Benjamin G. Neel

Wolfram Syndrome protein, Miner1, regulates sulphydryl redox status, the unfolded protein response, and Ca2+ homeostasis

Miner1 is a redox‐active 2Fe2S cluster protein. Mutations in Miner1 result in Wolfram Syndrome, a metabolic disease associated with diabetes, blindness, deafness, and a shortened lifespan. Embryonic fibroblasts from Miner1−/− mice displayed ER stress and showed hallmarks of the unfolded protein response. In addition, loss of Miner1 caused a depletion of ER Ca2+ stores, a dramatic increase in mitochondrial Ca2+ load, increased reactive oxygen and nitrogen species, an increase in the GSSG/GSH and NAD+/NADH ratios, and an increase in the ADP/ATP ratio consistent with enhanced ATP utilization. Furthermore, mitochondria in fibroblasts lacking Miner1 displayed ultrastructural alterations, such as increased cristae density and punctate morphology, and an increase in O2 consumption. Treatment with the sulphydryl anti‐oxidant N‐acetylcysteine reversed the abnormalities in the Miner1 deficient cells, suggesting that sulphydryl reducing agents should be explored as a treatment for this rare genetic disease.

Megakaryocyte-specific deletion of the protein-tyrosine phosphatases Shp1 and Shp2 causes abnormal megakaryocyte development, platelet production and function

The SH2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2 have been implicated in regulating signaling from a variety of platelet and megakaryocyte receptors. In this study, we investigate the functions of Shp1 and Shp2 in megakaryocytes and platelets. Megakaryocyte/platelet (MP)-specific deletion of Shp1 in mice resulted in platelets being less responsive to collagen-related peptide due to reduced GPVI expression and signaling via the Src family kinase (SFK)-Syk-PLCγ2 pathway, and fibrinogen due to reduced SFK activity. By contrast, deletion of Shp2 in the MP lineage resulted in macrothrombocytopenia and platelets being hyper-responsive to anti-CLEC-2 antibody and fibrinogen. Shp1- and Shp2-deficient megakaryocytes had partial blocks at 2N/4N ploidy; however, only the latter exhibited reduced proplatelet formation, thrombopoietin, and integrin signaling. Mice deficient in both Shp1 and Shp2 were severely macrothrombocytopenic and had reduced platelet surface glycoprotein expression, including GPVI, αIIbβ3, and GPIbα. Megakaryocytes from these mice were blocked at 2N/4N ploidy and did not survive ex vivo. Deletion of the immunoreceptor tyrosine-based inhibition motif-containing receptor G6b-B in the MP lineage phenocopied multiple features of Shp1/2-deficient mice, suggesting G6b-B is a critical regulator of Shp1 and Shp2. This study establishes Shp1 and Shp2 as major regulators of megakaryocyte development, platelet production, and function.

Erk1 and Erk2 are required for maintenance of hematopoietic stem cells and adult hematopoiesis

Extracellular signal-regulated kinase 1 (Erk1) and Erk2 play crucial roles in cell survival, proliferation, cell adhesion, migration, and differentiation in many tissues. Here, we report that the absence of Erk1 and Erk2 in murine hematopoietic cells leads to bone marrow aplasia, leukopenia, anemia, and early lethality. Mice doubly-deficient in Erk1 and Erk2 show rapid attrition of hematopoietic stem cells and immature progenitors in a cell-autonomous manner. Reconstitution studies show that Erk1 and Erk2 play redundant and kinase-dependent functions in hematopoietic progenitor cells. Moreover, in cells transformed by the oncogenic KRas(G12D) allele, the presence of either Erk1 or Erk2 with intact kinase activity is sufficient to promote cytokine-independent proliferation.

PI3K p110δ uniquely promotes gain-of-function Shp2-induced GM-CSF hypersensitivity in a model of JMML

Although hyperactivation of the Ras-Erk signaling pathway is known to underlie the pathogenesis of juvenile myelomonocytic leukemia (JMML), a fatal childhood disease, the PI3K-Akt signaling pathway is also dysregulated in this disease. Using genetic models, we demonstrate that inactivation of phosphatidylinositol-3-kinase (PI3K) catalytic subunit p110δ, but not PI3K p110α, corrects gain-of-function (GOF) Shp2-induced granulocyte macrophage-colony-stimulating factor (GM-CSF) hypersensitivity, Akt and Erk hyperactivation, and skewed hematopoietic progenitor distribution. Likewise, potent p110δ-specific inhibitors curtail the proliferation of GOF Shp2-expressing hematopoietic cells and cooperate with mitogen-activated or extracellular signal-regulated protein kinase kinase (MEK) inhibition to reduce proliferation further and maximally block Erk and Akt activation. Furthermore, the PI3K p110δ-specific inhibitor, idelalisib, also demonstrates activity against primary leukemia cells from individuals with JMML. These findings suggest that selective inhibition of the PI3K catalytic subunit p110δ could provide an innovative approach for treatment of JMML, with the potential for limiting toxicity resulting from the hematopoietic-restricted expression of p110δ.

