Faculty Bibliography

Unveiling the transcriptome of human blastocysts can provide a wealth of important information regarding early embryonic ontology. Comparing the mRNA production of embryos with normal and abnormal karyotypes allows for a deeper understanding of the protein pathways leading to viability and aberrant fetal development. In addition, identifying transcripts specific for normal or abnormal chromosome copy number could aid in the search for secreted substances that could be used to non-invasively identify embryos best suited for IVF embryo transfer. Using RNA-seq, we characterized the transcriptome of 71 normally developing human blastocysts that were karyotypically normal vs. trisomic or monosomic. Every monosomy and trisomy of the autosomal and sex chromosomes were evaluated, mostly in duplicate. We first mapped the transcriptome of three normal embryos and found that a common core of more than 3,000 genes is expressed in all embryos. These genes represent pathways related to actively dividing cells, such as ribosome biogenesis and function, spliceosome, oxidative phosphorylation, cell cycle and metabolic pathways. We then compared transcriptome profiles of aneuploid embryos to those of normal embryos. We observed that non-viable embryos had a large number of dysregulated genes, some showing a hundred-fold difference in expression. On the contrary, sex chromosome abnormalities, XO and XXX displayed transcriptomes more closely mimicking those embryos with 23 normal chromosome pairs. Intriguingly, we identified a set of commonly deregulated genes in the majority of both trisomies and monosomies. This is the first paper demonstrating a comprehensive transcriptome delineation of karyotypic abnormalities found in the human pre-implantation embryo. We believe that this information will contribute to the development of new pre-implantation genetic screening methods as well as a better understanding of the underlying developmental abnormalities of abnormal embryos, fetuses and children.

Visual inspection of histopathology slides is one of the main methods used by pathologists to assess the stage, type and subtype of lung tumors. Adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) are the most prevalent subtypes of lung cancer, and their distinction requires visual inspection by an experienced pathologist. In this study, we trained a deep convolutional neural network (inception v3) on whole-slide images obtained from The Cancer Genome Atlas to accurately and automatically classify them into LUAD, LUSC or normal lung tissue. The performance of our method is comparable to that of pathologists, with an average area under the curve (AUC) of 0.97. Our model was validated on independent datasets of frozen tissues, formalin-fixed paraffin-embedded tissues and biopsies. Furthermore, we trained the network to predict the ten most commonly mutated genes in LUAD. We found that six of them-STK11, EGFR, FAT1, SETBP1, KRAS and TP53-can be predicted from pathology images, with AUCs from 0.733 to 0.856 as measured on a held-out population. These findings suggest that deep-learning models can assist pathologists in the detection of cancer subtype or gene mutations. Our approach can be applied to any cancer type, and the code is available at https://github.com/ncoudray/DeepPATH .

KRAS mutation is present in approximately 30% of human lung adenocarcinomas. Although recent advances in targeted therapy have shown great promise, effective targeting of KRAS remains elusive, and concurrent alterations in tumor suppressors render KRAS-mutant tumors even more resistant to existing therapies. Contributing to the refractoriness of KRAS-mutant tumors are immunosuppressive mechanisms, such as increased presence of suppressive regulatory T cells (Tregs) in tumors and elevated expression of the inhibitory receptor PD-1 on tumor-infiltrating T cells. Treatment with BET bromodomain inhibitors is beneficial for hematologic malignancies, and they have Treg-disruptive effects in a non-small cell lung cancer (NSCLC) model. Targeting PD-1 inhibitory signals through PD-1 antibody blockade also has substantial therapeutic impact in lung cancer, although these outcomes are limited to a minority of patients. We hypothesized that the BET bromodomain inhibitor JQ1 would synergize with PD-1 blockade to promote a robust antitumor response in lung cancer. In the present study, using Kras+/LSL-G12D; Trp53L/L (KP) mouse models of NSCLC, we identified cooperative effects between JQ1 and PD-1 antibody. The numbers of tumor-infiltrating Tregs were reduced and activation of tumor-infiltrating T cells, which had a T-helper type 1 (Th1) cytokine profile, was enhanced, underlying their improved effector function. Furthermore, lung tumor-bearing mice treated with this combination showed robust and long-lasting antitumor responses compared to either agent alone, culminating in substantial improvement in the overall survival of treated mice. Thus, combining BET bromodomain inhibition with immune checkpoint blockade offers a promising therapeutic approach for solid malignancies such as lung adenocarcinoma.

