also generated 16 cell lines with acquired resistance to MRTX1133, derived from KPC PDAC or KPP LUAD tumors. Transcriptomic analysis identified a large set of genes differentially expressed in cell with acquired resistance relative to controls. GSEA indicated that reactivation of the MAPK cascade as a common trait in resistant cells. To test if ERK inhibition synergizes with KRASi, we quantified the combinatorial response to MRTX1133 and Temuterkib, a specific inhibitor of ERK1 and ERK2, in a set of KPC and KPP derived cell lines, revealing significant synergy and reduced clonogenic potential in a majority of cell lines. A majority of cell lines with acquired resistance to MRTX1133 remained sensitive to Temuterkib. Finally, we tested combinatorial treatment of MRTX1133 and Temuterkib in a syngenetic subcutaneous model of PDAC, which showed at minimum additive effect. In conclusion we have identified ERK inhibition as a promising combination with KRAS inhibition in KRAS-mutant PDAC and LUAD. Citation Format: Fredrik I Thege, Amber Hoskins, Sonja M Woermann, Anirban Maitra. Identifying synergistic combinations with KRAS inhibition in PDAC [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 PR-15.

Abstract 458: Nanoparticle-mediated delivery of miR-590-3p decreases recurrent GBM tumor growth by inhibits multiple oncogenic nodes

Abstract Glioblastoma (GBM) is the most lethal and aggressive form of brain cancer. Despite aggressive therapy consisting of surgery followed by radio/chemotherapy GBM recurs in almost all patients and, currently, there are no proven therapies to treat recurrent GBM (rGBM). Recent developments in nanomedicine provide new and promising opportunities to develop new targeted therapeutics to treat brain tumors. We have recently developed PBAE nanoparticle formulations optimized for in vivo miRNA delivery to brain tumor stem cells, or GSCs. Carefully selecting one miRNA to target several mRNAs dysregulated within a pathway can allow the targeting of multiple nodes using a single agent, mimicking combinatorial therapies. To test this hypothesis, we performed bioinformatic analysis of RNA sequencing from GSCs and clinical rGBM specimens combined with gene set enrichment analysis (GSEA) and identified TGFBR2 signaling as a targetable pathway in rGBM. We show that SMAD2 signaling, a downstream effector of TGFBR2, is enriched in rGBM and that patients with elevated TGFBR2 transcripts respond poorly to current standard of care. Utilizing patient-derived xenograft lines from rGBM, we show that blocking TGFBR2 signaling using siRNAs or ITD1, a selective TGFBR2 inhibitor, decreases the stem cell capacity, cell viability and re-sensitizes clinical rGBM cells to temozolomide (TMZ) in-vitro. In vivo, we show that ITD1 is modestly effective in subcutaneous and ineffective in orthotopic rGBM models. Not surprisingly, these tumors exhibited classical growth patterns associated with acquisition of resistance. To overcome this obstacle, we performed a miRNA-based network analysis in rGBM and identified miR-590-3p as a putative tumor suppressor that targets SMAD2 signaling at multiple nodes in rGBM. In-vitro, transgenic miR-590-3p robustly decreased the expression of 37 putative oncogenes simultaneously concurrent with decreasing stem cell capacity, cell viability and re-sensitizing rGBM PDX cells to TMZ. In vivo delivery of miR-590-3p to established PDX-derived rGBM orthotopic tumors using novel PBAE nanocarriers robustly decreased tumor sizes and increased tumor necrosis. These results show that the promiscuous nature of miRNAs, in combination with cutting-edge nucleic acid delivery vehicles, can be leveraged as advanced targeted therapeutics for rGBM bypasses the resistance that is developed to standard of care and available small molecules. Citation Format: Hernando Lopez-Bertoni, Sophie Sall, Harmon Khela, Jack Korleski, Katherine Luly, Maya Johnson, Bachchu Lal, Amanda Johnson, Stephany Tzeng, Jordan Green, John Laterra. Nanoparticle-mediated delivery of miR-590-3p decreases recurrent GBM tumor growth by inhibits multiple oncogenic nodes [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 458.

A comprehensive integrated gene network construction to explore the essential role of Notch 1 in lung adenocarcinoma (LUAD)

The heterogeneous biological landscape of non-small cell lung cancer (NSCLC) is largely attributed to the activation of Notch signalling pathway. Among the Notch family transmembrane proteins, neurogenic locus notch homolog protein1 (NOTCH1) is a putative oncogene in NSCLC which activates the pathway as negative prognostic factor. This study aims to explore integrated network approach in lung adenocarcinoma (LUAD) especially linked to the notch pathway and its receptors. Our gene set enrichment analysis reveals the key Notch pathway genes are predominantly down regulated in LUAD. There were 675 genes with a total of 6517 functional interactions and 6 densely connected clusters of 38 miRNAs, 84 transcription factors with 156 edges identified through network construction. Here we report five key genes namely NOTCH1, CDH1, ERBB2, GAPDH and COL1A1 significantly enriched in Notch pathway which are further validated through the KM plot, box plots, stage plots and TIMER analysis. In addition, the NOTCH1 receptor is strongly linked to the immune checkpoint inhibitor CD274 (PD-L1) and can be considered as prognostic marker and tumour suppressor gene in LUAD which surely provide the basis for early diagnosis and futuristic immunotherapeutic targets for LUAD. Communicated by Ramaswamy H. Sarma

