Faculty Bibliography

RAS-ERK signaling pathway regulates large numbers of biological processes. Thus, germline mutations in RAS-ERK signaling pathway are associated with developmental disorders, collectively called RASopathy. BRAF is a direct downstream effector of RAS and germline mutations of BRAF accounts for over 70% of cardio-facio-cutaneous (CFC) syndrome which severely impairs cognitive functions. However, the cellular and molecular basis for the brain abnormalities in CFC syndrome remains largely unknown. Here, we investigated how the dysregulated RAS-ERK signaling affects malformation of central nervous system (CNS) and cognitive deficits by using conditional knockin mice harboring RASopathy-associated mutations in Braf. Neural stem cell-specific expression of Braf mutations significantly impaired hippocampus-dependent learning and memory in Morris water maze. Histological examination and transcriptome analyses revealed changes in specific cell types although mutants were expressed in neural stem cells, suggesting that the impacts of expressing Braf mutations are sensitive to specific cell types as previously shown in other RASopathy mouse models. Our study will contribute to untangle the complexity of RAS-ERK signaling network in brain development and cognitive functions.
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SHP2, encoded by the PTPN11 gene, is a ubiquitous protein tyrosine phosphatase that is a critical regulator of signal transduction. Germline mutations in PTPN11 gene responsible for catalytic gain- or loss- of function of SHP2 cause two disorders with multiple organ defects, respectively Noonan syndrome (NS) and NS with Multiple Lentigines (NSML). Bleeding anomalies have been frequently reported in NS, but causes remain unclear. This study investigates platelet activation in patients with NS and NSML and in two mouse models carrying PTPN11 mutations responsible for these two syndromes. Platelets from NS mice and patients displayed a significant reduction in aggregation induced by low concentrations of GPVI and CLEC-2 agonists, and a decrease in thrombus growth on a collagen surface under arterial shear stress. This was associated with deficiencies in GPVI and a_IIbb₃ integrin signaling, platelet secretion and TXA₂ generation. Similarly, arterial thrombus formation was significantly reduced in response to a local carotid injury in NS mice associated with a significant increase in the tail bleeding time. In contrast, NSML mice platelets exhibited increased platelet activation following GPVI and CLEC-2 stimulation and enhanced platelet thrombotic phenotype on collagen matrix under shear stress. Blood samples from NSML patients also showed a shear stress-dependent elevation of platelet responses on collagen matrix. This study brings new insights into the understanding of SHP2 function in platelets, points to new thrombopathies linked to platelet signaling defects and provides important information for the medical care of patients with NS in situations at risk of bleeding.

Reactive oxygen species (ROS) can act as second messengers in various signaling pathways, and abnormal oxidation contributes to multiple diseases, including cancer. Detecting and quantifying protein oxidation is crucial for a detailed understanding of reduction-oxidation reaction (redox) signaling. We developed an Activated Thiol Sepharose-based proteomic (ATSP) approach to quantify reversible protein oxidation. ATSP can enrich H₂O₂-sensitive thiol peptides, which are more likely to contain reactive cysteines involved in redox signaling. We applied our approach to analyze hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a type of kidney cancer that harbors fumarate hydratase (FH)-inactivating mutations and has elevated ROS levels. Multiple proteins were oxidized in FH-deficient cells, including many metabolic proteins such as the pyruvate kinase M2 isoform (PKM2). Treatment of HLRCC cells with dimethyl fumarate or PKM2 activators altered PKM2 oxidation levels. Finally, we found that ATSP could detect Src homology region 2 domain-containing phosphatase-2 and PKM2 oxidation in cells stimulated with platelet-derived growth factor. This newly developed redox proteomics workflow can detect reversible oxidation of reactive cysteines and can be employed to analyze multiple physiologic and pathologic conditions.-Xu, Y., Andrade, J., Ueberheide, B., Neel, B. G. Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.