An unbiased platelet transcriptome profile identified ATP binding cassette subfamily C member 4 (ABCC4) as a novel mediator of platelet activity in virologically suppressed human immunodeficiency virus (HIV)-infected subjects on antiretroviral therapy. Using ex vivo and in vitro cellular and molecular assays we demonstrated that ABCC4 regulated platelet activation by altering granule release and cyclic nucleotide homeostasis through a cAMP-protein kinase A (PKA)-mediated mechanism. Platelet ABCC4 inhibition attenuated platelet activation and effector cell function by reducing the release of inflammatory mediators, such as sphingosine-1-phosphate. ABCC4 inhibition may represent a novel antithrombotic strategy in HIV-infected subjects on antiretroviral therapy.

Cellular transformation is accompanied by extensive rewiring of many biological processes leading to augmented levels of distinct types of cellular stress, including proteotoxic stress. Cancer cells critically depend on stress-relief pathways for their survival. However, the mechanisms underlying the transcriptional initiation and maintenance of the oncogenic stress response remain elusive. Here, we show that the expression of heat shock transcription factor 1 (HSF1) and the downstream mediators of the heat shock response is transcriptionally upregulated in T cell acute lymphoblastic leukemia (T-ALL). Hsf1 ablation suppresses the growth of human T-ALL and eradicates leukemia in mouse models of T-ALL, while sparing normal hematopoiesis. HSF1 drives a compact transcriptional program and among the direct HSF1 targets, specific chaperones and co-chaperones mediate its critical role in T-ALL. Notably, we demonstrate that the central T-ALL oncogene NOTCH1 hijacks the cellular stress response machinery by inducing the expression of HSF1 and its downstream effectors. The NOTCH1 signaling status controls the levels of chaperone/co-chaperone complexes and predicts the response of T-ALL patient samples to HSP90 inhibition. Our data demonstrate an integral crosstalk between mediators of oncogene and non-oncogene addiction and reveal critical nodes of the heat shock response pathway that can be targeted therapeutically.

Peripheral T lymphocytes share many functional properties with hematopoietic stem cells (HSCs), including long-term maintenance, quiescence, and latent proliferative potential. In addition, peripheral T cells retain the capacity for further differentiation into a variety of subsets, much like HSCs. While the similarities between T cells and HSC have long been hypothesized, the potential common genetic regulation of HSCs and T cells has not been widely explored. We have studied the T cell-intrinsic role of Zfx, a transcription factor specifically required for HSC maintenance. We report that T cell-specific deletion of Zfx caused age-dependent depletion of naïve peripheral T cells. Zfx-deficient T cells also failed to undergo homeostatic proliferation in a lymphopenic environment, and showed impaired antigen-specific expansion and memory response. In addition, the invariant natural killer T cell compartment was severely reduced. RNA-Seq analysis revealed that the most dysregulated genes in Zfx-deficient T cells were similar to those observed in Zfx-deficient HSC and B cells. These studies identify Zfx as an important regulator of peripheral T cell maintenance and expansion and highlight the common molecular basis of HSC and lymphocyte homeostasis.

