Global and context-specific transcriptional consequences of oncogenic Fbw7 mutations.

The Fbw7 ubiquitin ligase targets many proteins for proteasomal degradation, which include oncogenic transcription factors (TFs) (e.g., c-Myc, c-Jun, and Notch). Fbw7 is a tumor suppressor and tumors often contain mutations in FBXW7, the gene that encodes Fbw7. The complexity of its substrate network has obscured the mechanisms of Fbw7-associated tumorigenesis, yet this understanding is needed for developing therapies. We used an integrated approach employing RNA-Seq and high-resolution mapping (cleavage under target and release using nuclease) of histone modifications and TF occupancy (c-Jun and c-Myc) to examine the combinatorial effects of misregulated Fbw7 substrates in colorectal cancer (CRC) cells with engineered tumor-associated FBXW7 null or missense mutations. Both Fbw7 mutations caused widespread transcriptional changes associated with active chromatin and altered TF occupancy: some were common to both Fbw7 mutant cell lines, whereas others were mutation specific. We identified loci where both Jun and Myc were coregulated by Fbw7, suggesting that substrates may have synergistic effects. One coregulated gene was CIITA, the master regulator of MHC Class II gene expression. Fbw7 loss increased MHC Class II expression and Fbw7 mutations were correlated with increased CIITA expression in TCGA colorectal tumors and cell lines, which may have immunotherapeutic implications for Fbw7-associated cancers. Analogous studies in neural stem cells in which FBXW7 had been acutely deleted closely mirrored the results in CRC cells. Gene set enrichment analyses revealed Fbw7-associated pathways that were conserved across both cell types that may reflect fundamental Fbw7 functions. These analyses provide a framework for understanding normal and neoplastic context-specific Fbw7 functions.

Two diphosphorylated degrons control c-Myc degradation by the Fbw7 tumor suppressor.

c-Myc (hereafter, Myc) is a cancer driver whose abundance is regulated by the SCFFbw7 ubiquitin ligase and proteasomal degradation. Fbw7 binds to a phosphorylated Myc degron centered at threonine 58 (T58), and mutations of Fbw7 or T58 impair Myc degradation in cancers. Here, we identify a second Fbw7 phosphodegron at Myc T244 that is required for Myc ubiquitylation and acts in concert with T58 to engage Fbw7. While Ras-dependent Myc serine 62 phosphorylation (pS62) is thought to stabilize Myc by preventing Fbw7 binding, we find instead that pS62 greatly enhances Fbw7 binding and is an integral part of a high-affinity degron. Crystallographic studies revealed that both degrons bind Fbw7 in their diphosphorylated forms and that the T244 degron is recognized via a unique mode involving Fbw7 arginine 689 (R689), a mutational hotspot in cancers. These insights have important implications for Myc-associated tumorigenesis and therapeutic strategies targeting Myc stability.

Proteasomal degradation of the tumour suppressor FBW7 requires branched ubiquitylation by TRIP12.

The tumour suppressor FBW7 is a substrate adaptor for the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), that targets several oncoproteins for proteasomal degradation. FBW7 is widely mutated and FBW7 protein levels are commonly downregulated in cancer. Here, using an shRNA library screen, we identify the HECT-domain E3 ubiquitin ligase TRIP12 as a negative regulator of FBW7 stability. We find that SCFFBW7-mediated ubiquitylation of FBW7 occurs preferentially on K404 and K412, but is not sufficient for its proteasomal degradation, and in addition requires TRIP12-mediated branched K11-linked ubiquitylation. TRIP12 inactivation causes FBW7 protein accumulation and increased proteasomal degradation of the SCFFBW7 substrate Myeloid Leukemia 1 (MCL1), and sensitizes cancer cells to anti-tubulin chemotherapy. Concomitant FBW7 inactivation rescues the effects of TRIP12 deficiency, confirming FBW7 as an essential mediator of TRIP12 function. This work reveals an unexpected complexity of FBW7 ubiquitylation, and highlights branched ubiquitylation as an important signalling mechanism regulating protein stability.

FOXM1 drives HPV+ HNSCC sensitivity to WEE1 inhibition.

