Identification of acquired Notch3 dependency in metastatic Head and Neck Cancer.

During cancer development, tumor cells acquire changes that enable them to invade surrounding tissues and seed metastasis at distant sites. These changes contribute to the aggressiveness of metastatic cancer and interfere with success of therapy. Our comprehensive analysis of "matched" pairs of HNSCC lines derived from primary tumors and corresponding metastatic sites identified several components of Notch3 signaling that are differentially expressed and/or altered in metastatic lines and confer a dependency on this pathway. These components were also shown to be differentially expressed between early and late stages of tumors in a TMA constructed from over 200 HNSCC patients. Finally, we show that suppression of Notch3 improves survival in mice in both subcutaneous and orthotopic models of metastatic HNSCC. Novel treatments targeting components of this pathway may prove effective in targeting metastatic HNSCC cells alone or in combination with conventional therapies.

Impact of neoadjuvant chemotherapy on somatic mutation status in high-grade serous ovarian carcinoma.

BACKGROUND: Patients treated with neoadjuvant chemotherapy (NACT) for advanced high-grade serous ovarian carcinoma (HGSC) have a higher rate and shorter time to platinum-resistant recurrence compared to patients treated with primary cytoreductive surgery (PCS) and adjuvant chemotherapy. The purpose of this study is to determine the impact of NACT on somatic mutation status in platinum-sensitive and resistant HGSC. Patients with advanced HGSC who had a documented response to platinum-based NACT, a banked blood sample, and a banked tumor sample before and after NACT were identified. Whole exome and/or targeted deep sequencing was performed in matched normal and pre/post-NACT tumor samples from 3 platinum-resistant and 2 platinum-sensitive patients to identify somatic non-synonymous mutations at each time point. RESULTS: When comparing exonic non-synonymous mutations in pre-NACT and post-NACT samples from the same patient, an average of 41% (1-68%) of genes were mutated at both time points. There were no trends detected in the mutational burden following exposure to NACT in platinum-resistant vs. platinum-sensitive cases. The majority of mutated genes were unique to each case. We identified several genes that were commonly mutated in pre-NACT samples specific to platinum-resistant (CSPG4, SLC35G5, TUBA3D) or sensitive (CYP2D6, NUTM1, DNAH5) cases. Four mutated genes emerged exclusively in the platinum-resistant cases (ADGRV1, MUC17, MUC20, PAK2) following NACT. CONCLUSIONS: Patients with advanced HGSC present with significant intra-tumor heterogeneity. NACT significantly impacts the somatic mutation status irrespective of the time to recurrence. The mutated genes detected in chemo-naive pre-NACT tumor samples from either resistant or sensitive cases could potentially have a role in the prediction of chemotherapy response in patients scheduled to receive NACT; larger studies are required to further validate these genes.

Patient consent preferences on sharing personal health information during the COVID-19 pandemic: "the more informed we are, the more likely we are to help".

BACKGROUND: Rapid ethical access to personal health information (PHI) to support research is extremely important during pandemics, yet little is known regarding patient preferences for consent during such crises. This follow-up study sought to ascertain whether there were differences in consent preferences between pre-pandemic times compared to during Wave 1 of the COVID-19 global pandemic, and to better understand the reasons behind these preferences. METHODS: A total of 183 patients in the pandemic cohort completed the survey via email, and responses were compared to the distinct pre-pandemic cohort (n = 222); all were patients of a large Canadian cancer center. The survey covered (a) broad versus study-specific consent; (b) opt-in versus opt-out contact approach; (c) levels of comfort sharing with different recipients; (d) perceptions of commercialization; and (e) options to track use of information and be notified of results. Four focus groups (n = 12) were subsequently conducted to elucidate reasons motivating dominant preferences. RESULTS: Patients in the pandemic cohort were significantly more comfortable with sharing all information and biological samples (90% vs. 79%, p = 0.009), sharing information with the health care institution (97% vs. 83%, p < 0.001), sharing information with researchers at other hospitals (85% vs. 70%, p < 0.001), sharing PHI provincially (69% vs. 53%, p < 0.002), nationally (65% vs. 53%, p = 0.022) and internationally (48% vs. 39%, p = 0.024) compared to the pre-pandemic cohort. Discomfort with sharing information with commercial companies remained unchanged between the two cohorts (50% vs. 51% uncomfortable, p = 0.58). Significantly more pandemic cohort patients expressed a wish to track use of PHI (75% vs. 61%, p = 0.007), and to be notified of results (83% vs. 70%, p = 0.012). Thematic analysis uncovered that transparency was strongly desired on outside PHI use, particularly when commercialization was involved. CONCLUSIONS: In pandemic times, patients were more comfortable sharing information with all parties, except with commercial entities, where levels of discomfort (~ 50%) remained unchanged. Focus groups identified that the ability to track and receive results of studies using one's PHI is an important way to reduce discomfort and increase trust. These findings meaningfully inform wider discussions on the use of personal health information for research during global crises.

