Jian-Jun Wei

Preclinical Evaluation of NTX-301, a Novel DNA Hypomethylating Agent in Ovarian Cancer

Abstract Purpose: DNA methylation causes silencing of tumor-suppressor and differentiation-associated genes, being linked to chemoresistance. Previous studies demonstrated that hypomethylating agents (HMA) resensitize ovarian cancer to chemotherapy. NTX-301 is a highly potent and orally bioavailable HMA, in early clinical development. Experimental Design: The antitumor effects of NTX-301 were studied in ovarian cancer models by using cell viability, stemness and ferroptosis assays, RNA sequencing, lipidomic analyses, and stimulated Raman spectroscopy. Results: Ovarian cancer cells (SKOV3, IC50 = 5.08 nmol/L; OVCAR5 IC50 = 3.66 nmol/L) were highly sensitive to NTX-301 compared with fallopian tube epithelial cells. NTX-301 downregulated expression of DNA methyltransferases 1–3 and induced transcriptomic reprogramming with 15,000 differentially expressed genes (DEG, P < 0.05). Among them, Gene Ontology enrichment analysis identified regulation of fatty acid biosynthesis and molecular functions related to aldehyde dehydrogenase (ALDH) and oxidoreductase, known features of cancer stem cells. Low-dose NTX-301 reduced the ALDH(+) cell population and expression of stemness-associated transcription factors. Stearoyl-coenzyme A desaturase 1 (SCD), which regulates production of unsaturated fatty acids (UFA), was among the top DEG downregulated by NTX-301. NTX-301 treatment decreased levels of UFA and increased oxidized lipids, and this was blunted by deferoxamine, indicating cell death via ferroptosis. NTX-301–induced ferroptosis was rescued by oleic acid. In vivo, monotherapy with NTX-301 significantly inhibited ovarian cancer and patient-derived xenograft growth (P < 0.05). Decreased SCD levels and increased oxidized lipids were detected in NTX-301–treated xenografts. Conclusions: NTX-301 is active in ovarian cancer models. Our findings point to a new mechanism by which epigenetic blockade disrupts lipid homeostasis and promotes cancer cell death.

Methylation Signatures Identify Two Distinct Clusters of Uterine Leiomyosarcoma With Unique Histologic and Clinical Behaviors

Uterine leiomyosarcoma (uLMS) is a rare and deadly gynecologic malignancy. uLMS is histologically heterogeneous and presents with a wide spectrum of tumor differentiation, with a broad range of genomic DNA instability, which can make the diagnosis and prognosis of uLMS challenging. Methylation has emerged as a useful molecular tool in tumor classification and diagnosis in certain neoplasms. We initiated this study to investigate the role of global methylation in the differential diagnosis of uLMS from its mimics in correlation with pathologic characteristics and clinical outcomes. In this study, we performed array-based global methylation profiling analysis in a total of 71 uLMS and compared the methylation signatures of uLMS with several other uterine mesenchymal tumors and soft tissue leiomyosarcoma. We found that uLMS demonstrated distinct methylation patterns differing from all other tumor types. Notably, methylation profiling defines 2 distinct subgroups of uLMS with differing copy number alterations, resulting in unique histologic and clinical behaviors, further emphasized by differences in methylation pathway analysis. This study is the first to report methylation profiling as a useful diagnostic tool in differentiating uLMS from mimics and defines 2 subtypes of uLMS based on methylation signatures.

Genomic characterization and histologic analysis of uterine leiomyosarcoma arising from leiomyoma with bizarre nuclei

Abstract Leiomyoma with bizarre nuclei (LM‐BN) is a rare variant of leiomyoma with a benign clinical course. In contrast, leiomyosarcoma (LMS) is a high‐grade, malignant neoplasm characterized by high recurrence rates and poor survival. While LM‐BN and LMS show distinct morphologies, they share similar immunoprofiles and molecular alterations, with both considered ‘genomically unstable’. Rare cases of LM‐BN associated with LMS have been reported; however, the histogenesis and molecular relationship between these two tumors remains unclear. In this study, we assessed 11 cases of LMS arising in conjunction with LM‐BN confirmed by histology and immunohistochemistry (IHC), further analyzed by clinical, histologic, and molecular characteristics of these distinct components. LM‐BN and LMS had similar p16 and p53 IHC patterns, but LMS had a higher Ki‐67 index and lower estrogen and progesterone eceptor expression. Digital image analysis based on cytologic features revealed spatial relationships between LMS and LM‐BN. Genomic copy number alterations (CNAs) demonstrated the same clonal origin of LMS arising from existing LM‐BN through conserved CNAs. LMS harbored highly complex CNAs and more frequent losses of the TP53 , RB1 , and PTEN genomic regions than LM‐BN ( p  = 0.0031), with CDKN2A/B deletion identified in LMS only. Mutational profiling revealed many shared oncogenic alterations in both LM‐BN and LMS; however, additional mutations were present within LMS, indicative of tumor progression through progressive genomic instability. Analysis of spatial transcriptomes defined uniquely expressed gene signatures that matched the geographic distribution of LM‐BN, LMS, and other cell types. Our findings indicate for the first time that a subset of LMS arises from an existing LM‐BN, and highly complex genomic alterations could be potential high risks associated with disease progression in LM‐BN. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Somatic MED12 Mutations in Myometrial Cells

