Faculty Bibliography

Loss-of-function mutations in TET2 occur frequently in patients with clonal hematopoiesis, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML) and are associated with a DNA hypermethylation phenotype. To determine the role of TET2 deficiency in leukemia stem cell maintenance, we generated a reversible transgenic RNAi mouse to model restoration of endogenous Tet2 expression. Tet2 restoration reverses aberrant hematopoietic stem and progenitor cell (HSPC) self-renewal in vitro and in vivo. Treatment with vitamin C, a co-factor of Fe2+ and alpha-KG-dependent dioxygenases, mimics TET2 restoration by enhancing 5-hydroxymethylcytosine formation in Tet2-deficient mouse HSPCs and suppresses human leukemic colony formation and leukemia progression of primary human leukemia PDXs. Vitamin C also drives DNA hypomethylation and expression of a TET2-dependent gene signature in human leukemia cell lines. Furthermore, TET-mediated DNA oxidation induced by vitamin C treatment in leukemia cells enhances their sensitivity to PARP inhibition and could provide a safe and effective combination strategy to selectively target TET deficiency in cancer.

Plakophilin-2 (PKP2) is a component of the desmosome and known for its role in cell-cell adhesion. Mutations in human PKP2 associate with a life-threatening arrhythmogenic cardiomyopathy, often of right ventricular predominance. Here, we use a range of state-of-the-art methods and a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mouse to demonstrate that in addition to its role in cell adhesion, PKP2 is necessary to maintain transcription of genes that control intracellular calcium cycling. Lack of PKP2 reduces expression of Ryr2 (coding for Ryanodine Receptor 2), Ank2 (coding for Ankyrin-B), Cacna1c (coding for CaV1.2) and Trdn (coding for triadin), and protein levels of calsequestrin-2 (Casq2). These factors combined lead to disruption of intracellular calcium homeostasis and isoproterenol-induced arrhythmias that are prevented by flecainide treatment. We propose a previously unrecognized arrhythmogenic mechanism related to PKP2 expression and suggest that mutations in PKP2 in humans may cause life-threatening arrhythmias even in the absence of structural disease.It is believed that mutations in desmosomal adhesion complex protein plakophilin 2 (PKP2) cause arrhythmia due to loss of cell-cell communication. Here the authors show that PKP2 controls the expression of proteins involved in calcium cycling in adult mouse hearts, and that lack of PKP2 can cause arrhythmia in a structurally normal heart.

Melanoma patients with BRAFV600E -mutant tumors display striking responses to BRAF inhibitors (BRAFi); however, almost all invariably relapse with drug-resistant disease. Here we report that microRNA-125a (miR-125a) expression is upregulated in human melanoma cells and patient tissues upon acquisition of BRAFi resistance. We show that miR-125a induction confers resistance to BRAFV600E melanoma cells to BRAFi by directly suppressing pro-apoptotic components of the intrinsic apoptosis pathway, including BAK1 and MLK3. Apoptotic suppression and prolonged survival favor reactivation of the MAPK and AKT pathways by drug-resistant melanoma cells. We demonstrate that miR-125a inhibition suppresses the emergence of resistance to BRAFi and, in a subset of resistant melanoma cell lines, leads to partial drug re-sensitization. Finally, we show that miR-125a upregulation is mediated by TGFbeta signaling. In conclusion, the identification of this novel role for miR-125a in BRAFi resistance exposes clinically relevant mechanisms of melanoma cell survival that can be exploited therapeutically

Background and Rationale: Mutations in Plakophilin-2 (PKP2) are the most common cause of ARVC, an inherited disease characterized by fibro- or fibrofatty infiltration of RV predominance, ventricular arrhythmias and sudden death in the young. The relation between PKP2 expression and the heart transcriptome in vivo, is unknown. Furthermore, while splicing misregulation has been associated with other inherited diseases, PKP2-dependent exon usage differences remain unexplored. We generated a murine line of cardiac-restricted, tamoxifen activated PKP2 deficiency ("PKP2-cKO") and defined PKP2- dependent exon usage in adult non-failing hearts. Methods and Results: The first disease manifestation was an increase in RV area, detected by echocardiography 14 days after tamoxifen injection (14 days post-injection or "dpi"), followed by marked RV dilation and reparative fibrosis (21 dpi), then bi-ventricular dilated cardiomyopathy (28 dpi), heart failure and death (30-50 dpi). To capture the earliest molecular events, hearts 14 dpi were used for RNAseq and exon usage. Comparing RV vs LV revealed minor changes in transcript abundance, but significant differences in alternative splicing (AS) program. We found ~75% of differentially spliced exons flanked by sequences that bind RBFox2, an RNA-binding protein that acts as central AS regulator of the adult heart, and that is necessary to maintain cardiac structure. Western blot analysis at 14 dpi and thereafter showed reduced abundance of RBFox2. RNAseq at 21 dpi showed that in addition to RBFox2, transcripts were reduced for RBFox1, MBNL1, MBNL2 and RBM20 (also molecules that control the AS program). Exon usage analysis at 21 dpi identified massive AS misregulation, similar to that of a failing heart, even though ejection fraction at this stage was ~50%. Misregulated genes included several involved in electrical rhythm and intracellular calcium homeostasis. Conclusion: We generated a model of PKP2-dependent ARVC. Our studies point to a previously unrecognized association between a desmosomal molecule, a splicing regulator, and the control of electrical and mechanical function. AS misregulation may be a substrate for sudden unexpected arrhythmic death in the young

