E2F Signaling Research Articles

Multiomics and machine learning-based analysis of pancancer pseudouridine modifications

Pseudouridine widely affects the stability and function of RNA. However, our knowledge of pseudouridine properties in tumors is incomplete. We systematically analyzed pseudouridine synthases (PUSs) expression, genomic aberrations, and prognostic features in 10907 samples from 33 tumors. We found that the pseudouridine-associated pathway was abnormal in tumors and affected patient prognosis. Dysregulation of the PUSs expression pattern may arise from copy number variation (CNV) mutations and aberrant DNA methylation. Functional enrichment analyses determined that the PUSs expression was closely associated with the MYC, E2F, and MTORC1 signaling pathways. In addition, PUSs are involved in the remodeling of the tumor microenvironment (TME) in solid tumors, such as kidney and lung cancers. Particularly in lung cancer, increased expression of PUSs is accompanied by increased immune checkpoint expression and Treg infiltration. The best signature model based on more than 112 machine learning combinations had good prognostic ability in ACC, DLBC, GBM, KICH, MESO, THYM, TGCT, and PRAD tumors, and is expected to guide immunotherapy for 19 tumor types. The model was also effective in identifying patients with tumors amenable to etoposide, camptothecin, cisplatin, or bexarotene treatment. In conclusion, our work highlights the dysregulated features of PUSs and their role in the TME and patient prognosis, providing an initial molecular basis for future exploration of pseudouridine. Studies targeting pseudouridine are expected to lead to the development of potential diagnostic strategies and the evaluation and improvement of antitumor therapies.

Genomic profiling in a subgroup analysis of patients (pts) with diffuse large B-cell lymphoma (DLBCL) and extranodal (EN) sites of involvement in the phase 3 Pola-R-CHP versus R-CHOP (POLARIX) study.

7061 Background: The prognosis and survival outcomes of pts with EN involvement remain a challenge for DLBCL management. In the Phase 3 POLARIX study (NCT03274492), Pola-R-CHP demonstrated significantly improved progression-free survival (PFS) compared with R-CHOP in previously untreated DLBCL (Tilly et al. 2022). This retrospective analysis assessed clinical outcomes and genomic patterns of pts with DLBCL and EN disease. Methods: POLARIX methods were previously described (Tilly et al. 2022). Using a data cutoff of June 15, 2022, this analysis assessed pts with EN involvement as determined by investigators. Additional outcomes and genomics analyses were performed on pts whose EN status was confirmed from documented radiographic lesions or bone marrow biopsy with denotation of EN disease sites. Hazard ratios (HRs) for investigator-assessed PFS were adjusted for age (≤60 vs >60) and sex. Cell of origin (COO) by NanoString, mutation analysis by whole exome sequencing, and global gene expression profiling by RNAseq were performed centrally. Results: In POLARIX, 616/879 pts (70%) had identified EN involvement. After a median follow-up of 39.7 months, pts showed improved PFS with Pola-R-CHP vs R-CHOP (HR 0.72, 95% confidence interval [CI]: 0.55–0.96), with a 2-year PFS of 75% vs 66%. A total of 556 pts (Pola-R-CHP, n=279; R-CHOP, n=277) had confirmed sites of EN involvement, defined as pts with EN radiographic lesions or bone marrow disease. In these pts, PFS favored Pola-R-CHP vs R-CHOP but the difference was not statistically significant (HR 0.84, 95% CI: 0.63–1.13). Of pts with confirmed EN disease, 429 had COO results (activated B-cell, 145 [34%]; germinal center B-cell, 223 [52%]; unclassified DLBCL, 61 [14%]). Mutation data and frequencies of select clinically relevant mutations (Ptashkin, 2023) across EN sites were available for 364 pts (Table). Gene expression data were available for 418 pts; genes involved in proliferation (E2F or PI3K signaling targets and checkpoint genes) and regulated by IRF4, OCT2 and XBP1, were commonly upregulated in pts with EN disease. Conclusions: Results in pts with DLBCL with EN involvement suggest superior PFS with Pola-R-CHP vs R-CHOP. Ongoing analyses will further assess the independent impact of EN status on outcome. POLARIX represents the largest cohort of pts with annotated EN disease and systematic genomic analysis, providing new insights into the genomics of these pts. Clinical trial information: NCT03274492 . [Table: see text]

Dual Inhibition of CDK4/6 and XPO1 Induces Senescence With Acquired Vulnerability to CRBN-Based PROTAC Drugs

Background and aimsDespite the increasing number of treatment options available for liver cancer, only a small proportion of patients achieve long-term clinical benefits. Herein, we aim to develop new therapeutic approaches for liver cancer. MethodsA compound screen was conducted to identify inhibitors that could synergistically induce senescence when combined with CDK4/6 inhibitor. The combination effects of CDK4/6 inhibitor and XPO1 inhibitor on cellular senescence were investigated in a panel of human liver cancer cell lines and multiple liver cancer models. A senolytic-drug screen was performed to identify drugs that selectively killed senescent liver cancer cells. ResultsThe combination of CDK4/6 inhibitor and XPO1 inhibitor synergistically induces senescence of liver cancer cells in vitro and in vivo. The XPO1 inhibitor acts by causing accumulation of RB1 in the nucleus, leading to decreased E2F signaling and promoting senescence induction by the CDK4/6 inhibitor. Through a senolytic-drug screen, CRBN-based PROTAC ARV-825 was identified as an agent that can selectively kill senescent liver cancer cells. Upregulation of CRBN was a vulnerability of senescent liver cancer cells, making them sensitive to CRBN-based PROTAC drugs. Mechanistically, we find that USP2 directly interacts with CRBN, leading to the deubiquitination and stabilization of CRBN in senescent liver cancer cells. ConclusionOur study demonstrates a striking synergy in senescence induction of liver cancer cells through the combination of CDK4/6 inhibitor and XPO1 inhibitor. These findings also shed light on the molecular processes underlying the vulnerability of senescent liver cancer cells to CRBN-based PROTAC therapy.

