Jonathan E. Grim

FBW7 mutations in leukemic cells mediate NOTCH pathway activation and resistance to γ-secretase inhibitors

γ-secretase inhibitors (GSIs) can block NOTCH receptor signaling in vitro and therefore offer an attractive targeted therapy for tumors dependent on deregulated NOTCH activity. To clarify the basis for GSI resistance in T cell acute lymphoblastic leukemia (T-ALL), we studied T-ALL cell lines with constitutive expression of the NOTCH intracellular domain (NICD), but that lacked C-terminal truncating mutations in NOTCH1. Each of the seven cell lines examined and 7 of 81 (8.6%) primary T-ALL samples harbored either a mutation or homozygous deletion of the gene FBW7, a ubiquitin ligase implicated in NICD turnover. Indeed, we show that FBW7 mutants cannot bind to the NICD and define the phosphodegron region of the NICD required for FBW7 binding. Although the mutant forms of FBW7 were still able to bind to MYC, they do not target it for degradation, suggesting that stabilization of both NICD and its principle downstream target, MYC, may contribute to transformation in leukemias with FBW7 mutations. In addition, we show that all seven leukemic cell lines with FBW7 mutations were resistant to the MRK-003 GSI. Most of these resistant lines also failed to down-regulate the mRNA levels of the NOTCH targets MYC and DELTEX1 after treatment with MRK-003, implying that residual NOTCH signaling in T-ALLs with FBW7 mutations contributes to GSI resistance.

Multisite Phosphorylation by Cdk2 and GSK3 Controls Cyclin E Degradation

Autophosphorylation-triggered ubiquitination has been proposed to be the major pathway regulating cyclin E protein abundance: phosphorylation of cyclin E on T380 by its associated CDK allows binding to the receptor subunit, Fbw7, of the SCFFbw7 ubiquitin ligase. We have tested this model in vivo and found it to be an inadequate representation of the pathways that regulate cyclin E degradation. We show that assembly of cyclin E into cyclin E-Cdk2 complexes is required in vivo for turnover by the Fbw7 pathway; that Cdk2 activity is required for cyclin E turnover in vivo because it phosphorylates S384; that phosphorylation of T380 in vivo does not require Cdk2 and is mediated primarily by GSK3; and that two additional phosphorylation sites, T62 and S372, are also required for turnover. Thus, cyclin E turnover is controlled by multiple biological inputs and cannot be understood in terms of autophosphorylation alone.

Notch Activation Induces Endothelial Cell Cycle Arrest and Participates in Contact Inhibition: Role of p21Cip1 Repression

ABSTRACT Although previous studies demonstrate that appropriate Notch signaling is required during angiogenesis and in vascular homeostasis, the mechanisms by which Notch regulates vascular function remain to be elucidated. Here, we show that activation of the Notch pathway by the ligand Jagged1 reduces the proliferation of endothelial cells. Notch activation inhibits proliferation of endothelial cells in a cell-autonomous manner by inhibiting phosphorylation of the retinoblastoma protein (Rb). During cell cycle entry, p21Cip1 is upregulated in endothelial cells. Activated Notch inhibits mitogen-induced upregulation of p21Cip1 and delays cyclin D-cdk4-mediated Rb phosphorylation. Notch-dependent repression of p21Cip1 prevents nuclear localization of cyclin D and cdk4. The necessity of p21Cip1 for nuclear translocation of cyclin D-cdk4 and S-phase entry in endothelial cells was demonstrated by targeted downregulation of p21Cip1 by using RNA interference. We further demonstrate that when endothelial cells reach confluence, Notch is activated and p21Cip1 is downregulated. Inhibition of the Notch pathway at confluence prevents p21Cip1 downregulation and induces Rb phosphorylation. We suggest that Notch activation contributes to contact inhibition of endothelial cells, in part through repression of p21Cip1 expression.

