Biology subjects

Ronnekleiv-Kelly, S. M.

Publications and source records attributed to Ronnekleiv-Kelly, S. M..

5 recordsLinked to original sources

bioRxiv2025

Single-cell RNA sequencing of murine liver reveals an aligned circadian clock and cell-population specific circadian regulated pathways

The circadian clock is tightly connected to metabolism, which is evident in various metabolic processes performed by the liver. Perturbation of these processes due to circadian dysregulation leads to liver specific pathology. The liver is composed of multiple different cell populations each with distinct functions contributing to organ homeostasis, but individual cell population contributions to circadian clock function is not yet known. Single-cell RNA sequencing provides the opportunity to understand clock function and oscillating gene expression within an organ system at the individual cell population level that would allow for better understanding of the crosstalk between the circadian clock and metabolic pathways within the liver. In the past, barriers to achieving this goal included complexity associated with generating single-cell RNA sequencing time series data as well as the complexity of data analysis. Here, we established a protocol that enabled the generation of murine liver cell population time series data, as well as a methodological approach to evaluate the core molecular clock and oscillating gene expression in individual cell populations. Using a combination of normalized coefficient of variation, clock-correlation and aggregate pseudobulk, we found a robust and aligned circadian clock in each of the cell populations. We then employed a pseudoreplicate / pseudobulk strategy to identify oscillating gene expression and benchmarked against bulk RNA sequencing data; we demonstrated that many metabolic genes were oscillating in several of the cell populations, including non-hepatocyte clusters. Finally, we identified oscillating genes unique to specific cell populations that play critical roles in liver function. The findings in this study lay an important foundation for understanding clock function and contributions of oscillating gene function at the individual cell population level in liver.

molecular biology↗

bioRxiv2024

Single-cell multi-omic analysis of fibrolamellar carcinoma reveals rewired cell-to-cell communication patterns and unique vulnerabilities

Fibrolamellar carcinoma (FLC) is a rare malignancy disproportionately affecting adolescents and young adults with no standard of care. FLC is characterized by thick stroma, which has long suggested an important role of the tumor microenvironment. Over the past decade, several studies have revealed aberrant markers and pathways in FLC. However, a significant drawback of these efforts is that they were conducted on bulk tumor samples. Consequently, identities and roles of distinct cell types within the tumor milieu, and the patterns of intercellular communication, have yet to be explored. In this study we unveil cell-type specific gene signatures, transcription factor networks, and super-enhancers in FLC using a multi-omics strategy that leverages both single-nucleus ATAC-seq and single-nucleus RNA-seq. We also infer completely rewired cell-to-cell communication patterns in FLC including signaling mediated by SPP1-CD44, MIF-ACKR3, GDF15-TGFBR2, and FGF7-FGFR. Finally, we validate findings with loss-of-function studies in several models including patient tissue slices, identifying vulnerabilities that merit further investigation as candidate therapeutic targets in FLC.

cancer biology↗

bioRxiv2023

CDK7 is a Novel Therapeutic Vulnerability in Fibrolamellar Carcinoma

Fibrolamellar carcinoma (FLC) is a rare and lethal cancer that afflicts young individuals. The tumor arises in the background of a healthy liver, and patients typically present with advanced cancer at the time of diagnosis. Unfortunately, for these patients with advanced or recurrent cancer, no proven systemic therapies exist resulting in only 30-45% of patients surviving to 5 years. Investigations into the molecular underpinning of FLC have revealed a unique gene fusion between heat shock protein 40 (DNAJB1) and the catalytic subunit alpha of protein kinase A (PRKACA), leading to the formation of an oncoprotein (DNAJ-PKAc) that retains kinase activity and is a proven tumor-causing event in FLC. To uncover potential therapeutic targets, we engineered an FLC cell line by introducing the DNAJB1-PRKACA oncogene rearrangement into human hepatocellular cells using CRISPR/Cas9. We identified aberrant cell cycle progression, and follow-up molecular analysis revealed evidence of enhanced cyclin dependent kinase 7 (CDK7) activation in the DNAJB1-PRKACA expressing FLC cells. These findings were confirmed in human samples of FLC. In turn, targeting CDK7 with selective inhibitors demonstrated efficacy in several patient-derived models of FLC, with minimal toxicity to normal liver. Collectively, this work uncovers a novel candidate therapeutic vulnerability in FLC.

