Comprehensive characterization of a Canadian cohort of von Hippel-Lindau disease patients.

Von Hippel-Lindau disease (VHL) is a heritable condition caused by pathogenic variants in VHL and is characterized by benign and malignant lesions in the central nervous system (CNS) and abdominal viscera. Due to its variable expressivity, existing efforts to collate VHL patient data do not adequately capture all VHL manifestations. We developed a comprehensive and standardized VHL database in the web-based application, REDCap, that thoroughly captures all VHL manifestation data. As an initial trial, information from 86 VHL patients from the University Health Network/Hospital for Sick Children was populated into the database. Analysis of this cohort showed missense variants occurring with the greatest frequency, with all variants localizing to the α- or ß-domains of VHL. The most prevalent manifestations were central nervous system (CNS), renal, and retinal neoplasms, which were associated with frameshift variants and large deletions. We observed greater age-related penetrance for CNS hemangioblastomas with truncating variants compared to missense, while the reverse was true for pheochromocytomas. We demonstrate the utility of a comprehensive VHL database, which supports the standardized collection of clinical and genetic data specific to this patient population. Importantly, we expect that its web-based design will facilitate broader international collaboration and lead to a better understanding of VHL.

The Chromatin Regulator Ankrd11 Controls Palate and Cranial Bone Development.

Epigenetic and chromatin regulation of craniofacial development remains poorly understood. Ankyrin Repeat Domain 11 (ANKRD11) is a chromatin regulator that has previously been shown to control neural stem cell fates via modulation of histone acetylation. ANKRD11 gene variants, or microdeletions of the 16q24.3 chromosomal region encompassing the ANKRD11 gene, cause KBG syndrome, a rare autosomal dominant congenital disorder with variable neurodevelopmental and craniofacial involvement. Craniofacial abnormalities include a distinct facial gestalt, delayed bone age, tooth abnormalities, delayed fontanelle closure, and frequently cleft or submucosal palate. Despite this, the dramatic phenotype and precise role of ANKRD11 in embryonic craniofacial development remain unexplored. Quantitative analysis of 3D images of KBG syndromic subjects shows an overall reduction in the size of the middle and lower face. Here, we report that mice with heterozygous deletion of Ankrd11 in neural crest cells (Ankrd11nchet) display a mild midfacial hypoplasia including reduced midfacial width and a persistent open fontanelle, both of which mirror KBG syndrome patient facial phenotypes. Mice with a homozygous Ankrd11 deletion in neural crest cells (Ankrd11ncko) die at birth. They show increased severity of several clinical manifestations described for KBG syndrome, such as cleft palate, retrognathia, midfacial hypoplasia, and reduced calvarial growth. At E14.5, Ankrd11 expression in the craniofacial complex is closely associated with developing bony structures, while expression at birth is markedly decreased. Conditional deletion of Ankrd11 leads to a reduction in ossification of midfacial bones, with several ossification centers failing to expand and/or fuse. Intramembranous bones show features of delayed maturation, with bone remodeling severely curtailed at birth. Palatal shelves remain hypoplastic at all developmental stages, with a local reduction in proliferation at E13.5. Our study identifies Ankrd11 as a critical regulator of intramembranous ossification and palate development and suggests that Ankrd11nchet and Ankrd11ncko mice may serve as pre-clinical models for KBG syndrome in humans.

Single allele loss-of-function mutations select and sculpt conditional cooperative networks in breast cancer.

The most common events in breast cancer (BC) involve chromosome arm losses and gains. Here we describe identification of 1089 gene-centric common insertion sites (gCIS) from transposon-based screens in 8 mouse models of BC. Some gCIS are driver-specific, others driver non-specific, and still others associated with tumor histology. Processes affected by driver-specific and histology-specific mutations include well-known cancer pathways. Driver non-specific gCIS target the Mediator complex, Ca++ signaling, Cyclin D turnover, RNA-metabolism among other processes. Most gCIS show single allele disruption and many map to genomic regions showing high-frequency hemizygous loss in human BC. Two gCIS, Nf1 and Trps1, show synthetic haploinsufficient tumor suppressor activity. Many gCIS act on the same pathway responsible for tumor initiation, thereby selecting and sculpting just enough and just right signaling. These data highlight ~1000 genes with predicted conditional haploinsufficient tumor suppressor function and the potential to promote chromosome arm loss in BC.

