Molecular profiles and urinary biomarkers of upper tract urothelial carcinomas associated with aristolochic acid exposure.

Recurrent upper tract urothelial carcinomas (UTUCs) arise in the context of nephropathy linked to exposure to the herbal carcinogen aristolochic acid (AA). Here we delineated the molecular programs underlying UTUC tumorigenesis in patients from endemic aristolochic acid nephropathy (AAN) regions in Southern Europe. We applied an integrative multiomics analysis of UTUCs, corresponding unaffected tissues and of patient urines. Quantitative microRNA (miRNA) and messenger ribonucleic acid (mRNA) expression profiling, immunohistochemical analysis by tissue microarrays and exome and transcriptome sequencing were performed in UTUC and nontumor tissues. Urinary miRNAs of cases undergoing surgery were profiled before and after tumor resection. Ribonucleic acid (RNA) and protein levels were analyzed using appropriate statistical tests and trend assessment. Dedicated bioinformatic tools were used for analysis of pathways, mutational signatures and result visualization. The results delineate UTUC-specific miRNA:mRNA networks comprising 89 miRNAs associated with 1,862 target mRNAs, involving deregulation of cell cycle, deoxyribonucleic acid (DNA) damage response, DNA repair, bladder cancer, oncogenes, tumor suppressors, chromatin structure regulators and developmental signaling pathways. Key UTUC-specific transcripts were confirmed at the protein level. Exome and transcriptome sequencing of UTUCs revealed AA-specific mutational signature SBS22, with 68% to 76% AA-specific, deleterious mutations propagated at the transcript level, a possible basis for neoantigen formation and immunotherapy targeting. We next identified a signature of UTUC-specific miRNAs consistently more abundant in the patients' urine prior to tumor resection, thereby defining biomarkers of tumor presence. The complex gene regulation programs of AAN-associated UTUC tumors involve regulatory miRNAs prospectively applicable to noninvasive urine-based screening of AAN patients for cancer presence and recurrence.

SF3B1 homeostasis is critical for survival and therapeutic response in T cell leukemia.

The production of noncanonical mRNA transcripts is associated with cell transformation. Driven by our previous findings on the sensitivity of T cell acute lymphoblastic leukemia (T-ALL) cells to SF3B1 inhibitors, we identified that SF3B1 inhibition blocks T-ALL growth in vivo with no notable associated toxicity. We also revealed protein stabilization of the U2 complex component SF3B1 via deubiquitination. Our studies showed that SF3B1 inhibition perturbs exon skipping, leading to nonsense-mediated decay and diminished levels of DNA damage response-related transcripts, such as the serine/threonine kinase CHEK2, and impaired DNA damage response. We also identified that SF3B1 inhibition leads to a general decrease in R-loop formation. We further demonstrate that clinically used SF3B1 inhibitors synergize with CHEK2 inhibitors and chemotherapeutic drugs to block leukemia growth. Our study provides the proof of principle for posttranslational regulation of splicing components and associated roles and therapeutic implications for the U2 complex in T cell leukemia.

Posttranslational Regulation of the Exon Skipping Machinery Controls Aberrant Splicing in Leukemia.

Splicing alterations are common in diseases such as cancer, where mutations in splicing factor genes are frequently responsible for aberrant splicing. Here we present an alternative mechanism for splicing regulation in T-cell acute lymphoblastic leukemia (T-ALL) that involves posttranslational stabilization of the splicing machinery via deubiquitination. We demonstrate there are extensive exon skipping changes in disease, affecting proteasomal subunits, cell-cycle regulators, and the RNA machinery. We present that the serine/arginine-rich splicing factors (SRSF), controlling exon skipping, are critical for leukemia cell survival. The ubiquitin-specific peptidase 7 (USP7) regulates SRSF6 protein levels via active deubiquitination, and USP7 inhibition alters the exon skipping pattern and blocks T-ALL growth. The splicing inhibitor H3B-8800 affects splicing of proteasomal transcripts and proteasome activity and acts synergistically with proteasome inhibitors in inhibiting T-ALL growth. Our study provides the proof-of-principle for regulation of splicing factors via deubiquitination and suggests new therapeutic modalities in T-ALL. SIGNIFICANCE: Our study provides a new proof-of-principle for posttranslational regulation of splicing factors independently of mutations in aggressive T-cell leukemia. It further suggests a new drug combination of splicing and proteasomal inhibitors, a concept that might apply to other diseases with or without mutations affecting the splicing machinery.This article is highlighted in the In This Issue feature, p. 1241.

Early pathological characterization of murine dissecting abdominal aortic aneurysms.

We report here on the early pathology of a well-established murine model of dissecting abdominal aortic aneurysms (AAAs). Continuous infusion of angiotensin II (AngII) into apolipoprotein E-deficient mice induces the formation of aortic dissection and expansion at some point after implantation of miniosmotic pumps containing AngII. While this model has been studied extensively at a chronic stage, we investigated the early pathology of dissecting AAA formation at multiple scales. Using high-frequency ultrasound, we screened 12-week-old male mice daily for initial formation of these aneurysmal lesions between days 3 and 10 post-implantation. We euthanized animals on the day of diagnosis of a dissecting AAA or at day 10 if no aneurysmal lesion developed. Aortic expansion and reduced vessel wall strain occurred in animals regardless of whether a dissecting AAA developed by day 10. The aortas of mice that did not develop dissecting AAAs showed intermediate changes in morphology and biomechanical properties. RNA sequencing and gene expression analysis revealed multiple proinflammatory and matrix remodeling genes to be upregulated in the suprarenal aorta of AngII-infused mice as compared to saline-infused controls. Histology and immunohistochemistry confirmed that extracellular matrix remodeling and inflammatory cell infiltration, notably neutrophils and macrophages, occurred in AngII-infused mice with and without dissecting AAAs but not saline-infused controls. Understanding early disease processes is a critical step forward in translating experimental results in cardiovascular disease research. This work advances our understanding of this well-established murine model with applications for improving early diagnosis and therapy of acute aortic syndrome in humans.