Faculty Bibliography

Cutaneous T cell lymphoma is a heterogeneous group of lymphomas characterized by the accumulation of malignant T cells in the skin. The molecular and cellular etiology of this malignancy remains enigmatic and what role antigenic stimulation plays in the initiation and/or progression of the disease remains to be elucidated. Deep sequencing of the tumor genome revealed a highly heterogeneous landscape of genetic perturbations and transcriptome analysis of transformed T cells further highlighted the heterogeneity of this disease. Nonetheless, using data harvested from high-throughput transcriptional profiling allowed us to develop a reliable signature of this malignancy. Focusing on a key cytokine signaling pathway, previously implicated in CTCL pathogenesis, JAK/STAT signaling, we used conditional gene targeting to develop a fully penetrant small animal model of this disease that recapitulates many key features of mycosis fungoides, a common variant of CTCL. Using this mouse model, we demonstrate that T cell receptor engagement is critical for malignant transformation of the T lymphocytes and that progression of the disease is dependent on microbiota.

Glioblastoma (GBM) is the most lethal primary adult brain tumor and its pathology is hallmarked by distorted neovascularization, diffuse tumor-associated macrophage infiltration, and potent immunosuppression. Reconstituting organotypic tumor angiogenesis models with biomimetic cell heterogeneity and interactions, pro-/anti-inflammatory milieu and extracellular matrix (ECM) mechanics is critical for preclinical anti-angiogenic therapeutic screening. However, current in vitro systems do not accurately mirror in vivo human brain tumor microenvironment. Here, we engineered a three-dimensional (3D), microfluidic angiogenesis model with controllable and biomimetic immunosuppressive conditions, immune-vascular and cell-matrix interactions. We demonstrate in vitro, GL261 and CT-2A GBM-like tumors steer macrophage polarization towards a M2-like phenotype for fostering an immunosuppressive and proangiogenic niche, which is consistent with human brain tumors. We distinguished that GBM and M2-like immunosuppressive macrophages promote angiogenesis, while M1-like pro-inflammatory macrophages suppress angiogenesis, which we coin "inflammation-driven angiogenesis." We observed soluble immunosuppressive cytokines, predominantly TGF-β1, and surface integrin (αvβ3) endothelial-macrophage interactions are required in inflammation-driven angiogenesis. We demonstrated tuning cell-adhesion receptors using an integrin (αvβ3)-specific collagen hydrogel regulated inflammation-driven angiogenesis through Src-PI3K-YAP signaling, highlighting the importance of altered cell-ECM interactions in inflammation. To validate the preclinical applications of our 3D organoid model and mechanistic findings of inflammation-driven angiogenesis, we screened a novel dual integrin (αvβ3) and cytokine receptor (TGFβ-R1) blockade that suppresses GBM tumor neovascularization by simultaneously targeting macrophage-associated immunosuppression, endothelial-macrophage interactions, and altered ECM. Hence, we provide an interactive and controllable GBM tumor microenvironment and highlight the importance of macrophage-associated immunosuppression in GBM angiogenesis, paving a new direction of screening novel anti-angiogenic therapies.

Background: Microglandular adenosis (MGA) is a rare borderline lesion of the breast characterized by an infiltrative collection of small glands that characteristically lack a myoepithelial cell layer. MGA is associated with invasive carcinoma in 20-30% of cases, and has been proposed as a non-obligate precursor to basal-like breast cancers. Somatic TP53 mutation of MGA and its associated carcinoma has been previously reported. We identified a case of triple negative metaplastic carcinoma with mesenchymal differentiation with morphologic and immunohistochemical evidence of progression from MGA to atypical MGA (AMGA), carcinoma in situ (CIS) and invasive carcinoma. We performed laser microdissection and whole exome sequencing of each four components (MGA, AMGA, CIS and cancer) with a matched benign sample to characterize the mutational landscape of these foci. Design: We selected a case of a metaplastic carcinoma with mesenchymal differentiation in juxtaposition to foci of MGA, AMGA and CIS. Immunohistochemically, all four foci were negative for estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (her2) and positive for S-100. The distinct morphologic components and a matched control lymph node were separately microdissected from formalin-fixed paraffin embedded blocks. DNA extraction was performed and subjected to whole-exome sequencing on Illumina HiSeq platform. The results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. Singlenucleotide and small indel somatic variants were called with MuTect2. Copy number profiles were calculated using Control-FREEC. Clonal populations were identified and quantified using PyClone. Results: Sequencing data resulted in mean coverage of 96-134X of targeted exome regions. Our results found a recurrent stop-gain R213* TP53 mutation in MGA, atypical MGA, CIS and metaplastic carcinoma. In addition, through variant allele frequency analysis, we identified two putative clonal clusters shared by all foci indicating a common molecular signature that is preserved in the morphologic spectrum of MGA-AMGA-CIS-metaplastic carcinoma. Conclusions: MGA is a molecularly advanced lesion with somatic mutation of TP53. We postulate that TP53 is an early event in the progression of MGA through AMGA, CIS and its associated metaplastic carcinoma. We report significant genetic overlap between MGA and its associated cancer