Biomarker Driven Strategies for Targeted Elimination of Hypoxia Adapted Lymphoma Cells

Large body of evidence suggests that hypoxia drives aggressive molecular features irrespective of cancer type. Non-Hodgkin lymphomas (NHL) are the most common hematologic malignancies characterized by widespread disease and frequent involvement of diverse hypoxic microenvironments (e.g., bone marrow, malignant effusions, large lymphoma masses). Impact of long-term hypoxia on the biology of lymphoma cells and its potential role in mediating drug resistance and disease relapses due to adaptive changes shaped by the hypoxic microenvironment remain only partially understood. In this study we analyzed impact of short- and long-term hypoxia on a panel of six lymphoma cell lines including diffuse large B-cell lymphoma (UPF4D, UPF8D), Burkitt lymphoma (Ramos, UPF9T), and mantle cell lymphoma (HBL2, MINO). Only 2 out of 8 tested lymphoma cell lines (Ramos, and HBL2) survived longer than 4 weeks under deep hypoxia (1% O2). The remaining tested lymphoma cell lines died by hypoxia-induced apoptosis (which could be inhibited by co-cultivation with pan-caspase inhibitor Z-VAD-FMK). The hypoxia-adapted (HA) Ramos and HBL2 cells had severely decreased proliferation rate accompanied by complex changes of the transcriptome, proteome, and metabolome. Seahorse analysis revealed significant inhibition of both oxidative phosphorylation and glycolytic pathways ( Figure 1). Of note, the whole-proteome profiling confirmed significant downregulation of proteins regulating oxidative phosphorylation, but significant upregulation of proteins regulating glycolysis. Sensitivity of HA cells to 2-deoxyglucose, an inhibitor of glycolysis was markedly increased ( Figure 1). The data suggest that under long-term deep hypoxia, lymphoma cells try to compensate for the decrease in ATP production from the oxidative phosphorylation process by boosting structural machinery of glycolysis, on which they became vitally dependent for survival. Despite the increased glycolytic machinary, however, the level of glycolysis decreases due to severe lack of oxygen. In our opinion, this is a clear representation of the Warburg effect, a hallmark of cancer based on the metabolic reprogramming of the cancer cells under hypoxia. Sensitivity of HA cells to a panel of the tested cytotoxic drugs (cytarabin, cisplatin, bortezomib) and targeted agents (venetoclax, S63545, A1155463, TRAIL, polatuzumab vedotin, IM-156, copanlisib) was either unchanged or increased (compared to sensitivity of the original cell lines cultured under normoxia). In contrast to so far published data, long-term hypoxia was not associated with acquired drug resistance in case of any of the tested agent. Of note, HA cells became significantly more sensitive to A1155463, an inhibitor of anti-apoptotic protein BCL-XL ( Figure 1). Unchanged levels of BCL-XL protein in HA cells and the respective normoxic cell lines (detected by western blotting) suggest that more complex mechanisms plausibly underlie the observed markedly increased sensitivity to A1155463. The whole-transcriptome and proteome analyses of both HA cell lines (compared to the parental cell lines cultured under normoxic conditions) detected among other complex changes signficant upregulation of P4HA1 (both mRNA, and protein), a prolyl hydroxylase involved in the stabilization of hypoxia-induced factor (HIF) 1 alpha, and putative oncogene associated with tumor aggressiveness. Transgenic (over)expression of P4HA1 in hypoxia-sensitive MINO cells effectively inhibited the hypoxia-induced apoptosis. Western blot analysis implemented on a panel of primary lymphoma cells revealed markedly higher expression of P4HA1 protein in the lymphoma cells obtained from malignant effusions (putative hypoxic microenvironment) compared to lymphoma cells obtained from leukemized blood (putative normoxic microenvironment). Our data suggest that P4HA1 positively impacts survival of lymphoma cells under hypoxia. In translation, P4HA1, BCL-XL, and structural proteins of the glycolytic pathway may represent novel drugable targets for more effective elimination of hypoxia-adapted lymphoma cells. Financial Support: Ministry of Health of the Czech Republic AZV NU23-03-00172, Grant Agency of the Czech Republic GA23-05377S, and National Institute for Cancer Research (EXCELES) LX22NPO5102.