Investigation of the Synergistic Combination of a Novel Celmod (CC-99282) and BET Inhibitors in Preclinical DLBCL

Background: Golcadomide (GOLCA; CC-99282) is a novel cereblon E3 ligase modulator (CELMoD), which binds to cereblon (CRBN), a substrate receptor of CRL4 CRBN E3 ubiquitin ligase, and induces proteasome-dependent degradation of Ikaros and Aiolos, transcription factors essential for B cell malignancies. GOLCA was shown to exhibit promising cell of origin independent activity in pre-clinical DLBCL models and is currently being investigated in early clinical trials (NCT03930953) for relapsed/refractory non-Hodgkin lymphomas (R/R NHL). Ikaros and Aiolos have previously been shown to have occupancy on the promoter of the proto-oncogene MYC, which is subsequently downregulated by CELMoDs in DLBCL models. Given MYC is a well-studied downstream target of BET inhibitors and a driver of lymphoma disease, we investigated the mechanistic synergy of anti-proliferative and pro-apoptotic effects of GOLCA in combination with BMS-986158, a potent BET inhibitor. Results: Dose titration and analysis of the anti-proliferative effects of DLBCL cell lines (SU-DHL-4, WSU-DLCL2 and CARNAVAL) treated with either GOLCA or BMS-986158 suggests significant single agent activity of either agent. Bliss synergy score was determined and strong synergism of the two drugs was observed on day 5, with maximal bliss score (14.3-29.8) centered at low doses (1.25 and 2.5 nM) of BMS-986158 and sub-lethal doses of GOLCA (5-50 nM). Similar synergistic effects were found when combining GOLCA with JQ1, another tool compound BET inhibitor, confirming BET inhibition potentiates the anti-DLBCL effects of the CELMoD agent. Incucyte-based live cell image confirmed the enhanced anti-proliferative capacity of the combination compared to single agents. EdU (5-ethynyl-2′-deoxyuridine)-based cell cycle analysis revealed an increased proportion of G1-phase cells in combination treatment (67%) compared to GOLCA (38%), BMS-986158 (43%) and DMSO (35.4%) in the GOLCA resistant SU-DHL-4 cells. To identify key pathways responsible for the observed synergistic combination effect, we performed transcriptomic analyses of SU-DHL-4 and WSU-DLCL2 cells treated with GOLCA/BMS-986158 for 72 hrs. Compared to either single agents, the combination treatment downregulated significantly more genes (~1000 and 500 genes in SU-DHL-4 and WSU-DLCL2, respectively), which were enriched with cell cycle pathways. Gene set enrichment analysis (GSEA) showed the top downregulated gene sets in combination treatment were E2F targets such as CHEK1 and G2M checkpoint molecules including AURKA and CDC45. Given the critical role of CDKN1A (gene encodes p21) in cell cycle arrest, we examined its expression by real-time PCR. We found treatment of GOLCA induced CDKN1A mRNA expression (16.4 and 25.1-fold in SU-DHL-4 and WSU-DLCL2, respectively); remarkably, this induction was further enhanced by the combination with BMS-986158 (40.1 and 128.5-fold in SU-DHL-4 and WSU-DLCL2, respectively). Western blot confirmed the synergistic induction of p21 in combination compared to single agents, with a concomitant reduction of phosphorylated Rb, E2F target genes (Chk1, SKP2 and E2F1) and MYC. Since the main targets of GOLCA and BMS-986158 are transcription factors (Ikaros and Aiolos) and transcriptional regulator (Brd4), respectively, we performed chromatin immunoprecipitation followed by sequencing (ChIP-seq) with antibodies targeting these three proteins along with various histone marks including H3K27ac and H3K4me3. Interestingly, the chromatin occupancies of Ikaros/Aiolos/Brd4 were highly overlapping in genes that are upregulated ( CDKN1A) and downregulated (e.g. CHEK1 and MYC) by the combination treatment. Further characterization of the interplay between Ikaros/Aiolos/Brd4 and their loci-specific interactomes may help elucidate the synergism at epigenetic levels. Conclusions: BET inhibition potentiates anti-proliferative effects of GOLCA in DLBCL cells through synergistic induction of p21 and inhibition of E2F signaling. The synergetic effects of BMS-986158 and GOLCA suggest potential clinical benefits of combining lower doses of single agents to achieve better efficacy with reduced risk of adverse effects.

CSIG-10. SEX-DIFFERENCES IN NF-ΚB SIGNALING DRIVE SEX-BIASED RESPONSES TOWARDS SENESCENCE AND INFLAMMATION IN GLIOBLASTOMA

Abstract Male glioblastoma (GBM) patients have a higher incidence rate and worse median survival in comparison to females. The molecular mechanisms that drive these sex differences in incidence and survival are not well understood. Using a murine in vitro GBM model, we identified that male and female cells tend to segregate into two unique groups based on differential gene expression. Male-enriched processes included myc, E2F and G2-M checkpoint signaling. Female-enriched processes included epithelial-mesenchymal transition, interferon responses and TNF-α signaling by NF-κB. Reports from human GBM patients corroborate an increased enrichment for inflammatory and innate immune responses in females. Since the NF-κB pathway regulates senescence and its associated inflammatory response, the senescence-associated secretory phenotype, we examined if there are sex differences in NF-κB signaling as we have reported that female GBM cells undergo senescence more readily. We found that low levels of NF-κB activity were correlated with higher levels of senescence, and that cells from the female-enriched group correspondingly had lower levels of NF-κB activity. Interestingly, p-p65 S536 expression (a marker of increased NF-κB transcriptional activity) was negatively correlated with NF-κB activity in cells within the female-enriched group. Furthermore, female cells with low NF-κB activity responded more readily to TNF-α treatment. These results suggest that female cells can more readily modulate the NF-κB pathway in response to stimuli, thereby poising them towards a pro-inflammatory state. Since inflammation and senescence regulate therapeutic responses in GBM, and we have previously reported that female GBM cells undergo senescence more readily following radiation, these results suggest that further studies to understand the sex differences in NF-κB signaling may improve patient response to radiation and chemotherapy treatment.