Over recent years, multiple groups have shown that a large number of structural variants, repeats, or problems with the underlying genome assembly have dramatic effects on the mapping, calling, and overall reliability of single nucleotide polymorphism calls. This project endeavored to develop an easy-to-use track for looking at structural variant and repeat regions. This track, DangerTrack, can be displayed alongside the existing Genome Reference Consortium assembly tracks to warn clinicians and biologists when variants of interest may be incorrectly called, of dubious quality, or on an insertion or copy number expansion. While mapping and variant calling can be automated, it is our opinion that when these regions are of interest to a particular clinical or research group, they warrant a careful examination, potentially involving localized reassembly. DangerTrack is available at https://github.com/DCGenomics/DangerTrack.

Pluripotent embryonic stem cells (ESCs) self-renew or differentiate into all tissues of the developing embryo and cell-specification factors are necessary to balance gene expression. Here we delineate the function of the PHD-finger protein 5a (Phf5a) in ESC self-renewal and ascribe its role in regulating pluripotency, cellular reprogramming and myoblast specification. We demonstrate that Phf5a is essential for maintaining pluripotency, since depleted ESCs exhibit hallmarks of differentiation. Mechanistically, we attribute Phf5a function to the stabilization of the Paf1 transcriptional complex and control of RNA polymerase II elongation on pluripotency loci. Apart from an ESC-specific factor, we demonstrate that Phf5a controls differentiation of adult myoblasts. Our findings suggest a potent mode of regulation by Phf5a in stem cells, which directs their transcriptional programme, ultimately regulating maintenance of pluripotency and cellular reprogramming.

Successful pregnancy requires delicate control over the immunological and inflammatory properties of the maternal/fetal interface. For example, inflammation within the pregnant uterus is likely to be a major instigator of preterm birth, while inadequate immune surveillance of the maternal/fetal interface likely increases the risk of fetal and placental infection. We will discuss our recent work on the molecular and cellular pathways that regulate immune cell trafficking and inflammation within the pregnant mouse uterus. This work points to the seminal importance of the decidua, i.e. the specialized endometrial stromal tissue that encases the implanted embryo, and in particular an epigenetic program active in decidual stromal cells that we previously found transcriptionally silences the expression of chemokine genes that control effector T cell trafficking. Our recent results suggest that this program also silences a multitude of other important genes, including ones whose misexpression might be expected to generate uterine inflammation and lead to a variety of pregnancy complications including preterm birth

Glioblastoma (GBM) is a deadly primary brain malignancy with extensive intratumoral hypoxia. Hypoxic regions of GBM contain stem-like cells and are associated with tumor growth and angiogenesis. The molecular mechanisms that regulate tumor growth in hypoxic conditions are incompletely understood. Here, we use primary human tumor biospecimens and cultures to identify GPR133 (ADGRD1), an orphan member of the adhesion family of G-protein-coupled receptors, as a critical regulator of the response to hypoxia and tumor growth in GBM. GPR133 is selectively expressed in CD133+ GBM stem cells (GSCs) and within the hypoxic areas of PPN in human biospecimens. GPR133 mRNA is transcriptionally upregulated by hypoxia in hypoxia-inducible factor 1alpha (Hif1alpha)-dependent manner. Genetic inhibition of GPR133 with short hairpin RNA reduces the prevalence of CD133+ GSCs, tumor cell proliferation and tumorsphere formation in vitro. Forskolin rescues the GPR133 knockdown phenotype, suggesting that GPR133 signaling is mediated by cAMP. Implantation of GBM cells with short hairpin RNA-mediated knockdown of GPR133 in the mouse brain markedly reduces tumor xenograft formation and increases host survival. Analysis of the TCGA data shows that GPR133 expression levels are inversely correlated with patient survival. These findings indicate that GPR133 is an important mediator of the hypoxic response in GBM and has significant protumorigenic functions. We propose that GPR133 represents a novel molecular target in GBM and possibly other malignancies where hypoxia is fundamental to pathogenesis.