Functional Genomics of Novel Rhabdomyosarcoma Fusion-Oncogenes Using Zebrafish.

Clinical sequencing efforts continue to identify novel putative oncogenes with limited strategies to perform functional validation in vivo and study their role in tumorigenesis. Here, we present a pipeline for fusion-driven rhabdomyosarcoma (RMS) in vivo modeling using transgenic zebrafish systems. This strategy originates with novel fusion-oncogenes identified from patient samples that require functional validation in vertebrate systems, integrating these genes into the zebrafish genome, and then characterizing that they indeed drive rhabdomyosarcoma tumor formation. In this scenario, the human form of the fusion-oncogene is inserted into the zebrafish genome to understand if it is an oncogene, and if so, the underlying mechanisms of tumorigenesis. This approach has been successful in our models of infantile rhabdomyosarcoma and alveolar rhabdomyosarcoma, both driven by respective fusion-oncogenes, VGLL2-NCOA2 and PAX3-FOXO1. Our described zebrafish platform is a rapid method to understand the impact of fusion-oncogene activity, divergent and shared fusion-oncogenebiology, and whether any analyzed pathways converge for potential clinically actionable targets.

ETV5 facilitates tumor progression in head-neck squamous cell carcinoma.

E26 transformation-specific (ETS) factors have emerged as key mediators underlying human tumorigenesis. Here, we sought to characterize the expression pattern, biological roles, and clinical significance of ETS Variant Transcription Factor 5 (ETV5) in head neck squamous cell carcinoma (HNSCC). ETV5 expression pattern in HNSCC was determined by bioinformatics interrogations and immunohistochemical staining in primary samples. The associations between its abundance with clinicopathological parameters, and patient survival were evaluated. Colony formation, CCK-8, flow cytometry, wound healing, and Transwell invasion assays, as well as xenograft models, were utilized to determine the phenotypic changes after ETV5 silencing in vitro and vivo. The potential binding of ETV5 in the Slug promoter was determined by ChIP-qPCR. ETV5 was significantly overexpressed in HNSCC samples. Its overexpression is significantly associated with aggressiveness features and reduced survival. ETV5 knockdown significantly inhibited cell proliferation, migration, invasion, and induced apoptosis in vitro, and impaired tumor growth in vivo. Moreover, ETV5-activated Slug transcription by binding its promoter region in HNSCC cells. Patients with ETV5highSlughigh had the worst survival across multiple HNSCC cohorts. Our findings reveal that ETV5 serves as a novel prognostic biomarker and putative oncogene for HNSCC progression likely by activating Slug transcription.

An integrated genomic approach identifies follistatin as a target of the p63-epidermal growth factor receptor oncogenic network in head and neck squamous cell carcinoma.

Although numerous putative oncogenes have been associated with the etiology of head and neck squamous cell carcinoma (HNSCC), the mechanisms by which these oncogenes and their downstream targets mediate tumor progression have not been fully elucidated. We performed an integrative analysis to identify a crucial set of targets of the oncogenic transcription factor p63 that are common across multiple transcriptomic datasets obtained from HNSCC patients, and representative cell line models. Notably, our analysis revealed FST which encodes follistatin, a secreted glycoprotein that inhibits the transforming growth factor TGFβ/activin signaling pathways, to be a direct transcriptional target of p63. In addition, we found that FST expression is also driven by epidermal growth factor receptor EGFR signaling, thus mediating a functional link between the TGF-β and EGFR pathways. We show through loss- and gain-of-function studies that FST predominantly imparts a tumor-growth and migratory phenotype in HNSCC cells. Furthermore, analysis of single-cell RNA sequencing data from HNSCC patients unveiled cancer cells as the dominant source of FST within the tumor microenvironment and exposed a correlation between the expression of FST and its regulators with immune infiltrates. We propose FST as a prognostic biomarker for patient survival and a compelling candidate mediating the broad effects of p63 on the tumor and its associated microenvironment.

The genomic landscape and prognostic significance of 11.q.13 amplification in breast, head & neck, and lung cancer.