Impaired Plakophilin-2 in obesity breaks cell cycle dynamics to breed adipocyte senescence

Plakophilin-2 (PKP2) is a key component of desmosomes, which, when defective, is known to promote the fibro-fatty infiltration of heart muscle. Less attention has been given to its role in adipose tissue. We report here that levels of PKP2 steadily increase during fat cell differentiation, and are compromised if adipocytes are exposed to a pro-inflammatory milieu. Accordingly, expression of PKP2 in subcutaneous adipose tissue diminishes in patients with obesity, and normalizes upon mild-to-intense weight loss. We further show defective PKP2 in adipocytes to break cell cycle dynamics and yield premature senescence, a key rheostat for stress-induced adipose tissue dysfunction. Conversely, restoring PKP2 in inflamed adipocytes rewires E2F signaling towards the re-activation of cell cycle and decreased senescence. Our findings connect the expression of PKP2 in fat cells to the physiopathology of obesity, as well as uncover a previously unknown defect in cell cycle and adipocyte senescence due to impaired PKP2.

Decoding the prognostic significance of integrator complex subunit 9 (INTS9) in glioma: links to TP53 mutations, E2F signaling, and inflammatory microenvironments

IntroductionGliomas, a type of brain neoplasm, are prevalent and often fatal. Molecular diagnostics have improved understanding, but treatment options are limited. This study investigates the role of INTS9 in processing small nuclear RNA (snRNA), which is crucial to generating mature messenger RNA (mRNA). We aim to employ advanced bioinformatics analyses with large-scale databases and conduct functional experiments to elucidate its potential role in glioma therapeutics.Materials and methodsWe collected genomic, proteomic, and Whole-Exon-Sequencing data from The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) for bioinformatic analyses. Then, we validated INTS9 protein expression through immunohistochemistry and assessed its correlation with P53 and KI67 protein expression. Gene Set Enrichment Analysis (GSEA) was performed to identify altered signaling pathways, and functional experiments were conducted on three cell lines treated with siINTS9. Then, we also investigate the impacts of tumor heterogeneity on INTS9 expression by integrating single-cell sequencing, 12-cell state prediction, and CIBERSORT analyses. Finally, we also observed longitudinal changes in INTS9 using the Glioma Longitudinal Analysis (GLASS) dataset.ResultsOur findings showed increased INTS9 levels in tumor tissue compared to non-neoplastic components, correlating with high tumor grading and proliferation index. TP53 mutation was the most notable factor associated with upregulated INTS9, along with other potential contributors, such as combined chromosome 7 gain/10 loss, TERT promoter mutation, and increased Tumor Mutational Burden (TMB). In GSEA analyses, we also linked INTS9 with enhanced cell proliferation and inflammation signaling. Downregulating INTS9 impacted cellular proliferation and cell cycle regulation during the function validation. In the context of the 12 cell states, INTS9 correlated with tumor-stem and tumor-proliferative-stem cells. CIBERSORT analyses revealed increased INTS9 associated with increased macrophage M0 and M2 but depletion of monocytes. Longitudinally, we also noticed that the INTS9 expression declined during recurrence in IDH wildtype.ConclusionThis study assessed the role of INTS9 protein in glioma development and its potential as a therapeutic target. Results indicated elevated INTS9 levels were linked to increased proliferation capacity, higher tumor grading, and poorer prognosis, potentially resulting from TP53 mutations. This research highlights the potential of INTS9 as a promising target for glioma treatment.

Investigation of enzalutamide, docetaxel, and cabazitaxel resistance in the castration resistant prostate cancer cell line C4 using genome-wide CRISPR/Cas9 screening

Enzalutamide, docetaxel, and cabazitaxel treatment resistance is a major problem in metastatic castration resistant prostate cancer (mCRPC), but the underlying genetic determinants are poorly understood. To identify genes that modulate treatment response to these drugs, we performed three genome-wide CRISPR/Cas9 knockout screens in the mCRPC cell line C4. The screens identified seven candidates for enzalutamide (BCL2L13, CEP135, E2F4, IP6K2, KDM6A, SMS, and XPO4), four candidates for docetaxel (DRG1, LMO7, NCOA2, and ZNF268), and nine candidates for cabazitaxel (ARHGAP11B, DRG1, FKBP5, FRYL, PRKAB1, RP2, SMPD2, TCEA2, and ZNF585B). We generated single-gene C4 knockout clones/populations for all genes and could validate effect on treatment response for five genes (IP6K2, XPO4, DRG1, PRKAB1, and RP2). Altered enzalutamide response upon IP6K2 and XPO4 knockout was associated with deregulation of AR, mTORC1, and E2F signaling, and deregulated p53 signaling (IP6K2 only) in C4 mCRPC cells. Our study highlights the necessity of performing individual validation of candidate hits from genome-wide CRISPR screens. Further studies are needed to assess the generalizability and translational potential of these findings.

Identification and Validation of Anoikis-Related Signatures for Predicting Prognosis in Lung Adenocarcinoma with Machine Learning.