A clinical laboratory-developed LSC17 stemness score assay for rapid risk assessment of patients with acute myeloid leukemia.

Leukemia stem cells (LSCs) are linked to relapse in acute myeloid leukemia (AML). The LSC17 gene expression score robustly captures LSC stemness properties in AML and can be used to predict survival outcomes and response to therapy, enabling risk-adapted, upfront treatment approaches. The LSC17 score was developed and validated in a research setting. To enable widespread use of the LSC17 score in clinical decision making, we established a laboratory-developed test (LDT) for the LSC17 score that can be deployed broadly in clinical molecular diagnostic laboratories. We extensively validated the LSC17 LDT in a College of American Pathologists/Clinical Laboratory Improvements Act (CAP/CLIA)-certified laboratory, determining specimen requirements, a synthetic control, and performance parameters for the assay. Importantly, we correlated values from the LSC17 LDT to clinical outcome in a reference cohort of patients with AML, establishing a median assay value that can be used for clinical risk stratification of individual patients with newly diagnosed AML. The assay was established in a second independent CAP/CLIA-certified laboratory, and its technical performance was validated using an independent cohort of patient samples, demonstrating that the LSC17 LDT can be readily implemented in other settings. This study enables the clinical use of the LSC17 score for upfront risk-adapted management of patients with AML.

The Prevent Ovarian Cancer Program (POCP): Identification of women at risk for ovarian cancer using complementary recruitment approaches.

BACKGROUND: Up to 20% of high-grade serous ovarian carcinomas (HGSOC) are hereditary; however, historical uptake of genetic testing is low. We used a unique combination of approaches to identify women in Ontario, Canada, with a first-degree relative (FDR) who died from HGSOC without prior genetic testing, and offer them multi-gene panel testing. METHODS: From May 2015-Sept 2019, genetic counseling and testing was provided to eligible participants. Two recruitment strategies were employed, including self-identification in response to an outreach campaign and direct targeting of FDRs of deceased HGSOC patients treated at our institution. The rate of pathogenic variants (PV) in established/potential ovarian cancer risk genes and the benefits/challenges of each approach were assessed. RESULTS: A total of 564 women enrolled in response to our outreach campaign (n = 473) or direct recruitment (n = 91). Mean age at consent was 52 years and 96% did not meet provincial testing criteria. Genetic results were provided to 528 individuals from 458 families. The rate of PVs in ovarian cancer risk genes was highest when FDRs were diagnosed with HGSOC <60 years (9.4% vs. 3.9% ≥ 60y, p = 0.0160). Participants in the outreach vs. direct recruitment cohort had a similar rate of PVs; however, uptake of genetic testing (97% vs. 89%; p = 0.0036) and study completion (95% vs. 87%; p = 0.0062) rates were higher in the former. Eleven participants with pathogenic variants have completed risk-reducing gynecologic surgery, with one stage I HGSOC and two breast cancers identified. CONCLUSION: Overall PV rates in this large cohort were lower than expected; however, we provide evidence that genetic testing criteria in Ontario should include individuals with a deceased FDR diagnosed with HGSOC <60 years of age.

Implementation and Outcomes of Virtual Care Across a Tertiary Cancer Center During COVID-19.

