MYC Pathway Research Articles

Abstract 2475: Differential sensitivity of mouse PDAC KrasG12D cells to Ref-1/APE1 redox signalling inhibitors: Role of NFkB as a primary target of Ref-1/APE1 in Kras driven pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, with a five-year survival rate of less than ten percent. Kras is a common driver mutation of PDAC, contributing to progression and chemotherapy resistance. Additionally, inflammation is implicated in the development of PDAC and NFκB is recognized as a key mediator of inflammation and has been frequently observed to be upregulated in PDAC. Ref-1/APE1 (redox effector factor 1/apurinic endonuclease 1), a multifunctional protein involved in redox signaling of selected transcription factors (TF), is an upstream activator of NFκB and a therapeutic target. We have developed inhibitors to prevent Ref-1/APE1's redox signaling function, including activation of NFkB. We screened eight murine PDAC KC cell lines, which contain the KrasG12D mutation, to determine if there are subsets of these tumor cells that would respond differentially to Ref-1/APE1 redox signaling which recently completed phase I clinical trials for solid tumors. Additionally, we tested a series of second-generation inhibitors, including APX2009, APX2014 and others. The cell lines split into two distinct groups of sensitive and resistant to the APX compounds. In RNAseq analysis, the sensitive cell lines displayed enriched pathway genes and increased expression of TNFα-NFkB, epithelial to mesenchymal transition, inflammatory response and JAK/STAT3 signaling pathways as well as others, while the resistant lines were enriched for mTORC1, MYC and estrogen response pathways. Previous studies by our group have demonstrated the regulation of the JAK/STAT3 and NFkB pathways by Ref-1/APE1 and inhibition by APX compounds. To further tease out which of the downstream TFs and pathways were most dominant in the sensitivity response, we used KC3590 cells containing the KrasG12D mutation and expressing only a truncated, inactive form of NFκB. Rescue cells were created which have a full-length NFκB. When challenged with Ref-1/APE1 inhibitors APX3330, APX2009, and APX2014, cells containing the full-length, active NFκB were more sensitive to the APX compounds. To ascertain whether NFkB is the dominant TF reflecting APX inhibitor sensitivity, we studied whether knocking down RelA and STAT3, using siRNA, would alter the response of the cells to the APX compounds. STAT3 is known to be regulated by Ref-1/APE1 redox signaling. In these studies, the cellular response to APX inhibitors was unaffected following siRNA knock-down of RelA or STAT3. Overall, these experiments reveal suppression of NFκB as a primary determinant of Ref-1/APE1 cytotoxicity in KrasG12D cells. These findings allow us to begin to determine whether a specific gene signature within KRas driven PDAC tumors will inform which tumors would be more sensitive to Ref-1/APE1 redox signaling inhibitors. Citation Format: Rachel A. Caston, Randy Wireman, Lee Armstrong, Silpa Gampala, Olivia Babb, Nayela Chowdhury, Zonera Hassan, Christian Schneeweis, Gunter Schneider, Melissa L. Fishel, Mark R. Kelley. Differential sensitivity of mouse PDAC KrasG12D cells to Ref-1/APE1 redox signalling inhibitors: Role of NFkB as a primary target of Ref-1/APE1 in Kras driven pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2475.

Abstract 1227: Oncofusion driven de novo enhancer assembly promotes malignancy in Ewing sarcomavia aberrant expression of the stereociliary protein LOXHD1