The Dynamic International Prognostic Scoring System for myelofibrosis predicts outcomes after hematopoietic cell transplantation

Studies by the International Working Group showed that the prognosis of myelofibrosis patients is predicted by the Dynamic International Prognostic Scoring System (DIPSS) risk categorization, which includes patient age, constitutional symptoms, hemoglobin, leukocyte count, and circulating blasts. We evaluated the prognostic usefulness of the DIPSS in 170 patients with myelofibrosis, 12 to 78 years of age (median, 51.5 years of age), who received hematopoietic cell transplantation (HCT) between 1990 and 2009 from related (n = 86) or unrelated donors (n = 84). By DIPSS, 21 patients had low-risk disease, 48 had intermediate-1, 50 had intermediate-2, and 51 had high-risk disease. Five-year incidence of relapse, relapse-free survival, overall survival, and nonrelapse mortality for all patients were 10%, 57%, 57%, and 34%, respectively. Among patients with DIPSS high-risk disease, the hazard ratio for post-HCT mortality was 4.11 (95% CI, 1.44-11.78; P = .008), and for nonrelapse mortality was 3.41 (95% CI, 1.15-10.09; P = .03) compared with low-risk patients. After a median follow-up of 5.9 years, the median survivals have not been reached for DIPSS risk groups low and intermediate-1, and were 7 and 2.5 years for intermediate-2 and high-risk patients, respectively. Thus, HCT was curative for a large proportion of patients with myelofibrosis, and post-HCT success was dependent on pre-HCT DIPSS classification.

Isoform- and cell cycle–dependent substrate degradation by the Fbw7 ubiquitin ligase

The SCFFBW7 ubiquitin ligase degrades proteins involved in cell division, growth, and differentiation and is commonly mutated in cancers. The Fbw7 locus encodes three protein isoforms that occupy distinct subcellular localizations, suggesting that each has unique functions. We used gene targeting to create isoform-specific Fbw7-null mutations in human cells and found that the nucleoplasmic Fbw7α isoform accounts for almost all Fbw7 activity toward cyclin E, c-Myc, and sterol regulatory element binding protein 1. Cyclin E sensitivity to Fbw7 varies during the cell cycle, and this correlates with changes in cyclin E–cyclin-dependent kinase 2 (CDK2)–specific activity, cyclin E autophosphorylation, and CDK2 inhibitory phosphorylation. These data suggest that oscillations in cyclin E–CDK2-specific activity during the cell cycle regulate the timing of cyclin E degradation. Moreover, they highlight the utility of adeno-associated virus–mediated gene targeting in functional analyses of complex loci.

Fbw7 dimerization determines the specificity and robustness of substrate degradation

The Fbw7 tumor suppressor targets a broad network of proteins for ubiquitylation. Here we show critical functions for Fbw7 dimerization in regulating the specificity and robustness of degradation. Dimerization enables Fbw7 to target substrates through concerted binding to two suboptimal and independent recognition sites. Accordingly, an endogenous dimerization-deficient Fbw7 mutation stabilizes suboptimal substrates. Dimerization increases Fbw7s robustness by preserving its function in the setting of mutations that disable Fbw7 monomers, thereby buffering against pathogenic mutations. Finally, dimerization regulates Fbw7 stability, and this likely involves Fbw7 trans-autoubiquitylation. Our study reveals novel functions of Fbw7 dimerization and an unanticipated complexity in substrate degradation.

Fbxw7 hotspot mutations and human colon cancer: mechanistic insights from new mouse models

The recent explosion in cancer genomics provides an unprecedented view of the genetic changes present in solid tumours. The characterisation of colorectal cancer samples via DNA sequencing has confirmed the importance of established oncogene and tumour suppressor pathways (including the ‘mountains’ of Apc, Tp53 and Ras) but has also identified novel cancer genes that are mutated in a small percentage of patient samples (hills).1 ,2 Furthermore, these studies give a new appreciation of the spectrum of gene mutations present in cancers, especially in candidate tumour suppressor genes (TSGs). While classic genetics teaches that two hits are required to inactivate TSGs, sequencing studies show that heterozygous mutations are also quite frequent. Some of these are loss of function alleles, while others are recurrent missense mutations known as ‘hotspots’. Now, researchers are charged with the task of understanding how these genetic changes contribute to cancer development, invasion and spread. Why are heterozygous mutations in TSGs commonly found in cancer genomes? While many of these are passenger mutations, some single allele mutations can significantly alter tumour suppressor protein expression or function. This, in turn, leads to incremental changes in growth or to other hallmarks of cancer. The concept of haploinsufficiency refers to such situations where decreased dosage of certain gene products is sufficient to promote tumorigenesis.3 However, the impact of most heterozygous mutations is largely unknown, because they …

Regulation of cell proliferation in a stratified culture system of epithelial cells from prostate tissue

