Kosinsky, Robyn Laura

Abstract Transcriptional condensates (TCs) are dynamic, membrane-less assemblies of proteins and nucleic acids that form at specific genomic loci to control gene expression. The primary drivers of TC formation and function are long stretches of polypeptide chains that lack well-defined tertiary structure, known as the intrinsically disordered regions (IDRs) of Transcription Factors (TFs). However, the role of well-structured domains such as the DNA-binding domain (DBD) in condensate formation and downstream regulation remains poorly understood. Here, we investigated whether the DBD of the Progesterone Receptor (PR), a model TF regulated by hormone binding, contributes to the formation of PR TCs. By combining Single Particle Tracking (SPT) with deep-learning-based single-trajectory analysis, we demonstrated that both the IDR and the DBD are required for the formation of PR condensates. Furthermore, transcriptomic analysis showed that mutations of either of the two domains result in the expression of a distinct set of genes, with functional assays corroborating these findings and linking domain-specific transcription to differential effects on cell proliferation and migration. Together, our results show that domains beyond IDRs also play a role in TC formation and function and uncover domain-specific regulation of transcriptional and oncogenic programs.

Abstract Cutaneous T-cell lymphoma (CTCL) is a rare malignancy of skin-resident T cells characterized by an aberrant cytokine profile, notably elevated Interleukin-17A (IL-17A) levels. However, the functional importance of IL-17A dysregulation in the CTCL pathogenesis remains poorly understood. Our analysis revealed increased IL-17A receptor (IL-17RA) expression in CTCL patient samples, supporting the importance of IL-17A signaling in CTCL progression. Proteomic and metabolomic profiling of IL-17A-treated malignant T cells revealed mitochondrial electron transport chain (ETC) complex I as a key downstream target of IL-17A signaling. Furthermore, we confirmed the elevated expression of ETC complex I in CTCL patient samples compared to healthy control T cells, hence highlighting the clinical relevance of the IL-17A signaling-ETC complex 1 axis. Altogether, our data unveil the function of IL-17A signaling in the metabolic axis in CTCL progression, which could be useful for developing advanced therapeutic strategies. Significance statement: We uncover an IL-17A-induced electron transport chain (ETC) complex I pro-tumorigenic circuit and provide proof of concept evidence that ETC complex I can be targeted to combat the challenges posed by Cutaneous T-cell Lymphoma aggressiveness.

Cells expressing features of senescence, including upregulation of p21 and p16, appear transiently following tissue injury, yet the properties of these cells or how they contrast with age-induced senescent cells remains unclear. Here, we used skeletal injury as a model and identified the rapid appearance following fracture of p21 + cells expressing senescence markers, mainly as osteochondroprogenitors (OCHs) and neutrophils. Targeted genetic clearance of p21 + cells suppressed senescence-associated signatures within the fracture callus and accelerated fracture healing. By contrast, p21 + cell clearance did not alter bone loss due to aging; conversely, p16 + cell clearance, known to alleviate skeletal aging, did not affect fracture healing. Following fracture, p21 + neutrophils were enriched in signaling pathways known to induce paracrine stromal senescence, while p21 + OCHs were highly enriched in senescence-associated secretory phenotype factors known to impair bone formation. Further analysis revealed an injury-specific stem cell–like OCH subset that was p21 + and highly inflammatory, with a similar inflammatory mesenchymal population (fibro-adipogenic progenitors) evident following muscle injury. Thus, intercommunicating senescent-like neutrophils and mesenchymal progenitor cells were key regulators of tissue repair in bone and potentially across tissues. Moreover, our findings established contextual roles of p21 + versus p16 + senescent/senescent-like cells that may be leveraged for therapeutic opportunities.

Abstract A major hurdle to the application of precision oncology in pancreatic cancer is the lack of molecular stratification approaches and targeted therapy for defined molecular subtypes. In this work, we sought to gain further insight and identify molecular and epigenetic signatures of the Basal-like A pancreatic ductal adenocarcinoma (PDAC) subgroup that can be applied to clinical samples for patient stratification and/or therapy monitoring. We generated and integrated global gene expression and epigenome mapping data from patient-derived xenograft models to identify subtype-specific enhancer regions that were validated in patient-derived samples. In addition, complementary nascent transcription and chromatin topology (HiChIP) analyses revealed a Basal-like A subtype-specific transcribed enhancer program in PDAC characterized by enhancer RNA (eRNA) production that is associated with more frequent chromatin interactions and subtype-specific gene activation. Importantly, we successfully confirmed the validity of eRNA detection as a possible histologic approach for PDAC patient stratification by performing RNA-ISH analyses for subtype-specific eRNAs on pathologic tissue samples. Thus, this study provides proof-of-concept that subtype-specific epigenetic changes relevant for PDAC progression can be detected at a single-cell level in complex, heterogeneous, primary tumor material. Implications: Subtype-specific enhancer activity analysis via detection of eRNAs on a single-cell level in patient material can be used as a potential tool for treatment stratification.