Abstract Ewing sarcoma (EwS) is a highly aggressive tumor of bone and soft tissues that mostly affects children and adolescents. The pathognomonic oncofusion EWSR1-ETS (EWS-FLI1/EWS-ERG) transcription factors drive EwS by orchestrating an oncogenic transcription program through de novo enhancers. Pharmacological targeting of these oncofusions has been challenged by unstructured prion-like domains and common DNA binding motifs in the EWSR1 and ETS, respectively. Alternatively, identification and characterization of mediators and downstream targets of EWS-FLI1 dependent or independent function could offer novel therapeutic options. By integrative analysis of thousands of transcriptomic datasets representing pan-cancer cell lines, primary cancer, metastasis, and normal tissues, we have identified a 32 gene signature (ESS32) in EwS that could stratify EwS from pan-cancer. Of the ESS32, LOXHD1 - coding for a stereociliary protein was the most exquisite gene expressed in EwS. CRISPR-Cas9 mediated deletion or silencing of EWS-FLI1 bound upstream de novo enhancer elements in EwS cells led to the loss of LOXHD1 expression and altered the EWS-FLI1, MYC, and HIF1a pathway genes, resulting in decreased proliferation and invasion in vitro and in vivo. These observations open up new avenues for developing LOXHD1-targeted small molecules or cellular therapies for this deadly disease. Citation Format: Qu Deng, Ramakrishnan Natesan, Florencia Cidre-Aranz, Shehbeel Arif, Ying Liu, Reyaz Rasool, Pei Wang, Zvi Crammer, Margaret Chou, Chandan Kumar, Kristy Weber, Karin Eisinger, Nicolas Grillet, Thomas Grünewald, Irfan Asangani. Oncofusion driven de novo enhancer assembly promotes malignancy in Ewing sarcomavia aberrant expression of the stereociliary protein LOXHD1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1227.

Abstract 2961: The novel roles of fatty acid binding proteins in multiple myeloma

Abstract Background: Our goal was to determine the role of the fatty acid binding protein (FABP) family in multiple myeloma (MM) using cancer dependency mapping and RNA sequencing, and to assess the potential for combined targeting of FABPs with other MM therapies. We reported at the ASH 2020 Meeting that targeting FABPs in vitro induces apoptosis, cell cycle arrest, and cell death using the FABP inhibitors BMS309403 and SBFI-26. These inhibitors alone, or in combination, reduced tumor bioluminescence and increased survival in a SCID-beige MM1S mouse model. Lastly, we showed the clinical relevancy and excellent potential for targeting the FABPs by analyzing published human MM datasets. Methods: Herein, we searched Crispr screen data from The Cancer Dependency Map (DepMap.org) to quantify MM cell dependency on all the FABP members. We also performed RNA sequencing of human MM1S MM cells treated with BMS309403, SBFI-26, or both for 24 hours and analyzed the gene and pathway changes using dbString analysis. Lastly, we tested dexamethasone (dex) with SBFI-26 in the SCID-beige GFP+Luc+MM1S in vivo model. Results: Using DepMap, MM cells were most highly dependent on FABP5, which fell within the top 25% of essential genes for MM cells. FABP5 was also the most highly expressed FABP in 3 cell lines (MM1S, OPM2 and RPMI8226) in our RNA sequencing data, and was expressed by primary MM cells. Our RNA sequencing data revealed many changes in MM1S cells after treatment with BMS309403, SBFI-26, or both; the greatest number of genes changed was with the combination treatment BMS309403 + SBFI-26 (either up- or down-regulated) versus vehicle controls. Interestingly, the central node in the dbString analysis of downregulated genes after combination treatment (FC<-1.5, p<0.05) out of 154 genes was Myc, suggesting that disruption of the Myc pathway caused the cell cycle arrest, decreased proliferation, and increased apoptosis in MM cells treated with FABP inhibitors. 196 upregulated genes (p<0.05, FC>1.5) were identified in MM cells with the combination treatment including CD44, PECAM, and IGFBP7; the “cellular response to interferon gamma” gene ontology term was an up-regulated biological process. Lastly, although our in vitro data supported the combination of SBFI-26 + dex, we did not observe a decrease in tumor bioluminescence using the combination of 1 or 9 mg/kg dex plus 1 mg/mL SBFI-26 3X/week in the MM1S mouse model. Future studies using different doses of these and other drugs (eg. lenalidomide and bortezomib) to more thoroughly investigate the combination potential for FABP inhibitors with common anti-MM therapies are planned. Conclusion: Targeting the FABPs holds great potential as a novel, safe therapy for MM patients. We identified the Myc signaling pathway as a potential mediator of FABP action. As Myc is a commonly upregulated oncogene in MM and other cancers, we believe there may be great untapped potential for targeting FABPs, especially FABP5, in MM and other cancers. Citation Format: Mariah Farrell, Anastasia D'Amico, Heather Fairfield Campbell, Connor Murphy, Michaela Ruth Reagan. The novel roles of fatty acid binding proteins in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2961.