e18037 Background: The 11.q.13 (11q) chromosomal band is amplified in many cancers and is associated with poor outcomes in Head & Neck (HN) cancer. The 11q amplicon contains several putative oncogenes, but it’s unclear which amplified genes drive clinical outcomes. We compared the genomic landscape and prognostic significance of 11q amplification in Breast (BC), HN, and non-small cell lung cancer (NSCLC) tumors and identify genes whose expression may be driving clinical outcomes. Methods: DNA (592 genes or WES)/RNA (WTS) sequencing was performed for BC (N = 6,967), HN (N = 2,337), and NSCLC (N = 13,433) tumors submitted to Caris Life Sciences (Phoenix, AZ). 11q amplification (CNA) was defined as tumors with concurrent copy number amplification with copies of FGF3, FGF4, FGF19, and CCND1. Non-amplified tumors (copy number normal, CNN) were defined as a < 4 copies for all four genes. Her2/Neu (+: ≥3+ and > 10%) and HR (ER or PR+: ≥1+ and ≥1%) expression was tested by IHC. tests were applied as appropriate, with P-values adjusted for multiple comparisons. Real-world overall survival (OS) data was obtained from insurance claims, and Kaplan-Meier estimates were calculated for a molecularly defined subset of tumors. 11q genes that were two-fold over expressed in CNA vs CNN were further investigated. The hazard ratio (HR) was calculated for subgroups stratified by median expression of 11q genes. The genes with the five largest HRs (worse OS for CNA, p < .05) were identified as potential driver genes. Results: 11q CNA was observed in 10% of BC, 12% of HN, and 3% of NSCLC. 11q CNA rates were enriched in HR+ BC (HR+/HER2-: 17%, HR+/HER2+: 19%) as compared to HR- tumors (HR-/HER2+: 6%, TNBC: 3%, p < .05). TP53 mutations were less frequent in CNA BC (32 vs 53% CNN) but more frequent in CNA HN (91 vs 52%) and NSCLC (89 vs 66%, p < .05 all). In NSCLC, CNA tumors had a lower rate of EGFR (4.3 vs 12%), KRAS (6.7 vs 28%) and STK11 (5.7 vs 13%) mutations ( p < .05). Over 34 gene were amplified as compared to CNN ( > 3% increase, p < .05), including FGFR3 (11 vs 0.2%) and PDCD1 (10 vs 0). MEN1 (5 vs 0%) was the only 11q gene amplified. CNA was associated with worse OS in HR-/HER2+ (HR 6.3 [2.6-15.5], p = .001), while no difference in OS was observed in BC (HR 1.0 [.9-1.1], p = .62). Compared to CNN, CNA had worse OS in HN (HR 1.5 [1.3-1.7], p < .001) and NSCLC (HR 1.3 [1.2-1.4], p < .001). Stratification by 11q gene expression identified DHCR7, GAL, ANO1, MRPL21, and FADD as the strongest predictors of OS in HN tumors, while GAL, MYEOV, MRPL21, OVOL1, and DHCR7 were strongest in NSCLC (HR: 1.3-1.2). Conclusions: 11q CNA shows differential association with key driver mutations across cancer types that provides new insight to patient subpopulations harboring these alterations. Co-amplification of non-11q genes in NSCLC suggests a unique mechanism of genome instability. The role of DHCR7 and GAL as predictors of clinical outcomes in HN and NSCLC CNA tumors warrants further investigation.

Discovery of Potent, Selective, and Orally Bioavailable Small-Molecule Inhibitors of CDK8 for the Treatment of Cancer.

Cyclin-dependent kinase 8 (CDK8), as a kinase subunit of the Mediator complex, is involved in the regulation of RNA polymerase II-mediated transcription, thereby modulating multiple signaling pathways and multiple transcription factors involved in oncogenic control. CDK8 deregulation has been implicated in human diseases, particularly in acute myeloid leukemia (AML) and advanced solid tumors, where it has been reported as a putative oncogene. Here, we report the successful optimization of an azaindole series of CDK8 inhibitors that were identified and further progressed through a structure-based generative chemistry approach. In several optimization cycles, we improved in vitro microsomal stability, kinase selectivity, and in vivo pharmacokinetic profile cross-species, leading to the discovery of compound 23, which demonstrated robust tumor growth inhibition in multiple in vivo efficacy models after oral administration.

R2R3-type LoMYB21 affects jasmonate-regulated development and dehiscence of anthers in lily (Lilium oriental hybrids)

Lilies are widely cultivated for cut flowers, but their large anthers carry a considerable amount of colored pollen that is dispersed easily. Studying the molecular mechanism of anther development and dehiscence could help solve this problem. LoMYB21, encoding a putative R2R3 v-myb avian myeloblastosis viral oncogene homolog (MYB) transcription factor, was identified from oriental lilies (Lilium ‘Siberia’). Real-time quantitative PCR analysis showed that LoMYB21 was mainly expressed in the anther, filament and stigma and had high expression during the late stages of lily anther development. LoMYB21 had transactivation activity and was located in the nucleus through yeast one-hybrid assays and transient expression in Nicotiana benthamiana. Suppression of LoMYB21 by virus-induced gene silencing (VIGS) in Lilium ‘Siberia’ led to anther indehiscence and reduced the expression of genes related to Jasmonate acid (JA) biosynthesis and signal transduction. Induction of LoMYB21 in DEX::LoMYB21 transgenic Arabidopsis caused procumbent inflorescences that became infertile, accompanied by higher expression of JA biosynthetic and signaling genes. These results demonstrated that JA content and signaling were abnormal in silenced lily and transgenic LoMYB21 Arabidopsis, which affected anther development. Our study indicated that LoMYB21 could regulate lily anther dehiscence through JA biosynthesis and signaling during the late stages of anther development.