cancer biology↗

bioRxiv2022

Chronic Jetlag Accelerates Pancreatic Neoplasia in Conditional Kras-Mutant Mice

Misalignment of the circadian clock compared to environmental cues causes circadian desynchrony, which is pervasive in humans. Clock misalignment can lead to various pathologies including obesity and diabetes, both of which are associated with pancreatic ductal adenocarcinoma - a devastating cancer with an 80% five-year mortality rate. Although circadian desynchrony is associated with an increased risk of several solid-organ cancers, the correlation between clock misalignment and pancreas cancer is unclear. Using a chronic jetlag model, we investigated the impact of clock misalignment on pancreas cancer initiation in mice harboring a pancreas-specific activated Kras mutation. We found that chronic jetlag accelerated the development of pancreatic cancer precursor lesions, with a concomitant increase in precursor lesion grade. Cell-autonomous knock-out of the clock in pancreatic epithelial cells of Kras-mutant mice demonstrated no acceleration of precursor lesion formation, indicating non-cell-autonomous clock dysfunction was responsible for the expedited tumor development. Therefore, we applied single-cell RNA sequencing over time and identified fibroblasts as the cell population manifesting the greatest clock-dependent changes, with enrichment of specific cancer-associated fibroblast pathways due to circadian misalignment. Collectively, these results suggest fibroblasts as the putative target of chronic jetlag-induced accelerated pancreas cancer initiation.

cancer biology↗

bioRxiv2022

Pdx1 expression in hematopoietic cells activates Kras-mutation to drive leukemia in KC (Pdx1-Cre ; LSL-KrasG12D/+) mice

BackgroundPdx1 expression in pancreatic lineage cells underlies the utility of the KC mouse model (Pdx1-Cre; LSL-KrasG12D/+) for understanding how KrasG12D-mutation drives formation of pancreas cancer precursor lesions and carcinoma. The highly utilized KC model has a reported mortality rate of about 30%, which has been attributed to pancreas cancer, despite lack of substantive evidence. This study describes a novel cause of the early deaths, in which KC mice develop Kras-driven T-cell acute lymphoblastic leukemia (T-ALL). MethodsKC mice and control mice underwent histopathologic examination including thymus, liver, spleen, bone marrow and pancreas, and immunohistochemistry (IHC) was used to confirm leukemia development. A reporter strain (Ai14) was used to identify location of Pdx1-Cre expression and concomitant mutant-Kras activation, which was confirmed using flow cytometry, IHC, immunofluorescence, mRNA analysis, and bone marrow transplant studies. ResultsPdx1 expression in the hematopoietic compartment of KC mice resulted in Cre-recombinase mediated excision of lox-stop and activation of mutant-Kras gene (KrasG12D/+) in the multipotent progenitor cells (MPP), and subsequent development of Kras-mutant T-ALL creating thymic tumors in a subset of mice. Overall, 20% (5/25) of KC mice developed a large thymic tumor due to T-ALL by 9 months of age. Moreover, through isolation and transplantation of pooled bone marrow from KC mice into CD45 congenic mice, 100% of recipient mice were found to develop T-ALL. These results further confirm mutant-Kras expression in the hematologic compartment is driving the development of T-ALL in the KC mouse model. ConclusionsThese results are an essential consideration for investigators while utilizing this model in pancreas cancer studies, particularly when evaluating factors that may coincidentally enhance the formation of KrasG12D-driven T-ALL (e.g. transcription factors impacting hematopoietic cells). Finally, the lower penetrance of T-ALL development in KC mice (compared to existing leukemia models) suggest that the KC mouse could be considered as an alternative research model to evaluate onset and factors that exacerbate development of T-ALL.

cancer biology↗