Abstract 614: Transcriptomic analysis of BCG-treated T1HG bladder cancer patients identifies an EMT-basal subgroup with immune suppressive characteristics at high risk of BCG-failure

Abstract Intravesical instillations with Bacillus Calmette-Guérin (BCG) are the recommended treatment in T1 high-grade (HG) bladder cancer (BC) patients. However, even with BCG treatment, 50% of patients develop a HG recurrence or progression to advanced disease. Current risk stratification is insufficient to identify patients at risk of BCG failure. We aimed to identify molecular predictors of BCG failure in T1HG BC patients with the objective to improve clinical decision-making. Primary T1HG BC patients treated with ≥5/6 BCG induction instillations were retrospectively included. RNA-sequencing (50 million paired-end reads, ≥80% tumor) was performed on centrally reviewed tumors from: 1) BCG-naïve tumors and recurrent tumors from patients that failed BCG therapy (BCG non-responders), matched to an equal number of 2) patients who had an ongoing complete response until study inclusion (BCG responders). Endpoints were HG recurrence-free survival defined as BCG-failure (RFS) and progression-free survival (PFS), assessed by survival analyses. BCG-failure was defined as development of a biopsy-proven HG recurrence or progression to muscle-muscle invasion. Using RNA-seq, we sought to identify genes, pathways and signatures that stratified patients with respect to these endpoints. 132 BCG-naïve T1HG tumors were sequenced and 45 post-BCG tumors. Median follow-up for all patients was 75 months (IQR 48-106), 99 months for n=63 BCG-responders (IQR 74-120) and 49 months for n=69 BCG-non-responders (IQR 25-79). Three molecular subtypes were identified: BCG1 (32%), BCG2 (40%) and BCG3 (28%). BCG3 patients had a significantly worse RFS (HR 2.4 p<0.01) and PFS (HR 2.7; p<0.01) compared to BCG1 or BCG2 patients. BCG3 tumors expressed high EMT and basal markers and had an immune suppressive tumor microenvironment. Immune deconvolution revealed significantly increased immune cell infiltration (i.e. B cells, T regulatory cells and tumor associated macrophages), while CD14 and FOXP3 were identified as significant regulons in BCG3 patients. BCG1 patients showed a favorable PFS and overexpression of genes associated with BCG processing and antigen presentation. BCG2 patients overexpressed luminal BC markers, such as FGFR3, showed MYC pathway activity and were mostly luminal papillary (consensus muscle-invasive BC classifier). Interestingly, post-BCG tumor recurrences were strongly enriched for the BCG3 subtype; 30/45 (67%) of recurring tumors were classified as BCG3. Gene expression profiling of BCG-naïve primary T1HG BC patients identified three molecular subtypes that corresponded to clinical outcome and response to BCG therapy. The EMT-basal/immune suppressive BCG3 patients are not candidates for treatment with BCG given the poor RFS and PFS and might be candidates for early radical cystectomy or immune-modulating therapy. Citation Format: Florus C. De Jong, Teemu D. Laajala, Robert F. Hoedemaeker, Sébastien Rinaldetti, Jolien T. Mensink, Angelique C. Van der Made, Deric K. Van der Schoot, Egbert R. Boevé, Ellen C. Zwarthoff, Joost L. Boormans, Dan Theodorescu, James C. Costello, Tahlita C. Zuiverloon. Transcriptomic analysis of BCG-treated T1HG bladder cancer patients identifies an EMT-basal subgroup with immune suppressive characteristics at high risk of BCG-failure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 614.

IRF5 Loss Impairs TLR9/BCR-Induced B Cell Activation, Proliferation and Plasmablast Differentiation through Erk1-Myc Signaling