Objective Pancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel mechanistic approaches to overcome or prevent resistance in PDAC. Design We used in vitro and in vivo models of resistant PDAC and integrated epigenomic, transcriptomic, nascent RNA and chromatin topology data. We identified a JunD-driven subgroup of enhancers, called interactive hubs (iHUBs), which mediate transcriptional reprogramming and chemoresistance in PDAC. Results iHUBs display characteristics typical for active enhancers (H3K27ac enrichment) in both therapy sensitive and resistant states but exhibit increased interactions and production of enhancer RNA (eRNA) in the resistant state. Notably, deletion of individual iHUBs was sufficient to decrease transcription of target genes and sensitise resistant cells to chemotherapy. Overlapping motif analysis and transcriptional profiling identified the activator protein 1 (AP1) transcription factor JunD as a master transcription factor of these enhancers. JunD depletion decreased iHUB interaction frequency and transcription of target genes. Moreover, targeting either eRNA production or signaling pathways upstream of iHUB activation using clinically tested small molecule inhibitors decreased eRNA production and interaction frequency, and restored chemotherapy responsiveness in vitro and in vivo. Representative iHUB target genes were found to be more expressed in patients with poor response to chemotherapy compared with responsive patients. Conclusion Our findings identify an important role for a subgroup of highly connected enhancers (iHUBs) in regulating chemotherapy response and demonstrate targetability in sensitisation to chemotherapy.

Emergency hematopoiesis is a concerted response aimed toward enhanced protection from infection, involving multiple cell types and developmental stages across the immune system. Despite its importance, the underlying molecular regulation remains poorly understood. The deubiquitinase USP22 regulates the levels of monoubiquitinated histone H2B (H2Bub1), which is associated with activation of interferon responses upon viral infection. Here, we show that in the absence of infection or inflammation, mice lacking Usp22 in all hematopoietic cells display profound systemic emergency hematopoiesis, evident by increased hematopoietic stem cell proliferation, myeloid bias, and extramedullary hematopoiesis. Functionally, loss of Usp22 results in elevated phagocytosis by neutrophilic granulocytes and enhanced innate protection against Listeria monocytogenes infection. At the molecular level, we found this state of emergency hematopoiesis associated with transcriptional signatures of myeloid priming, enhanced mitochondrial respiration, and innate and adaptive immunity and inflammation. Augmented expression of many inflammatory genes was linked to elevated locus-specific H2Bub1 levels. Collectively, these results demonstrate the existence of a tunable epigenetic state that promotes systemic emergency hematopoiesis in a cell-autonomous manner to enhance innate protection, identifying potential paths toward immune enhancement.

Abstract Human aging is associated with molecular changes and cellular degeneration, resulting in a significant increase in cancer incidence with age. Whether aging as its own entity can be initiator of putative cancerous developments, the transcriptional changes underlying the cancerous development itself are largely unknown. Here, we analyzed publicly available bulk RNA sequencing data of 328 control vs. 453 cancer samples in combination with single cell sequencing datasets from 49 control vs. 105 cancer patients. Using widely accepted aging-associated gene sets, we assembled key features of aging and their reflection in cancer development in five distinct cancer entities with an age-dependent increase of incidence, i.e. chronic myeloid leukemia (CML), colorectal cancer (CRC), hepatocellular carcinoma (HCC), lung cancer (LC) and pancreatic ductal adenocarcinoma (PDAC). After confirmation of the aging/senescence-induced gene (ASIG) expression to be upregulated in malignant diseases compared to healthy controls, we elucidated the importance of ASIGs during single cell development, making use of pseudotime analyses. In each entity, distinct temporally late enrichment states were carved out, and their transcriptional connectivity next to epigenetic control verified. Remarkably, we were able to demonstrate that all cancer entities analyzed in this study comprised cell populations expressing ASIGs. Except for pancreatic cancer, all entities accounted for subpopulations with aging associated patterns. Using a novel pipeline to repurpose FDA-approved drugs and combinations on a single cell level, we discovered a path for novel treatment regimen on a tumor-specific basis. These respective cellular subpopulations demonstrate pillars of susceptibility within the complex tumor microenvironment and serve as a basis for future studies on the role of aging and senescence in human malignancies. Citation Format: Dominik Saul, Robyn Laura Kosinsky. Aging patterns in five disparate cancer entities indicate a novel senescent cell population with therapeutic targetability [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 5024.

