spatially-compact-arrangement-larval-zebrafish-sections-for-spatial

Spatially Compact Arrangement of Larval Zebrafish Sections for Spatial Transcriptomic Analysis

Spatial transcriptomic techniques are a sophisticated tool in biomedical research to visualize spatially registered gene expression patterns. Imaging and ...

Dartmouth Cancer Center

Pancreatic ductal adenocarcinoma (PDAC) is driven by co-existing mutations in KRAS and TP53. However, how these mutations collaborate to promote this cancer is unknown. Here, we uncover sequence-specific changes in RNA splicing enforced by mutant p53 which enhance KRAS activity. Mutant p53 increases expression of splicing regulator hnRNPK to promote inclusion of cytosine-rich exons within GTPase-activating proteins (GAPs), negative regulators of RAS family members. Mutant p53-enforced GAP isoforms lose cell membrane association, leading to heightened KRAS activity. Preventing cytosine-rich exon inclusion in mutant KRAS/p53 PDACs decreases tumor growth. Moreover, mutant p53 PDACs are sensitized to inhibition of splicing via spliceosome inhibitors. These data provide insight into co-enrichment of KRAS and p53 mutations and therapeutics targeting this mechanism in PDAC.

Altered RNA Splicing by Mutant p53 Activates Oncogenic RAS Signaling in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is typically diagnosed at an advanced stage, which limits surgical options and portends a dismal prognosis. Current oncologic PDAC therapies confer marginal benefit and, thus, a significant unmet clinical need exists for new therapeutic strategies. To identify effective PDAC therapies, we leveraged a syngeneic orthotopic PDAC transplant mouse model to perform a large-scale, in vivo screen of 16 single-agent and 41 two-drug targeted therapy combinations in mice. Among 57 drug conditions screened, combined inhibition of heat shock protein (Hsp)-90 and MEK was found to produce robust suppression of tumor growth, leading to an 80% increase in the survival of PDAC-bearing mice with no significant toxicity. Mechanistically, we observed that single-agent MEK inhibition led to compensatory activation of resistance pathways, including components of the PI3K/AKT/mTOR signaling axis, which was overcome with the addition of HSP90 inhibition. The combination of HSP90(i) + MEK(i) was also active in vitro in established human PDAC cell lines and in vivo in patient-derived organoid PDAC transplant models. These findings encourage the clinical development of HSP90(i) + MEK(i) combination therapy and highlight the power of clinically relevant in vivo model systems for identifying cancer therapies.

Unbiased in vivo preclinical evaluation of anticancer drugs identifies effective therapy for the treatment of pancreatic adenocarcinoma.

Despite progress in developing learning health systems (LHS) and associated metrics of success, a gap remains in identifying measures to guide the implementation and assessment of the impact of an oncology LHS. Our aim was to identify a balanced set of measures to guide a person-centered oncology LHS.

Prioritizing Measures That Matter Within a Person-Centered Oncology Learning Health System.

Little is known about the distal excretory component of the urinary tract in Danio rerio (zebrafish). This component is affected by many human diseases and disorders of development. Here, we undertook multi-level analyses to determine the structure and composition of the distal urinary tract in the zebrafish. In silico searches identified uroplakin 1a, 2-like and 3-like genes in the zebrafish genome (orthologues to genes that encode urothelium-specific proteins in humans). In situ hybridization demonstrated uroplakin-1a expression in the zebrafish pronephros and cloaca from 96 hours post-fertilisation. H&E sectioning of adult zebrafish demonstrated two mesonephric ducts uniting into a urinary bladder which leads to a distinct urethral opening. Immunohistochemistry identified Uroplakin 1a and 2 expression and Gata3 expression in zebrafish urinary bladder cell layers that match human urothelial expression. Fluorescent dye injections demonstrated zebrafish urinary bladder function, including urine storage, intermittent micturition, and a urethral orifice separate from the larger anal canal and rectum. Our findings reveal homology between the urinary tracts of zebrafish and humans and offer the former as a model system to study disease.

Analysis of the distal urinary tract in larval and adult zebrafish reveals homology to the human system.

Chemoresistance is a primary cause of treatment failure in pancreatic cancer. Identifying cell surface markers specifically expressed in chemoresistant cancer cells (CCCs) could facilitate targeted therapies to overcome chemoresistance. We performed an antibody-based screen and found that TRA-1-60 and TRA-1-81, two 'stemness' cell surface markers, are highly enriched in CCCs. Furthermore, TRA-1-60+/TRA-1-81+ cells are chemoresistant compared to TRA-1-60-/TRA-1-81- cells. Transcriptome profiling identified UGT1A10, shown to be both necessary and sufficient to maintain TRA-1-60/TRA-1-81 expression and chemoresistance. From a high-content chemical screen, we identified Cymarin, which downregulates UGT1A10, eliminates TRA-1-60/TRA-1-81 expression, and increases chemosensitivity both in vitro and in vivo. Finally, TRA-1-60/TRA-1-81 expression is highly specific in primary cancer tissue and positively correlated with chemoresistance and short survival, which highlights their potentiality for targeted therapy. Therefore, we discovered a novel CCC surface marker regulated by a pathway that promotes chemoresistance, as well as a leading drug candidate to target this pathway.

A targetable pathway to eliminate TRA-1-60+/TRA-1-81+ chemoresistant cancer cells.

KRAS mutations, mainly G12D and G12V, are found in more than 90% of pancreatic ductal adenocarcinoma (PDAC) cases. The success of drugs targeting KRAS suggests the potential for drugs specifically targeting these alternative PDAC-associated KRAS mutations. Here, we report a high-throughput drug-screening platform using a series of isogenic murine pancreatic organoids that are wild type (WT) or contain common PDAC driver mutations, representing both classical and basal PDAC phenotypes. We screened over 6,000 compounds and identified perhexiline maleate, which can inhibit the growth and induce cell death of pancreatic organoids carrying the Kras mutation both in vitro and in vivo and primary human PDAC organoids. scRNA-seq analysis suggests that the cholesterol synthesis pathway is upregulated specifically in the KRAS mutant organoids, including the key cholesterol synthesis regulator SREBP2. Perhexiline maleate decreases SREBP2 expression levels and reverses the KRAS mutant-induced upregulation of the cholesterol synthesis pathway.

Article	Year
Altered RNA Splicing by Mutant p53 Activates Oncogenic RAS Signaling in Pancreatic Cancer. Cancer cell\| PubMed ID: 32559497	2020
Unbiased in vivo preclinical evaluation of anticancer drugs identifies effective therapy for the treatment of pancreatic adenocarcinoma. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 33199632	2020
Prioritizing Measures That Matter Within a Person-Centered Oncology Learning Health System. JNCI cancer spectrum\| PubMed ID: 35736219	2022
Analysis of the distal urinary tract in larval and adult zebrafish reveals homology to the human system. Disease models & mechanisms\| PubMed ID: 37293698	2023
A targetable pathway to eliminate TRA-1-60+/TRA-1-81+ chemoresistant cancer cells. Journal of molecular cell biology\| PubMed ID: 37327088	2023
A pancreatic cancer organoid platform identifies an inhibitor specific to mutant KRAS. Cell stem cell\| PubMed ID: 38151022	2024

Steven D. Leach

Biography

JoVE Journal Publications

Other Publications