Abstract 3136: HYENA detects non-coding genes activated by distal enhancers in cancer

Abstract Genome instability is a hallmark of cancer, resulting in the accumulation of mutation. Among different types of mutations, structural variants (SVs) widely spread in cancer genomes and can be important cancer drivers, demonstrated in multiple cancer types. SVs can induce oncogene activation by redirecting enhancers to promoter regions or disrupting chromatin topology. These events are called “enhancer hijacking” and known to upregulate oncogenes in multiple cancers. Enhancers are among the best understood cis-regulatory elements and are essential regulators for precise gene expression in normal tissue. Since SV associated gene deregulation has been shown in multiple cancer types, it is a feasible way to infer putative oncogenes by detecting enhancer hijacking events. Taking advantage of next generation sequencing, bioinformatic tools were developed to study pan-histology protein-coding gene deregulation caused by SVs. However, algorithms that can unbiasedly detect enhancer hijacking in both coding and non-coding genes are yet to be developed. Here we present a computational algorithm “HYENA” to identify putative oncogenes activated by enhancer hijacking using whole-genome sequencing and RNA sequencing data. It deploys rank-based regression model to test whether an SV breakpoint located close to a gene significantly increases the gene expression. Permutation tests were used to generate a false discovery rate for each gene. By benchmarking with existing tools serving similar purposes, we confirmed that HYENA has superior capabilities to infer enhancer hijacking events. We detected 192 oncogene candidates in 1,148 tumor samples from 25 cancer types. While confirming the known cases of enhancer hijacking, we also identified numerous novel oncogene candidates, of which a substantial proportion was non-coding genes. Specifically, we found that a long non-coding RNA TOB1-AS1 was activated by rearranged enhancers in 10% of pancreatic cancer samples. Furthermore, experimental validation with pancreatic cancer cell lines and immunodeficient mice indicated that high expression of TOB1-AS1 is capable to promote cancer cell invasion and tumor metastasis, suggesting it is a novel cancer driver. As the existing treatments for pancreatic cancer are largely inefficacious and patients still have poor prognosis, new targets are urgently needed to improve pancreatic cancer treatments. Although the molecular mechanisms underlying TOB1-AS1 promoted metastasis remain to be unveiled, this non-coding gene can be a promising target in the patients carrying this enhancer hijacking event. In summary, our results demonstrated that HYENA is a sensitive and reliable tool to infer enhancer hijacking activated oncogenes and spot new potential targets to treat cancer. Citation Format: Anqi Yu, Ali E. Yesilkanal, Ashish Thakur, Fan Wang, Xiaoyang Wu, Alexander Muir, Xin He, Francois Spitz, Lixing Yang. HYENA detects non-coding genes activated by distal enhancers in 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 3136.

Abstract 3750: miR-590-3p inhibits multiple oncogenic nodes within the TGFBR2:SMAD2/3 pathway in recurrent GBM cells

Abstract Glioblastoma (GBM) is the most lethal and aggressive primary brain malignancy with high recurrence rates and virtually no long-term survival. Despite our expanding knowledge regarding GBM at the genomic/molecular level, we still don’t fully comprehend the molecular mechanisms responsible for GBM recurrence. Current knowledge implicates tumor-propagating Glioma Stem Cells (GSCs) in the evolution of treatment resistance and tumor recurrence. Understanding these cellular/molecular events will inform strategies for preventing and/or treating recurrent GBM (rGBM). Gene set enrichment analysis (GSEA) of RNA sequencing datasets obtained from GSCs and clinical rGBM specimens identified TGFBR2 signaling as a high frequency and potentially targetable pathway in rGBM. We show that SMAD2/3 signaling, a downstream effector of TGFBR2, is enriched in rGBM and that patients with elevated TGFBR2 have statistically shorter long-term survival outlooks. The functional relevance of these findings was supported using ITD1, a selective TGFBR2 inhibitor, which decreased the GSC phenotype and cell viability of cells derived from clinical rGBM (rGBM cells), while simultaneously re-sensitizing them to temozolomide (TMZ) in-vitro. Since GBM is known to efficiently develop resistance to receptor-targeting monotherapies, we sought to simultaneously target multiple putative oncogenic drivers downstream of TGFBR2. To achieve this, we applied an unbiased miRNA-based network analysis that identified miR-590-3p as an inhibitor of SMAD2/3 signaling at multiple nodes and thus a potential tumor suppressor in rGBM. Transgenic miR-590-3p robustly decreased the expression of >30 putative oncogenes, reduced the GSC phenotype and cell viability, as well as re-sensitized rGBM cells to TMZ. These results predict that blocking TGFBR2 signaling will negatively impact the tumor phenotype of rGBM as well as its GSC capacity. Ongoing directions for this research include comparing the efficacy of ITD1 monotherapy with miR-590-3p therapy in rGBM models in-vivo. These results identify a significant role of TGFBR2 signaling in both TMZ resistance and the GSC phenotype in rGBM and provide a miRNA-based strategy for simultaneously targeting multiple oncogenic nodes within the TGFBR2 and potentially other oncogenic signaling pathways. Citation Format: Sophie Sall, Jack Korleski, Amanda Johnson, Maya Johnson, Hernando Lopez-Bertoni. miR-590-3p inhibits multiple oncogenic nodes within the TGFBR2:SMAD2/3 pathway in recurrent GBM cells. [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 3750.

