SCF Ubiquitin Ligase Research Articles

3q26 Amplifications in Cervical Squamous Carcinomas.

Background: Squamous carcinomas of the uterine cervix often carry mutations of the gene encoding for the catalytic sub-unit of kinase PI3K, PIK3CA. The locus of this gene at chromosome 3q26 and neighboring loci are also commonly amplified. The landscape of 3q26-amplified cases have not been previously characterized in detail in cervical cancer. Methods: Published genomic data and associated clinical data from TCGA cervical cancer cohort were analyzed at cBioportal for amplifications in genes at 3q26. The clinical and molecular characteristics of the group of patients with 3q26 amplifications was compared with the group without 3q26 amplifications. Comparative prevalence of amplification and expression of genes at 3q26 in amplified squamous cervical cancer cases were surveyed as well as 3q26 amplifications in cervical cancer cell line databases. Results: Amplification of 3q26 locus is a prevalent molecular lesion in cervical squamous cell carcinomas encountered in about 15% of cases in TCGA cohort of 247 patients. Cancer-related genes commonly amplified from 3q26 include PIK3CA, TBL1XR1, DCUN1D1, SOX2, MECOM, PRKCI, and TERC. Amplified cases do not completely overlap with PIK3CA mutant cases. Differences exist between 3q26-amplified and non-amplified carcinomas in the frequency of mutations and frequency of other amplifications. Most commonly over-expressed genes in 3q26 amplified cases include PIK3CA, TBL1XR1, DCUN1D1, and less commonly SOX2 and PRKCI. Conclusion: The subset of squamous cervical carcinomas with 3q26 amplifications is not overlapping with cancers carrying PIK3CA mutations and contains, besides PIK3CA, other cancer-associated genes that are over-expressed at the mRNA level, including TBL1XR1 and DCUN1D1. DCUN1D1, a regulator of SCF ubiquitin ligase activity, may be a relevant pathogenic player given the importance of ubiquitination and the proteasome in the disease. These observations could form the basis for therapeutic exploitation in this subset of squamous cervical carcinomas.

Novel Mechanisms Underlying IMiDs Resistance in Multiple Myeloma: Therapeutic Implications