Importance: The coronavirus disease 2019 (COVID-19) pandemic has burdened health care resources and disrupted care of patients with cancer. Virtual care (VC) represents a potential solution. However, few quantitative data support its rapid implementation and positive associations with service capacity and quality. Objective: To examine the outcomes of a cancer center-wide virtual care program in response to the COVID-19 pandemic. Design, Setting, and Participants: This cohort study applied a hospitalwide agile service design to map gaps and develop a customized digital solution to enable at-scale VC across a publicly funded comprehensive cancer center. Data were collected from a high-volume cancer center in Ontario, Canada, from March 23 to May 22, 2020. Main Outcomes and Measures: Outcome measures were care delivery volumes, quality of care, patient and practitioner experiences, and cost savings to patients. Results: The VC solution was developed and launched 12 days after the declaration of the COVID-19 pandemic. A total of 22 085 VC visits (mean, 514 visits per day) were conducted, comprising 68.4% (range, 18.8%-100%) of daily visits compared with 0.8% before launch (P < .001). Ambulatory clinic volumes recovered a month after deployment (3714-4091 patients per week), whereas chemotherapy and radiotherapy caseloads (1943-2461 patients per week) remained stable throughout. No changes in institutional or provincial quality-of-care indexes were observed. A total of 3791 surveys (3507 patients and 284 practitioners) were completed; 2207 patients (82%) and 92 practitioners (72%) indicated overall satisfaction with VC. The direct cost of this initiative was CAD$ 202 537, and displacement-related cost savings to patients totaled CAD$ 3 155 946. Conclusions and Relevance: These findings suggest that implementation of VC at scale at a high-volume cancer center may be feasible. An agile service design approach was able to preserve outpatient caseloads and maintain care quality, while rendering high patient and practitioner satisfaction. These findings may help guide the transformation of telemedicine in the post COVID-19 era.

Myxoid smooth muscle neoplasia of the uterus: comprehensive analysis by next-generation sequencing and nucleic acid hybridization.

Uterine myxoid smooth muscle tumors, including myxoid leiomyosarcoma, are rare and their genomic profile has not been fully characterized. With the discovery of uterine sarcomas with ZC3H7B-BCOR fusion and BCOR internal tandem duplications, the differential diagnosis of myxoid smooth muscle lesions is expanding to include molecularly-defined tumors. Thus, we aimed to explore the genomic landscape of myxoid smooth muscle tumor using comprehensive tools. We performed whole exome next-generation sequencing and a pan-sarcoma RNA fusion assay in tumoral paraffin-embedded tissue from nine well-characterized uterine myxoid smooth muscle tumors (seven myxoid leiomyosarcomas and two myxoid smooth muscle tumors of unknown malignant potential). By immunohistochemistry, all tumors were strongly positive for smooth muscle markers and negative for BCOR staining; 4/6 expressed PLAG1. None of the tumors harbored known fusions including ZC3H7B-BCOR, TRPS1-PLAG1, and RAD51B-PLAG1. None harbored exon 15 BCOR internal tandem duplications; however, four tumors contained BCOR internal tandem duplications of unknown significance (mostly intronic). Mutational burden was low (median 3.8 mutations/megabase). DNA damage repair pathway gene mutations, including TP53 and BRCA2, were found. Copy number variation load, inferred from sequencing data, was variable with genomic indexes ranging from 2.2 to 74.7 (median 25.7), with higher indexes in myxoid leiomyosarcomas than myxoid smooth muscle tumors of unknown malignant potential. The absence of clear driver mutations suggests myxoid smooth muscle tumors to be genetically heterogeneous group of tumours and that other genetic (eg., undiscovered translocation) or epigenetic events drive the pathogenesis of uterine myxoid smooth muscle neoplasia.

Somatic Tumor Variant Filtration Strategies to Optimize Tumor-Only Molecular Profiling Using Targeted Next-Generation Sequencing Panels.

