Cuddling worms: Characterization of Caenorhabditis elegans mutants exhibiting social feeding/foraging/aggregation behavior to identify the causal mutation for the phenotype

Social feeding/foraging/aggregation behavior in Caenorhabditis elegans is a simple system for studying neural circuitry and molecular players that regulate a complex behavior. npr-1 and daf-7 pathways regulate the social feeding behavior in parallel in response to various sensory cues such as noxious stimuli, population density, food availability, oxygen level and pheromones. Here the role of pdk-1, a candidate gene identified from a mutagenesis screen performed to isolate molecular players responsible for aggregation behavior in C. elegans, was investigated in contributing to the social feeding behavior in the worms. The candidacy for causal mutation was examined by creating transgenic lines carrying extrachromosal array with GFP marker that contained genomic fragment of wildtype pdk-1 gene under the endogenous promoter, gut promoter (elt-2) or pan-neuronal promoter (h20). A fluorescence microscope setup for a normal dissecting scope was also constructed to sort out GFP positive worms. After assaying the transgenic lines for aggregation phenotype, we concluded that wildtype pdk-1 gene rescued the phenotype and mutation in pdk-1 is the causal mutation in both of our mutant lines

BMI1 is a context-dependent tumor suppressor that is a barrier to dedifferentiation in non-small cell lung adenocarcinoma

This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2019Cataloged from student-submitted PDF version of thesis. "May 2019."Includes bibliographical references.Predictive value is expected when preclinical models of disease are used for research. However, not all models appropriately mimic the disease progression or the treatment paradigm in the clinic. This thesis addresses an epigenetic regulator, Bmi1, which acts in stem cells to maintain their proliferative and self-renewal capacity primarily through silencing of the Ink4a/Arf locus. Bmi1 has been proposed as a good therapeutic candidate in cancer because of its presumed role in maintaining tumor propagating cells (TPCs). This conclusion is based on the observed tumor suppressive effects of Bmi1 deletion in in vitro cell culture models, in vivo transplant models, and autochthonous models in which Bmi1 was absent throughout development. However, to date, no one has assessed the consequences of deleting Bmi1 in existing autochthonous tumors, to mimic patient treatment in the clinic.To accomplish this, we have generated a mouse model that allows induction of autochthonous lung adenocarcinoma, driven by oncogenic Kras and Tp53 loss (KP LUAD), and subsequent deletion of Bmi1 specifically within the tumor cells once more than half the tumors progress to grade 3 or higher. We confirmed that this model yielded Bmi1 loss that was tumor-specific and almost complete. We then aged tumor bearing mice for up to seven weeks post Bmi1 deletion to determine the impact on LUAD. Unexpectedly, Bmi1 deletion did not yield significant tumor suppression. Instead, gene expression analyses of Bmi1 deficient tumor cells revealed upregulation of a gastric gene expression program that is a known marker of lung tumor progression towards a more aggressive state in the KP LUAD model. Additionally, single cell sequencing showed that Bmi1 deficient tumors contained a higher frequency of cells that expressed previously described markers of TPCs and metastasis.We also extended these findings to colorectal cancer where we show that deletion of Bmi1 is not tumor suppressive in either in vitro organoids or orthotopic transplants. Given these findings, we conclude that deletion, or inhibition, of BMI1 in existing tumors will be ineffective for cancer treatment in the contexts examined, and potentially deleterious because it can enable acquisition of alternate differentiation states that promote tumor progression.by Rachit Neupane.Ph. D.Ph.D. Massachusetts Institute of Technology, Department of Biolog

Coordinated Splicing of Regulatory Detained Introns within Oncogenic Transcripts Creates an Exploitable Vulnerability in Malignant Glioma

Glioblastoma (GBM) is a devastating malignancy with few therapeutic options. We identify PRMT5 in an in vivo GBM shRNA screen and show that PRMT5 knockdown or inhibition potently suppresses in vivo GBM tumors, including patient-derived xenografts. Pathway analysis implicates splicing in cellular PRMT5 dependency, and we identify a biomarker that predicts sensitivity to PRMT5 inhibition. We find that PRMT5 deficiency primarily disrupts the removal of detained introns (DIs). This impaired DI splicing affects proliferation genes, whose downregulation coincides with cell cycle defects, senescence and/or apoptosis. We further show that DI programs are evolutionarily conserved and operate during neurogenesis, suggesting that they represent a physiological regulatory mechanism. Collectively, these findings reveal a PRMT5-regulated DI-splicing program as an exploitable cancer vulnerability. Braun et al. show that glioblastoma is selectively sensitive to the inhibition of PRMT5 and identify a predictive biomarker for this sensitivity. PRMT5 inhibition primarily disrupts the removal of detained introns, which results in the reduction of functional transcripts of mainly proliferation-associated genes. Keywords: splicing addiction; GBM; PRMT5; EPZ015666; biomarker; CLNS1A; RIOK1National Institutes of Health (U.S.) (Grant PO1-CA42063)National Cancer Institute (U.S.) (Grant PO1-CA42063)National Institutes of Health (U.S.) (Grant R01GM034277)National Cancer Institute (U.S.) (Grant R01GM034277)National Institutes of Health (U.S.) (Grant P30-CA14051

In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.

In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis, which rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;Kras(G12D/+);Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5(+) stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.</p