Lung adenocarcinoma (LUAD) is an aggressive cancer that has an extremely poor prognosis. As well as facilitating the detachment of cancer cells from the primary tumor site, anoikis plays an important role in cancer metastasis. Few studies to date, however, have examined the role of anoikis in LUAD, in patient prognosis. A total of 316 anoikis-related genes (ANRGs) integrated from Genecards and Harmonizome portals. LUAD transcriptome data were retrieved from the Genotype-Tissue Expression Project (GEO) and The Cancer Genome Atlas (TCGA). Anoikis-related prognostic genes (ANRGs) were primarily screened by univariate Cox regression. All ANRGs were included in the Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression model to construct the powerful prognostic signature. This signature was validated and assessed using the Kaplan-Meier method as well as univariate and multivariate Cox regression analyses. Anoikis-related regulators of risk score were identified using a XG-boost machine learning model. The expression of ITGB4 protein was examined in a ZhengZhou University (ZZU) tissue cohort by immunohistochemistry, and the potential mechanisms of action of ITGB4 in LUAD were explored by GO, KEGG, and ingenuity pathway analyses and by GSEA. A risk score signature was constructed based on eight ANRGs, with high risk scores found to closely correlate with unfavorable clinical features. ITGB4 expression may be associated with 5-year over survival, with immunohistochemistry showed that the expression of ITGB4 was higher in LUAD than in nontumor tissues. Enrichment analysis suggested that ITGB4 may promote LUAD development by targeting E2F, MYC, and oxidative phosphorylation signaling pathways. Our anoikis-related signature from RNA-seq data may be a novel prognostic biomarker in patients with LUAD. It may help physicians develop personalized LUAD treatments in clinical practice. Moreover, ITGB4 may affect the development of LUAD through the oxidative phosphorylation pathway.

Silencing of RAB42 down-regulated PD-L1 expression to inhibit the immune escape of hepatocellular carcinoma cells through inhibiting the E2F signaling pathway

ObjectiveTo investigate the mechanistic role of RAB42 and corresponding regulatory path in hepatocellular carcinoma (HCC). MethodsThe expression of RAB42 in HCC tissue was checked by RT-qPCR and immunohistochemical staining assay. Cell proliferation was checked by colony formation and CCK-8 assay. Cell apoptosis and cycle distribution were analyzed with flow cytometry. The relevance of RAB42 and PD-L1 was analyzed from TCGA database. The binding of E2F1 to PD-L1 was detected by JASPAR database, luciferase and ChIP assay. The expression of PD-L1, cell apoptosis- and E2F pathway-related proteins were checked by western blotting. ResultsRAB42 was highly expressed in HCC tissue. RAB42 silencing could inhibit proliferation and induce G1 phase arrest and apoptosis of HCC cells. TCGA database disclosed that PD-L1 was highly associated with RAB42 expression. Silencing of RAB42 could retard PD-L1 expression in HCC cells. GSEA analysis showed RAB42 could activate E2F signaling pathway. Silencing of RAB42 could observably weaken the expression of E2F1, CDK1 and CDC20 in HCC cells. JASPAR database predicted the binding site between E2F1 and PD-L1, and E2F1 overexpression could promote PD-L1 expression. Overexpression of E2F1 could reverse the biological function of RAB42 silencing in HCC cells. ConclusionSilencing of RAB42 could down-regulate PD-L1 expression to inhibit immune escape through inhibiting E2F signaling pathway in HCC cells. RAB42 may become a novel clinical diagnostic and therapy marker for HCC.

Abstract P4-02-28: EMP1 low expressing tumor is associated with cell proliferation and poor overall survival of ER-positive breast cancer patients

Abstract Background: Epithelial membrane protein 1 (EMP1) belongs to PMP22 gene family and is reported to be expressed low in numerous gastrointestinal cancers. However, little is known about its role in breast cancer. EMP1 functions to promote cell proliferation. In this study, we hypothesized that low EMP1 expressing breast cancer is associated with high proliferative characteristics and poor survival. Material and Methods: The clinicopathological data and transcriptome data of breast cancer patients from two independent large publicly available databases, The Cancer Genome Atlas (TCGA, n = 1090) and The Molecular Taxonomy of Breast Cancer International Consortium (METARBRIC, n = 1904), were utilized in the current study. Survival analyses; Overall survival (OS), Disease-specific survival (DSS) and Disease-free survival (DFS) were performed by comparing the high and low expression groups. CYT score, xCell, and other immunological factors were used to evaluate intratumoral immune cell composition. Also, gene set enrichment analysis (GSEA) was performed between EMP1 high and low expression groups. Results: We divided each cohort into EMP1 expression high and low groups by utilizing median cutoff. The expression levels of EMP1 were not associated with clinical stage in any subtypes. However, interestingly lower expression of EMP1 was significantly associated with more advanced grades in ER positive/HER2 negative (ER+/HER2) subtype in both TCGA and METABRIC cohorts (p< 0.001 and p< 0.001, respectively). Also, low EMP1 expressing tumors demonstrated higher MKI67 expression levels in ER+/HER2- subtype consistently in both TCGA (p< 0.001) and METABRIC cohorts (p< 0.001). Furthermore, GSEA demonstrated that low EMP1 expressing tumors enriched the gene sets associated with cell proliferation such as MYC Targets, E2F signaling, and G2M Checkpoint signaling, compared with low EMP1 expressing tumors in ER+/HER2- of both TCGA and METABRIC cohorts. On the contrary, high EMP1 expressing tumors enriched the immune related gene sets such as Coagulation, Inflammatory_Response, Complement, and IL-6_JAK_STAT3 Signaling in ER+/HER2- of both TCGA and METABRIC cohorts. To this end, we further hypothesized that high EMP1 tumors were associated with favorable tumor immune microenvironment (TIME) and analyzed TIME utilizing xCell. However, to our surprise high EMP1 expressing tumors were not associated with favorable TIME. Low EMP1 expressing tumors demonstrated worse DFS, DSS, and OS compared with high EMP1 expressing tumors in ER+/HER2- breast cancer patients (p=0.013, p=0.003, and p=0.006) which was not the case in the other subtypes in METABRIC cohort. Conclusion: Low EMP1 expressing tumors were associated with improved OS, DSS, DFS in ER-positive breast cancer patients. Also, low EMP1 expressing tumors were found to associate with advanced grades and enriched gene sets related to cell proliferation and cell cycle, which may explain the poor survival of patients with low EMP1 expressing ER+/HER2- breast cancer. Citation Format: Junko Ukai, Yoshihisa Tokumaru, Masanori Oshi, Yoshimi Niwa, Ryutaro Mori, Kazuaki Takabe, Manabu Futamura. EMP1 low expressing tumor is associated with cell proliferation and poor overall survival of ER-positive breast cancer patients [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-28.

