Uterine leiomyosarcoma (uLMS) is the most common type of uterine sarcoma associated with poor prognosis, high rates of recurrence, and metastasis. There is currently limited information about uLMS molecular mechanisms of origin and development. Bromodomain (BRD)-containing proteins are involved in many biological processes, most notably epigenetic regulation of transcription, and BRD protein dysfunction has been linked to many diseases including tumorigenesis. However, the role of BRD proteins in the pathogenesis of uLMS is unknown. Here, we show for the first time that BRD9 is aberrantly overexpressed in uLMS tissues compared to adjacent myometrium. BRD9 expression is also upregulated in uLMS cell lines compared to benign uterine fibroid and myometrium cell lines. Inhibition of BRD9 using the specific inhibitor (TP-472) suppressed uLMS cell proliferation via inducing apoptosis and cell cycle arrest. To further characterize the mechanistic basis for TP-472 inhibition of uLMS cell growth, we performed a comparative RNA-seq analysis of vehicle-treated and TP-472-treated uLMS cells (n = 4 each). Bioinformatics analysis revealed that TP-472 treatment distinctly altered the uLMS cell transcriptome. Gene set enrichment analysis identified critical pathways altered by BRD9 inhibition, including interferon-alpha response, KRAS signaling, MYC targets, TNF-a signaling via NFkB, and MTORC1 signaling. Parsimonious gene correlation network analysis identified nine enriched modules, including cell cycle and apoptosis modules. Moreover, the ENCODE Histone Modifications gene set and TargetScan microRNA analysis in Enrichr suggested that TP-472-induced BRD9 inhibition may alter the uLMS cell transcriptome by reprograming the oncogenic epigenome and inducing miRNA-mediated gene regulation. Therefore, BRD9 constitutes a specific vulnerability in malignant uLMS, and targeting non-BET BRD proteins in uLMS may provide a promising and novel strategy for treating patients with this aggressive uterine cancer.

Uterine leiomyosarcoma (uLMS) is the most frequent subtype of uterine sarcoma that presents a poor prognosis, high rates of recurrence, and metastasis. Currently, the molecular mechanism of the origin and development of uLMS is unknown. Class I histone deacetylases (including HDAC1, 2, 3, and 8) are one of the major classes of the HDAC family and catalyze the removal of acetyl groups from lysine residues in histones and cellular proteins. Class I HDACs exhibit distinct cellular and subcellular expression patterns and are involved in many biological processes and diseases through diverse signaling pathways. However, the link between class I HDACs and uLMS is still being determined. In this study, we assessed the expression panel of Class I HDACs in uLMS and characterized the role and mechanism of class I HDACs in the pathogenesis of uLMS. Immunohistochemistry analysis revealed that HDAC1, 2, and 3 are aberrantly upregulated in uLMS tissues compared to adjacent myometrium. Immunoblot analysis demonstrated that the expression levels of HDAC 1, 2, and 3 exhibited a graded increase from normal and benign to malignant uterine tumor cells. Furthermore, inhibition of HDACs with Class I HDACs inhibitor (Tucidinostat) decreased the uLMS proliferation in a dose-dependent manner. Notably, gene set enrichment analysis of differentially expressed genes (DEGs) revealed that inhibition of HDACs with Tucidinostat altered several critical pathways. Moreover, multiple epigenetic analyses suggested that Tucidinostat may alter the transcriptome via reprogramming the oncogenic epigenome and inducing the changes in microRNA-target interaction in uLMS cells. In the parallel study, we also determined the effect of DL-sulforaphane on the uLMS. Our study demonstrated the relevance of class I HDACs proteins in the pathogenesis of malignant uLMS. Further understanding the role and mechanism of HDACs in uLMS may provide a promising and novel strategy for treating patients with this aggressive uterine cancer.

DNA of many breast tumors is barraged by C-to-T/G mutations within TCW (W:T,A). These mutations are attributed to the aberrant expression and activity of APOBEC3 enzymes. They have been shown to account for many driver mutations in genes such as PIK3CA, ERBB2, and PPP2R1A, however their precise source and also their roles in tumor development, evolution, and patient survival are debated. Currently, quantification of APOBEC3 expression changes in tumor cells is confounded by the ubiquitous expression of these enzymes in immune infiltrating cells. In this study, we used a novel quantitative biology approach to determine the expression profiles of APOBEC3 enzymes in breast tumor and tumor microenvironment cells from >1,000 patients. We combined diverse datasets including tumor/matched normal RNAseqs, tumor somatic mutations, cell line RNAseqs and mutations, estimates of tumor purities and immune cell compositions, and expression of purified cell populations to show that in breast cancer there is only a single APOBEC3 dysregulation process. This process is subtype-independent and is represented by APOBEC3B upregulation and and extreme APOBEC3C downregulation. Compared to all other tumor types, breast tumors are affected the most by this process. Citation Format: Hamid Hamidi, Azad Khosh, Hamzeh Rahimi, Diako Ebrahimi. Profiling of APOBEC3 dysregulation in 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 P5-13-05.

The zinc finger antiviral protein (ZAP) is known to restrict viral replication by binding to the CpG rich regions of viral RNA, and subsequently inducing viral RNA degradation. This enzyme has recently been shown to be capable of restricting SARS-CoV-2. These data have led to the hypothesis that the low abundance of CpG in the SARS-CoV-2 genome is due to an evolutionary pressure exerted by the host ZAP. To investigate this hypothesis, we performed a detailed analysis of many coronavirus sequences and ZAP RNA binding preference data. Our analyses showed neither evidence for an evolutionary pressure acting specifically on CpG dinucleotides, nor a link between the activity of ZAP and the low CpG abundance of the SARS-CoV-2 genome.

DNA of many tumors is barraged by C-to-T/G mutations within TCW (W:T,A). These mutations are attributed to the aberrant expression and activity of APOBEC3 enzymes. They have been shown to account for many driver mutations in genes such as PIK3CA, ERBB2, and PPP2R1A, however their precise source and also their roles in tumor development, evolution, and patient survival are debated. Currently, quantification of APOBEC3 expression changes in tumor cells is confounded by the ubiquitous expression of these enzymes in infiltrating immune cells. In this study, we used quantitative biology approaches to separate the expression profiles of APOBEC3 enzymes in tumor and tumor microenvironment cells and determine their associations with tumor mutational signatures. For this purpose, we analyzed diverse datasets including TCGA tumor/matched normal RNAseqs, tumor somatic mutations, cell line RNAseqs and mutations, estimates of tumor purities and immune cell compositions, and expression of purified cell populations to precisely determine how APOBEC3 enzymes are dysregulated across tumors and whether their dysregulations are proportional to tumor mutational signatures. Unexpectedly, we found that dysregulation of APOBEC3 enzymes is independent of tumor C-to-T/G mutational burden. Importantly, our data suggest that this disconnect is likely not due to the episodic bursts of APOBEC3-induced mutations in cancer. Citation Format: Azad Khosh, Hamid Hamidi, Hamzeh Rahimi, Diako Ebrahimi. Disconnect between APOBEC3 expressions and mutations across cancers. [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 3733.