Upon recognition of antigen, B cells undergo rapid proliferation followed by differentiation to specialized antibody secreting cells (ASCs). During this transition, B cells are reliant upon a multilayer transcription factor network to achieve a dramatic remodeling of the B cell transcriptional landscape. Increased levels of ASCs are seen in autoimmune diseases, such as sytemic lupus erythematosus (SLE), and certain B cell-associated malignancies. Studies suggest that altered expression of regulatory transcription factors play a role in the observed imbalance of B cell subsets in these diseases. The transcription factor interferon regulatory factor 5 (IRF5) is one such candidate as polymorphisms in IRF5 associate with risk of numerous autoimmune diseases and correlate with elevated IRF5 expression. Furthermore, dysregulated IRF5 expression and activation have been implicated in a variety of B cell lymphoproliferative disorders. It remains unclear, however, whether IRF5 has a role in normal B cell function. To date, little is known of IRF5 function in human B cells. We utilized human primary naïve B cells combined with IRF5 knockdown to identify a critical new intrinsic role for IRF5 in B cell activation, proliferation and plasma cell differentiation. IRF5 knockdown resulted in significant IgD retention, reduced proliferation, plasmablast differentiation, and IgG secretion. The observed decreases were due to impaired B cell activation and clonal expansion. Distinct from murine studies, we identify and confirm new IRF5 target genes, IRF4, ERK1, and MYC, and signaling pathways that mediate IRF5 B cell-intrinsic function. Together, these results identify IRF5 as an early regulator of human B cell activation and provide the first dataset in human primary B cells to map IRF5 dysfunction in disease. DisclosuresNo relevant conflicts of interest to declare.

Preliminary Results from an Ongoing Phase 1/2 Study of INCB057643, a Bromodomain and Extraterminal (BET) Protein Inhibitor, in Patients (pts) with Advanced Malignancies

Preliminary Results from an Ongoing Phase 1/2 Study of INCB057643, a Bromodomain and Extraterminal (BET) Protein Inhibitor, in Patients (pts) with Advanced Malignancies

Restoration of Aged Hematopoietic Cells by Their Young Counterparts Through Instructive Microvesicle Release

This study addresses the potential to reverse age-associated morbidity by establishing methods to restore the aged hematopoietic system. Parabiotic animal models indicated that young secretome could restore aged tissues, leading us to establish a heterochronic transwell system with aged mobilized peripheral blood (MPB), co-cultured with young MPB or umbilical cord blood (UCB) cells. Functional studies and omics approaches indicate that the miRNA cargo of microvesicles (MVs) restores the aged hematopoietic system. The in vitro findings were validated in immune deficient (NSG) mice carrying an aged hematopoietic system, improving aged hallmarks such as increased lymphoid:myeloid ratio, decreased inflammation and cellular senescence. Elevated MYC and E2F pathways, and decreased p53 were key to hematopoietic restoration. These processes require four restorative miRs that target the genes for transcription/differentiation, namely PAX and phosphatase PPMIF. These miRs when introduced in aged cells were sufficient to restore the aged hematopoietic system in NSG mice. The aged MPBs were the drivers of their own restoration, as evidenced by the changes from distinct baseline miR profiles in MPBs and UCB to comparable expressions after exposure to aged MPBs. Restorative natural killer cells eliminated dormant breast cancer cells in vivo, indicating the broad relevance of this cellular paradigm - preventing and reversing age-associated disorders such as clearance of early malignancies and enhanced responses to vaccine and infection.

Prognostic Implication of the m6A RNA Methylation Regulators in Rectal Cancer.

N6-methyladenosine (m6A) is a very common and abundant RNA modifications occurring in nearly all types of RNAs. Although the dysregulated expression of m6A regulators is implicated in cancer progression, our understanding of the prognostic value of the m6A regulators in rectal cancer is still quite limited. In this study, we analyzed the RNA expression levels of the 17 m6A regulator genes of 95 rectal cancer and 10 normal rectal samples from the The Cancer Genome Atlas Rectum Adenocarcinoma (TCGA-READ) dataset. Lasso regression analysis was conducted to build a prognostic model and calculate the risk score. The rectal cancer patients were then devided into the high-risk and low-risk groups according to the mean risk score. The prognostic value of the identified model was separately evaluated in the TCGA-READ and GSE87211 datasets. GSEA was conducted to analyze the functional difference of high-risk and low-risk rectal cancer patients. Our analysis revealed that rectal cancer patients with lower expression of YTHDC2 and METTL14 had a remarkable worse overall survival (P < 0.05). The prognostic value of the model was validated in GSE87211 datasets, with AUC = 0.612 for OS and AUC = 0.651 for RFS. Furthermore, the m6A modification-based risk score system is associated with activation of distinct signaling pathways, such as DNA repair, epithelial-mesenchymal transition, G2M checkpoint and the MYC pathway, that may contribute to the progression of rectal cancer. In conclusion, our findings demonstrated that the m6A RNA methylation regulators, specifically YTHDC2 and METTL14, were significantly down-regulated and might be potential prognostic biomarkers in rectal cancer.