IntroductionImmunomodulatory drugs (IMiDs) including lenalidomide and pomalidomide bind to the CRL4CRBN ubiquitin ligase and trigger proteasomal degradation of IKZF1/3 and cytotoxicity of multiple myeloma (MM) cells. Previous studies show that downregulation of CRL4CRBN abrogates sensitivity to IMiDs in MM cell lines. Clinical studies, show that IMiDs achieve better clinical responses in MM patients when combined with proteasome inhibitor bortezomib, which would in principle inhibit proteasomal degradation of IMiDs-targeted proteins. Moreover, some MM cells are resistant to IMiDs treatment despite harboring high CRBN expression levels. Thus, the molecular mechanisms of intrinsic resistance to IMiDs are not yet fully understood. In this study, we found that dual roles of IMiDs in activating CRL4CRBN and a mechanism of action for combination therapy of IMiDs with bortezomib. We also show that non-canonical NF-κB pathway, independent of CRBN, is involved in the IMiDs resistance in MM.Methods and ResultsTo study the molecular mechanisms underlying IMiDs resistance, we first performed genome-wide knockout screening in IMiDs-sensitive MM.1S cells, using a CRISPR-Cas9 GeCKOv2 library containing 6 unique sgRNAs against each of 19,050 genes and 4 sgRNAs against each of 1,864 miRNAs. Twenty-eight genes and one miRNA were identified which were associated with resistance to IMiDs. All six sgRNAs targeting CRBN were also identified, consistent with CRBN as the direct target of IMiDs and required for their MM cytotoxicity. Among these validated genes, 7 of 9 components of the COP signalosome (CSN) subunits were found to be required for MM cell sensitivity to IMiDs. Deletion of any of these CSN subunits in MM cells led to a dramatic decrease of CRBN protein level thereby inhibiting IKZF1/3 degradation induced by IMiDs.To investigate whether the CSN regulates CRBN level through the ubiquitin-proteasome pathway, we carried out ubiquitination assays and found that CRBN can be degraded by proteasomes via cullin E3, rather than CRL4, ubiquitin ligase. Moreover, we identified the interaction of CRBN with SCFFbxo7 complex, and demonstrated that CRBN interacted with both CRL4 and SCFFbxo7 with distinct domains. To determine if CRBN is a SCFFbxo7 substrate, we examined the ubiquitination and degradation of CRBN after altering SCFFbxo7 complex. CRBN, when unbound to CRL4, is ubiquitinated by SCFFbxo7, followed by proteasomal degradation. Importantly, deletion of Cul1 or Fbxo7 completely restored the CRBN protein in CSN2 or CSN6 knockout MM cells to levels found in wild type cells, as well as re-sensitized these CSN mutant MM cells to pomalidomide-induced cytotoxicity.Since CRBN protein turnover is controlled by a SCF ubiquitin ligase, we next tested whether proteasome inhibitor, in addition to their inherent anti-MM activities, might similarly enhance IMiDs-induced MM cell cytotoxicity. As expected, sequential treatment with bortezomib and pomalidomide primed MM cells to pomalidomide-induced downregulation of IKZF3 and cell death, associated with synergistic cytotoxicity. We also found that lenalidomide potently inhibited the ubiquitination of CRBN, which was abrogated by loss of lenalidomide binding on CRBN, downregulation of Fbxo7, or Cul1 expression. These data suggest that IMiDs stabilize the CRBN protein both by shifting its assembly with the SCF and CRL4 complexes and by directly blocking its ubiquitination and degradation targeted by SCFFbxo7.From the genome-scale CRISPR-Cas9 knockout screening, we also found that knock-out of TRAF2, a member of the TNF receptor associated factor protein family, conferred resistance of MM cells to IMiDs treatment, without effecting CRBN level and degradation of IKZF1/3 induced by IMiDs. TRAF2 -KO MM cells displayed increased processing of p100 NF-κB2 protein to p52, resulting in hyperactivation of the non-canonical NF-κB pathway. This finding suggests that the non-canonical NF-kB pathway may be involved in the intrinsic resistance of MM to IMiDs.ConclusionsTaken together, our findings therefore identify novel mechanisms of IMiDs resistance, involving the regulation of CRBN and the non-canonical NF-κB pathway, and provide a mechanistic framework to inform combination therapy strategies to overcome IMiDs resistance in MM. DisclosuresZhou:WuXi AppTec Inc.: Employment. Hideshima:C4 Therapeutics: Equity Ownership; Acetylon: Consultancy. Anderson:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder; MedImmune: Membership on an entity's Board of Directors or advisory committees.

The tumor suppressor archipelago E3 ligase is required for spermatid differentiation in Drosophila testis

The human orthologue of the tumor suppressor protein FBW7 is encoded by the Drosophila archipelago (ago) gene. Ago is an F-box protein that gives substrate specificity to its SCF ubiquitin ligase complex. It has a central role in multiple biological processes in a tissue-specific manner such as cell proliferation, cellular differentiation, hypoxia-induced gene expression. Here we present a previously unknown tissue-specific role of Ago in spermatid differentiation. We identified a classical mutant of ago which is semi-lethal and male-sterile. During the characterization of ago function in testis, we found that ago plays role in spermatid development, following meiosis. We confirmed spermatogenesis defects by silencing ago by RNAi in testes. The ago mutants show multiple abnormalities in elongating and elongated spermatids, including aberration of the cyst morphology, malformed mitochondrial structures, and individualization defects. Additionally, we determined the subcellular localization of Ago protein with mCherry-Ago transgene in spermatids. Our findings highlight the potential roles of Ago in different cellular processes of spermatogenesis, like spermatid individualization, and regulation of mitochondrial morphology.

FBXO11 is a candidate tumor suppressor in the leukemic transformation of myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a heterogeneous myeloid malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of transformation to secondary acute myeloid leukemia (sAML). A number of genetic abnormalities have been identified in MDS and sAML, but sensitive sequencing methods can detect these mutations in nearly all healthy individuals by 60 years of age. To discover novel cellular pathways that accelerate MDS and sAML, we performed a CRISPR/Cas9 screen in the human MDS-L cell line. We report here that loss of the F-Box protein FBXO11, a component of the SCF ubiquitin ligase complex, confers cytokine independent growth to MDS-L cells, suggesting a tumor suppressor role for FBXO11 in myeloid malignancies. Putative FBXO11 substrates are enriched for proteins with functions in RNA metabolism and, of note, spliceosome mutations that are commonly found in MDS/sAML are rare in patients with low FBXO11 expression. We also reveal that loss of FBXO11 leads to significant changes in transcriptional pathways influencing leukocyte proliferation, differentiation, and apoptosis. Last, we find that FBXO11 expression is reduced in patients with secondary AML. We conclude that loss of FBXO11 is a mechanism for disease transformation of MDS into AML, and may represent a future therapeutic target.

The E3 ubiquitin ligase SCF(Fbxo7) mediates proteasomal degradation of UXT isoform 2 (UXT-V2) to inhibit the NF-κB signaling pathway

BackgroundUbiquitously eXpressed Transcript isoform 2 (UXTV2) is a prefoldin-like protein involved in NF-κB signaling, apoptosis, and the androgen and estrogen response. UXT-V2 is a cofactor in the NF-κB transcriptional enhanceosome, and its knockdown inhibits TNF-α -induced NF-κB activation. Fbxo7 is an F-box protein that interacts with SKP1, Cullin1 and RBX1 proteins to form an SCF(Fbxo7) E3 ubiquitin ligase complex. Fbxo7 negatively regulates NF-κB signaling through TRAF2 and cIAP1 ubiquitination. MethodsWe combine co-immunoprecipitation, ubiquitination in vitro and in vivo, cycloheximide chase assay, ubiquitin chain restriction analysis and microscopy to investigate interaction between Fbxo7 and overexpressed UXT-V2-HA. ResultsThe Ubl domain of Fbxo7 contributes to interaction with UXTV2. This substrate is polyubiquitinated by SCF(Fbxo7) with K48 and K63 ubiquitin chain linkages in vitro and in vivo. This post-translational modification decreases UXT-V2 stability and promotes its proteasomal degradation. We further show that UXTV1, an alternatively spliced isoform of UXT, containing 12 additional amino acids at the N-terminus as compared to UXTV2, also interacts with and is ubiquitinated by Fbxo7. Moreover, FBXO7 knockdown promotes UXT-V2 accumulation, and the overexpression of Fbxo7-ΔF-box protects UXT-V2 from proteasomal degradation and enhances the responsiveness of NF-κB reporter. We find that UXT-V2 colocalizes with Fbxo7 in the cell nucleus. ConclusionsTogether, our study reveals that SCF(Fbxo7) mediates the proteasomal degradation of UXT-V2 causing the inhibition of the NF-κB signaling pathway. General significanceDiscovering new substrates of E3 ubiquitin-ligase SCF(Fbxo7) contributes to understand its function in different diseases such as cancer and Parkinson.

Functional characterization of a gibberellin F-box protein, PslSLY1, during plum fruit development.

Fruit development is orchestrated by a complex network of interactions between hormone signaling pathways. The phytohormone gibberellin (GA) is known to regulate a diverse range of developmental processes; however, the mechanisms of GA action in perennial fruit species are yet to be elucidated. In the current study, a GA signaling gene PslSLY1, encoding a putative F-box protein that belongs to the SLY1 (SLEEPY1)/GID2 (gibberellin-insensitive dwarf2) gene family, was isolated from Japanese plum (Prunus salicina). PslSLY1 transcript abundance declined as fruit development progressed, along with potential negative feedback regulation of PslSLY1 by GA. Subcellular localization and protein-protein interaction assays suggested that PslSLY1 functions as an active GA signaling component that interacts with the ASK1 (Arabidopsis SKP1) subunit of an SCF-ubiquitin ligase complex and with PslDELLA repressors, in a GA-independent manner. By using a domain omission strategy, we illustrated that the F-box and C-terminal domains of PslSLY1 are essential for its interactions with the downstream GA signaling components. PslSLY1 overexpression in wild-type and Arabidopsissly1.10 mutant backgrounds resulted in a dramatic enhancement in overall plant growth, presumably due to triggered GA signaling. This includes germination characteristics, stem elongation, flower structure, and fertility. Overall, our findings shed new light on the GA strategy and signaling network in commercially important perennial crops.

E3 Ligase FBXW2 Is a New Therapeutic Target in Obesity and Atherosclerosis.

Chronic low‐grade inflammation orchestrated by macrophages plays a critical role in metabolic chronic diseases, like obesity and atherosclerosis. However, the underlying mechanism remains to be elucidated. Here, the E3 ubiquitin ligase F‐box/WD Repeat‐Containing Protein 2 (FBXW2), the substrate‐binding subunit of E3 ubiquitin ligase SCF (a complex of FBXW2, SKP1, and cullin‐1), as an inflammatory mediator in macrophages, is identified. Myeloid‐specific FBXW2 gene deficiency improves both obesity‐associated with insulin resistance and atherosclerosis in murine models. The beneficial effects by FBXW2 knockout are accompanied by decreased proinflammatory responses and macrophage infiltration in the microenvironment. Mechanistically, it is identified that KH‐type splicing regulatory protein (KSRP) is a new bona fide ubiquitin substrate of SCFFBXW2. Inhibition of KSRP prevents FBXW2‐deficient macrophages from exerting a protective effect on inflammatory reactions, insulin resistance and plaque formation. Furthermore, it is demonstrated that the C‐terminus (P3) of FBXW2 competitively ablates the function of FBXW2 in KSRP degradation and serves as an effective inhibitor of obesity and atherogenesis progression. Thus, the data strongly suggest that SCFFBXW2 is an important mediator in the context of metabolic diseases. The development of FBXW2 (P3)‐mimicking inhibitors and small‐molecular drugs specifically abrogating KSRP ubiquitination‐dependent inflammatory responses are viable approaches for obesity and atherosclerosis treatment.

An Amyotrophic Lateral Sclerosis-Associated Mutant of C21ORF2 Is Stabilized by NEK1-Mediated Hyperphosphorylation and the Inability to Bind FBXO3.

SummaryC21ORF2 and NEK1 have been identified as amyotrophic lateral sclerosis (ALS)-associated genes. Both genes are also mutated in certain ciliopathies, suggesting that they might contribute to the same signaling pathways. Here we show that FBXO3, the substrate receptor of an SCF ubiquitin ligase complex, binds and ubiquitylates C21ORF2, thereby targeting it for proteasomal degradation. C21ORF2 stabilizes the kinase NEK1, with the result that loss of FBXO3 stabilizes not only C21ORF2 but also NEK1. Conversely, NEK1-mediated phosphorylation stabilizes C21ORF2 by attenuating its interaction with FBXO3. We found that the ALS-associated V58L mutant of C21ORF2 is more susceptible to phosphorylation by NEK1, with the result that it is not ubiquitylated by FBXO3 and therefore accumulates together with NEK1. Expression of C21ORF2(V58L) in motor neurons induced from mouse embryonic stem cells impaired neurite outgrowth. We suggest that inhibition of NEK1 activity is a potential therapeutic approach to ALS associated with C21ORF2 mutation.

Abstract A32: The small GTPase Rac1 controls the stability of Yes-Associated Protein (YAP) independently of the LATS1/2 kinases

Abstract Activating mutations in the K-Ras gene are the earliest and most common genetic alterations detected in human pancreatic cancer (PC) specimens. In genetically engineered mouse models of PC, oncogenic K-Ras drives the formation of PanIN precursor lesions and their progression to invasive PC. Oncogenic K-Ras signals through at least four effector pathways that play distinct roles in malignant transformation: MAPK pathway, PI3K pathway, Ral-GDS, and the Rac1 GTPase (Ras-related C3 botulinum toxin substrate 1). Among these effectors, the least well understood is the Rac1 pathway. Yet, this pathway has now been shown to be critical for Ras-driven PC development and malignant transformation. However, the specific mechanisms by which Rac1 promotes anchorage-independent growth and tumorigenicity are still unclear. In recent studies, we have discovered that the Rac1 pathway regulates the stability of the Yes-Associated Protein (YAP). YAP is a transcriptional coactivator that controls the transcription of genes involved in proliferation and survival. In mouse models of Ras-driven PC, YAP is required for the formation of PanIN precursor lesions and their progression to invasive PC. In our telomerase-immortalized human pancreatic ductal cells, overexpressed YAP alone is sufficient to allow anchorage-independent growth. Our results show that in PC cells treated with Rac1 inhibitors (NSC23766, EHT-1864, AZA1, and Rac1T17N), YAP is rapidly and almost completely degraded within 24 hours. Importantly, this degradation of YAP is blocked by the preincubation of the cells with proteasome inhibitor MG132, thereby implying the involvement of a ubiquitin ligase complex. YAP contains a phosphodegron that controls its ubiqutination and subsequent degradation by the proteasome. Activation of this degron recognized by SCF-βTRCP ubiquitin ligase complexes requires its phosphorylation by the LATS1/2 kinases. To identify the molecular signals that control the degradation of YAP after Rac1 inhibition, Flag-tagged mutants of YAP were tested that were missing either a functional degron (ΔβTRCP mutant) or all of their LATS1/2 phosphorylation sites (5SA mutant). Our results show that while the functionality of the degron is required for YAP degradation after Rac1 inhibition, the phosphorylation of this degron by the LATS1/2 kinases is needed. Not only was the 5SA YAP mutant still degraded after Rac1 inhibition, but the knockdown of the LATS1/2 kinases also did not prevent the degradation of YAP. These findings suggest the existence of a novel SCF ubiquitin ligase complex that regulates YAP stability independently of the LATS1/2 kinases. The screening of a siRNA library has now been undertaken to identify the F-box protein involved. The proposed work is expected to shed light on novel mechanisms underlying the development of PC and of other Ras-driven malignancies. Citation Format: Chitra Palanivel, Bailey M. Gabler, Ying Yan, Surinder K. Batra, Michel M. Ouellette. The small GTPase Rac1 controls the stability of Yes-Associated Protein (YAP) independently of the LATS1/2 kinases [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A32.

Chemical Inhibition of PTEN‐Induced Kinase 1 (PINK1) Degradation Confers Neuroprotection in Culture Models of Parkinson’s Disease

PTEN‐induced kinase 1 (PINK1) loss of function is linked to familial recessive Parkinson’s disease and, more recently, sporadic Alzheimer’s disease. After mitochondrial processing and release, PINK1 is rapidly degraded by the proteasome.ObjectiveWe hypothesized that stabilization of PINK1 by interfering with binding to its SCF ubiquitin ligase complex would elevate PINK1 expression levels and confer neuroprotection.Methods & ResultsAfter identifying an F‐box protein that interacted with PINK1 and regulated PINK1 expression levels, we used computational binding simulations to screen compounds likely to interfere with this interaction. We identified a small molecule that stabilized PINK1 expression and reduced both cell death and dendritic retraction in several models of Parkinsonian injury in primary cortical neurons and patient‐derived cells.ConclusionsOur identification of an F‐box protein‐targeted small molecule antagonist capable of elevating endogenous PINK1 levels sets the stage for additional target validation for the potential treatment of neurodegenerative diseases and other disorders characterized by mitochondrial dysfunction.Support or Funding InformationGrant support: NIH grants AG026389, NS101628 (co‐funded by NINDS/NIA) and NS065789 (C.T.C.), HL096376, HL097376, HL098174, HL081784, P01 HL114453 and Merit Review Award from the United States Department of Veterans Affairs (R. K. M.), HL139860 and HL133184 (B.B.C.), HL142777 and AHA 16SDG27650008 (Y.L.). The content is solely the responsibility of the authors and does not represent the official views of the National Institutes of Health.

The ubiquitin ligase FBXW7 targets the centriolar assembly protein HsSAS-6 for degradation and thereby regulates centriole duplication

Formation of a single new centriole from a pre-existing centriole is strictly controlled to maintain correct centrosome number and spindle polarity in cells. However, the mechanisms that govern this process are incompletely understood. Here, using several human cell lines, immunofluorescence and structured illumination microscopy methods, and ubiquitination assays, we show that the E3 ubiquitin ligase F-box and WD repeat domain-containing 7 (FBXW7), a subunit of the SCF ubiquitin ligase, down-regulates spindle assembly 6 homolog (HsSAS-6), a key protein required for procentriole cartwheel assembly, and thereby regulates centriole duplication. We found that FBXW7 abrogation stabilizes HsSAS-6 and increases its recruitment to the mother centriole at multiple sites, leading to supernumerary centrioles. Ultrastructural analyses revealed that FBXW7 is broadly localized on the mother centriole and that its presence is reduced at the site where the HsSAS-6-containing procentriole is formed. This observation suggested that FBXW7 restricts procentriole assembly to a specific site to generate a single new centriole. In contrast, during HsSAS-6 overexpression, FBXW7 strongly associated with HsSAS-6 at the centriole. We also found that SCFFBXW7 interacts with HsSAS-6 and targets it for ubiquitin-mediated degradation. Further, we identified putative phosphodegron sites in HsSAS-6, whose substitutions rendered it insensitive to FBXW7-mediated degradation and control of centriole number. In summary, SCFFBXW7 targets HsSAS-6 for degradation and thereby controls centriole biogenesis by restraining HsSAS-6 recruitment to the mother centriole, a molecular mechanism that controls supernumerary centrioles/centrosomes and the maintenance of bipolar spindles.

FBW7 inhibits nucleus pulposus cells proliferation by downregulation of cyclin E in the intervertebral disc degeneration.

TF-box and WD repeat domain-containing 7 (FBW7), a component of SCF ubiquitin ligase complex, usually acts as a tumor suppressor because it has an ability in the inhibition of cell proliferation. Nevertheless, the role of FBW7 in intervertebral disc degeneration (IDD) is not quite understood. The total protein and RNA were isolated from patients' disc tissues. WB was carried out to analyze the collagen II and FBW7 protein levels of different Pfirrmann grades disc degeneration. Reverse transcription-polymerase chain reaction (RT-PCR) was used to test the collagen II and FBW7 mRNA expression in these disc samples. NP cells were transfected with siRNA-FWB7 to downregulate the FBW7 expression. SiRNA-NC was used as the sham group. Cyclin E, E2F1, and E2F2 were analyzed with WB and RT-PCR. In this study, different kinds of degenerated disc tissues were analyzed, and it was found that FBW7 was overexpressed in much severe degeneration condition, which was also proved by the IL-1β stimuli nucleus pulposus (NP) cells degeneration model in vitro. Interestingly, the results showed that FBW7 suppression could reverse the degeneration of NP cells. Furthermore, we found that FBW7 induced NP cell cycle arrest in the G1 phase of and inhibited cell proliferation by upregulating p27 expression in vitro. The overexpression of p27 resulted in the inhibition of cyclin E, which promotes cell proliferation. Taken together, our study uncovered that FBW7 played an essential inhibitory role in NP cells proliferation, providing new insights that FBW7 may be a potential strategy for IDD treatments.

FBXO11 Is a Candidate Tumor Suppressor in the Transformation of MDS to Secondary AML