Over 70% of leiomyoma (LM) harbor MED12 mutations, primarily in exon 2 at c.130-131 (GG). Myometrial cells are the cell origin of leiomyoma, but the MED12 mutation status in non-neoplastic myometrial cells is unknown. In this study, we investigated the mutation burden of MED12 in myometrium. As traditional Sanger or even NGS sequencing may not be able to detect MED12 mutations that are lower than 0.1% in the testing sample, we used duplex deep sequencing analysis (DDS) to overcome this limitation. Tumor-free myometria (confirmed by pathology evaluation) were dissected, and genomic DNA from MED12 exon 2 (test) and TP53 exon 5 (control) were captured by customer-designed probe sets, followed by DDS. Notably, DDS demonstrated that myometrial cells harbored a high frequency of mutations in MED12 exon 2 and predominantly in code c.130-131. In contrast, the baseline mutations in other coding sequences of MED12 exon 2 as well as in the TP53 mutation hotspot, c.477-488 were comparably low in myometrial cells. This is the first report demonstrating a non-random accumulation of MED12 mutations at c.130-131 sites in non-neoplastic myometrial cells which provide molecular evidence of early somatic mutation events in myometrial cells. This early mutation may contribute to the cell origin for uterine LM development in women of reproductive age.

Racial disparity in uterine leiomyoma: new insights of genetic and environmental burden in myometrial cells

Abstract Uterine leiomyoma (LM), also known as uterine fibroids, are common gynecological tumors and can reach a prevalence of 70% among women by the age of 50 years. Notably, the LM burden is much higher in Black women with earlier onset, a greater tumor number, size, and severity compared to White women. Published knowledge shows that there are genetic, environmental, and lifestyle-based risk factors associated with racial disparity for LM. Significant strides have been made on genomic, epigenomic, and transcriptomic data levels in Black and White women to elucidate the underlying pathomolecular reasons of racial disparity in LM development. However, racial disparity of LM remains a major area of concern in gynecological research. This review highlights risk factors of LM and their role in different races. Furthermore, we discuss the genetics and uterine myometrial microenvironment in LM development. Comparative findings revealed that a major racial difference in the disease is linked to myometrial oxidative burden and altered ROS pathways which is relevant to the oxidized guanine in genomic DNA and MED12 mutations that drive the LM genesis. Considering the burden and morbidity of LM, we anticipate that this review on genetic risk and myometrial microenvironment will strengthen understanding and propel the growth of research to address the racial disparity of LM burden.

MYC-regulated pseudogene HMGA1P6 promotes ovarian cancer malignancy via augmenting the oncogenic HMGA1/2

AbstractPseudogenes have long been considered as nonfunctional genomic sequences. Recent studies have shown that they can potentially regulate the expression of protein-coding genes and are dysregulated in diseases including cancer. However, the potential roles of pseudogenes in ovarian cancer have not been well studied. Here we characterized the pseudogene expression profile in HGSOC (high-grade serous ovarian carcinoma) by microarray. We identified 577 dysregulated pseudogenes and most of them were up-regulated (538 of 577). HMGA1P6 (High mobility group AT-hook 1 pseudogene 6) was one of the overexpressed pseudogenes and its expression was inversely correlated with patient survival. Mechanistically, HMGA1P6 promoted ovarian cancer cell malignancy by acting as a ceRNA (competitive endogenous RNA) that led to enhanced HMGA1 and HMGA2 expression. Importantly, HMGA1P6 was transcriptionally activated by oncogene MYC in ovarian cancer. Our findings reveal that MYC may contribute to oncogenesis through transcriptional regulation of pseudogene HMGA1P6 in ovarian cancer.

Primary HPV-Associated Enteric-Type Adenocarcinoma of Vagina: A Case Report

Primary enteric type adenocarcinomas of the vagina are extremely rare. We present a 63-year-old woman who had a polypoid mass localized to the distal vagina. The lesion was composed of a columnar glandular cell proliferation with focal cribriforming, reminiscent of tubular adenoma. Immunohistochemical stains were notable for expression of enteric markers (CDX2 and KRT20), as well as negativity for Mullerian markers (PAX8, ER, and PR), diffuse expression for p16, and positivity for high-risk HPV mRNA expression. Ultimately, a diagnosis of vaginal primary HPV-associated enteric type adenocarcinoma was rendered for this unusual lesion. To our knowledge, no prior cases of HPV-associated enteric type adenocarcinomas of the vagina have been described before.

Splice-switching of the oncogenic BCS1L isoform suppresses ovarian cancer progression by disrupting mitochondrial function

Abstract Increasing evidences demonstrate that mitochondrial function is essential for cancer cell survival and metastasis. However, the role of mitochondrial metabolic reprogramming in ovarian cancer progression remains largely unknown. Here, we report that mitochondrial chaperone BCS1L generates two major alternative-spliced isoforms, a full-length isoform ( BCS1L-L ) and a short isoform lacking exon 2 ( BCS1L-S ). Interestingly, BCS1L-L is elevated in several human cancers, and it significantly increased oxidative phosphorylation and ATP production in the present work, which is required for the survival of cancer cells. In contrast, BCS1L-S was unable to localize to the mitochondria as BCS1L-L did, and this led to impaired metabolic function. Mechanistically, splicing factor USP39 promoted exon 2 inclusion, thus facilitating the generation of oncogenic BCS1L-L and, thereby, maintaining mitochondrial homeostasis and survival of ovarian cancer cells. Importantly, we developed splice-switch antisense oligonucleotides (ASOs) that successfully induced exon 2 skipping and decreased BCS1L-L abundance, resulting in impaired tumor growth. These findings suggest that targeting oncogenic BCS1L-L by ASOs is a novel approach for ovarian cancer treatment.