Uncovering the causes of pregnancy complications such as preterm labor requires greater insight into how the uterus remains in a noncontractile state until term and then surmounts this state to enter labor. Here, we show that dynamic generation and erasure of the repressive histone modification tri-methyl histone H3 lysine 27 (H3K27me3) in decidual stromal cells dictate both elements of pregnancy success in mice. In early gestation, H3K27me3-induced transcriptional silencing of select gene targets ensured uterine quiescence by preventing the decidua from expressing parturition-inducing hormone receptors, manifesting type 1 immunity, and most unexpectedly, generating myofibroblasts and associated wound-healing responses. In late gestation, genome-wide H3K27 demethylation allowed for target gene upregulation, decidual activation, and labor entry. Pharmacological inhibition of H3K27 demethylation in late gestation not only prevented term parturition, but also inhibited delivery while maintaining pup viability in a noninflammatory model of preterm parturition. Immunofluorescence analysis of human specimens suggested that similar regulatory events might occur in the human decidua. Together, these results reveal the centrality of regulated gene silencing in the uterine adaptation to pregnancy and suggest new areas in the study and treatment of pregnancy disorders.

Single-cell RNA sequencing (sc-RNASeq) is a recently developed technique used to evaluate the transcriptome of individual cells. As opposed to conventional RNASeq in which entire populations are sequenced in bulk, sc-RNASeq can be beneficial when trying to better understand gene expression patterns in markedly heterogeneous populations of cells or when trying to identify transcriptional signatures of rare cells that may be underrepresented when using conventional bulk RNASeq. In this method, we describe the generation and analysis of cDNA libraries from single patient-derived glioblastoma cells using the C1 Fluidigm system. The protocol details the use of the C1 integrated fluidics circuit (IFC) for capturing, imaging and lysing cells; performing reverse transcription; and generating cDNA libraries that are ready for sequencing and analysis.

Squamous cell carcinomas (SCCs) are heterogeneous tumors sustained by tumor-propagating cancer cells (TPCs). SCCs frequently resist chemotherapy through still unknown mechanisms. Here, we combine H2B-GFP-based pulse-chasing with cell-surface markers to distinguish quiescent from proliferative TPCs within SCCs. We find that quiescent TPCs resist DNA damage and exhibit increased tumorigenic potential in response to chemotherapy, whereas proliferative TPCs undergo apoptosis. Quiescence is regulated by TGF-beta/SMAD signaling, which directly regulates cell-cycle gene transcription to control a reversible G1 cell-cycle arrest, independent of p21CIP function. Indeed, genetic or pharmacological TGF-beta inhibition increases the susceptibility of TPCs to chemotherapy because it prevents entry into a quiescent state. These findings provide direct evidence that TPCs can reversibly enter a quiescent, chemoresistant state and thereby underscore the need for combinatorial approaches to improve treatment of chemotherapy-resistant SCCs.

Treating KRAS-mutant lung adenocarcinoma (LUAD) remains a major challenge in cancer treatment given the difficulties associated with directly inhibiting the KRAS oncoprotein. One approach to addressing this challenge is to define mutations that frequently co-occur with those in KRAS, which themselves may lead to therapeutic vulnerabilities in tumors. Approximately 20% of KRAS-mutant LUAD tumors carry loss-of-function mutations in the KEAP1 gene encoding Kelch-like ECH-associated protein 1 (refs. 2, 3, 4), a negative regulator of nuclear factor erythroid 2-like 2 (NFE2L2; hereafter NRF2), which is the master transcriptional regulator of the endogenous antioxidant response. The high frequency of mutations in KEAP1 suggests an important role for the oxidative stress response in lung tumorigenesis. Using a CRISPR-Cas9-based approach in a mouse model of KRAS-driven LUAD, we examined the effects of Keap1 loss in lung cancer progression. We show that loss of Keap1 hyperactivates NRF2 and promotes KRAS-driven LUAD in mice. Through a combination of CRISPR-Cas9-based genetic screening and metabolomic analyses, we show that Keap1- or Nrf2-mutant cancers are dependent on increased glutaminolysis, and this property can be therapeutically exploited through the pharmacological inhibition of glutaminase. Finally, we provide a rationale for stratification of human patients with lung cancer harboring KRAS/KEAP1- or KRAS/NRF2-mutant lung tumors as likely to respond to glutaminase inhibition.

We report here an update to the draft genome sequence of Kluyvera intestini sp. nov. strain GT-16, generated using MinION long-read sequencing technology. The complete genome sequence of the human-derived strain GT-16 measured 5,768,848 bp. An improved high-quality complete genome sequence provides insights into the mobility potential of resistance genes in this species.