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.

Noonan syndrome (NS) is caused by mutations in RAS/ERK pathway genes, and is characterized by craniofacial, growth, cognitive and cardiac defects. NS patients with kinase-activating RAF1 alleles typically develop pathological left ventricular hypertrophy (LVH), which is reproduced in Raf1L613V/+ knock-in mice. Here, using inducible Raf1L613V expression, we show that LVH results from the interplay of cardiac cell types. Cardiomyocyte Raf1L613V enhances Ca2+ sensitivity and cardiac contractility without causing hypertrophy. Raf1L613V expression in cardiomyocytes or activated fibroblasts exacerbates pressure overload-evoked fibrosis. Endothelial/endocardial (EC) Raf1L613V causes cardiac hypertrophy without affecting contractility. Co-culture and neutralizing antibody experiments reveal a cytokine (TNF/IL6) hierarchy in Raf1L613V-expressing ECs that drives cardiomyocyte hypertrophy in vitro. Furthermore, postnatal TNF inhibition normalizes the increased wall thickness and cardiomyocyte hypertrophy in vivo. We conclude that NS-cardiomyopathy involves cardiomyocytes, ECs and fibroblasts, TNF/IL6 signalling components represent potential therapeutic targets, and abnormal EC signalling might contribute to other forms of LVH.

BACKGROUND: Advancing structural and functional maturation of stem cell-derived cardiomyocytes remains a key challenge for applications in disease modeling, drug screening, and heart repair. Here, we sought to advance cardiomyocyte maturation in engineered human myocardium (EHM) toward an adult phenotype under defined conditions. METHODS: We systematically investigated cell composition, matrix, and media conditions to generate EHM from embryonic and induced pluripotent stem cell-derived cardiomyocytes and fibroblasts with organotypic functionality under serum-free conditions. We used morphological, functional, and transcriptome analyses to benchmark maturation of EHM. RESULTS: EHM demonstrated important structural and functional properties of postnatal myocardium, including: (1) rod-shaped cardiomyocytes with M bands assembled as a functional syncytium; (2) systolic twitch forces at a similar level as observed in bona fide postnatal myocardium; (3) a positive force-frequency response; (4) inotropic responses to beta-adrenergic stimulation mediated via canonical beta1- and beta2-adrenoceptor signaling pathways; and (5) evidence for advanced molecular maturation by transcriptome profiling. EHM responded to chronic catecholamine toxicity with contractile dysfunction, cardiomyocyte hypertrophy, cardiomyocyte death, and N-terminal pro B-type natriuretic peptide release; all are classical hallmarks of heart failure. In addition, we demonstrate the scalability of EHM according to anticipated clinical demands for cardiac repair. CONCLUSIONS: We provide proof-of-concept for a universally applicable technology for the engineering of macroscale human myocardium for disease modeling and heart repair from embryonic and induced pluripotent stem cell-derived cardiomyocytes under defined, serum-free conditions.

Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the membrane yeast two-hybrid (MYTH) and the mammalian membrane two-hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provides a broad and deep view of RTK signaling.

Growing evidence suggests a major role for Src-homology-2-domain-containing phosphatase 2 (SHP2/PTPN11) in MYCN-driven high-risk neuroblastoma, although biologic confirmation and a plausible mechanism for this contribution are lacking. Using a zebrafish model of MYCN-overexpressing neuroblastoma, we demonstrate that mutant ptpn11 expression in the adrenal gland analog of MYCN transgenic fish promotes the proliferation of hyperplastic neuroblasts, accelerates neuroblastomagenesis, and increases tumor penetrance. We identify a similar mechanism in tumors with wild-type ptpn11 and dysregulated Gab2, which encodes a Shp2 activator that is overexpressed in human neuroblastomas. In MYCN transgenic fish, Gab2 overexpression activated the Shp2-Ras-Erk pathway, enhanced neuroblastoma induction, and increased tumor penetrance. We conclude that MYCN cooperates with either GAB2-activated or mutant SHP2 in human neuroblastomagenesis. Our findings further suggest that combined inhibition of MYCN and the SHP2-RAS-ERK pathway could provide effective targeted therapy for high-risk neuroblastoma patients with MYCN amplification and aberrant SHP2 activation.