Abstract 5009: Inhibition of TGFBR2 signaling re-sensitizes TMZ-resistant glioblastoma cells to therapy

Abstract Glioblastoma (GBM), the most aggressive and lethal form of brain tumors, recurs in 90% of cases and the standard protocol of care—temozolomide (TMZ)—is an ineffective method to combat chemo-resistant GBM. It is therefore imperative that we identify, develop, and design novel therapeutics to effectively combat recurrent GBM (rGBM). Multi-potent stem-like cell subpopulations within GBM (also referred to as glioma stem cells or GSCs) play important roles in tumor propagation, therapeutic resistance, and recurrence following treatment. Understanding the molecular mechanisms that drive resistance in GSCs can lead to novel therapeutic approaches for rGBM. Bioinformatics analyses of transcriptome data sets from rGBM clinical specimens identified transforming growth factor-beta receptor type 2 (TGFBR2) as a putative driver of resistance and recurrence in GBM. We show that TMZ-resistant cells become more stem-like concurrent with higher expression of TGFBR2 protein and mRNA. Importantly, shRNA-mediated inhibition of TGFBR2 decreases proliferation of TMZ-resistant cells and enhances the effects of TMZ. We hypothesize that by developing targeted therapeutics to reduce TGFBR2 expression we will inhibit the growth capacity and/or re-sensitize rGBM cells to ionizing radiation (IR) or TMZ treatment by simultaneously targeting multiple putative oncogenes implicated in the induction of stem-like resistant GBM cell subsets. Future directions include inhibiting TGFBR2 signaling in clinical specimens derived from rGBM patients using two translatable approaches: (1) ITD1, a small molecule TGFBR2 inhibitor predicted to cross the blood-brain-barrier and (2) microRNAs to target multiple TGFBR2 target genes up-regulated in rGBM. Citation Format: Maya K. Johnson, Jack Korleski, Amanda Johnson, Sophie Sall, John Laterra, Hernando Lopez-Bertoni. Inhibition of TGFBR2 signaling re-sensitizes TMZ-resistant glioblastoma cells to therapy. [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 5009.

A pan-cancer analysis reveals CHD1L as a prognostic and immunological biomarker in several human cancers.

Introduction: Several recent studies pointed out that chromodomain-helicase-DNA-binding protein 1-like (CHD1L) is a putative oncogene in many human tumors. However, up to date, there is no pan-cancer analysis performed to study the different aspects of this gene expression and behavior in tumor tissues. Methods: Here, we applied several bioinformatics tools to make a comprehensive analysis for CHD1L. Firstly we assessed the expression of CHD1L in several types of human tumors and tried to correlate that with the stage and grade of the analyzed tumors. Following that, we performed a survival analysis to study the correlation between CHD1L upregulation in tumors and the clinical outcome. Additionally, we investigated the mutation forms, the correlation with several immune cell infiltration, and the potential molecular mechanisms of CHD1L in the tumor tissue. Result and discussion: The results demonstrated that CHD1L is a highly expressed gene across several types of tumors and that was correlated with a poor prognosis for most cancer patients. Moreover, it was found that CHD1L affects the tumor immune microenvironment by influencing the infiltration level of several immune cells. Collectively, the current study provides a comprehensive overview of the oncogenic roles of CHD1L where our results nominate CHD1L as a potential prognostic biomarker and target for antitumor therapy development.

Abstract C056: Prognostic relevance of SUV420H2 (KMT5C) gene expression in clear cell renal cell carcinoma: Implications for patients of African ancestry