A common approach in clinical diagnostic laboratories to variant assessment from tumor molecular profiling is sequencing of genomic DNA extracted from both tumor (somatic) and normal (germline) tissue, with subsequent variant comparison to identify true somatic variants with potential impact on patient treatment or prognosis. However, challenges exist in paired tumor-normal testing, including increased cost of dual sample testing and identification of germline cancer predisposing variants. Alternatively, somatic variants can be identified by in silico tumor-only variant filtration precluding the need for matched normal testing. The barrier to tumor-only variant filtration is defining a reliable approach, with high sensitivity and specificity to identify somatic variants. In this study, we used retrospective data sets from paired tumor-normal samples tested on small (48 gene) and large (555 gene) targeted next-generation sequencing panels, to model algorithms for tumor-only variants classification. The optimal algorithm required an ordinal filtering approach using information from variant population databases (1000 Genomes Phase 3, ESP6500, ExAC), clinical mutation databases (ClinVar), and information on recurring clinically relevant somatic variants. Overall the tumor-only variant filtration strategy described in this study can define clinically relevant somatic variants from tumor-only analysis with sensitivity of 97% to 99% and specificity of 87% to 94%, and with significant potential utility for clinical laboratories implementing tumor-only molecular profiling.

BRCA1 Mutation Status and Follicular Fluid Exposure Alters NFκB Signaling and ISGylation in Human Fallopian Tube Epithelial Cells.

Germline BRCA1 or BRCA2 mutations (mtBRCA1 and mtBRCA2) increase risk for high-grade serous ovarian cancer (HGSOC), the most commonly diagnosed epithelial ovarian cancer histotype. Other identified risk factors for this cancer, which originates primarily in the distal fallopian tube epithelium (FTE), implicate ovulation, during which the FTE cells become transiently exposed to follicular fluid (FF). To test whether mtBRCA1 or mtBRCA2 nonmalignant FTE cells respond differently to periovulatory FF exposure than control patient FTE cells, gene expression profiles from primary FTE cultures derived from BRCA1 or BRCA2 mutation carriers or control patients were compared at baseline, 24 hours after FF exposure, and 24 hours after FF replacement with culture medium. Hierarchical clustering revealed both FF exposure and BRCA mutation status affect gene expression, with BRCA1 mutation having the greatest impact. Gene set enrichment analysis revealed increased NFκB and EGFR signaling at baseline in mtBRCA1 samples, with increased interferon target gene expression, including members of the ISGylation pathway, observed after recovery from FF exposure. Gene set enrichment analysis did not identify altered pathway signaling in mtBRCA2 samples. An inverse relationship between EGFR signaling and ISGylation with BRCA1 protein levels was verified in an immortalized FTE cell line, OE-E6/E7, stably transfected with BRCA1 cDNA. Suppression of ISG15 and ISGylated protein levels by increased BRCA1 expression was found to be mediated by decreased NFκB signaling. These studies indicate that increased NFκB signaling associated with decreased BRCA1 expression results in increased ISG15 and protein ISGylation following FF exposure, which may be involved in predisposition to HGSOC.

Gene expression signatures prognostic for relapse in stage I testicular germ cell tumours.

OBJECTIVES: To identify differentially expressed genes between relapsed and non-relapsed clinical stage I testicular germ cell tumours (TGCTs). MATERIALS AND METHODS: We reviewed patients with clinical stage I non-seminoma and seminoma from an institutional database (2000-2012) who were managed by active surveillance. Patients with non-relapsed non-seminoma and non-relapsed seminoma were defined as being relapse-free after 2 and 3 years of surveillance, respectively. RNA extraction and gene expression analysis was performed on archival primary tumour samples and gene-set enrichment analysis (GSEA) was conducted in order to identify differentiating biological pathways. RESULTS: A total of 57 patients (relapsed non-seminoma, n = 12; relapsed seminoma, n =15; non-relapsed non-seminoma, n = 15; non-relapsed seminoma, n = 15) were identified, with a median (range) relapse time of 5.6 (2.5-18.1) and 19.3 (4.7-65.3) months in the relapsed non-seminoma and relapsed seminoma cohorts, respectively. A total of 1 039 differentially expressed genes were identified that separated relapsed and non-relapsed groups. In patients with relapse, GSEA revealed enrichment in pathways associated with differentiation, such as skeletal development (i.e. FGFR1, BMP4, GLI2, SPARC, COL2A1), tissue (i.e. BMP4, SPARC, COL13A1) and bone remodelling (i.e. CARTPT, GLI2, MGP). A discriminative gene expression profile between relapsed and non-relapsed cases was discovered when combining non-seminoma and seminoma samples using 10- and 30-probe signatures; however, this profile was not observed in the seminoma and non-seminoma cohorts individually. CONCLUSION: A discriminating signature for relapsed disease was identified for clinical stage I TGCT that we were not able to identify by histology alone. Further validation is required to determine if this signature provides independent prognostic information to standard pathological risk factors.