AutoEncoder-Based Computational Framework for Tumor Microenvironment Decomposition and Biomarker Identification in Metastatic Melanoma.

Melanoma is one of the most aggressive cancer types whose prognosis is determined by both the tumor cell-intrinsic and -extrinsic features as well as their interactions. In this study, we performed systematic and unbiased analysis using The Cancer Genome Atlas (TCGA) melanoma RNA-seq data and identified two gene signatures that captured the intrinsic and extrinsic features, respectively. Specifically, we selected genes that best reflected the expression signals from tumor cells and immune infiltrate cells. Then, we applied an AutoEncoder-based method to decompose the expression of these genes into a small number of representative nodes. Many of these nodes were found to be significantly associated with patient prognosis. From them, we selected two most prognostic nodes and defined a tumor-intrinsic (TI) signature and a tumor-extrinsic (TE) signature. Pathway analysis confirmed that the TE signature recapitulated cytotoxic immune cell related pathways while the TI signature reflected MYC pathway activity. We leveraged these two signatures to investigate six independent melanoma microarray datasets and found that they were able to predict the prognosis of patients under standard care. Furthermore, we showed that the TE signature was also positively associated with patients’ response to immunotherapies, including tumor vaccine therapy and checkpoint blockade immunotherapy. This study developed a novel computational framework to capture the tumor-intrinsic and -extrinsic features and identified robust prognostic and predictive biomarkers in melanoma.

Comprehensive genomic profiling and immune characterization of adenoid cystic carcinoma.

e18050 Background: Adenoid cystic carcinoma (ACC) is a rare malignancy of glandular tissue with a high rate of local recurrence and metastatic disease for which clinical behavior varies widely. Currently, no standard therapy exists, and available therapies have low response rates. Therefore, prognostic biomarkers to determine optimal treatment strategies are urgently needed. Here we investigate the molecular landscape and therapeutic targets of ACC. Methods: ACC samples were analyzed using next generation sequencing (NGS) (NextSeq, 592 gene panel) or whole exome sequencing (NovaSeq) (Caris Life Sciences, Phoenix, AZ). Pathogenic fusion events were detected using whole transcriptome sequencing (NovaSeq). Statistical significance was determined using the Chi-square test and adjusted for multiple comparisons. Tumor infiltrating Immune cell fractions were determined using the QuanTIseq deconvolution algorithm and WTS data. Results: 327 ACC patients were identified, median age of 58 years, 62% female and 62% metastatic disease. The most frequent mutations (excluding indeterminate results) involved genes in the NOTCH pathway ( NOTCH1 16% [41 of 298 cases]), chromatin remodeling ( ARID1A 24.3% [27 of 138 cases], KDM6A 12.5% [28 of 224 cases], KMT2C 10.7% [21 of 197 cases]) and TP53 10.2% (25 of 246 cases). Chromatin remodeling genes were co-mutated with NOTCH1. NOTCH1 mutations were found in 41 cases, 18 primary and 23 metastatic were associated with reduced median overall survival (mOS 1.8 years vs 3.8 years, p = 0.01). Mutations were most frequent in the PEST (n = 35), NRR (n = 18), PEST+NRR (n = 16) and EGF-like domains (n = 11). PEST domain mutations were found exclusively in ACC originating from the head and neck (H&amp;N). GSEA showed that MYC, E2F and G2M target pathways were enriched in NOTCH1 mutated ACC regardless of the presence of MYB fusions. All three pathways were enriched in PEST domain mutations whereas only MYC targets were enriched in NRR and NRR+PEST domain mutations indicating that PEST domain mutations drive transcriptomic changes in NOTCH1 mutated ACC. MYC gene expression was significantly upregulated in NOTCH1 mutated ACC. Fusion events were detected in 45% (104/231). MYB:NFIB was the most common fusion (40%) and was more frequent in H&amp;N origin than other sites. There was no difference in the mOS between MYB fusion positive and negative ACC (4.4 years vs 5.5 years, p = 0.14). M2-polarized macrophages, myeloid dendritic cells and neutrophils were significantly higher in metastatic compared to primary ACC while NK cells were more abundant in primary tumor. Conclusions: ACC is a genetically heterogenous disease with an immune-excluded microenvironment. NOTCH1 mutations were associated with worse, while MYB:NFIB fusion was not associated with prognosis. Our study shows that M2 macrophages and MYC are potential novel therapeutic targets in ACC.