The degree of genetic aberrations characteristic of high-grade serous ovarian cancer (HGSC) makes identification of the molecular features that drive tumor progression difficult. Here, we perform genome-wide RNAi screens and comprehensive expression analysis of cell-surface markers in a panel of HGSC cell lines to identify genes that are critical to their survival. We report that the tetraspanin CD151 contributes to survival of a subset of HGSC cell lines associated with a ZEB transcriptional program and supports the growth of HGSC tumors. Moreover, we show that high CD151 expression is prognostic of poor clinical outcome. This study reveals cell-surface vulnerabilities associated with HGSC, provides a framework for identifying therapeutic targets, and reports a role for CD151 in HGSC.

Reversible protein-tyrosine phosphorylation is catalyzed by the antagonistic actions of protein-tyrosine kinases (PTKs) and phosphatases (PTPs), and represents a major form of cell regulation. Acute myeloid leukemia (AML) is an aggressive hematological malignancy that results from the acquisition of multiple genetic alterations, which in some instances are associated with deregulated protein-phosphotyrosine (pY)-mediated signaling networks. However, although individual PTKs and PTPs have been linked to AML and other malignancies, analysis of protein-pY networks as a function of activated PTKs and PTPs has not been done. In this study, mass spectrometry was used to characterize AML proteomes, and phospho-proteome-subsets including pY proteins, PTKs, and PTPs. AML proteomes resolved into two groups related to high or low degrees of maturation according to French-American-British (FAB) classification, and reflecting differential expression of cell surface antigens. AML pY proteomes reflect canonical, spatially organized signaling networks, unrelated to maturation, with heterogeneous expression of activated receptor and non-receptor PTKs. We present the first integrated analysis of the pY-proteome, activated PTKs, and PTPs. Every PTP and most PTKs have both positive and negative associations with the pY-proteome. pY proteins resolve into groups with shared PTK and PTP correlations. These findings highlight the importance of pY turnover and the PTP phosphatome in shaping the pY-proteome in AML

RAS-like protein expressed in many tissues 1 (RIT1) is a disease-associated RAS subfamily small guanosine triphosphatase (GTPase). Recent studies revealed that germ-line and somatic RIT1 mutations can cause Noonan syndrome (NS), and drive proliferation of lung adenocarcinomas, respectively, akin to RAS mutations in these diseases. However, the locations of these RIT1 mutations differ significantly from those found in RAS, and do not affect the three mutational "hot spots" of RAS. Moreover, few studies have characterized the GTPase cycle of RIT1 and its disease-associated mutants. Here we developed a real-time NMR-based GTPase assay for RIT1 and investigated the effect of disease-associated mutations on GTPase cycle. RIT1 exhibits an intrinsic GTP hydrolysis rate similar to that of H-RAS, but its intrinsic nucleotide exchange rate is ∼4-fold faster, likely as a result of divergent residues near the nucleotide binding site. All of the disease-associated mutations investigated increased the GTP-loaded, activated state of RIT1 in vitro, but they could be classified into two groups with different intrinsic GTPase properties. The S35T, A57G, and Y89H mutants exhibited more rapid nucleotide exchange, whereas F82V and T83P impaired GTP hydrolysis. A RAS-binding domain pulldown assay indicated that RIT1 A57G and Y89H were highly activated in HEK293T cells, whereas T83P and F82V exhibited more modest activation. All five mutations are associated with NS, whereas two (A57G and F82V) have also been identified in urinary tract cancers and myeloid malignancies. Characterization of the effects on the GTPase cycle of RIT1 disease-associated mutations should enable better understanding of their role in disease processes.