A Genomically Characterized Collection of High-Grade Serous Ovarian Cancer Xenografts for Preclinical Testing.

High-grade serous ovarian cancer (HGSC) is the leading cause of morbidity and mortality from gynecologic malignant tumors. Overall survival remains low because of the nearly ubiquitous emergence of platinum resistance and the paucity of effective next-line treatments. Current cell culture-based models show limited similarity to HGSC and are therefore unreliable predictive models for preclinical evaluation of investigational drugs. This deficiency could help explain the low overall rate of successful drug development and the decades of largely unchanged approaches to HGSC treatment. We used gene expression, copy number variation, and exome sequencing analyses to credential HGSC patient-derived xenografts (PDXs) as effective preclinical models that recapitulate the features of human HGSC. Mice bearing PDXs were also treated with standard-of-care carboplatin therapy. PDXs showed similar sensitivity to carboplatin as the patient's tumor at the time of sampling. PDXs also recapitulated the diversity of genomic alterations (copy number variation and mutation profiles) previously described in large data sets that profiled HGSC. Furthermore, mRNA profiling showed that the PDXs represent all HGSC subtypes with the exception of the immunoreactive group. Credentialing of PDX models of HGSC should aid progress in HGSC research by providing improved preclinical models of HGSC that can be used to test novel targets and more accurately evaluate their likelihood of success.

Exome-Wide Association Study of Pancreatic Cancer Risk.

We conducted a case-control exome-wide association study to discover germline variants in coding regions that affect risk for pancreatic cancer, combining data from 5 studies. We analyzed exome and genome sequencing data from 437 patients with pancreatic cancer (cases) and 1922 individuals not known to have cancer (controls). In the primary analysis, BRCA2 had the strongest enrichment for rare inactivating variants (17/437 cases vs 3/1922 controls) (P = 3.27x10-6; exome-wide statistical significance threshold P < 2.5x10-6). Cases had more rare inactivating variants in DNA repair genes than controls, even after excluding 13 genes known to predispose to pancreatic cancer (adjusted odds ratio, 1.35; P = .045). At the suggestive threshold (P < .001), 6 genes were enriched for rare damaging variants (UHMK1, AP1G2, DNTA, CHST6, FGFR3, and EPHA1) and 7 genes had associations with pancreatic cancer risk, based on the sequence-kernel association test. We confirmed variants in BRCA2 as the most common high-penetrant genetic factor associated with pancreatic cancer and we also identified candidate pancreatic cancer genes. Large collaborations and novel approaches are needed to overcome the genetic heterogeneity of pancreatic cancer predisposition.

SOX2 and PI3K Cooperate to Induce and Stabilize a Squamous-Committed Stem Cell Injury State during Lung Squamous Cell Carcinoma Pathogenesis.

