Ras GTPases are mutated at codons 12, 13, and 61, with different frequencies in KRas, HRas, and NRas and in a cancer-specific manner. The G13D mutant appears in 25% of KRas-driven colorectal cancers, while observed only rarely in HRas or NRas. Structures of Ras G13D in the three isoforms show an open active site, with adjustments to the D13 backbone torsion angles and with disconnected switch regions. KRas G13D has unique features that destabilize the nucleotide-binding pocket. In KRas G13D bound to GDP, A59 is placed in the Mg2+ binding site, as in the HRas-SOS complex. Structure and biochemistry are consistent with an intermediate level of KRas G13D bound to GTP, relative to wild-type and KRas G12D, observed in genetically engineered mouse models. The results explain in part the elevated frequency of the G13D mutant in KRas over the other isoforms of Ras.

The key negative regulatory gene of the RAS pathway, NF1, is mutated or deleted in numerous cancer types and is associated with increased cancer risk and drug resistance. Even though women with neurofibromatosis (germline NF1 mutations) have a substantially increased breast cancer risk at a young age and NF1 is commonly mutated in sporadic breast cancers, we have a limited understanding of the role of NF1 in breast cancer. We utilized CRISPR–Cas9 gene editing to create Nf1 rat models to evaluate the effect of Nf1 deficiency on tumorigenesis. The resulting Nf1 indels induced highly penetrant, aggressive mammary adenocarcinomas that express estrogen receptor (ER) and progesterone receptor (PR). We identified distinct Nf1 mRNA and protein isoforms that were altered during tumorigenesis. To evaluate NF1 in human breast cancer, we analyzed genomic changes in a data set of 2000 clinically annotated breast cancers. We found NF1 shallow deletions in 25% of sporadic breast cancers, which correlated with poor clinical outcome. To identify biological networks impacted by NF1 deficiency, we constructed gene co-expression networks using weighted gene correlation network analysis (WGCNA) and identified a network connected to ESR1 (estrogen receptor). Moreover, NF1-deficient cancers correlated with established RAS activation signatures. Estrogen-dependence was verified by estrogen-ablation in Nf1 rats where rapid tumor regression was observed. Additionally, Nf1 deficiency correlated with increased estrogen receptor phosphorylation in mammary adenocarcinomas. These results demonstrate a significant role for NF1 in both NF1-related breast cancer and sporadic breast cancer, and highlight a potential functional link between neurofibromin and the estrogen receptor.

Malignant peripheral nerve sheath tumors (MPNST) are highly resistant sarcomas that occur in up to 13% of individuals with neurofibromatosis type I (NF1). Genomic analysis of longitudinally collected tumor samples in a case of MPNST disease progression revealed early hemizygous microdeletions in NF1 and TP53, with progressive amplifications of MET, HGF, and EGFR. To examine the role of MET in MPNST progression, we developed mice with enhanced MET expression and Nf1 ablation (Nf1^fl/ko;lox-stop-loxMET^tg/+;Plp-creERT^tg/+; referred to as NF1-MET). NF1-MET mice express a robust MPNST phenotype in the absence of additional mutations. A comparison of NF1-MET MPNSTs with MPNSTs derived from Nf1^ko/+;p53^R172H;Plp-creERT^tg/+ (NF1-P53) and Nf1^ko/+;Plp-creERT^tg/+ (NF1) mice revealed unique Met, Ras, and PI3K signaling patterns. NF1-MET MPNSTs were uniformly sensitive to the highly selective MET inhibitor, capmatinib, whereas a heterogeneous response to MET inhibition was observed in NF1-P53 and NF1 MPNSTs. Combination therapy of capmatinib and the MEK inhibitor trametinib resulted in reduced response variability, enhanced suppression of tumor growth, and suppressed RAS/ERK and PI3K/AKT signaling. These results highlight the influence of concurrent genomic alterations on RAS effector signaling and therapy response to tyrosine kinase inhibitors. Moreover, these findings expand our current understanding of the role of MET signaling in MPNST progression and identify a potential therapeutic niche for NF1-related MPNSTs. Significance: Longitudinal genomic analysis reveals a positive selection for MET and HGF copy number gain early in malignant peripheral nerve sheath tumor progression. Cancer Res; 78(13); 3672–87. ©2018 AACR.

Giant cell tumor of bone (GCTB) is a benign, locally destructive neoplasm, with tumors comprised of mesenchymal fibroblast-like stromal cells; monocytic, mononuclear cells of myeloid lineage; and the characteristic osteoclast-like, multinucleated giant cells. Hampering the study of the complex interaction of its constituent cell types is the propensity of longstanding, repeatedly passaged cell cultures to undergo phenotypic alteration and loss of osteoclast-inducing capacities. In this study, we employed a novel, single-step technique to purify freshly harvested stromal cells using a CD14-negative selection column. Using 9 freshly harvested GCTB specimens and the purified stromal cell component, we performed analyses for markers of osteoblast lineage and analyzed the capacity of the stromal cells to undergo osteoblastic differentiation and induce osteoclastogenesis in co-cultures with monocytic cells. Successful purification of the CD14-negative stromal cells was confirmed via flow cytometric analysis and immunocytochemistry. Osteogenic media upregulated the expression of osteocalcin, suggesting an osteoblastic lineage of the GCTB stromal cells. The effects of the Wnt pathway agonist, SB415286, and recombinant human bone morphogenetic protein (BMP)-2 on osteoblastogenesis varied among samples. Notably, osteogenic media and SB415286 reversed the receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) expression ratio resulting in diminished osteoclastogenic capacity. Recombinant human BMP2 had the opposite effect, resulting in enhanced and sustained support of osteoclastogenesis. Targeting the giant cell tumor stromal cell may be an effective adjunct to existing anti-resorptive strategies.

Osteosarcoma (OS) is the most common type of solid bone cancer and remains the second leading cause of cancer-related death for children and young adults. Hypoxia is an element intrinsic to most solid-tumor microenvironments, including that of OS, and is associated with resistance to therapy, poor survival, and a malignant phenotype. Cells respond to hypoxia through alterations in gene expression, mediated most notably through the hypoxia-inducible factor (HIF) class of transcription factors. Here we investigate hypoxia-induced changes in the Wnt/β-catenin signaling pathway, a key signaling cascade involved in OS pathogenesis. We show that hypoxia results in increased expression and signaling activation of HIF proteins in human osteosarcoma cells. Wnt/β-catenin signaling is down-regulated by hypoxia in human OS cells, as demonstrated by decreased active β-catenin protein levels and axin2 mRNA expression (p<0.05). This down-regulation appears to rely on both HIF-independent and HIF-dependent mechanisms, with HIF-1α standing out as an important regulator. Finally, we show that hypoxia results in resistance of human OS cells to doxorubicin-mediated toxicity (6–13 fold increase, p<0.01). These hypoxic OS cells can be sensitized to doxorubicin treatment by further inhibition of the Wnt/β-catenin signaling pathway (p<0.05). These data support the conclusion that Wnt/β-catenin signaling is down-regulated in human OS cells under hypoxia and that this signaling alteration may represent a viable target to combat chemoresistant OS subpopulations in a hypoxic niche.