Abstract African Americans are second only to Native Americans in having the highest incidence of renal cancer. Despite this statistic, very few studies have examined whether there are racial/ethnic differences in gene and protein expression. Since higher stage/grade ccRCC is uniformly lethal, it is imperative that candidate genes and their corresponding protein products be found that can identify patients across various demographics who are at greater risk of progressing to advanced stage/grade, drug-resistant clear cell renal carcinoma; and that can point to the drivers of ccRCC progression in different demographic groups. Using KIRC (clear cell renal carcinoma) TCGA dataset, we observed that SUV420H2 (KMT5C), a Lysine N-Methyltransferase, is significantly overexpressed in ccRCC (p<1E-12). Moreover, SUV420H2 transcripts were significantly elevated in higher stages and grades (p<.001). Higher SUV420H2 transcripts levels were also correlated with metastasis (p<0.01) and were associated with the more aggressive ccB Renal Csrcinoma phenotype (p<0.001). Overexpression of SUV420H2 was also associated with worst overall patient survival (p<0.0001; HR = 2.05 (1.5151 to 2.79) with a mean survival of 2208 days for the high expression group and 3256 days for the low expression group. Elevated levels were also associated with lower Disease Specific Survival [p<0.0001 HR= 2.7658 (1.8406 to 4.1560)] with a mean survival for the high expression group of 2208 versus 3871 days for the low expression group. Significantly shorter progression free disease times [p<0.0001, HR= 1.9830 (1.4389 to 2.7328) were also observed in the higher expression group (2189 days) relative to the low expression group (2971 days). In addition, in silico data analysis revealed that African Americans exhibited higher expression of SUV420H2 transcripts relative to Whites (p=5.94E-09) and that expression was associated with lower survival outcome (p<0.01, HR=4.33 (1.29 to 14.51). Taken collectively, our data suggests that higher levels of SUV420H2(KMT5C) is associated with cancer progression and the more aggressive ccRCC subtypes. Moreover, SUV420H2 behaves in a manner that suggest it may be a possible oncogene and that it is an unfavorable ccRCC biomarker. Analysis of CPTAC database and Human Protein Atlas failed to yield any information of protein expression in relationship to ccRCC. Given that SUV420H2 functions as a histone methyltransferase that specifically trimethylates histone H4, it is possible that dysregulation could result in altered epigenetic processes and inappropriate repression of genes in ccRCC. Lastly, our data underscores the need for proteomics studies that analyze protein expression differences of putative oncogenes or tumor suppressor genes and proteins across racial and ethnic demographic groups. Citation Format: Romonia Renee Reams, Simone Heyliger, Marilyn Saulsbury. Prognostic relevance of SUV420H2 (KMT5C) gene expression in clear cell renal cell carcinoma: Implications for patients of African ancestry [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C056.

PATH-31. ECDNA PROMOTES TUMORIGENICITY AND INTRATUMORAL HETEROGENEITY IN MEDULLOBLASTOMA

Abstract Extrachromosomal circular DNA (ecDNA) is an important driver of aggressive cancers, including medulloblastoma (MB), the most common pediatric brain tumor. To assess the clinical importance of ecDNA in MB, we applied computational methods to detect ecDNA in the genomes of a cohort of 468 MB patients and 31 MB model systems. Among patients, ecDNA was detected in 18% of tumors and carried over twofold greater risk of mortality. Affected genomic loci harbor up to hundredfold amplification of oncogenes including MYC, MYCN, TERT, and other novel putative oncogenes. Between sequential patient biopsies at initial diagnosis and subsequent relapse, we observed structural variation at ecDNA loci and generation of new ecDNA sequences. Among model systems, ecDNA was found in 19 of 31 genomes (61%). Although ecDNA was far more prevalent among MB models than patients, the ecDNA genomic sequences were conserved between most patient-derived xenograft (PDX) models and the human tumors from which they were made. To elucidate the functional regulatory landscapes of ecDNAs in MB, we generated transcriptional (RNA-seq), accessible chromatin (ATAC-seq), and chromatin interaction (Hi-C) profiles of 6 MB tumor samples. In each case, we identified regulatory interactions that cross fusion breakpoints on the ecDNA, representing potential “enhancer rewiring” events which may contribute to transcriptional activation of co-amplified oncogenes. To test this hypothesis, we conducted an in vitro CRISPRi screen targeting regulatory regions on the ecDNA of a MB cell line and identified distal enhancers which promote proliferation. Using single-cell sequencing, we have also developed methods to explore intratumoral heterogeneity of ecDNA in a p53-mutant SHH MB patient tumor and its corresponding PDX model. In summary, our study analyzes the frequency, diversity, and functional relevance of ecDNA across MB subgroups and provides strong justification for continued mechanistic studies of ecDNA in MB with the potential to uncover new therapeutic approaches.

Genomic and biological study of fusion genes as resistance mechanisms to EGFR inhibitors

The clinical significance of gene fusions detected by DNA-based next generation sequencing remains unclear as resistance mechanisms to EGFR tyrosine kinase inhibitors in EGFR mutant non-small cell lung cancer. By studying EGFR inhibitor-resistant patients treated with a combination of an EGFR inhibitor and a drug targeting the putative resistance-causing fusion oncogene, we identify patients who benefit and those who do not from this treatment approach. Through evaluation including RNA-seq of potential drug resistance-imparting fusion oncogenes in 504 patients with EGFR mutant lung cancer, we identify only a minority of them as functional, potentially capable of imparting EGFR inhibitor resistance. We further functionally validate fusion oncogenes in vitro using CRISPR-based editing of EGFR mutant cell lines and use these models to identify known and unknown drug resistance mechanisms to combination therapies. Collectively, our results partially reveal the complex nature of fusion oncogenes as potential drug resistance mechanisms and highlight approaches that can be undertaken to determine their functional significance.