Although cancers are considered stem cell diseases, mechanisms involving stem cell alterations are poorly understood. Squamous cell carcinoma (SQCC) is the second most common lung cancer, and its pathogenesis appears to hinge on changes in the stem cell behavior of basal cells in the bronchial airways. Basal cells are normally quiescent and differentiate into mucociliary epithelia. Smoking triggers a hyperproliferative response resulting in progressive premalignant epithelial changes ranging from squamous metaplasia to dysplasia. These changes can regress naturally, even with chronic smoking. However, for unknown reasons, dysplasias have higher progression rates than earlier stages. We used primary human tracheobronchial basal cells to investigate how copy number gains in SOX2 and PIK3CA at 3q26-28, which co-occur in dysplasia and are observed in 94% of SQCCs, may promote progression. We find that SOX2 cooperates with PI3K signaling, which is activated by smoking, to initiate the squamous injury response in basal cells. This response involves SOX9 repression, and, accordingly, SOX2 and PI3K signaling levels are high during dysplasia, while SOX9 is not expressed. By contrast, during regeneration of mucociliary epithelia, PI3K signaling is low and basal cells transiently enter a SOX2LoSOX9Hi state, with SOX9 promoting proliferation and preventing squamous differentiation. Transient reduction in SOX2 is necessary for ciliogenesis, although SOX2 expression later rises and drives mucinous differentiation, as SOX9 levels decline. Frequent coamplification of SOX2 and PIK3CA in dysplasia may, thus, promote progression by locking basal cells in a SOX2HiSOX9Lo state with active PI3K signaling, which sustains the squamous injury response while precluding normal mucociliary differentiation. Surprisingly, we find that, although later in invasive carcinoma SOX9 is generally expressed at low levels, its expression is higher in a subset of SQCCs with less squamous identity and worse clinical outcome. We propose that early pathogenesis of most SQCCs involves stabilization of the squamous injury state in stem cells through copy number gains at 3q, with the pro-proliferative activity of SOX9 possibly being exploited in a subset of SQCCs in later stages.

Molecular profiling of advanced solid tumors and patient outcomes with genotype-matched clinical trials: the Princess Margaret IMPACT/COMPACT trial.

BACKGROUND: The clinical utility of molecular profiling of tumor tissue to guide treatment of patients with advanced solid tumors is unknown. Our objectives were to evaluate the frequency of genomic alterations, clinical "actionability" of somatic variants, enrollment in mutation-targeted or other clinical trials, and outcome of molecular profiling for advanced solid tumor patients at the Princess Margaret Cancer Centre (PM). METHODS: Patients with advanced solid tumors aged ≥18 years, good performance status, and archival tumor tissue available were prospectively consented. DNA from archival formalin-fixed paraffin-embedded tumor tissue was tested using a MALDI-TOF MS hotspot panel or a targeted next generation sequencing (NGS) panel. Somatic variants were classified according to clinical actionability and an annotated report included in the electronic medical record. Oncologists were provided with summary tables of their patients' molecular profiling results and available mutation-specific clinical trials. Enrolment in genotype-matched versus genotype-unmatched clinical trials following release of profiling results and response by RECIST v1.1 criteria were evaluated. RESULTS: From March 2012 to July 2014, 1893 patients were enrolled and 1640 tested. After a median follow-up of 18 months, 245 patients (15 %) who were tested were subsequently treated on 277 therapeutic clinical trials, including 84 patients (5 %) on 89 genotype-matched trials. The overall response rate was higher in patients treated on genotype-matched trials (19 %) compared with genotype-unmatched trials (9 %; p < 0.026). In a multi-variable model, trial matching by genotype (p = 0.021) and female gender (p = 0.034) were the only factors associated with increased likelihood of treatment response. CONCLUSIONS: Few advanced solid tumor patients enrolled in a prospective institutional molecular profiling trial were treated subsequently on genotype-matched therapeutic trials. In this non-randomized comparison, genotype-enrichment of early phase clinical trials was associated with an increased objective tumor response rate. TRIAL REGISTRATION: NCT01505400 (date of registration 4 January 2012).

PTP1B controls non-mitochondrial oxygen consumption by regulating RNF213 to promote tumour survival during hypoxia.

Tumours exist in a hypoxic microenvironment and must limit excessive oxygen consumption. Hypoxia-inducible factor (HIF) controls mitochondrial oxygen consumption, but how/if tumours regulate non-mitochondrial oxygen consumption (NMOC) is unknown. Protein-tyrosine phosphatase-1B (PTP1B) is required for Her2/Neu-driven breast cancer (BC) in mice, although the underlying mechanism and human relevance remain unclear. We found that PTP1B-deficient HER2(+) xenografts have increased hypoxia, necrosis and impaired growth. In vitro, PTP1B deficiency sensitizes HER2(+) BC lines to hypoxia by increasing NMOC by α-KG-dependent dioxygenases (α-KGDDs). The moyamoya disease gene product RNF213, an E3 ligase, is negatively regulated by PTP1B in HER2(+) BC cells. RNF213 knockdown reverses the effects of PTP1B deficiency on α-KGDDs, NMOC and hypoxia-induced death of HER2(+) BC cells, and partially restores tumorigenicity. We conclude that PTP1B acts via RNF213 to suppress α-KGDD activity and NMOC. This PTP1B/RNF213/α-KGDD pathway is critical for survival of HER2(+) BC, and possibly other malignancies, in the hypoxic tumour microenvironment.