Rapid impulse propagation is a defining attribute of the pectinated atrial myocardium and His-Purkinje system (HPS) that safeguards against atrial and ventricular arrhythmias, conduction block, and myocardial dyssynchrony. The complex transcriptional circuitry that dictates rapid conduction remains incompletely understood. Here, we demonstrate that ETV1 (ER81)-dependent gene networks dictate the unique electrophysiological characteristics of atrial and His-Purkinje myocytes. Cardiomyocyte-specific deletion of ETV1 results in cardiac conduction abnormalities, decreased expression of rapid conduction genes (Nkx2-5, Gja5, and Scn5a), HPS hypoplasia, and ventricularization of the unique sodium channel properties that define Purkinje and atrial myocytes in the adult heart. Forced expression of ETV1 in postnatal ventricular myocytes (VMs) reveals that ETV1 promotes a HPS gene signature while diminishing ventricular and nodal gene networks. Remarkably, ETV1 induction in human induced pluripotent stem cell-derived cardiomyocytes increases rapid conduction gene expression and inward sodium currents, converting them towards a HPS phenotype. Our data identify a cardiomyocyte-autonomous, ETV1-dependent pathway that is responsible for specification of rapid conduction zones in the heart and demonstrate that ETV1 is sufficient to promote a HPS transcriptional and functional program upon VMs.

The IRF8-dependent subset of classical dendritic cells (cDCs), termed cDC1, is important for cross-priming cytotoxic T cell responses against pathogens and tumors. Culture of hematopoietic progenitors with DC growth factor FLT3 ligand (FLT3L) yields very few cDC1s (in humans) or only immature "cDC1-like" cells (in the mouse). We report that OP9 stromal cells expressing the Notch ligand Delta-like 1 (OP9-DL1) optimize FLT3L-driven development of cDC1s from murine immortalized progenitors and primary bone marrow cells. Co-culture with OP9-DL1 induced IRF8-dependent cDC1s with a phenotype (CD103⁺ Dec205⁺ CD8α⁺) and expression profile resembling primary splenic cDC1s. OP9-DL1-induced cDC1s showed preferential migration toward CCR7 ligands in vitro and superior T cell cross-priming and antitumor vaccination in vivo. Co-culture with OP9-DL1 also greatly increased the yield of IRF8-dependent CD141⁺ cDC1s from human bone marrow progenitors cultured with FLT3L. Thus, Notch signaling optimizes cDC generation in vitro and yields authentic cDC1s for functional studies and translational applications.

BACKGROUND:In lung cancer, upregulation of the PI3K pathway is an early event that contributes to cell proliferation, survival, and tissue invasion. Upregulation of this pathway was recently described as associated with enrichment of the lower airways with bacteria identified as oral commensals. We hypothesize that host-microbe interactions in the lower airways of subjects with lung cancer affect known cancer pathways. METHODS:Airway brushes were collected prospectively from subjects with lung nodules at time of diagnostic bronchoscopy, including 39 subjects with final lung cancer diagnoses and 36 subjects with non-cancer diagnosis. Additionally, samples from 10 healthy control subjects were included. 16S rRNA gene amplicon sequencing and paired transcriptome sequencing (RNAseq) were performed on all airway samples. In addition, an in vitro model with airway epithelial cells exposed to bacteria/bacterial products was performed. RESULTS:The composition of the lower airway transcriptome in the cancer patients was significantly different from the controls, which included upregulation of ERK and PI3K signaling pathways. The lower airways of lung cancer patients were enriched for oral taxa (Streptococcus and Veillonella), which was associated with upregulation of the ERK and PI3K signaling pathways. In vitro exposure of airway epithelial cells to Veillonella, Prevotella, and Streptococcus led to upregulation of these same signaling pathways. CONCLUSIONS:The data presented here shows that several transcriptomic signatures previously identified as relevant to lung cancer pathogenesis are associated with enrichment of the lower airway microbiota with oral commensals.

The transcription factor AhR modulates immunity at multiple levels. Here we report that phagocytes exposed to apoptotic cells exhibited rapid activation of AhR, which drove production of the cytokine IL-10. Activation of AhR was dependent on interactions between apoptotic-cell DNA and the pattern-recognition receptor TLR9 that was required for the prevention of immune responses to DNA and histones in vivo. Moreover, disease progression in mouse systemic lupus erythematosus (SLE) correlated with strength of the AhR signal, and the disease course could be altered by modulation of AhR activity. Deletion of AhR in the myeloid lineage caused systemic autoimmunity in mice, and an enhanced AhR transcriptional signature correlated with disease in patients with SLE. Thus, AhR activity induced by apoptotic cell phagocytes maintains peripheral tolerance.