Role of PTPN11 (SHP2) in Cancer

Src homology-2 domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 gene, is a highly conserved, non-transmembrane protein-tyrosine phosphatase (PTP), found in all metazoans. The molecular details of SHP2 regulation by phosphotyrosyl (pTyr) peptide ligand binding are well-understood, and knowledge of these details is critical to understanding SHP2 function in health and disease. Studies using mice with gain- or loss-of-function alleles of Ptpn11 have provided much detail about the physiological functions and signaling pathways regulated by SHP2 at the cellular and whole organism levels. Germline mutations in PTPN11 cause Noonan syndrome, Noonan syndrome with multiple lentigines (previously, LEOPARD syndrome), as well as the cartilage tumor syndrome, metachondromatosis. Somatic PTPN11 mutations occur in several types of hematologic malignancy, most notably juvenile myelomonocytic leukemia and, more rarely, in neuroblastoma and other solid tumors. PTPN11 is crucial for transformation initiated by mutant receptor-tyrosine kinases (RTKs) and is an important effector of H. pylori virulence. However, the direct target(s) of SHP2 responsible for its physiological and pathological effects remain controversial and their identification remains a major goal for the future research.

From an orphan disease to a generalized molecular mechanism: PTPN11 loss-of-function mutations in the pathogenesis of metachondromatosis.

Recently, loss-of-function mutations in PTPN11 were linked to the cartilage tumor syndrome metachondromatosis (MC), a rare inherited disorder featuring osteochondromas, endochondromas and skeletal deformation. However, the underlying molecular and cellular mechanism for MC remained incompletely understood. By studying the role of the Src homology-2 domain-containing protein tyrosine phosphatase Shp2 (encoded by mouse Ptpn11) in cathepsin K-expressing cells, we identified a novel cell population in the perichondrial groove of Ranvier. In the absence of Shp2, these cells exhibit elevated Indian hedgehog (Ihh) signaling, proliferate excessively and cause ectopic cartilage formation and tumors. Our findings establish a critical role for a protein-tyrosine phosphatase (PTP) family member, in addition to the well-known roles of receptor tyrosine kinases (RTKs), in cartilage development and homeostasis. However, whether Shp2 deficiency in other epiphyseal chondroid cells and whether signaling pathways in addition to the IHH/Parathyroid Hormone–related Peptide (PTHrP) axis attribute to the formation of enchondromas and osteochondromas remains elusive. Understanding how chondrogenic events are regulated by SHP2 could aid in the development of novel therapeutic approaches to prevent and treat cartilage diseases, such as MC and osteoarthritis (OA).

Redox Regulation of PTPs in Metabolism: Focus on Assays

Protein-tyrosine phosphatases (PTPs), along with protein-tyrosine kinases (PTKs), are the key regulators of phosphotyrosine signaling, and therefore are important contributors to normal metabolism and metabolic disease. Over the past 10 years, reactive oxygen species (ROS), which had long been viewed as toxic by-products of metabolism, have been recast as important second messengers, which act, at least in part, to regulate PTP activity by reversible oxidation. For example, ROS-catalyzed PTP oxidation can transiently inhibit PTP enzymatic activity and facilitate ligand-induced receptor tyrosine kinase (RTK) signaling. Identifying ROS-inactivated PTPs represents a key challenge to understanding the role of PTPs and redox regulation in physiology and pathology. Here, we briefly review ROS regulation of PTPs, focusing on existing assays and new approaches to identify and quantify PTP oxidation.