Ovarian tumour growth is characterized by mevalonate pathway gene signature in an orthotopic, syngeneic model of epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer and often is not detected until late stages when cancer cells transcoelomically metastasize to the abdomen and typically become resistant to therapy resulting in very low survival rates. We utilize an orthotopic, syngeneic mouse model to study late stage disease and have discovered that the tumor cells within the abdominal ascites are irreversibly re-programmed, with an increased tumorigenicity and resistance to apoptosis. The goal of this study was to characterize the reprogramming that occurred in the aggressive ascites-derived cells (28-2 cells) compared to the original cell line used for tumor induction (ID8 cells). Microarray experiments showed that the majority of genes upregulated in the 28-2 cells belonged to the mevalonate pathway, which is involved in cholesterol biosynthesis, protein prenylation, and activation of small GTPases. Upregulation of mevalonate appeared to be associated with the acquisition of a p53 mutation in the ascites-derived cells. Treatment with simvastatin to inhibit HMG CoA reductase, the rate limiting enzyme of this pathway, induced apoptosis in the 28-2 cell line. Rescue experiments revealed that mevalonate, but not cholesterol, could inhibit the simvastatin-mediated effects. In vivo, daily intraperitoneal simvastatin treatment significantly regressed advanced stage disease and induced death of metastatic tumor cells. These data suggest that ovarian cancer cells become reprogrammed, with genetic mutations, and upregulation of the mevalonate pathway, which facilitates the development of advanced stage disease. The use of statins to inhibit HMGCR may provide novel therapeutic opportunities for the treatment of advanced stage EOC.

Functional Genomic Landscape of Human Breast Cancer Drivers, Vulnerabilities, and Resistance.

Large-scale genomic studies have identified multiple somatic aberrations in breast cancer, including copy number alterations and point mutations. Still, identifying causal variants and emergent vulnerabilities that arise as a consequence of genetic alterations remain major challenges. We performed whole-genome small hairpin RNA (shRNA) "dropout screens" on 77 breast cancer cell lines. Using a hierarchical linear regression algorithm to score our screen results and integrate them with accompanying detailed genetic and proteomic information, we identify vulnerabilities in breast cancer, including candidate "drivers," and reveal general functional genomic properties of cancer cells. Comparisons of gene essentiality with drug sensitivity data suggest potential resistance mechanisms, effects of existing anti-cancer drugs, and opportunities for combination therapy. Finally, we demonstrate the utility of this large dataset by identifying BRD4 as a potential target in luminal breast cancer and PIK3CA mutations as a resistance determinant for BET-inhibitors.

Distinct GAB2 signaling pathways are essential for myeloid and lymphoid transformation and leukemogenesis by BCR-ABL1.

Tyrosine kinase inhibitors (TKIs) directed against BCR-ABL1, the product of the Philadelphia (Ph) chromosome, have revolutionized treatment of patients with chronic myeloid leukemia (CML). However, acquired resistance to TKIs is a significant clinical problem in CML, and TKI therapy is much less effective against Ph(+)B-cell acute lymphoblastic leukemia (B-ALL). BCR-ABL1, via phosphorylated Tyr177, recruits the adapter GRB2-associated binding protein 2 (GAB2) as part of a GRB2/GAB2 complex. We showed previously that GAB2 is essential for BCR-ABL1-evoked myeloid transformation in vitro. Using a genetic strategy and mouse models of CML and B-ALL, we show here that GAB2 is essential for myeloid and lymphoid leukemogenesis by BCR-ABL1. In the mouse model, recipients of BCR-ABL1-transducedGab2(-/-)bone marrow failed to develop CML-like myeloproliferative neoplasia. Leukemogenesis was restored by expression of GAB2 but not by GAB2 mutants lacking binding sites for its effectors phosphatidylinositol 3-kinase (PI3K) or SRC homology 2-containing phosphotyrosine phosphatase 2 (SHP2). GAB2 deficiency also attenuated BCR-ABL1-induced B-ALL, but only the SHP2 binding site was required. The SHP2 and PI3K binding sites were differentially required for signaling downstream of GAB2. Hence, GAB2 transmits critical transforming signals from Tyr177 to PI3K and SHP2 for CML pathogenesis, whereas only the GAB2-SHP2 pathway is essential for lymphoid leukemogenesis. Given that GAB2 is dispensable for normal hematopoiesis, GAB2 and its effectors PI3K and SHP2 represent promising targets for therapy in Ph(+)hematologic neoplasms.