Head and neck squamous cell carcinoma (HNSCC) associated with high-risk human papilloma virus (HPV) infection is a growing clinical problem. The WEE1 kinase inhibitor AZD1775 (WEE1i) overrides cell cycle checkpoints and is being studied in HNSCC regimens. We show that the HPV16 E6/E7 oncoproteins sensitize HNSCC cells to single-agent WEE1i treatment through activation of a FOXM1-CDK1 circuit that drives mitotic gene expression and DNA damage. An isogenic cell system indicated that E6 largely accounts for these phenotypes in ways that extend beyond p53 inactivation. A targeted genomic analysis implicated FOXM1 signaling downstream of E6/E7 expression and analyses of primary tumors and The Cancer Genome Atlas (TCGA) data revealed an activated FOXM1-directed promitotic transcriptional signature in HPV+ versus HPV- HNSCCs. Finally, we demonstrate the causality of FOXM1 in driving WEE1i sensitivity. These data suggest that elevated basal FOXM1 activity predisposes HPV+ HNSCC to WEE1i-induced toxicity and provide mechanistic insights into WEE1i and HPV+ HNSCC therapies.

A novel landscape of nuclear human CDK2 substrates revealed by in situ phosphorylation.

Cyclin-dependent kinase 2 (CDK2) controls cell division and is central to oncogenic signaling. We used an "in situ" approach to identify CDK2 substrates within nuclei isolated from cells expressing CDK2 engineered to use adenosine 5'-triphosphate analogs. We identified 117 candidate substrates, ~40% of which are known CDK substrates. Previously unknown candidates were validated to be CDK2 substrates, including LSD1, DOT1L, and Rad54. The identification of many chromatin-associated proteins may have been facilitated by labeling conditions that preserved nuclear architecture and physiologic CDK2 regulation by endogenous cyclins. Candidate substrates include proteins that regulate histone modifications, chromatin, transcription, and RNA/DNA metabolism. Many of these proteins also coexist in multi-protein complexes, including epigenetic regulators, that may provide new links between cell division and other cellular processes mediated by CDK2. In situ phosphorylation thus revealed candidate substrates with a high validation rate and should be readily applicable to other nuclear kinases.

Insertional mutagenesis using the Sleeping Beauty transposon system identifies drivers of erythroleukemia in mice.

Insertional mutagenesis is a powerful means of identifying cancer drivers in animal models. We used the Sleeping Beauty (SB) transposon/transposase system to identify activated oncogenes in hematologic cancers in wild-type mice and mice that express a stabilized cyclin E protein (termed cyclin ET74AT393A). Cyclin E governs cell division and is misregulated in human cancers. Cyclin ET74AT393A mice develop ineffective erythropoiesis that resembles early-stage human myelodysplastic syndrome, and we sought to identify oncogenes that might cooperate with cyclin E hyperactivity in leukemogenesis. SB activation in hematopoietic precursors caused T-cell leukemia/lymphomas (T-ALL) and pure red blood cell erythroleukemias (EL). Analysis of >12,000 SB integration sites revealed markedly different oncogene activations in EL and T-ALL: Notch1 and Ikaros were most common in T-ALL, whereas ETS transcription factors (Erg and Ets1) were targeted in most ELs. Cyclin E status did not impact leukemogenesis or oncogene activations. Whereas most SB insertions were lost during culture of EL cell lines, Erg insertions were retained, indicating Erg's key role in these neoplasms. Surprisingly, cyclin ET74AT393A conferred growth factor independence and altered Erg-dependent differentiation in EL cell lines. These studies provide new molecular insights into erythroid leukemia and suggest potential therapeutic targets for human leukemia.

Merkel cell polyomavirus Tumor antigens expressed in Merkel cell carcinoma function independently of the ubiquitin ligases Fbw7 and ß-TrCP.

Merkel cell polyomavirus (MCPyV) accounts for 80% of all Merkel cell carcinoma (MCC) cases through expression of two viral oncoproteins: the truncated large T antigen (LT-t) and small T antigen (ST). MCPyV ST is thought to be the main driver of cellular transformation and has also been shown to increase LT protein levels through the activity of its Large-T Stabilization Domain (LSD). The ST LSD was reported to bind and sequester several ubiquitin ligases, including Fbw7 and ß-TrCP, and thereby stabilize LT-t and several other Fbw7 targets including c-Myc and cyclin E. Therefore, the ST LSD is thought to contribute to transformation by promoting the accumulation of these oncoproteins. Targets of Fbw7 and ß-TrCP contain well-defined, conserved, phospho-degrons. However, as neither MCPyV LT, LT-t nor ST contain the canonical Fbw7 phospho-degron, we sought to further investigate the proposed model of ST stabilization of LT-t and transformation. In this study, we provide several lines of evidence that fail to support a specific interaction between MCPyV T antigens and Fbw7 or ß-TrCP by co-immunoprecipitation or functional consequence. Although MCPyV ST does indeed increase LT protein levels through its Large-T Stabilization domain (LSD), this is accomplished independently of Fbw7. Therefore, our study indicates a need for further investigation into the role and mechanism(s) of MCPyV T antigens in viral replication, latency, transformation, and tumorigenesis.

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

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.

RAD54 N-terminal domain is a DNA sensor that couples ATP hydrolysis with branch migration of Holliday junctions.

In eukaryotes, RAD54 catalyzes branch migration (BM) of Holliday junctions, a basic process during DNA repair, replication, and recombination. RAD54 also stimulates RAD51 recombinase and has other activities. Here, we investigate the structural determinants for different RAD54 activities. We find that the RAD54 N-terminal domain (NTD) is responsible for initiation of BM through two coupled, but distinct steps; specific binding to Holliday junctions and RAD54 oligomerization. Furthermore, we find that the RAD54 oligomeric state can be controlled by NTD phosphorylation at S49, a CDK2 consensus site, which inhibits RAD54 oligomerization and, consequently, BM. Importantly, the effect of phosphorylation on RAD54 oligomerization is specific for BM, as it does not affect stimulation of RAD51 recombinase by RAD54. Thus, the transition of the oligomeric states provides an important control of the biological functions of RAD54 and, likely, other multifunctional proteins.

The miR-106a~363Xpcl1 miRNA cluster induces murine T cell lymphoma despite transcriptional activation of the p27Kip1 cell cycle inhibitor.

The miR-106a~363 cluster encodes 6 miRNAs on the X-chromosome which are abundant in blood cells and overexpressed in a variety of malignancies. The constituent miRNA of miR-106a~363 have functional activities in vitro that are predicted to be both oncogenic and tumor suppressive, yet little is known about their physiological functions in vivo. Mature miR-106a~363 (Mirc2) miRNAs are processed from an intragenic, non-protein encoding gene referred to as Xpcl1 (or Kis2), situated at an X-chromosomal locus frequently targeted by retroviruses in murine lymphomas. The oncogenic potential of miR-106a~363 Xpcl1 has not been proven, nor its potential role in T cell development. We show that miR106a~363 levels normally drop at the CD4+/CD8+ double positive (DP) stage of thymocyte development. Forced expression of Xpcl1 at this stage impairs thymocyte maturation and induces T-cell lymphomas. Surprisingly, miR-106a~363 Xpcl1 also induces p27 transcription via Foxo3/4 transcription factors. As a haploinsufficient tumor suppressor, elevated p27 is expected to inhibit lymphomagenesis. Consistent with this, concurrent p27 Kip1 deletion dramatically accelerated lymphomagenesis, indicating that p27 is rate limiting for tumor development by Xpcl1. Whereas down-regulation of miR-106a~363 is important for normal T cell differentiation and for the prevention of lymphomas, eliminating p27 reveals Xpcl1's full oncogenic potential.

DGCR8 Mediates Repair of UV-Induced DNA Damage Independently of RNA Processing.

Ultraviolet (UV) radiation is a carcinogen that generates DNA lesions. Here, we demonstrate an unexpected role for DGCR8, an RNA binding protein that canonically functions with Drosha to mediate microRNA processing, in the repair of UV-induced DNA lesions. Treatment with UV induced phosphorylation on serine 153 (S153) of DGCR8 in both human and murine cells. S153 phosphorylation was critical for cellular resistance to UV, the removal of UV-induced DNA lesions, and the recovery of RNA synthesis after UV exposure but not for microRNA expression. The RNA-binding and Drosha-binding activities of DGCR8 were not critical for UV resistance. DGCR8 depletion was epistatic to defects in XPA, CSA, and CSB for UV sensitivity. DGCR8 physically interacted with CSB and RNA polymerase II. JNKs were involved in the UV-induced S153 phosphorylation. These findings suggest that UV-induced S153 phosphorylation mediates transcription-coupled nucleotide excision repair of UV-induced DNA lesions in a manner independent of microRNA processing.

Genome-wide CRISPR-Cas9 Screens Reveal Loss of Redundancy between PKMYT1 and WEE1 in Glioblastoma Stem-like Cells.

To identify therapeutic targets for glioblastoma (GBM), we performed genome-wide CRISPR-Cas9 knockout (KO) screens in patient-derived GBM stem-like cells (GSCs) and human neural stem/progenitors (NSCs), non-neoplastic stem cell controls, for genes required for their in vitro growth. Surprisingly, the vast majority GSC-lethal hits were found outside of molecular networks commonly altered in GBM and GSCs (e.g., oncogenic drivers). In vitro and in vivo validation of GSC-specific targets revealed several strong hits, including the wee1-like kinase, PKMYT1/Myt1. Mechanistic studies demonstrated that PKMYT1 acts redundantly with WEE1 to inhibit cyclin B-CDK1 activity via CDK1-Y15 phosphorylation and to promote timely completion of mitosis in NSCs. However, in GSCs, this redundancy is lost, most likely as a result of oncogenic signaling, causing GBM-specific lethality.

Tumor suppression by the Fbw7 ubiquitin ligase: mechanisms and opportunities.

Tumor suppressors with widespread impact on carcinogenesis control broad spectra of oncogenic pathways. Protein degradation is an emerging mechanism by which tumor suppressors regulate a diversity of pathways and is exemplified by the SCF(Fbw7) ubiquitin ligase. Rapidly accumulating data indicate that SCF(Fbw7) regulates a network of crucial oncoproteins. Importantly, the FBXW7 gene, which encodes Fbw7, is one of the most frequently mutated genes in human cancers. These studies are yielding important new insights into tumorigenesis and may soon enable therapies targeting the Fbw7 pathway. Here, we focus on the mechanisms and consequences of Fbw7 deregulation in cancers and discuss possible therapeutic approaches.

Mislocalization of p27 to the cytoplasm of breast cancer cells confers resistance to anti-HER2 targeted therapy.

As a cell cycle inhibitor and tumor suppressor, p27 is frequently misregulated in human cancers. Increased degradation is the most common mechanism of misregulation, however in some cancers, p27 is mislocalized from its cell cycle inhibitory location in the nucleus, to the cytoplasm. In normal cells cytoplasmic p27 has functions that are distinct from its cell cycle-regulatory nuclear functions. Therefore, an important question is whether localization of p27 to the cytoplasm in tumor cells is primarily a mechanism for cancelling its inhibitory effect on cell proliferation, or whether cytoplasmic p27 has more direct oncogenic actions. To study p27 mislocalization in human cancers we screened a panel of common breast cancer cell lines. We observed that p27 accumulated in the cytoplasm exclusively in cell lines that are Her2+. To address the significance of p27 mislocalization in Her2+ breast cancer cells we interrogated the cellular response to the dual-Her2/EGFR kinase inhibitor, lapatinib. Knockdown of p27 using shRNA sensitized Her2+ cells to lapatinib-induced apoptosis. Moreover, expression of a constitutively cytoplasmic form of p27 (p27ΔNLS) reversed the lapatinib-induced apoptosis, suggesting that cytoplasmic p27 contributed to lapatinib resistance in Her2+ breast cancer cells by suppressing apoptosis. Our results suggest that p27 localization may be useful as a predictive biomarker of therapeutic response in patients with Her2+ breast cancers.

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 Fbw7's 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.

Fbw7 repression by hes5 creates a feedback loop that modulates Notch-mediated intestinal and neural stem cell fate decisions.

FBW7 is a crucial component of an SCF-type E3 ubiquitin ligase, which mediates degradation of an array of different target proteins. The Fbw7 locus comprises three different isoforms, each with its own promoter and each suspected to have a distinct set of substrates. Most FBW7 targets have important functions in developmental processes and oncogenesis, including Notch proteins, which are functionally important substrates of SCF(Fbw7). Notch signalling controls a plethora of cell differentiation decisions in a wide range of species. A prominent role of this signalling pathway is that of mediating lateral inhibition, a process where exchange of signals that repress Notch ligand production amplifies initial differences in Notch activation levels between neighbouring cells, resulting in unequal cell differentiation decisions. Here we show that the downstream Notch signalling effector HES5 directly represses transcription of the E3 ligase Fbw7ß, thereby directly bearing on the process of lateral inhibition. Fbw7(Δ/+) heterozygous mice showed haploinsufficiency for Notch degradation causing impaired intestinal progenitor cell and neural stem cell differentiation. Notably, concomitant inactivation of Hes5 rescued both phenotypes and restored normal stem cell differentiation potential. In silico modelling suggests that the NICD/HES5/FBW7ß positive feedback loop underlies Fbw7 haploinsufficiency. Thus repression of Fbw7ß transcription by Notch signalling is an essential mechanism that is coupled to and required for the correct specification of cell fates induced by lateral inhibition.

The SCF-Fbw7 ubiquitin ligase degrades MED13 and MED13L and regulates CDK8 module association with Mediator.

The Mediator complex is an essential transcription regulator that bridges transcription factors with RNA polymerase II. This interaction is controlled by dynamic interactions between Mediator and the CDK8 module, but the mechanisms governing CDK8 module-Mediator association remain poorly understood. We show that Fbw7, a tumor suppressor and ubiquitin ligase, binds to CDK8-Mediator and targets MED13/13L for degradation. MED13/13L physically link the CDK8 module to Mediator, and Fbw7 loss increases CDK8 module-Mediator association. Our work reveals a novel mechanism regulating CDK8 module-Mediator association and suggests an expanded role for Fbw7 in transcriptional control and an unanticipated relationship with the CDK8 oncogene.

Phosphorylation of eukaryotic elongation factor 2 (eEF2) by cyclin A-cyclin-dependent kinase 2 regulates its inhibition by eEF2 kinase.

Protein synthesis is highly regulated via both initiation and elongation. One mechanism that inhibits elongation is phosphorylation of eukaryotic elongation factor 2 (eEF2) on threonine 56 (T56) by eEF2 kinase (eEF2K). T56 phosphorylation inactivates eEF2 and is the only known normal eEF2 functional modification. In contrast, eEF2K undergoes extensive regulatory phosphorylations that allow diverse pathways to impact elongation. We describe a new mode of eEF2 regulation and show that its phosphorylation by cyclin A-cyclin-dependent kinase 2 (CDK2) on a novel site, serine 595 (S595), directly regulates T56 phosphorylation by eEF2K. S595 phosphorylation varies during the cell cycle and is required for efficient T56 phosphorylation in vivo. Importantly, S595 phosphorylation by cyclin A-CDK2 directly stimulates eEF2 T56 phosphorylation by eEF2K in vitro, and we suggest that S595 phosphorylation facilitates T56 phosphorylation by recruiting eEF2K to eEF2. S595 phosphorylation is thus the first known eEF2 modification that regulates its inhibition by eEF2K and provides a novel mechanism linking the cell cycle machinery to translational control. Because all known eEF2 regulation is exerted via eEF2K, S595 phosphorylation may globally couple the cell cycle machinery to regulatory pathways that impact eEF2K activity.

Fbw7 and p53 cooperatively suppress advanced and chromosomally unstable intestinal cancer.

Colorectal cancer (CRC) remains a major cause of cancer mortality worldwide. Murine models have yielded critical insights into CRC pathogenesis, but they often fail to recapitulate advanced-disease phenotypes, notably metastasis and chromosomal instability (CIN). New models are thus needed to understand disease progression and to develop therapies. We sought to model advanced CRC by inactivating two tumor suppressors that are mutated in human CRCs, the Fbw7 ubiquitin ligase and p53. Here we report that Fbw7 deletion alters differentiation and proliferation in the gut epithelium and stabilizes oncogenic Fbw7 substrates, such as cyclin E and Myc. However, Fbw7 deletion does not cause tumorigenesis in the gut. In contrast, codeletion of both Fbw7 and p53 causes highly penetrant, aggressive, and metastatic adenocarcinomas, and allografts derived from these tumors form highly malignant adenocarcinomas. In vitro evidence indicates that Fbw7 ablation promotes genetic instability that is suppressed by p53, and we show that most Fbw7⁻/⁻; p53⁻/⁻ carcinomas exhibit a CIN⁺ phenotype. We conclude that Fbw7 and p53 synergistically suppress adenocarcinomas that mimic advanced human CRC with respect to histopathology, metastasis, and CIN. This model thus represents a novel tool for studies of advanced CRC as well as carcinogenesis associated with ubiquitin pathway mutations.