Faculty Bibliography

Introduction/Background Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumour heterogeneity of OC are limited and hard to establish. Methodology We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Results 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 tumour 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. Conclusion Taken together, this demonstrates their potential application for research and personalized medicine. (Figure Presented)

Background The cornerstone of the existing treatment of high-grade serous ovarian cancer (HGSOC) is DNA-damaging chemotherapy; however, practically all patients eventually develop the progressive disease and the 5-year survival is only 40%. Immunotherapy would seem to be an attractive alternative treatment to chemotherapy, yet existing immunotherapies perform poorly in ovarian cancer, with only ~10% of patients responding to checkpoint-blockade. Why this is the case remains poorly understood and there is a pressing need to understand the underlying biology of immune evasion in ovarian cancer. Unfortunately, the preclinical tools required to explore the relationship between the types of DNA damage repair deficiencies and immune evasion have been lacking. Hence, we have modeled the biology of ovarian cancer using patient-relevant mutational landscapes in an immune-proficient, syngeneic-mouse model to help us identify the contribution of common driver mutations to the immune repertoire in the tumor microenvironment, and thus to responses of HGSOC tumors to immunotherapy. Methods We hypothesize that the immune composition and gene expression signatures of the resulting tumors will vary based on the combination of genetic alterations and the DNA repair proficiency of the transformed cells. To this end, we have engineered novel syngeneic mouse models from murine fallopian tube epithelium using CRISPR/Cas9 technology. These tumors capture the most common combinations of cooccurring mutations observed in patients. These models can identify the contribution of common driver mutations to the heterotypic interactions between cancer and stromal/immune compartments and examine how DNA repair proficiency contributes to immunogenicity. Results To validate the DNA repair proficiency of the transformed cells, we measured Rad51 nuclear focus formation after ionizing radiation (IR) and PARPinhibitor and DNA-damaging-agent sensitivity. The HR-deficient cell lines had significantly fewer Rad51 nuclear foci and were more sensitive to PARP-inhibition in comparison to HR-proficient cells. Initial immune /stromal analysis using flow cytometry, scRNAseq transcriptomic and immunofluorescence analysis revealed substantial differences in the myeloid and T-cell regulatory compartments between HR-proficient and-deficient primary and metastatic tumors and within the ascitic fluid. Preliminary results also suggest that inhibition of the DNA damage response, checkpoint kinase 1 in combination with immune checkpoint inhibitors, potentiates antitumor effects and augments cytotoxic T-cell infiltration. Conclusions These results reveal how common mutational drivers determine the microenvironment of the tumor and its response to treatment. Understanding the genetic basis of these complex cellular interactions will be critical to better tailor combinations of existing targeted treatments and immunotherapies in ovarian cancer to fight this devastating disease

The cell-of-origin of high grade serous ovarian carcinoma (HGSOC) remains controversial, with fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE) both considered candidates. Here, by using genetically engineered mouse models and organoids, we assessed the tumor-forming properties of FTE and OSE harboring the same oncogenic abnormalities. Combined RB family inactivation and Tp53 mutation in Pax8 + FTE caused Serous Tubal Intraepithelial Carcinoma (STIC), which metastasized rapidly to the ovarian surface. These events were recapitulated by orthotopic injection of mutant FTE organoids. Engineering the same genetic lesions into Lgr5 + OSE or OSE-derived organoids also caused metastatic HGSOC, although with longer latency and lower penetrance. FTE- and OSE-derived tumors had distinct transcriptomes, and comparative transcriptomics and genomics suggest that human HGSOC arises from both cell types. Finally, FTE- and OSE-derived organoids exhibited differential chemosensitivity. Our results comport with a dualistic origin for HGSOC and suggest that the cell-of-origin might influence therapeutic response.

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.
Copyright

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.

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

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.