Activating Mutations Affecting the Dbl Homology Domain of SOS2 Cause Noonan Syndrome.

The RASopathies constitute a family of autosomal-dominant disorders whose major features include facial dysmorphism, cardiac defects, reduced postnatal growth, variable cognitive deficits, ectodermal and skeletal anomalies, and susceptibility to certain malignancies. Noonan syndrome (NS), the commonest RASopathy, is genetically heterogeneous and caused by functional dysregulation of signal transducers and regulatory proteins with roles in the RAS/extracellular signal-regulated kinase (ERK) signal transduction pathway. Mutations in known disease genes account for approximately 80% of affected individuals. Here, we report that missense mutations altering Son of Sevenless, Drosophila, homolog 2 (SOS2), which encodes a RAS guanine nucleotide exchange factor, occur in a small percentage of subjects with NS. Four missense mutations were identified in five unrelated sporadic cases and families transmitting NS. Disease-causing mutations affected three conserved residues located in the Dbl homology (DH) domain, of which two are directly involved in the intramolecular binding network maintaining SOS2 in its autoinhibited conformation. All mutations were found to promote enhanced signaling from RAS to ERK. Similar to NS-causing SOS1 mutations, the phenotype associated with SOS2 defects is characterized by normal development and growth, as well as marked ectodermal involvement. Unlike SOS1 mutations, however, those in SOS2 are restricted to the DH domain.

Human ortholog of Drosophila Melted impedes SMAD2 release from TGF-ß receptor I to inhibit TGF-ß signaling.

Drosophila melted encodes a pleckstrin homology (PH) domain-containing protein that enables normal tissue growth, metabolism, and photoreceptor differentiation by modulating Forkhead box O (FOXO), target of rapamycin, and Hippo signaling pathways. Ventricular zone expressed PH domain-containing 1 (VEPH1) is the mammalian ortholog of melted, and although it exhibits tissue-restricted expression during mouse development and is potentially amplified in several human cancers, little is known of its function. Here we explore the impact of VEPH1 expression in ovarian cancer cells by gene-expression profiling. In cells with elevated VEPH1 expression, transcriptional programs associated with metabolism and FOXO and Hippo signaling were affected, analogous to what has been reported for Melted. We also observed altered regulation of multiple transforming growth factor-ß (TGF-ß) target genes. Global profiling revealed that elevated VEPH1 expression suppressed TGF-ß-induced transcriptional responses. This inhibitory effect was verified on selected TGF-ß target genes and by reporter gene assays in multiple cell lines. We further demonstrated that VEPH1 interacts with TGF-ß receptor I (TßRI) and inhibits nuclear accumulation of activated Sma- and Mad-related protein 2 (SMAD2). We identified two TßRI-interacting regions (TIRs) with opposing effects on TGF-ß signaling. TIR1, located at the N terminus, inhibits canonical TGF-ß signaling and promotes SMAD2 retention at TßRI, similar to full-length VEPH1. In contrast, TIR2, located at the C-terminal region encompassing the PH domain, decreases SMAD2 retention at TßRI and enhances TGF-ß signaling. Our studies indicate that VEPH1 inhibits TGF-ß signaling by impeding the release of activated SMAD2 from TßRI and may modulate TGF-ß signaling during development and cancer initiation or progression.