EGR1 Addiction in Diffuse Large B-cell Lymphoma.

Early growth response gene (EGR1) is a transcription factor known to be a downstream effector of B-cell receptor signaling and Janus kinase 1 (JAK1) signaling in diffuse large B-cell lymphoma (DLBCL). While EGR1 is characterized as a tumor suppressor in leukemia and multiple myeloma, the role of EGR1 in lymphoma is unknown. Here we demonstrate that EGR1 is a potential oncogene that promotes cell proliferation in DLBCL. IHC analysis revealed that EGR1 expression is elevated in DLBCL compared with normal lymphoid tissues and the level of EGR1 expression is higher in activated B cell-like subtype (ABC) than germinal center B cell-like subtype (GCB). EGR1 expression is required for the survival and proliferation of DLBCL cells. Genomic analyses demonstrated that EGR1 upregulates expression of MYC and E2F pathway genes through the CBP/p300/H3K27ac/BRD4 axis while repressing expression of the type I IFN pathway genes by interaction with the corepressor NAB2. Genetic and pharmacologic inhibition of EGR1 synergizes with the BRD4 inhibitor JQ1 or the type I IFN inducer lenalidomide in growth inhibition of ABC DLBCL both in cell cultures and xenograft mouse models. Therefore, targeting oncogenic EGR1 signaling represents a potential new targeted therapeutic strategy in DLBCL, especially for the more aggressive ABC DLBCL. IMPLICATIONS: The study characterizes EGR1 as a potential oncogene that promotes cell proliferation and defines EGR1 as a new molecular target in DLBCL, the most common non-Hodgkin lymphoma.

Ubiquitin-related molecular classification and risk stratification of hepatocellular carcinoma

The roles of ubiquitin-related genes in hepatocellular carcinoma (HCC) have not been thoroughly investigated. This study aimed to systematically examine ubiquitin-related genes and identify subtypes and stratify prognosis of HCC by using ubiquitin-related signatures. Survival, biological processes, tumor microenvironment (TME), and genomic alterations of the HCC subtypes were investigated. Patients with HCC were classified into two subtypes (clusters 1 and 2) with distinct survival outcomes, pathways, and genomic alterations. Cluster 2 had better prognosis than did cluster 1. Hepatitis B, hepatitis C, Janus tyrosine kinase (JAK)-signal transducer and activator of transcription (STAT) pathway, and natural killer cell-mediated cytotoxicity were enriched in cluster 1. Moreover, cluster 2 had a higher immune score and immune cell infiltrations, whereas cluster 1 had a lower immune score and immune infiltrations. Additionally, mutations, amplifications, and deletions among the phosphatidylinositol 3-kinase (PI3K)-AKT, p53, and receptor tyrosine kinase (RTK)-RAS pathways more frequently occurred in cluster 1, while those among the Hippo, MYC, and Notch signaling pathways were found in cluster 2. Finally, a prognostic signature, consisting of eight ubiquitin-related genes, was established and validated. In brief, our study established a new classification and developed a prognostic signature for HCC.

AMBRA1 regulates cyclin D to guard S-phase entry and genomic integrity.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel-the MYC pathway and the cyclin D-cyclin-dependent kinase (CDK)-retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D-CDK-RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to Sphase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1-cyclinD pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis.

PLCG2 as a potential indicator of tumor microenvironment remodeling in soft tissue sarcoma.

The tumor microenvironment (TME) plays an important role in the occurrence and development of soft tissue sarcoma (STS). A number of studies have shown that to inhibit tumor growth, the TME can be remodeled into an environment unsuitable for tumor proliferation. However, a lack of understanding exists regarding the dynamic regulation of TME.In this study, we used CIBERSORT and ESTIMATE calculation methods from the Cancer Genome Atlas (TCGA) database to calculate the proportion of tumor infiltrating immune cells (TICs) and the number of immune and stromal components in 263 STS samples. Differential expression genes (DEGs) shared by Immune Score and Stromal Score were obtained via difference analysis. Univariate Cox regression analysis and construction of protein–protein interaction (PPI) networks were applied to the DEGs.Through intersection analysis of univariate COX and PPI, PLCG2 was determined as the indicator. Further analysis showed that PLCG2 expression was positively correlated with the survival of STS patients. Gene set enrichment analysis (GSEA) showed that genes in the highly expressed PLCG2 group were enriched in immune-related activities. In the low-expression PLCG2 group, genes were enriched in the E2F, G2M, and MYC pathways. Difference analysis and correlation analysis showed that CD8+ T cells, gamma delta T cells, monocytes, and M1 macrophages were positively correlated with PLCG2 expression, indicating that PLCG2 may represent the immune status of TME.Therefore, the level of PLCG2 may aid in determining the prognosis of STS patients, especially the status of TME. These data provide additional insights into the remodeling of TME.