Abstract P3-07-19: Anti-depressant Amitriptyline Augments the Efficacy of Tamoxifen in ER positive breast cancer

Abstract Anti-depressant Amitriptyline Augments the Efficacy of Tamoxifen in ER positive breast cancer Pitta Venkata Prabhakar1, Arhan D Rao1, Timilsina Santosh1, Deepika Singh1, Ratna Vadlamudi1,2, Virginia Kaklamani3, Manjeet Rao1,2. 1Greehey Children’s Cancer Research Institute, University of Texas Health San Antonio, San Antonio, TX, USA. 2Mays Cancer Center, University of Texas Health San Antonio, San Antonio, TX, USA. 3Department of Medicine, UT Health, San Antonio, USA. Background: Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-related deaths in women worldwide, with 1 out of 8 women being diagnosed with invasive breast cancer during their lifetime. The majority of breast cancer patients (80%) are diagnosed with Estrogen Receptor positive (ER+) breast cancer. The most commonly used treatment option for ER+ Breast cancer is endocrine therapy, such as Tamoxifen(TAM). Although survival of breast cancer patients has dramatically improved with the use of endocrine therapy, acquired resistance and debilitating side effects of treatment became major concerns. Therefore, safer treatment options that effectively suppress cancer progression, reduce treatment associated side effects, and improve efficacy of standard of care therapies are much needed. Repurposing of clinically approved or investigational drugs has become promising alternative approach for treating cancer. Therefore, we tested the repurposing potential of anti-depressant Amitriptyline for the treatment of ER+ breast cancer. Methods: The anti-cancer effect of Amitriptyline was determined in vitro using short and long-term viability, migration, and apoptosis assays. To substantiate the in vitro data, the effect of Amitriptyline on tumor growth was assessed using orthotopic xenograft model. RNA-seq analysis was performed in Vehicle and Amitriptyline treated breast cancer cells to understand the mechanism of action. Finally, to test whether amitriptyline can sensitize ER+ breast cancer cells to endocrine therapy, we performed cell viability assays after treatment with Amitriptyline and Tamoxifen. Results: Amitriptyline treatment resulted in significant reduction of short term and long term viability, as well as, migration of ER+ BC cells. Furthermore, Amitriptyline treatment significantly increased apoptosis in BC cells. Our RNA-seq analysis revealed that Amitriptyline treatment inhibited important genes involved in cancer growth and survival including E2F signaling, G2/M pathway, and DNA repair pathways. Confirming our in vitro findings, Amitriptyline treatment blocked the growth of ER+ BC growth in pre-clinical orthotopic xenograft model of breast cancer. Importantly, Amitriptyline treatment sensitized ER+ BC to TAM, showing highly synergistic effects. Amitriptyline treatment significantly improved the effects of TAM on cell viability, survival, migration, and apoptosis. Furthermore, Amitriptyline augmented the efficacy of Tamoxifen in TAM resistant BC cells. Conclusion: Our study establishes potential of Amitriptyline as a repurposable drug as safe and robust treatment option for ER+ patients, either as a monotherapy or in combination. We are poised to begin a clinical trial and test the therapeutic efficacy of Amitriptyline for treating breast cancer patients. Citation Format: Prabhakar Pitta Venkata, Arhan D. Rao, Santosh Timilsina, Deepika Singh, Ratna K. Vadlamudi, Virginia Kaklamani, Manjeet K. Rao. Anti-depressant Amitriptyline Augments the Efficacy of Tamoxifen in ER positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-07-19.

Genomic analysis to elucidate similarities and differences between primary colorectal cancers and colorectal cancer liver metastases.

25 Background: Whole transcriptome (WT) analysis of primary colorectal cancer (pCRC) has shown that the tumor microenvironment (TME) plays a critical role in disease progression and response to treatment. Colorectal cancer liver metastases (CRCLM) bear many genetic similarities to primary tumors, however differences in the microenvironment could effect drug response. Since 80% of metastatic CRC patients have liver metastases there is a critical need for further molecular analysis of CRCLM. Methods: Using a novel method to identify aberrant activity of signaling pathways, continuous scores (called pathway cluster scores, PCS) were derived from subsets of Hallmark gene sets of MSigDB. PCS scores that were prognostic in CRCLM were considered relevant to disease progression. Combined studies of such scores in CRCLM and pCRC were used to explore differing effects of the colon and liver TMEs. Analyses were performed on published WT datasets of pCRC (4 cohorts, n = 2,978) and CRCLM (3 cohorts, n = 498). Immune cell infiltration levels were estimated with Immunedeconv. Results: The most significantly prognostic PCS score in CRCLM cohort GSE159216 (n = 171) was one derived from a cluster of 28 KRAS signaling genes, called K-score (p = .0004). Subtypes K+ (good prognosis) and K- (poor prognosis) denote samples with K-score above, respectively below, the median. Gene set enrichment analysis showed that in all CRCLM cohorts, K- was enriched versus (v) K+ in proliferation-related pathways, e.g. E2F signaling and DNA Repair, while K+ was enriched v K- in multiple immune system pathways. In both CRCLM cohorts, K+ was enriched v K- in infiltration of neutrophils and CD4+ T cells, while infiltration levels of CD8+ T cell were not significantly different between K+ and K-. The T cell homing chemokines CXCL12, CCL19, CCL21 were elevated in K+ v K-. The aforementioned molecular features of K+ and K- also held in all pCRC cohorts; however in these cohorts, K+ was also enriched v K- in B cell and M2 macrophage infiltration. Also, in pCRC expression of the B cell attractor CXCL13 and biomarkers for tertiary lymphoid structures (TLS) were significantly elevated in K+ v K-. Of note, TLS have been associated with response to immune checkpoint inhibitors in multiple cancer types. In addition, in 18 pairs of matching pCRC and CRCLM samples (GSE49355), K-scores of pCRC samples were significantly correlated to K-scores of the matching CRCLM samples (p = .01). Chemokine expression levels differed in matching pairs: mean expression of CXCL12 was significantly higher in CRCLM than pCRC, while mean expression of CXCL13 was significantly higher in pCRC than CRCLM. Conclusions: In the study cohorts, K- tumors in both pCRC and CRCLM, were proliferative and lacked inflammation. However, while both pCRC and CRCLM K+ tumors were inflamed and enriched with CD4+ T cells, only pCRC K+ tumors were likely to contain TLS.