Abstract Osteosarcoma is the most common primary solid tumor of the bone, typically affecting children and adolescents. Surgery and adjuvant chemotherapy result in an unsatisfying 5-year survival rate of <25%. Although immune adjuvant strategies are promising, recent developments targeting the specialized tumor microenvironment by aiming at osteoclasts and checkpoint inhibitors focus on the individual immune biology. We aimed to mirror the tumor microenvironment and pseudotemporal development of osteoblasts during their transformation to osteoblastic osteosarcoma cells. For this purpose, single cell RNA-sequencing (scRNA-seq) profiles of 166,012 osteoblastic osteosarcoma and 49,555 healthy bone cells isolated from 28 patients were analyzed. In pseudotemporal analyses, we identified developmental divergence points with distinct transcriptional patterns purporting paths of devolution or giving rise to proliferative arrest. We found the biologically predicted clinging conjunction between osteoclasts, osteoblasts and B cells as regulatory T cells. In addition, we discovered intratumoral cellular communication patterns with ligand-receptor interactions and secreted signaling analysis between certain pillars of heterogeneous subpopulations, illustrating the importance of the local immune-genomic landscape. Upon several experimentally verified pathways, the TGF-β interaction was paramount. A mathematical assembly of degenerated cell populations revealed canonical markers of an osteoblastic cancerous state, out of which the vast majority has been associated with tumorigenesis in earlier studies. Intriguingly, nonnegative matrix factorization gave rise to a spatially localized senescent cell state, potentially accessible to new senolytic compounds which are currently in clinical trials for aging-related diseases. This senescent state with proliferative arrest and the secretion of a senescence-associated secretory phenotype (SASP), has not been described in osteosarcoma so far. In addition, we identified novel pathways of intertumoral communication in these distinct clusters, which can be exploited therapeutically, and validated these with a single-cell guided pipeline to repurpose drugs and their combinations. Thus, we were able to define a temporal pattern for the development of osteoblastic osteosarcoma. A local microenvironment with gradual states of devolution, and distinct cell-cell communicational patterns was established, out of which TGF-β warrants further experimental as therapeutical exploitation. Finally, we curtailed degenerated and, surprisingly, senescent subpopulations, each of which were predicted to vary in therapeutic response, mirroring the clinical observed intratumoral heterogeneity and potentially guiding the way to an individualized treatment regimen. Citation Format: Dominik Saul, Robyn Laura Kosinsky. Single cell RNA sequencing reveals degenerative mechanisms in osteoblastic osteosarcoma and targetability of senescent subpopulations [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 5025.

Based on the rapid increase in incidence of inflammatory bowel disease (IBD), the identification of susceptibility genes and cell populations contributing to this condition is essential. Previous studies suggested multiple genes associated with the susceptibility of IBD; however, due to the analysis of whole-tissue samples, the contribution of individual cell populations remains widely unresolved. Single-cell RNA sequencing (scRNA-seq) provides the opportunity to identify underlying cellular populations. We determined the enrichment of Crohn’s disease (CD)-induced genes in a publicly available Crohn’s disease scRNA-seq dataset and detected the strongest induction of these genes in innate lymphoid cells (ILC1), highly activated T cells and dendritic cells, pericytes and activated fibroblasts, as well as epithelial cells. Notably, these genes were highly enriched in IBD-associated neoplasia, as well as sporadic colorectal cancer (CRC). Indeed, the same six cell populations displayed an upregulation of CD-induced genes in a CRC scRNA-seq dataset. Finally, after integrating and harmonizing the CD and CRC scRNA-seq data, we demonstrated that these six cell types display a gradual increase in gene expression levels from a healthy state to an inflammatory and tumorous state. Together, we identified cell populations that specifically upregulate CD-induced genes in CD and CRC patients and could, therefore, contribute to inflammation-associated tumor development.