Faculty Bibliography

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.

Immunotherapy in ovarian cancer has been disappointing, with only ~10% of patients responding to checkpoint blockade. The determinants of this low response rate remain poorly understood, and there is a pressing need for immune-competent preclinical models to elucidate the biology of immune evasion in ovarian cancer. One critical area of interest is the role of homologous recombination (HR) repair in immune evasion. The types and abundance of potential antigens present on cancer cells may depend on the genotype of the tumor, its mutational burden, and the cellular state. Unfortunately, the preclinical tools required to explore the relationship between the types of DNA damage repair deficiencies and immune evasion have been lacking. 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 co-occurring mutations observed in homologous recombination-deficient and -proficient patient samples. These models can identify the contribution of common driver mutations, which are TP53, BRCA1, PTEN, Myc, Cyclin E1 (CCNE1), Akt2, and Kras, to the heterotypic interactions between cancer and stromal/immune compartments and examine how DNA repair proficiency contributes to immunogenicity. To validate the DNA repair proficiency of the transformed cells, we measured Rad51 nuclear focus formation after ionizing radiation (IR) and PARP inhibitor 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, single-cell RNA sequencing, and transcriptomic and immunofluorescence analysis revealed substantial differences in the myeloid and regulatory compartments between HR-proficient and -deficient tumors within the primary and metastatic tumors themselves and within the ascitic fluid. Preliminary results also suggest that inhibition of the DNA damage response (DDR), checkpoint kinase 1 (Chk1) in combination with immune checkpoint inhibitors, potentiates antitumor effects and augments cytotoxic T-cell infiltration. In conclusion, these results reveal how common mutational drivers, and particularly those associated with HR status, 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

High-grade serous ovarian cancer (HGSOC) is the most common, deadly subtype of ovarian epithelial cancer. HGSOC typically presents at an advanced stage, with widespread peritoneal metastasis. Surgical debulking and platinum/taxane-based chemotherapy can result in complete responses, but disease almost always recurs, eventually in drug-resistant form. The recent addition of Avastin and PARP inhibitors benefits a subset of patients, yet survival has improved marginally in the past 30 years. Responses to immune checkpoint inhibitors have been disappointing. Genomic studies (e.g., TCGA) show that HGSOC is a complex, diverse disease with widespread copy number abnormalities affecting multiple pathways. Nevertheless, most studies aimed at developing new therapies treat HGSOC as a single entity. The absence of genetically relevant, immune-competent HGSOC models poses a major barrier to developing new therapies. By exploiting a mouse fallopian tube organoid system that we developed, along with lentiviral gene transduction and/or CRISPR/Cas9 technology, we generated multiple new HGSOC models containing combinations of mutations seen in the human disease, including homologous recombination (HR)-proficient (Tp53-/-;Ccne1OE;Akt2OE and Tp53-/-;Ccne1OE;KrasOE) -deficient (Tp53-/-;Brca1-/-;Pten-/-and Tp53-/-; Brca1-/-;MycOE and unclassified (Tp53-/-;Pten-/-;Nf1-/-) models. These cells differ in proliferation, differentiation, and polarity/organoid structure in vitro, as well as tumorigenicity and metastatic spread upon orthotopic injection into syngeneic mice. Organoids with different mutations show differential sensitivity to current HGSOC drugs and evoke distinctly different immune microenvironments in vivo. Brca1-/- and CcneOE tumors show significant T-cell infiltration/Treg cells; the latter also have increased myeloid-derived suppressor cells (MDSCs). Pten-/- tumors have lower T-cell infiltration but high levels of MDSCs and macrophages. To test the utility of this platform for new therapeutic development, we focused on CcneOE tumors, because human CCNE-amplified HGSOC responds poorly to current therapy. CcneOE organoids were sensitive to gemcitabine (GEM), probably due to CCNE-evoked replication stress. GEM treatment reduced pro-MDSC cytokines in organoid-conditioned media, and decreased tumor burden in mice. Consistent with these in vitro data, residual tumors had decreased MDSCs (CD11b+Ly6CloLy6Ghi), but persistent Treg (CD24+CD25+FoxP3+) and "exhausted" CD8 T cells (CD8+PD1+). Intriguingly, a combination regimen aimed at attacking tumor cells (gemcitabine) and the residual microenvironment (CTLA4 and PDL1 antibodies) produced complete responses in mice with widespread metastatic disease. Our results argue that genotype-informed combination therapies will be necessary for better outcomes for this devastating disease

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.

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.

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.

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)

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

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