Identification and validation of an E2F-related gene signature for predicting recurrence-free survival in human prostate cancer

BackgroundIt is well-established that biochemical recurrence is detrimental to prostate cancer (PCa). In the present study, we explored the mechanisms underlying PCa progression.MethodsFive cohorts from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus databases were used to perform gene set variation analysis (GSVA) between nonrecurrent and recurrent PCa patients. We obtained the intersection of pathway enrichment results and extracted the corresponding gene list. LASSO Cox regression analysis was used to identify recurrence-free survival (RFS)-related significant genes and establish an RFS prediction gene signature and nomogram. MTT and colony formation assays were conducted to validate our findings.ResultsThe E2F signaling pathway was activated in recurrent PCa patients compared to nonrecurrent patients. We established an E2F-related gene signature for RFS prediction based on the four identified E2F-related genes (CDKN2C, CDKN3, RACGAP1, and RRM2) using LASSO Cox regression in the Memorial Sloan Kettering Cancer Center (MSKCC) cohort. The risk score of each patient in MSKCC was calculated based on the expression levels of CDKN2C, CDKN3, RACGAP1, and RRM2. PCa patients with low-risk scores exhibited higher RFS than those with high-risk scores. Receiver operating characteristic (ROC) curve analysis validated the good performance and prognostic accuracy of the E2F-related gene signature, which was validated in the TCGA-prostate adenocarcinoma (TCGA-PRAD) cohort. Compared to patients with low Gleason scores and early T stages, PCa patients with high Gleason scores and advanced T stages had high-risk scores. Moreover, the E2F-related gene signature-based nomogram yielded good performance in RFS prediction. Functional experiments further confirmed these results.ConclusionsThe E2F signaling pathway is associated with biochemical recurrence in PCa. Our established E2F-related gene signature and nomogram yielded good accuracy in predicting the biochemical recurrence in PCa.

Abstract A021: Lysine methylation in EHMT1/GLP acts as a molecular switch to reprogram transcription networks to drive prostate cancer progression

Abstract Background: Prostate Cancer (PCa) progresses to a metastatic form of cancer called Castration-Resistant Prostate Cancer (CRPC) after treatment with androgen deprivation therapies (ADTs). Epigenetic reprogramming through altered expression and activity of histone modifier proteins is one major mechanism of tumor resistance. In particular, histone lysine methyltransferases (KMTs) and demethylases (KDMs) are important epigenetic targets in CRPC. In this study, we have focused on studying the function and regulation of Euchromatic Histone Methyltransferase 1 protein (EHMT1/GLP), which is a well-known H3K9 methyltransferase and functions in repressing gene transcription. EHMT family genes are altered in ~2-3% of primary PCa and ~10% of CRPC (primarily gene amplification) and their expression levels are significantly increased in CRPC, suggesting these proteins may have oncogenic activities in driving CRPC progression. Methods: We conducted an integrated analysis of ChIP-seq and RNA-seq analysis in LNCaP PCa cells to characterize the transcription program of EHMT1. We then performed proteomics analyses in VCaP and LNCaP PCa cells to identify post-translational modifications of EHMT1 and functionally validated the findings by generating loss-of-function mutations. We performed a histone demethylase assay to discover if EHMT1 is a substrate of LSD1 (a member of the KDM family). Moreover, we also assessed the effects of EHMT1 silencing or inhibition (in multiple PCa cell lines) on cell proliferation and metastasis in vitro using cell cycle analysis and transwell migration assay, and tumor growth and metastasis in vivo using mouse subcutaneous injection and zebrafish embryo injection approaches. Results: Our data showed that EHMT1 can transcriptionally activate multiple oncogenic pathways, including E2F and MYC signaling. Proteomic analyses revealed that EHMT1 is methylated in PCa cell lines, with dual-lysine methylation occurring at the K450/K451 sites. The histone demethylase assay showed that methylated K450, but not K451 is a potential substrate of LSD1. By generating the K450R, K451R, and K450/451R mutants, we showed that the K450/451R mutant, but not any single lysine mutants, can greatly expand EHMT1 chromatin binding independent of its H3K9 methyltransferase activity. This mutant also significantly induced EHMT1 oncogenic activity by activating E2F and MYC pathways. Moreover, EHMT1 silencing, or inhibition can significantly suppress tumor growth and metastasis. Conclusion: We have demonstrated that EHMT1 can function to activate oncogenic transcriptional programs in PCa by an H3K9-independent mechanism, possibly mediated through dual-lysine demethylation at K450/451 sites. Our data also suggest that targeting EHMT1 in CRPC may be a potential therapeutic strategy to suppress tumor growth and metastasis. Citation Format: Anna Besschetnova, Wanting Han, Mingyu Liu, Yanfei Gao, Muqing Li, Zifeng Wang, Maryam Labaf, Susan Patalano, Kavita Venkataramani, Rachel Muriph, Jill Macoska, Kellee Siegfried-Harris, Jason Evans, Steven Balk, Shuai Gao, Dong Han, Changmeng Cai. Lysine methylation in EHMT1/GLP acts as a molecular switch to reprogram transcription networks to drive prostate cancer progression. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A021.

Construction of cuproptosis-related gene signature to predict the prognosis and immunotherapy efficacy of patients with bladder cancer through bioinformatics analysis and experimental validation.

Background: A new form of cell death, copper-dependent cell death (termed cuproptosis), was illustrated in a recent scientific study. However, the biological function or prognostic value of cuproptosis regulators in bladder cancer (BLCA) remains unknown. Materials and Methods: Sequencing data obtained from BLCA samples in TCGA and GEO databases were preprocessed for analysis. Biological function and immune cell infiltration levels evaluated by gene set variation analysis (GSVA) were employed to calculate enrichment scores. Iteration least absolute shrinkage and selection operator (LASSO) and COX regression model were employed to select feature genes and construct a novel cuproptosis-related (CR) score signature. The genomics of drug sensitivity in cancer (GDSC) and tumor immune dysfunction and exclusion (TIDE) analysis were used to predict the chemotherapy and immunotherapy efficacy for BLCA patients. The relative expression of the genes involved in the signature was also verified by real-time quantitative PCR (qRT-PCR) in cell lines and tissues. Results: Expression abundance and the prognostic value of cuproptosis regulators proved that cuproptosis might play a vital part in the carcinogenesis of BLCA. GSVA revealed that cuproptosis regulators might be associated with metabolism and metastasis-related pathways such as TGF-β, protein secretion, oxidative Phosphorylation, MYC targets, MTORC1, and adipogenesis pathways. CR scores could predict the prognosis and evaluate the chemotherapy and immunotherapy efficacies of BLCA. CR scores were positively correlated with EMT, MYC, MTORC1, HEDGEHOG, and E2F signaling pathways; meanwhile, they were negatively correlated with several immune cell infiltration levels such as CD8+ T cells, γδT cells, and activated dendritic cells. Several GEO datasets were used to validate the power of prognostic prediction, and a nomogram was also established for clinical use. The expressions of DDX10, RBM34, and RPL17 were significantly higher in BLCA cell lines and tissues in comparison with those in the corresponding normal controls. Conclusion: Cuproptosis might play an essential role in the progression of BLCA. CR scores could be helpful in the investigation of prognostic prediction and therapeutic efficacy and could make contributions to further studies in BLCA.

Functional Characterization of Cooperating MGA Deletions in RUNX1::RUNX1T1 Acute Myeloid Leukemia

Max-gene associated (MGA) is a dual-specificity transcription factor that is recurrently mutated in 2-5% of all cancers and commonly seen in lung adenocarcinoma, endometrial carcinoma, and colorectal cancer. Our group has previously described heterozygous loss-of-function mutations in 9% of RUNX1::RUNX1T1 pediatric AMLs. Similar loss-of-function mutations are seen in other hematopoietic malignancies, including chronic lymphocytic leukemia (CLL), natural killer/T-cell lymphoma, B-cell acute lymphoblastic leukemia (B-ALL), and T-cell acute lymphoblastic leukemia (T-ALL). MGA is a component of the non-canonical polycomb repressive complex (ncPRC1.6) containing an amino-terminal T-box domain and a carboxy-terminal Myc-like basic helix-loop-helix (bHLH) domain which are used to regulate MAX-network and T-box family targets. In hematological malignancies such as RUNX1::RUNX1T1 AMLs, MGA mutations disrupt or remove the Myc-like bHLH domain. The role of MGA in normal hematopoietic regulation and leukemic development are understudied. This is likely due to the lack of in vivo MGA deficiency models which are embryonic lethal. We hypothesize that the loss of MGA leads to disruption of the ncPRC1.6 complex, causing a loss in global transcriptional repression of many pathways, including Myc and E2F target genes, leading to enhanced hematopoietic cell growth and self-renewal, as well as shortening the latency of AML driven by RUNX1::RUNXT1. To test this hypothesis, we designed a multidisciplinary approach to characterize the hematopoietic function of MGA. IP-Westerns of overexpressed flag-tagged MGA and its subsequent variants in K562 cells revealed that MGA truncations disrupt interactions with components of the ncPRC1.6 complex. Because these truncations lead to the loss of MGA's molecular function, we next generated CRISPR/Cas9 to knockout MGA in Molm13 cells. Using RNA-Seq and CUT&RUN to assess the genomic localization of MGA, we determined that MGA binds to an average of 85,119 peaks and 17.7% of those sites are at transcription start sites, including cell cycle genes such as CCND2. Deletion of MGA led to the removal of repressive H3K27me3 marks and an increase in MYC binding as well as active histone marks such as H3K4me3 and H3K27Ac at the transcription start site of CCND2. This led to an increase in CCND2 expression (logFC 1.56, FDR 1.56E-12) which is supported by in vitro EdU assays showing a 10.43% increase in cells cycling into S-phase. To further define the hematopoietic function of MGA, we designed a conditional knockout Mga mouse model with LoxP sites flanking exon 3, leading to deletion of Mga in hematopoietic stem and progenitor cells (HSPCs) when crossed with Vav1-cre. Transcriptome profiling analysis of HSPCs isolated from heterozygous (Mga+/) and homozygous (Mga-/-) mice reveals that Mga-/- mice, but not Mga+/-, leads to up-regulation of cell cycle (FDR 4.2E-06), Myc (FDR 6.3E-10), and E2f (FDR 2.9E-17) signaling pathways. Ex vivo serial CFU replating and cell proliferation assays support this, showing that Mga-/- leads to increased stem cell self-renewal and cell growth. However, bone marrow and spleen analysis of both Mga+/- and Mga-/- mice reveal no changes in immunophenotype when compared to WT controls. Expression of RUNX1::RUNX1T1 isoform 9a (AML-ETO9a) in Mga+/- or Mga-/- HSPCs reveals a significant increase (69% and 74% respectively, p<0.01) in leukemic stem cell self-renewal, when compared to WT controls expressing AML-ETO9a at week 4. When transplanted in mice, Mga+/- and Mga-/- HSPCs expressing AML-ETO9a led to a rapid expansion of blasts (GFP+, lin-, cKit+) found in the peripheral blood as well as a shortened latency compared to wild-type controls expressing AML-ETO9a (Figure 1). These studies demonstrated MGA as a regulator of hematopoietic cell proliferation (Figure 2), and how MGA deficiency cooperates with oncogenes to create more aggressive leukemia. Because MGA mutations occur in many tumor types, these data also provide functional insights into ncPRC1.6 activity in leukemia and other tumor types with recurrent mutations in MGA or other components of this complex. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DUPILUMAB REBALANCES IMMUNE RESPONSE IN PEDIATRIC TYPE-2 DISORDERS: IMPACTS ON T-CELL CLONES AND FUNCTIONALITY