Background: Myeloproliferative neoplasms (MPN) are hematopoietic stem cell disorders with dysregulated JAK2 signaling. The limited effects of JAK inhibitors relate to compensatory MAPK activation. We hypothesized that ERK1/2 could be a favorable target given the distal position in the MAPK pathway and the essential role for hematopoiesis.
Method(s): We genetically targeted ERK1/2 in MPN by combining Jak2V617F with ERK1 and ERK2 knockout alleles and hematopoiesisspecific Mx-Cre.To assess engraftment dynamics and competitive performance of the MPN clone, CD45.2 Jak2V617F bone marrow (BM) +/-ERK1/2 double KO (dKO) was competitively transplanted 1:1 with CD45.1 Jak2 WT BM into WT recipients.
Result(s): Loss of ERK1/2 in Jak2V617F mice reduced splenomegaly at 3 months. Excessive erythropoiesis was moderated with decreased red cell and reticulocyte counts in peripheral blood and erythroid progenitors in BM. Red cell parameters remained slightly above normal over time without induction of anemia. The microcytic hypochromic red cell features of Jak2V617F mice normalized by ERK1/2 dKO and emergence of leukocytosis and neutrophilia was prevented. Platelets were normal in Jak2V617F mice, while ERK1/2 ablation moderated thrombopoiesis with reduced megakaryocyte progenitors and thrombocytopenia. Lin-Sca+Kit+ (LSK) hematopoietic stem/progenitor cells were decreased in ERK deficient Jak2V617F mice suggesting a reduced disease-initiating population. In competitive transplants, all recipients of Jak2V617F ERK1/2 dKO BM engrafted. The Jak2V617F clone as shown by CD45.2 chimerism, was reduced to 19% in mice with deficient vs. intact ERK1/2. The Jak2V617F clone was most prominent in neutrophils and gradually expanded, but remained low and stable in ERK deficient mice.(Figure presented)
Conclusion(s): Our data show that targeting ERK1/2 impairs the fitness of the MPN clone by restricting the stem/progenitor compartment and blunting clone expansion with reduced differentiated cell output and moderated cytoses. While targeting ERK could be a promising combination strategy with JAK2 inhibition, it should be restricted to settings of thrombocytosis to prevent induction of thrombocytopenia

The efficacy of prospective cancer treatments is routinely estimated by in vitro cell-line proliferation screens. However, it is unclear whether tumor aggressiveness and patient survival are influenced more by the proliferative or the migratory properties of cancer cells. To address this question, we experimentally measured proliferation and migration phenotypes across more than 40 breast cancer cell-lines. Based on the latter, we built and validated individual predictors of breast cancer proliferation and migration levels from the cells' transcriptomics. We then apply these predictors to estimate the proliferation and migration levels of more than 1000 TCGA breast cancer tumors. Reassuringly, both estimates increase with tumor's aggressiveness, as qualified by its stage, grade, and subtype. However, predicted tumor migration levels are significantly more strongly associated with patient survival than the proliferation levels. We confirmed these findings by conducting siRNA knock-down experiments on the highly migratory MDA-MB-231 cell lines and deriving gene knock-down based proliferation and migration signatures. We show that cytoskeletal drugs might be more beneficial in patients with high predicted migration levels. Taken together, these results testify to the importance of migration levels in determining patient survival.

Immunotherapy has shown modest activity in metastatic breast cancer (MBC). In this phase I dose escalation study, we assessed safety of tremelimumab, a humanized anti-CTLA4 monoclonal antibody, at starting dose 3 mg/kg, on the third day of palliative radiotherapy (2000cGy in 5 daily fractions) in patients with MBC. Primary objective was to determine the maximum tolerated dose (MTD) of tremelimumab combined with RT. Secondary objective was to assess response. Among 6 patients enrolled between July 2010 and October 2011, 5 had hormone receptor-positive MBC, 1 had triple negative MBC. Median age was 45 years. Common toxicities included lymphopenia (83%), fatigue (50%) and rash (33%). One dose-limiting toxicity occurred at 6 mg/kg, however the trial closed before MTD could be determined. One patient discontinued treatment due to a pathological fracture. Best response was stable disease (SD), 1 patient had SD for >6 months. Median follow up was 27.0 months. Median OS was 50.8 months, with 1 patient surviving >8 years. Peripheral blood mononuclear cell (PBMC) profiles showed increasing proliferating (Ki67+) Treg cells 1 week post treatment in 5 patients. Overall, tremelimumab at 3 mg/kg combined with RT appears to be a tolerable treatment strategy. Further studies are needed to optimize this combination approach.

Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have established 56 organoid lines from 32 patients, representing all main subtypes of OC. OC organoids recapitulate histological and genomic features of the pertinent lesion from which they were derived, illustrating intra- and interpatient heterogeneity, and can be genetically modified. We show that OC organoids can be used for drug-screening assays and capture different tumor subtype responses to the gold standard platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. Finally, OC organoids can be xenografted, enabling in vivo drug-sensitivity assays. Taken together, this demonstrates their potential application for research and personalized medicine.

RASopathies are a family of related syndromes caused by mutations in regulators of the RAS/Extracellular Regulated Kinase 1/2 (ERK1/2) signaling cascade that often result in neurological deficits. RASopathy mutations in upstream regulatory components, such as NF1, PTPN11/SHP2, and RAS have been well-characterized, but mutation-specific differences in the pathogenesis of nervous system abnormalities remain poorly understood, especially those involving mutations downstream of RAS. Here, we assessed cellular and behavioral phenotypes in mice expressing a Raf1L613V gain-of-function mutation associated with the RASopathy, Noonan Syndrome. We report that Raf1L613V/wt mutants do not exhibit a significantly altered number of excitatory or inhibitory neurons in the cortex. However, we observed a significant increase in the number of specific glial subtypes in the forebrain. The density of GFAP+ astrocytes was significantly increased in the adult Raf1L613V/wt cortex and hippocampus relative to controls. OLIG2+ oligodendrocyte progenitor cells were also increased in number in mutant cortices, but we detected no significant change in myelination. Behavioral analyses revealed no significant changes in voluntary locomotor activity, anxiety-like behavior, or sociability. Surprisingly, Raf1L613V/wt mice mutants performed better than controls in select aspects of the water radial-arm maze, Morris water maze, and cued fear conditioning tasks. Overall, these data show that increased astrocyte and oligodendrocyte progenitor cell (OPC) density in the cortex coincides with enhanced cognition in Raf1L613V/wt mutants and further highlight the distinct effects of RASopathy mutations on nervous system development and function.

High-grade serous ovarian carcinoma (HGSC) is the most common and lethal subtype of gynecologic malignancy in women. The current standard of treatment combines cytoreductive surgery and chemotherapy. Despite the efficacy of initial treatment, most patients develop cancer recurrence, and 70% of patients die within 5 years of initial diagnosis. CA125 is the current FDA-approved biomarker used in the clinic to monitor response to treatment and recurrence, but its impact on patient survival is limited. New strategies for the discovery of HGSC biomarkers are urgently needed. Here, we describe a proteomics strategy to detect tumor-associated proteins in serum of HGSC patient-derived xenograft models. We demonstrate proof-of-concept applicability using two independent, longitudinal serum cohorts from HGSC patients.

Conditional knockout (KO) mouse models are invaluable for elucidating the physiological roles of platelets. The Pf4-Cre transgenic mouse is the current model of choice for generating megakaryocyte/platelet-specific KO mice. Platelets and leukocytes work closely together in a wide range of disease settings, yet the specific contribution of platelets to these processes remains unclear. This is partially due to the Pf4-Cre transgene being expressed in a variety of leukocyte populations. To overcome this issue, we developed a Gp1ba-Cre transgenic mouse strain in which Cre expression in driven by the endogenous Gp1ba locus. By crossing Gp1ba-Cre and Pf4-Cre mice to the mT/mG dual-fluorescence reporter mouse and performing a head-to-head comparison, we demonstrate more stringent megakaryocyte lineage-specific expression of the Gp1ba-Cre transgene. Broader tissue expression was observed with the Pf4-Cre transgene, leading to recombination in many hematopoietic lineages, including monocytes, macrophages, granulocytes, dendritic, B and T cells. Direct comparison of phenotypes of Csk, Shp1 or CD148 conditional KO mice generated using either the Gp1ba-Cre or Pf4-Cre strains revealed similar platelet phenotypes. However, additional inflammatory and immunological anomalies were observed in Pf4-Cre-generated KO mice due to non-specific deletion in other hematopoietic lineages. By excluding leukocyte contributions to phenotypes, the Gp1ba-Cre mouse will advance our understanding of the role of platelets in inflammation and other pathophysiological processes where platelet-leukocyte interactions are involved.