NF1 Patients Receiving Breast Cancer Screening: Insights from The Ontario High Risk Breast Screening Program.

Neurofibromatosis Type I (NF1) is caused by variants in neurofibromin (NF1). NF1 predisposes to a variety of benign and malignant tumor types, including breast cancer. Women with NF1 <50 years of age possess an up to five-fold increased risk of developing breast cancer compared with the general population. Impaired emotional functioning is reported as a comorbidity that may influence the participation of NF1 patients in regular clinical surveillance despite their increased risk of breast and other cancers. Despite emphasis on breast cancer surveillance in women with NF1, the uptake and feasibility of high-risk screening programs in this population remains unclear. A retrospective chart review between 2014-2018 of female NF1 patients seen at the Elizabeth Raab Neurofibromatosis Clinic (ERNC) in Ontario was conducted to examine the uptake of high-risk breast cancer screening, radiologic findings, and breast cancer characteristics. 61 women with pathogenic variants in NF1 enrolled in the high-risk Ontario breast screening program (HR-OBSP); 95% completed at least one high-risk breast screening modality, and four were diagnosed with invasive breast cancer. Our findings support the integration of a formal breast screening programs in clinical management of NF1 patients.

shRNA kinome screen identifies TBK1 as a therapeutic target for HER2+ breast cancer.

HER2(+) breast cancer is currently treated with chemotherapy plus anti-HER2 inhibitors. Many patients do not respond or relapse with aggressive metastatic disease. Therefore, there is an urgent need for new therapeutics that can target HER2(+) breast cancer and potentiate the effect of anti-HER2 inhibitors, in particular those that can target tumor-initiating cells (TIC). Here, we show that MMTV-Her2/Neu mammary tumor cells cultured as nonadherent spheres or as adherent monolayer cells select for stabilizing mutations in p53 that "immortalize" the cultures and that, after serial passages, sphere conditions maintain TICs, whereas monolayer cells gradually lose these tumorigenic cells. Using tumorsphere formation as surrogate for TICs, we screened p53-mutant Her2/Neu(+) tumorsphere versus monolayer cells with a lentivirus short hairpin RNA kinome library. We identified kinases such as the mitogen-activated protein kinase and the TGFßR protein family, previously implicated in HER2(+) breast cancer, as well as autophagy factor ATG1/ULK1 and the noncanonical IκB kinase (IKK), TANK-binding kinase 1 (TBK1), which have not been previously linked to HER2(+) breast cancer. Knockdown of TBK1 or pharmacologic inhibition of TBK1 and the related protein, IKKε, suppressed growth of both mouse and human HER2(+) breast cancer cells. TBK1/IKKε inhibition promoted cellular senescence by suppressing p65-NF-κB and inducing p16(Ink4a). In addition, TBK1/IKKε inhibition cooperated with lapatinib, a HER2/EGFR1-targeted drug, to accelerate apoptosis and kill HER2(+) breast cancer cells both in culture and in xenografts. Our results suggest that patients with HER2(+) breast cancer may benefit from anti-TBK1/IKKε plus anti-HER2 combination therapies and establish conditions that can be used to screen for additional TIC-specific inhibitors of HER2(+) breast cancer.

Elevated PI3K signaling drives multiple breast cancer subtypes.

Most human breast tumors have mutations that elevate signaling through a key metabolic pathway that is induced by insulin and a number of growth factors. This pathway serves to activate an enzyme known as phosphatidylinositol 3' kinase (PI3K) as well as to regulate proteins that signal in response to lipid products of PI3K. The specific mutations that activate this pathway in breast cancer can occur in genes coding for tyrosine kinase receptors, adaptor proteins linked to PI3K, catalytic and regulatory subunits of PI3K, serine/threonine kinases that function downstream of PI3K, and also phosphatidylinositol 3' phosphatase tumor suppressors that function to antagonize this pathway. While each genetic change results in net elevation of PI3K pathway signaling, and all major breast cancer subtypes show pathway activation, the specific mutation(s) involved in any one tumor may play an important role in defining tumor subtype, prognosis and even sensitivity to therapy. Here, we describe mouse models of breast cancer with elevated PI3K signaling, and how they may be used to guide development of novel therapeutics.