Abstract PR01: Functional characterization of breast cancer using pooled lentivirus shRNA screens

Breast cancer is the most common invasive malignancy and the second leading cause of cancer death in U.S. women. Early detection and improved therapy have led to >85% 5-year survival, but still, half of currently affected women will succumb from metastatic disease. This poor outcome reflects our incomplete knowledge of essential genes driving tumor proliferation for each subtype. From genomic data alone, it can be difficult to access which genetic alterations drive pathogenesis because most of these are functionally irrelevant “passenger” mutations. Even if an oncogene or tumor suppressor is identified, these often are not amenable to targeted therapy. However, unanticipated gene/pathway dependencies can arise as a consequence of these genetic abnormalities in cancer cells (“synthetic lethality”). The recent development of lentiviral-based shRNA libraries enables genome-wide screening of cultured cancer cells in a pooled format, facilitating the identification of genes necessary for cancer cell proliferation and survival in cell culture as well as potential synthetic lethal interactions. The overall objectives of this project were to identify subtype-specific targets and synthetic lethal interactions for common mutations in human breast cancer using genome-wide shRNA screens, as well as to compare “functional genomic” and genomic classification schemes. We screened a panel of > 75 breast cancer lines using an 80,000 lentiviral shRNA library targeting 16,000 genes in a pooled format. We identified several classes of gene “dropouts,” including general essential genes, which are required for survival or growth in more than 70% of all cell lines, irrespective of subtype. Using a newly developed scoring algorithm that allows for precise measurement of statistical significance between two groups, we also identified several “subtype-specific” genes, whose essentiality is restricted to a defined subtype. These include well-known HER2 subtype-specific genes, ERBB2, ERBB3, and TFAP2C and luminal subtype-specific gene FOXA1, SPDEF, GATA3, ESR1, and CCDN1 as well as newly identified BRD4 and CHD4. These two genes were further validated as luminal-specific and results to explain their luminal subtype-specificity will be presented. In addition, the unprecedented number of lines allows the identification of synthetic lethal interaction with common breast cancer somatic mutation such as PIK3CA and PTEN. Finally, integration of gene expression, copy number variation, and functional screening results identified potential biomarkers with common genetic changes and functional drivers. Overall, our study represents an extensive functional genetic survey of four major breast cancer subtypes, reveals complexities between genomic and functional genomic results, and uncovers several unexpected gene dependencies and potential novel therapeutic target for each subtype. This abstract is also presented as Poster A22. Citation Format: Richard Marcotte, Azin Sayad, Maliha Haider, Kevin Brown, Troy Ketela, Jason Moffat, Benjamin G. Neel. Functional characterization of breast cancer using pooled lentivirus shRNA screens. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR01.

Abstract IA20: Analyzing the cellular basis for heterogeneity in serous ovarian carcinoma

Serous ovarian cancer (SOC) is the leading cause of morbidity/mortality from gynecologic cancer. Current therapies increase survival, yet the majority of SOC patients die of their disease. The cancer stem cell (CSC) hypothesis holds that only a subset of tumor cells can initiate/maintain the tumor and might be more resistant to chemotherapy. In the CSC model, it should be possible to purify a stable population with the ability to generate transplantable tumors that recreate the heterogeneity of the initial malignancy. We previously reported that CD133 marks all TIC from several primary SOC, but in some cases, TIC activity is also found in the CD133– fraction. Also, the TIC phenotype is unstable in xenografts. To address whether the expression of an unidentified protein marks all TIC in SOC, we performed high-throughput flow cytometry (profiling 365 cell surface markers) and combined these analyses with mass cytometry, a transformative technology allowing examination of 35 cell surface/intracellular markers on a single cell. Analysis of 40 primary SOC samples provided further evidence for inter- and intra-patient heterogeneity. Current analyses focus on functional validation of subpopulations marked by combinations of cell surface/stem cell genes. In the course of these studies, we have generated a large cohort of SOC xenografts. Xenografts recapitulate the inter- and intra-patient heterogeneity of SOC when their gene expression and copy number profiles are compared. Indeed, they also recapitulate the patient9s sensitivity to standard of care chemotherapy. These xenografts might provide a useful model l for more efficient and personalized testing of investigational drugs for SOC. Citation Format: Benjamin G. Neel. Analyzing the cellular basis for heterogeneity in serous ovarian carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr IA20.