Mechanisms controlling epithelial proliferation and differentiation in the prostate have been primarily investigated in mouse models. The regulation of proliferation and differentiation is poorly understood in human prostate epithelial cells. In vivo, the glandular prostate epithelium consists of a p63-positive proliferating basal cell layer and a post-mitotic p27-positive secretory cell layer. We have established an organized stratified culture system of human primary prostate epithelial cells to gain insight into mechanisms regulating proliferation and differentiation. In this system, expression of p63 is observed in the bottom layer. In addition, BrdU incorporation persists even though cells are confluent. In contrast, in the upper layer, p63 expression is greatly diminished, p27 is expressed, and the cells are growth arrested. Overexpression of cyclin D1 or knockdown of p27 does not increase proliferation. After inactivation of the nuclear phosphoprotein Rb, the cell layers remain organized and cell proliferation increases only in the bottom layer. Furthermore, the expression of p63 remains confined to the bottom layer after Rb inactivation. Altogether, this in vitro model recapitulates certain aspects of in vivo growth regulation and differentiation and suggests that the loss of Rb family proteins in human cells trigger hyperplasia but is not sufficient for transformation.

Pan-cancer transcriptional signatures predictive of oncogenic mutations reveal that Fbw7 regulates cancer cell oxidative metabolism

Significance Tumor suppression by the Fbw7 ubiquitin ligase remains poorly understood. Here, we used informatics and engineered cancer cells to show that Fbw7 mutations cause metabolic reprogramming by increasing oxidative phosphorylation and metabolic vulnerabilities that may represent therapeutic targets. Our approach may be applied to study other complex cancer genes. The Fbw7 (F-box/WD repeat-containing protein 7) ubiquitin ligase targets multiple oncoproteins for degradation and is commonly mutated in cancers. Like other pleiotropic tumor suppressors, Fbw7’s complex biology has impeded our understanding of how Fbw7 mutations promote tumorigenesis and hindered the development of targeted therapies. To address these needs, we employed a transfer learning approach to derive gene-expression signatures from The Cancer Gene Atlas datasets that predict Fbw7 mutational status across tumor types and identified the pathways enriched within these signatures. Genes involved in mitochondrial function were highly enriched in pan-cancer signatures that predict Fbw7 mutations. Studies in isogenic colorectal cancer cell lines that differed in Fbw7 mutational status confirmed that Fbw7 mutations increase mitochondrial gene expression. Surprisingly, Fbw7 mutations shifted cellular metabolism toward oxidative phosphorylation and caused context-specific metabolic vulnerabilities. Our approach revealed unexpected metabolic reprogramming and possible therapeutic targets in Fbw7-mutant cancers and provides a framework to study other complex, oncogenic mutations.

Abstract 1340: A mouse model of metastatic intestinal cancers associated with Fbw7- and p53-loss

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Colon cancer is a leading cause of cancer deaths in the USA, where it accounts for up to 50,000 deaths per year. Mouse models that recapitulate the genetic changes seen in human colon cancers are useful tools for biological and preclinical applications, but most available models develop adenomatous lesions that rarely metastasize. The Fbw7 tumor suppressor is a core component of a ubiquitin ligase complex that negatively regulates a number of proteins associated with genomic instability and cancer, including cyclin E, c-myc, Mcl-1, and Notch. Our previous work shows that Fbw7 cooperates with the p53 tumor suppressor to control genetic instability in vitro, and both Fbw7 and p53 are frequently deregulated in human colon cancers. We hypothesized that coordinate deletion of these two tumor suppressors would promote intestinal cancers in mice. To accomplish intestine specific deletion of these pathways, we interbred genetically engineered mice encoding conditional Fbw7 and p53 alleles with the VillinCre strain. Examination of normal intestines from the resulting Fbw7(fl/fl); p53(fl/fl); villinCre (FPV) animals showed abnormal proliferation and differentiation along the crypt-villus axis compared to control animals. Strikingly, the majority (55%) of FPV mice developed adenocarcinomas in the small intestine and cecum (median survival 70 weeks of age), and these tumors frequently metastasized to the lymph nodes and liver (in 36% and 14% of tumor bearing animals, respectively). Analysis of cancer tissues showed clear evidence of genetic instability in the majority of tumors. Finally, FPV tumors were readily grown as cell lines in vitro which could, in turn, grow as allografts in both immunodeficient and immunocompetent recipients. Notably, comparison of primary tumors and allografted cell lines showed similar pathological features and patterns of metastasis. This work confirms the critical role of the Fbw7 and p53 pathways in suppressing genetic instability and cancer development. Furthermore, the described FPV models recapitulate critical features of human colon cancer and should prove useful for studies of cancer biology, prevention, and therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1340. doi:1538-7445.AM2012-1340