MMP1 Overexpression Promotes Cancer Progression and Associates with Poor Outcome in Head and Neck Carcinoma.

Matrix metalloproteinase-1 (MMP1) has been reported to play key roles in a variety of cancers by degrading the extracellular matrix. However, its carcinogenic roles have not been shown yet in head and neck squamous cell carcinoma (HNSCC). This study aimed to elucidate its expression pattern and functional roles as well as clinical significance in HNSCC. The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and immunohistochemistry (IHC) were utilized to determine the MMP1 expression pattern and the associations between its expression and patients' outcome in HNSCC. Mice tongue squamous cell carcinoma model induced by 4-nitroquinoline 1-oxide (4NQO) and siRNA-mediated cellular assay in vitro were utilized to evaluate the oncogenic role of MMP1. The biological functions and cancer-related pathways involved in MMP1-related genes were found through bioinformatics analysis. Both mRNA and protein abundance of MMP1 were highly increased in HNSCC as compared to its non-tumor counterparts. MMP1 overexpression positively correlated with advanced tumor size, cervical node metastasis, and advanced pathological grade and lower patients' survival. In the 4NQO-induced animal model, MMP1 expression increased along with the progression of the disease. In HNSCC cells, siRNA-mediated knockdown of MMP1 significantly inhibited cell proliferation, migration, and invasion and activated apoptosis and epithelia-mesenchymal transition (EMT). GSEA, GO, and KEGG analyses showed that MMP1 expression was significantly related to cancer-related pathways and cancer-related functions. Together, our results demonstrated MMP1 serves as a novel prognostic biomarker and putative oncogene in HNSCC.

Small RNA-Seq Reveals Similar miRNA Transcriptome in Children and Young Adults with T-ALL and Indicates miR-143-3p as Novel Candidate Tumor Suppressor in This Leukemia.

We aimed to identify miRNAs and pathways specifically deregulated in adolescent and young adult (AYA) T-ALL patients. Small RNA-seq showed no major differences between AYA and pediatric T-ALL, but it revealed downregulation of miR-143-3p in T-ALL patients. Prediction algorithms identified several known and putative oncogenes targeted by this miRNA, including KRAS, FGF1, and FGF9. Pathway analysis indicated signaling pathways related to cell growth and proliferation, including FGFR signaling and PI3K-AKT signaling, with the majority of genes overrepresented in these pathways being predicted targets of hsa-miR-143-3p. By luciferase reporter assays, we validated direct interactions of this miRNA with KRAS, FGF1 and FGF9. In cell proliferation assays, we showed reduction of cell growth upon miR-143-3p overexpression in two T-ALL cell lines. Our study is the first description of the miRNA transcriptome in AYA T-ALL patients and the first report on tumor suppressor potential of miR-143-3p in T-ALL. Downregulation of this miRNA in T-ALL patients might contribute to enhanced growth and viability of leukemic cells. We also discuss the potential role of miR-143-3p in FGFR signaling. Although this requires more extensive validation, it might be an interesting direction, since FGFR inhibition proved promising in preclinical studies in various cancers.

Novel UHRF1-MYC Axis in Acute Lymphoblastic Leukemia.

Simple SummaryWe provided evidence that ubiquitin-like, containing PHD and RING finger domain, 1 (UHRF1) is overexpressed in acute lymphocytic leukemia (ALL). We further showed that UHRF1 directly interacts and regulates c-Myc expression to enable ALL cell growth through the cMYC-CDK4/6 phosphoRb-signaling axis.Ubiquitin-like, containing PHD and RING finger domain, (UHRF) family members are overexpressed putative oncogenes in several cancer types. We evaluated the protein abundance of UHRF family members in acute leukemia. A marked overexpression of UHRF1 protein was observed in ALL compared with AML. An analysis of human leukemia transcriptomic datasets revealed concordant overexpression of UHRF1 in B-Cell and T-Cell ALL compared with CLL, AML, and CML. In-vitro studies demonstrated reduced cell viability with siRNA-mediated knockdown of UHRF1 in both B-ALL and T-ALL, associated with reduced c-Myc protein expression. Mechanistic studies indicated that UHRF1 directly interacts with c-Myc, enabling ALL expansion via the CDK4/6-phosphoRb axis. Our findings highlight a previously unknown role of UHRF1 in regulating c-Myc protein expression and implicate UHRF1 as a potential therapeutic target in ALL.