MYC Activity Inference Captures Diverse Mechanisms of Aberrant MYC Pathway Activation in Human Cancers.

c-MYC (MYC) is deregulated in more than 50% of all cancers. While MYC amplification is the most common MYC-deregulating event, many other alterations can increase MYC activity. We thus systematically investigated MYC pathway activity across different tumor types. Using a logistic regression framework, we established tumor type-specific, transcriptomic-based MYC activity scores that can accurately capture MYC activity. We show that MYC activity scores reflect a variety of MYC-regulating mechanisms, including MYCL and/or MYCN amplification, MYC promoter methylation, MYC mRNA expression, lncRNA PVT1 expression, MYC mutations, and viral integrations near the MYC locus. Our MYC activity score incorporates all of these mechanisms, resulting in better prognostic predictions compared with MYC amplification status, MYC promoter methylation, and MYC mRNA expression in several cancer types. In addition, we show that tumor proliferation and immune evasion are likely contributors to this reduction in survival. Finally, we developed a MYC activity signature for liquid tumors in which MYC translocation is commonly observed, suggesting that our approach can be applied to different types of genomic alterations. In conclusion, we developed a MYC activity score that captures MYC pathway activity and is clinically relevant. IMPLICATIONS: By using cancer type-specific MYC activity profiles, we were able to assess MYC activity across many more tumor types than previously investigated. The range of different MYC-related alterations captured by our MYC activity score can be used to facilitate the application of future MYC inhibitors and aid physicians to preselect patients for targeted therapy.

Genomic Characterization of de novo Metastatic Breast Cancer

In contrast to recurrence after initial diagnosis of stage I-III breast cancer [recurrent metastatic breast cancer (rMBC)], de novo metastatic breast cancer (dnMBC) represents a unique setting to elucidate metastatic drivers in the absence of treatment selection. We present the genomic landscape of dnMBC and association with overall survival (OS). Targeted DNA sequencing (OncoPanel) was prospectively performed on either primary or metastatic tumors from 926 patients (212 dnMBC and 714 rMBC). Single-nucleotide variants, copy-number variations, and tumor mutational burden (TMB) in treatment-naïve dnMBC primary tumors were compared with primary tumors in patients who ultimately developed rMBC, and correlated with OS across all dnMBC. When comparing primary tumors by subtype, MYB amplification was enriched in triple-negative dnMBC versus rMBC (21.1% vs. 0%, P = 0.0005, q = 0.111). Mutations in KMTD2, SETD2, and PIK3CA were more prevalent, and TP53 and BRCA1 less prevalent, in primary HR+/HER2- tumors of dnMBC versus rMBC, though not significant after multiple comparison adjustment. Alterations associated with shorter OS in dnMBC included TP53 (wild-type: 79.7 months; altered: 44.2 months; P = 0.008, q = 0.107), MYC (79.7 vs. 23.3 months; P = 0.0003, q = 0.011), and cell-cycle (122.7 vs. 54.9 months; P = 0.034, q = 0.245) pathway genes. High TMB correlated with better OS in triple-negative dnMBC (P = 0.041). Genomic differences between treatment-naïve dnMBC and primary tumors of patients who developed rMBC may provide insight into mechanisms underlying metastatic potential and differential therapeutic sensitivity in dnMBC. Alterations associated with poor OS in dnMBC highlight the need for novel approaches to overcome potential intrinsic resistance to current treatments.

Inflammatory cell-derived CXCL3 promotes pancreatic cancer metastasis through a novel myofibroblast-hijacked cancer escape mechanism

ObjectivePancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy and lacks effective treatment. We aimed to understand molecular mechanisms of the intertwined interactions between tumour stromal components in metastasis and...