<h3>Introduction</h3> Dupilumab demonstrates efficacy in treating a variety of Type-2 disorders refractory to conventional therapies. Improvement in clinical outcomes establishes a need to investigate mechanistic pathways of potential immunomodulation. <h3>Methods</h3> 11 patients, two with moderate-to-severe asthma and 9 with moderate-top-severe AD were recruited to give blood samples pre and three months post-Dupilumab. Longitudinal phenotyping of leukocytes using mass cytometry (CyTOF) and lineage tracing of single-cells using mitochondrial single-cell ATAC-seq (mt-scATAC-seq) was conducted in PBMC and purified T-cells. Clones were inferred using a community-based detection algorithm to cluster cells based on mitochondrial variant allele-frequency (VAF). Gene scores were computed based on open-chromatin regions in the gene and promoter. Differential expression pre- and post-dupilumab was done for each cell cluster population. <h3>Results</h3> CyTOF immunophenotyping showed increased Th1 and Th17 cells following dupilumab, consistent with increased activity of IL-6, TNF, IFNa, and IFNg genes in lymphocytes by mt-scATAC-Seq. Rebalancing of adaptive immunity was accompanied by upregulation of Myc, E2F, MTORC1, PI3K-AKT, and p53 signaling intermediates, and was associated with upregulation of genes controlling apoptosis, unfolded protein response, and oxidative phosphorylation. Changes in frequency of T-cell clonal lineages <i>in vivo</i> were detected following dupilumab. <h3>Conclusion</h3> Data supports clone-specific effects of dupilumab in pediatric Type-2 disorders. This approach enables analysis of changes in gene networks in T-cell clones and populations following treatment. Upregulation of non-Type-2 signaling and changes at the clonal level suggest Dupilumab restores balance to the immune response.

NSD1 Mutations in Sotos Syndrome Induce Differential Expression of Long Noncoding RNAs, miR646 and Genes Controlling the G2/M Checkpoint.

An increasing amount of evidence indicates the critical role of the NSD1 gene in Sotos syndrome (SoS), a rare genetic disease, and in tumors. Molecular mechanisms affected by NSD1 mutations are largely uncharacterized. In order to assess the impact of NSD1 haploinsufficiency in the pathogenesis of SoS, we analyzed the gene expression profile of fibroblasts isolated from the skin samples of 15 SoS patients and of 5 healthy parents. We identified seven differentially expressed genes and five differentially expressed noncoding RNAs. The most upregulated mRNA was stratifin (SFN) (fold change, 3.9, Benjamini–Hochberg corrected p < 0.05), and the most downregulated mRNA was goosecoid homeobox (GSC) (fold change, 3.9, Benjamini–Hochberg corrected p < 0.05). The most upregulated lncRNA was lnc-C2orf84-1 (fold change, 4.28, Benjamini–Hochberg corrected p < 0.001), and the most downregulated lncRNA was Inc-C15orf57 (fold change, −0.7, Benjamini–Hochberg corrected p < 0.05). A gene set enrichment analysis reported the enrichment of genes involved in the KRAS and E2F signaling pathways, splicing regulation and cell cycle G2/M checkpoints. Our results suggest that NSD1 is involved in cell cycle regulation and that its mutation can induce the down-expression of genes involved in tumoral and neoplastic differentiation. The results contribute to defining the role of NSD1 in fibroblasts for the prevention, diagnosis and control of SoS.

Abstract 5827: Increased miR150 expression in tumors enhances lymphocytes infiltration causing immunosuppression through inflammatory chemokines

Abstract Dysregulated miRNAs play key roles in multiple biological processes and cancer. miR-150-5p, a cancer-related miRNA, has been reported to be aberrantly expressed in various cancers. We found that triple-negative and HER2+ breast cancer patients have elevated expression of miR150 in the TCGA and METABRIC patient cohort. Both cohorts showed significantly increased expression of miR150 in grade IV patients. Further studies explained that miR150 is strongly correlated with CD8+ T cells and B cells in accord with increased expression of immune-checkpoint markers PD-1, PDL-1, and CTLA4. We, therefore, hypothesized that higher expression of miR150 in tumors attracted more immune cells in the tumor microenvironment. We use two triple negative breast cancer cell lines MDA-231 and BT-549 cells and overexpressed miR150 followed by co-culture them with Jurkart and B cells. Our data revealed that both jurkart and B cells had increased migration towards cancer cells overexpressed miR150 mimic compared to miR150 mimic negative control. Interestingly, overexpressed miR150 mimic in cancer cells inhibited migration in both triple-negative breast cancer cells. Overexpression of miR150 mimic in cancer cells did not alter cell proliferation. To understand further the role of miR150 in breast cancer, we overexpressed miR150 mimic and its corresponding negative control in MDA-231 and did RNA seq analysis. RNA seq data explained that approximately 4927 genes are upregulated with miR150 mimic overexpression. Interestingly several chemokines and mediators of metastasis (CXCL2, CXCL1 CCL20, IGBP3, and ANGPTL4) are upregulated among the top 50 upregulated genes. In addition, gene enrichment analysis explained that TNF-α signaling pathways, Hypoxia, epithelial-mesenchymal pathways, E2F signaling, and Inflammatory pathways are a few upregulated pathways with miR150 mimic overexpression compared to the negative control. We also found miR150 high tumor enriched correlated with interferon-alpha and gamma signaling, inflammatory response pathways similar with cohort analysis data. In conclusion, miR150 in cancer plays a key role in interferon and inflammatory signaling pathways attracting more immune cells and suppressing the tumor microenvironment. Citation Format: Aparna Maiti, Masanori Oshi, Rongrong Wu, Nitai Hait, Kazuaki Takabe. Increased miR150 expression in tumors enhances lymphocytes infiltration causing immunosuppression through inflammatory chemokines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5827.