Faculty Bibliography

PTPRD is a receptor protein tyrosine phosphatase that is genetically associated with neurodevelopmental disorders. Here, we asked whether Ptprd mutations cause aberrant neural development by perturbing neurogenesis in the murine cortex. We show that loss of Ptprd causes increases in neurogenic transit-amplifying intermediate progenitor cells and cortical neurons and perturbations in neuronal localization. These effects are intrinsic to neural precursor cells since acute Ptprd knockdown causes similar perturbations. PTPRD mediates these effects by dephosphorylating receptor tyrosine kinases, including TrkB and PDGFRβ, and loss of Ptprd causes the hyperactivation of TrkB and PDGFRβ and their downstream MEK-ERK signaling pathway in neural precursor cells. Moreover, inhibition of aberrant TrkB or MEK activation rescues the increased neurogenesis caused by knockdown or homozygous loss of Ptprd. These results suggest that PTPRD regulates receptor tyrosine kinases to ensure appropriate numbers of intermediate progenitor cells and neurons, suggesting a mechanism for its genetic association with neurodevelopmental disorders.

The RASopathies are a group of genetic disorders that result from germline pathogenic variants affecting RAS-mitogen activated protein kinase (MAPK) pathway genes. RASopathies share RAS/MAPK pathway dysregulation and share phenotypic manifestations affecting numerous organ systems, causing lifelong and at times life-limiting medical complications. RASopathies may benefit from precision medicine approaches. For this reason, the Sixth International RASopathies Symposium focused on exploring precision medicine. This meeting brought together basic science researchers, clinicians, clinician scientists, patient advocates, and representatives from pharmaceutical companies and the National Institutes of Health. Novel RASopathy genes, variants, and animal models were discussed in the context of medication trials and drug development. Attempts to define and measure meaningful endpoints for treatment trials were discussed, as was drug availability to patients after trial completion.

The cell-of-origin of high grade serous ovarian carcinoma (HGSC) has been a focus of debate. Here, by using genetic mouse models as well as cognate organoid systems, we assessed the tumor forming capacity and properties of the fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE) bearing the same oncogenic abnormalities. Combined RB family inactivation (via T121 expression) and Tp53 mutation in Pax8+ fallopian tube secretory cells causes transformation and characteristics of Serous Tubal Intraepithelial Carcinoma (STIC). This genetically engineered mouse HGSC model is faithfully recapitulated in fallopian organoids, from which serous ovarian cancer with broad abdominal metastasis is generated upon orthotopic injection. The same genetic events in Lgr5+ OSE cells organdies derived from these cells also result in an apparent neoplastic process, expressing markers of early serous carcinoma (but not Pax8), which subsequently develop into serous-like tumors. Hence, both Pax8+ fallopian tube epithelial cells and Lgr5+ ovarian surface epithelial cells can undergo similar neoplastic transformation, suggesting that HGSC might derive from distinct cell and tissue sources. Similar organoid systems can be used to rapidly model other gene combinations associated with HGSC

High grade serous ovarian cancer (HGSC) is typically diagnosed at an advanced stage and the vast majority of patients relapse and die within 5 years of diagnosis. Significant clinical problems in HGSC include wide-spread abdominal dissemination of disease and chemotherapy resistance. Cancer-associated fibroblasts (CAFs) have been shown to play a role in promotion of cancer cell proliferation and invasion, and mediation of chemotherapy resistance. To interrogate the molecular properties of CAFs in HGSC we used fluorescence activated cell sorting to isolate CAFs directly from primary tumor samples and performed gene expression profiling. We found that patients stratify into two classes based on their CAF gene signatures: One with high expression of Fibroblast Activation Protein (FAP-High; FH) and one with low expression of FAP (FAP-Low; FL). FH CAFs express classical CAF genes whereas FL CAFs possesses a preadipocyte-like molecular signature. The FL phenotype has remained largely unnoticed as it is generally out-competed in vitro by FH cells when grown under classical CAF culture conditions. Patients from The Cancer Genome Atlas (TCGA), as well as from our own institute, can be stratified into FH and FL subtypes; in both cohorts patients with FH CAFs have a significantly shorter disease-free and overall survival. In vitro and in vivo functional assays performed with isolated CAFs of both types indicate that FH CAFs aggressively promote proliferation, invasion and therapy resistance of cancer cells, whereas FL CAFs do not. Finally, we identified TCF21, a transcriptional repressor, as a FL-specific transcription factor. Analysis of published TCF21 ChIP-Seq data indicates that TCF21 targets a large number of genes specific to FH CAFs. Overexpression of TCF21 in FH CAFs partially reversed their ability to promote cancer cell invasion and tumor growth. Our discovery of CAF heterogeneity in HGSC highlights the need to personalize patient treatment with respect to both cancer and stromal phenotypes. FH patients may benefit from inhibition of cancer-stroma interactions or from epigenetic modulators that reprogram cancer-promoting FH CAFs into the non-supportive FL state

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.