Prognostic Value and Molecular Mechanisms of Proteasome 26S Subunit, Non-ATPase Family Genes for Pancreatic Ductal Adenocarcinoma Patients after Pancreaticoduodenectomy

Objective: Pancreatic cancer (PC) is an extremely malignant tumor with similar morbidity and mortality and lack of an effective treatment. This study explored the prognostic value and molecular mechanisms of proteasome 26S subunit, non-ATPase (PSMD) family genes in pancreatic ductal adenocarcinoma (PDAC). Methods: Survival analyses were performed to elucidate the relationship between prognosis and the level of PSMD expression. ROC curves and nomograms were constructed to predict the prognosis. A bioinformatics analysis was used to explore the co-expression and complex interaction networks of PSMDs. The potential mechanisms were further explored via gene set enrichment analysis (GSEA). Results: We find high levels of PSMD6, PSMD9, PSMD11, and PSMD14 expression were significantly associated with a poorer OS. High PSMD6 and PSMD11 expression was associated with a poorer relapse-free survival (RFS). A risk score model was constructed based on prognosis-related genes. The area under ROC curves (AUC) was 53.3%, 59.3%, and 62.9% for 1-, 2-, 3 years, respectively. Conclusion: GSEA revealed that PSMD6 and PSMD11 play a role in PDAC through various biological processes and signaling pathways, including TP53, CDKN2A, MYC pathway, DNA repair, KRAS, cell cycle checkpoint, NIK, NF-κB signaling pathway, and proteasomes. This study demonstrated that PSMD6 and PSMD11 could serve as a potential prognostic and diagnostic biomarkers for patients with early-stage PDAC after pancreaticoduodenectomy.

Abstract PR006: 27-Hydroxycholesterol acts on myeloid immune cells to induce T cell dysfunction, promoting breast cancer progression

Abstract Breast cancer remains one of the leading causes of cancer mortality in the US. Elevated cholesterol is a major risk factor for breast cancer onset and recurrence, while cholesterol-lowering drugs, such as statins, are associated with a good prognosis. Previous work in murine models showed that cholesterol increases breast cancer metastasis, and the pro-metastatic effects of cholesterol were due to its primary metabolite, 27HC. In our prior work, myeloid cells were found to be required for the pro-metastatic effects of 27HC, but their precise contribution remains unclear. Here we report that 27HC impairs T cell expansion and cytotoxic function through its actions on myeloid cells, including macrophages, in an LXR-dependent manner. Many oxysterols and LXR ligands had similar effects on T cell expansion. Moreover, their ability to induce the LXR target gene ABCA1 was associated with their effectiveness in impairing T cell expansion. Interestingly, the enzyme responsible for the synthesis of 27HC, CYP27A1, is highly expressed in myeloid cells, suggesting that 27HC may have important autocrine or paracrine functions in these cells, a hypothesis supported by our finding that breast cancer metastasis was reduced in mice with a myeloid specific knockout of CYP27A1. Pharmacologic inhibition of CYP27A1 reduced metastatic growth and improved the efficacy of checkpoint inhibitor, anti-PD-L1. RNA sequencing of 27HC-treated macrophages and GSEA analysis provide further mechanistic insight, revealing an enrichment of MYC and NOTCH signaling pathways, both of which are documented pathways involved in tumor-associated macrophages. Taken together, our work suggests that targeting the CYP27A1 axis in myeloid cells may present therapeutic benefits and improve the response rate to immune therapies in breast cancer. Delineating the link between LXR and the known mechanisms of tumor-associated macrophages is important to fully understand 27HC-driven cancer metastasis. This work was supported by grants to ERN from the National Cancer Institute of the National Institutes of Health (R01CA234025) and METAvivor. This abstract is also being presented as PO050. Citation Format: Liqian Ma, Lawrence Wang, Adam T. Nelson, Chaeyeon Han, Sisi He, Madeline A. Henn, Karan Menon, Joy J. Chen, Amy E. Baek, Anna Vardanyan, Sayyed Hamed Shahoei, Sunghee Park, David J. Shapiro, Som G. Nanjappa, Erik R. Nelson. 27-Hydroxycholesterol acts on myeloid immune cells to induce T cell dysfunction, promoting breast cancer progression [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR006.

LncRNAs as key players in the MYC pathways

LncRNAs as key players in the MYC pathways