Functional and Genomic Analyses of Klebsiella Pneumoniae Population Dynamics in the Gastrointestinal Tract

The gastrointestinal tract is home to trillions of bacteria that interact with each other and with the host’s mucosal immune system. Obligate and facultative anaerobes thrive in the small and large intestine. While many of these bacteria have beneficial relationships with their host, opportunistic pathogens can bloom in times of inflammation and prolong disease. Klebsiella pneumoniae is an opportunistic pathogen that is part of the gut microbiota in many healthy individuals. Here we explore the population dynamics of K. pneumoniae in the gastrointestinal tract in mouse models of health and disease. To assess the role of intra-species genomic diversity in interactions with the host, a mouse K. pneumoniae isolate and three human clinical isolates from human stool, sputum, and urine were studied in the T-bet-/- Rag2-/- and dextran sodium sulfate models of ulcerative colitis and in a mouse model of systemic neonatal infection. Regardless of host origin, isolate site source, or genomic differences, all four K. pneumoniae isolates were able to stimulate colonic inflammation. However, only exposure to the murine K. pneumoniae isolate, and not human clinical isolates, led to neonatal death. In addition, this murine isolate correlated with differential shifts in levels of other Enterobacteriaceae species in the colon. Murine K. pneumoniae was found in higher amounts in host mesenteric lymph nodes compared to human isolates, suggesting subtle strain-based differences that affect response to K. pneumoniae in the gastrointestinal tract. A defining feature of K. pneumoniae is its mucoid capsular polysaccharide coat. To begin investigating a role for K. pneumoniae’s capsule in the gastrointestinal tract, K. pneumoniae biogeography was assessed in gnotobiotic mice. Encapsulated K. pneumoniae were outcompeted by naturally arising variants with reduced capsule throughout the gastrointestinal tract of gnotobiotic mice, except in the distal small intestine. This portion of the small intestine was also the region with the highest host Paneth cell antimicrobial peptide expression. Micro-injection of K. pneumoniae into small intestinal organoids revealed increased growth of encapsulated K. pneumoniae in the presence of α-defensins and decreased survival of K. pneumoniae with reduced capsule production. Capsule also conferred an advantage when K. pneumoniae was part of a diverse microbiota. Competition assays between K. pneumoniae and Escherichia coli showed enhanced survival of encapsulated K. pneumoniae compared to isolates with reduced capsule production. These data suggest that host AMPs, in combination with bacterial-bacterial interactions, shape population dynamics of K. pneumoniae and select for encapsulated K. pneumoniae throughout the gastrointestinal tract. Overall, these studies provide a foundation for understanding interactions between K. pneumoniae, the host, and other bacteria in the gastrointestinal tract.Medical Science

CK1ε Is Required for Breast Cancers Dependent on β-Catenin Activity

Background: Aberrant $\beta$-catenin signaling plays a key role in several cancer types, notably colon, liver and breast cancer. However approaches to modulate $\beta$-catenin activity for therapeutic purposes have proven elusive to date. Methodology: To uncover genetic dependencies in breast cancer cells that harbor active $\beta$-catenin signaling, we performed RNAi-based loss-of-function screens in breast cancer cell lines in which we had characterized $\beta$-catenin activity. Here we identify CSNK1E, the gene encoding casein kinase 1 epsilon (CK1$\varepsilon$) as required specifically for the proliferation of breast cancer cells with activated $\beta$-catenin and confirm its role as a positive regulator of $\beta$-catenin-driven transcription. Furthermore, we demonstrate that breast cancer cells that harbor activated $\beta$-catenin activity exhibit enhanced sensitivity to pharmacological blockade of Wnt/$\beta$-catenin signaling. We also find that expression of CK1$\varepsilon$ is able to promote oncogenic transformation of human cells in a $\beta$-catenin-dependent manner. Conclusions/Significance: These studies identify CK1$\varepsilon$ as a critical contributor to activated $\beta$-catenin signaling in cancer and suggest it may provide a potential therapeutic target for cancers that harbor active $\beta$-catenin. More generally, these observations delineate an approach that can be used to identify druggable synthetic lethal interactions with signaling pathways that are frequently activated in cancer but are difficult to target with the currently available small molecule inhibitors

SOX2 is an amplified lineage-survival oncogene in lung and esophageal squamous cell carcinomas

Lineage survival oncogenes are activated by somatic DNA alterations in cancers arising from the cell lineages in which these genes play a role in normal development.1,2 Here we show that a peak of genomic amplification on chromosome 3q26.33, found in squamous cell carcinomas (SCCs) of the lung and esophagus, contains the transcription factor gene SOX2—which is mutated in hereditary human esophageal malformations3 and necessary for normal esophageal squamous development4, promotes differentiation and proliferation of basal tracheal cells5 and co-operates in induction of pluripotent stem cells.6,7,8 SOX2 expression is required for proliferation and anchorage-independent growth of lung and esophageal cell lines, as shown by RNA interference experiments. Furthermore, ectopic expression of SOX2 cooperated with FOXE1 or FGFR2 to transform immortalized tracheobronchial epithelial cells. SOX2-driven tumors show expression of markers of both squamous differentiation and pluripotency. These observations identify SOX2 as a novel lineage survival oncogene in lung and esophageal SCC

COT drives resistance to RAF inhibition through MAP kinase pathway reactivation

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies