Modulating environmental signals to reveal mechanisms and vulnerabilities of cancer persisters.

Cancer persister cells are able to survive otherwise lethal doses of drugs through nongenetic mechanisms, which can lead to cancer regrowth and drug resistance. The broad spectrum of molecular differences observed between persisters and their treatment-naïve counterparts makes it challenging to identify causal mechanisms underlying persistence. Here, we modulate environmental signals to identify cellular mechanisms that promote the emergence of persisters and to pinpoint actionable vulnerabilities that eliminate them. We found that interferon-γ (IFNγ) can induce a pro-persistence signal that can be specifically eliminated by inhibition of type I protein arginine methyltransferase (PRMT) (PRMTi). Mechanistic investigation revealed that signal transducer and activator of transcription 1 (STAT1) is a key component connecting IFNγ's pro-persistence and PRMTi's antipersistence effects, suggesting a previously unknown application of PRMTi to target persisters in settings with high STAT1 expression. Modulating environmental signals can accelerate the identification of mechanisms that promote and eliminate cancer persistence.

Differential toxicity to murine small and large intestinal epithelium induced by oncology drugs.

Gastrointestinal toxicity is a major concern in the development of drugs. Here, we establish the ability to use murine small and large intestine-derived monolayers to screen drugs for toxicity. As a proof-of-concept, we applied this system to assess gastrointestinal toxicity of ~50 clinically used oncology drugs, encompassing diverse mechanisms of action. Nearly all tested drugs had a deleterious effect on the gut, with increased sensitivity in the small intestine. The identification of differential toxicity between the small and large intestine enabled us to pinpoint differences in drug uptake (antifolates), drug metabolism (cyclophosphamide) and cell signaling (EGFR inhibitors) across the gut. These results highlight an under-appreciated distinction between small and large intestine toxicity and suggest distinct tissue properties important for modulating drug-induced gastrointestinal toxicity. The ability to accurately predict where and how drugs affect the murine gut will accelerate preclinical drug development.

Transit-Amplifying Cells Coordinate Changes in Intestinal Epithelial Cell-Type Composition.

Renewing tissues have the remarkable ability to continually produce both proliferative progenitor and specialized differentiated cell types. How are complex milieus of microenvironmental signals interpreted to coordinate tissue-cell-type composition? Here, we investigate the responses of intestinal epithelium to individual and paired perturbations across eight epithelial signaling pathways. Using a high-throughput approach that combines enteroid monolayers and quantitative imaging, we identified conditions that enrich for specific cell types as well as interactions between pathways. Importantly, we found that modulation of transit-amplifying cell proliferation changes the ratio of differentiated secretory to absorptive cell types. These observations highlight an underappreciated role for transit-amplifying cells in the tuning of differentiated cell-type composition.

Generation and Quantitative Imaging of Enteroid Monolayers.

The intestinal epithelium is a single layer of cells that plays a critical role in digestion, absorbs nutrients from food, and coordinates the delicate interplay between microbes in the gut lumen and the immune system. Epithelial homeostasis is crucial for maintaining health; disruption of homeostasis results in disorders including inflammatory bowel disease and cancer. The advent of 3D intestinal epithelial organoids has greatly advanced our understanding of the molecular underpinnings of epithelial homeostasis and disease. Recently, we developed an enteroid monolayer (2D) culture system that recapitulates important features of 3D organoids and the in vivo intestinal epithelium such as tissue renewal, representation of diverse epithelial cell types, self-organization, and apical-basolateral polarization. Enteroid monolayers are cultured in microtiter plates, enabling high-throughput experiments. Furthermore, their 2D nature makes it easier to distinguish individual cells by fluorescent microscopy, enabling quantitative analysis of single cell behaviors within the epithelial tissue.Here we describe experimental methods for generating enteroid monolayers and computational methods for analyzing immunofluorescence images of enteroid monolayers. We outline experimental methods for generating enteroid monolayers from freshly isolated intestinal crypts, frozen intestinal crypts, and 3D organoids. Fresh crypts are easily obtained from murine or human intestinal samples, and the ability to derive enteroid monolayers from both frozen crypts and 3D organoids enables genetic modification and/or biobanking of patient samples for future studies. We outline computational methods for identifying distinct epithelial cell types (goblet, stem, EdU+) in immunofluorescence images of enteroid monolayers and, importantly, individual nuclei, enabling truly single cell measurements of epithelial cell behaviors to be made. Taken together, these methods will enable detailed studies of epithelial homeostasis and intestinal disease.

Small-Molecule and CRISPR Screening Converge to Reveal Receptor Tyrosine Kinase Dependencies in Pediatric Rhabdoid Tumors.

Cancer is often seen as a disease of mutations and chromosomal abnormalities. However, some cancers, including pediatric rhabdoid tumors (RTs), lack recurrent alterations targetable by current drugs and need alternative, informed therapeutic options. To nominate potential targets, we performed a high-throughput small-molecule screen complemented by a genome-scale CRISPR-Cas9 gene-knockout screen in a large number of RT and control cell lines. These approaches converged to reveal several receptor tyrosine kinases (RTKs) as therapeutic targets, with RTK inhibition effective in suppressing RT cell growth in vitro and against a xenograft model in vivo. RT cell lines highly express and activate (phosphorylate) different RTKs, creating dependency without mutation or amplification. Downstream of RTK signaling, we identified PTPN11, encoding the pro-growth signaling protein SHP2, as a shared dependency across all RT cell lines. This study demonstrates that large-scale perturbational screening can uncover vulnerabilities in cancers with "quiet" genomes.

A Direct Podocalyxin-Dynamin-2 Interaction Regulates Cytoskeletal Dynamics to Promote Migration and Metastasis in Pancreatic Cancer Cells.

The sialoglycoprotein podocalyxin is absent in normal pancreas but is overexpressed in pancreatic cancer and is associated with poor clinical outcome. Here, we investigate the role of podocalyxin in migration and metastasis of pancreatic adenocarcinomas using SW1990 and Pa03c as cell models. Although ezrin is regarded as a cytoplasmic binding partner of podocalyxin that regulates actin polymerization via Rac1 or RhoA, we did not detect podocalyxin-ezrin association in pancreatic cancer cells. Moreover, depletion of podocalyxin did not alter actin dynamics or modulate Rac1 and RhoA activities in pancreatic cancer cells. Using mass spectrometry, bioinformatics analysis, coimmunoprecipitation, and pull-down assays, we discovered a novel, direct binding interaction between the cytoplasmic tail of podocalyxin and the large GTPase dynamin-2 at its GTPase, middle, and pleckstrin homology domains. This podocalyxin-dynamin-2 interaction regulated microtubule growth rate, which in turn modulated focal adhesion dynamics and ultimately promoted efficient pancreatic cancer cell migration via microtubule- and Src-dependent pathways. Depletion of podocalyxin in a hemispleen mouse model of pancreatic cancer diminished liver metastasis without altering primary tumor size. Collectively, these findings reveal a novel mechanism by which podocalyxin facilitates pancreatic cancer cell migration and metastasis. SIGNIFICANCE: These findings reveal that a novel interaction between podocalyxin and dynamin-2 promotes migration and metastasis of pancreatic cancer cells by regulating microtubule and focal adhesion dynamics.