Abstract SY15-01: Cellular context-specific tumor suppression by PTPN11

Cartilage tumors, accounting for 22% of skeletal system tumors, are characterized by the formation of exostoses, enchondroma(s) or both, and cause significant morbidity and mortality. Both benign and malignant cartilaginous tumors can develop at various ages, and can show autosomal dominant inheritance or occur sporadically. The molecular mechanism underlying the development and progression of these cartilaginous lesions remains incompletely understood. Shp2, encoded by the Ptpn11 gene, is one of two Src homology 2 domain-containing protein-tyrosine phosphatases, and is required for most, if not all, receptor tyrosine kinase (RTK), cytokine, and integrin signaling pathways. Global deletion of Shp2 in mice results in early embryonic lethality, whereas postnatal Shp2 deficiency in various tissues/cells has diverse effects on their development and function. Several human malignancies, most notably childhood myeloproliferative disorders, are associated with Ptpn11 gain-of-function (GOF) mutations. Several lines of evidence indicate that Shp2 plays an important role in skeletal development and homoeostasis; however, little is known about its role in vivo. Recently Ptpn11 truncated mutations (presumably protein null) are reported to cause human metachondromatosis, a benign cartilage tumor syndrome with malignant potentials. By taking a tissue-specific gene knockout approach, we report here that Shp2 loss-of-function mutation, in contrast to its GOF mutants in other tissues/cells, causes widespread and severe cartilaginous tumors, strongly suggesting that Shp2 has a tissue specific-tumor suppressor function. These studies also identify the target of cartilage malignancies caused by Shp2 deficiency as a novel cell population with stem/progenitor properties located within the groove of Ranvier. Materials and Methods: Mice carrying Ptpn11 floxed (fl) or cathepsin K-Cre (Ctsk-Cre) were described previously. To generate Ctsk-expressing cell specific Shp2 deficient mice and study the role of Shp2 in these cells to regulate skeletal development and homeostasis, Ptpn11 floxed mice were bred to Ctsk-Cre mice to generate Ptpn11fl/fl;Ctsk-Cre (KO) and Ptpn11fl/+;Ctsk-Cre (Control) animals. To track the fate of Ctsk + cartilaginous cells in vivo in the presence or absence of Shp2, Roza26lacZ (R26lacZ) and Roza26EYFP (R26YFP) reporter alleles were bred to Control and KO mice to generate Control/R26YFP, KO/R26YFP, Control/R26lacZ and KO/R26lacZ compound mice, respectively. YFP-positive cartilaginous cells were isolated by serial enzymatic digestions of epiphyseal cartilage with hyaluronidase/Trypsin/ collagenase D and FACS sorting. Cell surface marker expression was determined by FACS analysis after staining with fluorescence-labeled antibodies. For histological analysis, skeletal tissues were removed from mice after euthanasia and fixed in 4% paraformaldehyde; after decalcification, embedding, and sectioning, they were stained with HELysMCre mcie, revealed that the cartilage phenotype in KO mice was not osteoclast-autonomous. Therefore to identify the cellular origin of the cartilage lesions in KO mice, we conducted a reporter study by using Ctsk-Cre mediated R26lacZ or YFP expression. Surprisingly, we found that the Ctsk promoter is active not only in mature osteoclasts, but also in a subset of cells that live in perichondrial groove of Ranvier. Deficiency of Shp2 in these cells causes unrestrained proliferation, chondrogenic differentiation, and exostoses/enchondroma(s) formation. These cells were found to share several mesenchymal/chondroprogenitor markers, such as CD44, CD90, Stro1 and jagged1. Biochemical analysis shows that Shp2 deficiency impaired Erk activation and caused elevated expression of Indian hedgehog (Ihh) and PTHrP in cartilage lesions. In summary, we specifically ablated Shp2 in Ctsk-expressing cells (primarily in mature osteoclasts and subsets of cartilaginous cells) in mice and found that Shp2, in addition to having a role in osteoclastogenesis in vitro and in vivo, negatively regulates the proliferation and chondrogenic differentiation of a unique population of ctsk+ perichondrial cartilaginous cells. Loss of Shp2 in these cells causes cell proliferation, chondrogenic differentiation, and cartilage tumorigenesis at the metaphyses of tubular bones. This phenotype is strikingly similar to the human disease metachondromatosis. Mechanistically, we believe that this pathogenic process is triggered by ectopic and/or elevated Ihh expression due to impaired Erk activation in Shp2- deficient perichondrial cells. If confirmed, these data identify hedgehog pathway inhibitors as potential therapeutic agents in metachromatosis patients. Our results, in concert with previous studies, show that, depending on the cellular context, Shp2 can act as an oncogene or a tumor suppressor gene. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY15-01. doi:1538-7445.AM2012-SY15-01