Myelodysplastic syndrome (MDS) is a heterogenous myeloid lineage malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of secondary transformation to acute myeloid leukemia (sAML). Genomic sequencing of large MDS cohorts has led to the identification of recurrent genetic abnormalities that carry independent prognostic significance and overlap with mutational changes in sAML. However, no set of mutations is sufficient to predict the transformation of MDS raising the question of how an identical genotype produces MDS in one patient and sAML in another? We hypothesize there are therapeutically targetable cellular processes altered by the initiating genetic changes in MDS that predict transformation to sAML. To uncover novel cellular pathways involved in MDS transformation, we performed an unbiased genome-wide CRISPR/Cas9 in the human MDS-L cell line. MDS-L was established from bone marrow mononuclear cells in a 52-year-old male patient and requires IL3-containing media for growth in vitro (Figure 1A). GFP expressing MDS-L cells were transduced with Cas9 and a sgRNA against GFP to confirm functional Cas9 expression in MDS-L (Figures 1B and C). In Cas9 expressing MDS-L cells, we then transduced the Brunello sgRNA CRISPR library and subjected the cells to IL-3 starvation for 4 weeks. Cells surviving IL-3 starvation were then expanded and harvested for genomic DNA. High throughput sequencing of the barcoded DNA produced raw reads that were analyzed using the PinAPL-Py web-based software. sgRNAs appearing in duplicate with absolute read counts over 1000 or in triplicate over 100 were considered significant. We identified 5 genes that conferred resistance to IL-3 starvation, which included FBXO11 (Figure 1D). The Fbox protein FBXO11 is a component of the SCF ubiquitin ligase complex and regulates its substrates via ubiquitination and proteasomal degradation. FBXO11 is mutated in up to 20% of diffuse large B-cell lymphomas and its loss in breast cancer models leads to increased metastases. Therefore, we hypothesized FBXO11 may also function as a tumor suppressor in the transformation of MDS to AML. We confirmed in the Bloodspot gene expression database that there are decreased levels of FBXO11 in a variety of AML samples, including complex karyotype, compared to normal HSCs. To validate the results of the screen, we synthesized two sgRNAs targeting FBXO11, transduced these into MDS-L cells, and detected reduced FBXO11 expression (Figure 1E). Loss of FBXO11 expression promoted survival in IL-3 free media, confirming the selection readout of the screen (Figure 1F). We then designed a silent mutation in the shorter isoform of FBXO11 (FBXO11v1sm1) that rendered resistance to CRISPR/Cas9 (Figure 1G) and observed that overexpression of FBXO11v1sm1 re-sensitized cells to cytokine starvation (Figure 1H). Whether there are different functions between FBXO11 variant 1 and 4 are currently being explored. We are actively performing RNA sequencing and ubiquitin proteomics in FBXO11 knockout cells to identify its downstream targets and assaying for reduced expression of FBXO11 in primary patient MDS and AML samples. Based on our studies, we predict that SCF ubiquitin ligase component FBXO11 is a tumor suppressor regulating the transformation of MDS to secondary AML. Figure 1 Disclosures Crispino: Sierra Oncology: Consultancy; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Forma Therapeutics: Research Funding; Scholar Rock: Research Funding.

Isoforms of RNF128 Regulate the Stability of Mutant P53 in Barrett's Esophageal Cells

Barrett's esophagus (BE) can progress to dysplasia and esophageal adenocarcinoma (EAC), accompanied by mutations in TP53 that increase the stability of its product, p53. We analyzed BE tissues for messenger RNAs (mRNAs) that associate with BE progression and identified one that affects the stabilization of p53. We obtained 54 BE samples collected from patients with high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC), from 1992 through 2015, and performed RNA sequence analyses, including isoform-specific analyses. We performed reverse-transcription polymerase chain reaction analyses of 166 samples and immunohistochemical analyses of tissue microarrays that contained BE tissues from 100 patients with HGD or EAC and normal esophageal squamous mucosa (controls). Proteins were expressed from transfected plasmids or knocked down with small interfering RNAs in BE cells and analyzed by immunoblots and in immunoprecipitation and ubiquitin ligase assays. Athymic nude mice bearing EAC xenograft tumors (grown from OE-33 cells) were given intraperitoneal injections of simvastatin; tumor growth was monitored and tumors were collected and analyzed by immunoblotting for levels of RNF128, p53, and acetylated p53. Progression of BE to HGD or EAC associated with changes in expression of mRNAs that encoded mucins and promoted inflammation and activation of ATM and the DNA damage response. As tissues progressed from BE to HGD to EAC, they increased expression of mRNAs encoding isoform 1 of RNF128 (Iso1) and decreased expression of Iso2 of RNF128. RNF128 is an E3 ubiquitin ligase that targets p53 for degradation. Incubation of BE cells with interferon gamma caused them to increase expression of Iso1 and reduce expression of Iso2. Iso1 was heavily glycosylated with limited ubiquitin ligase activity for p53, resulting in p53 stabilization. Knockdown of Iso1 in BE and EAC cells led to degradation of the mutant form of p53 and reduced clonogenic survival. In contrast, Iso2 was a potent ligase that reduced levels of the mutant form of p53 in BE cells. In BE cells, Iso2 was hypoglycosylated and degraded, via ATM and GSK3β-mediated phosphorylation and activation of the beta-TrCP1-containing SCF ubiquitin ligase complex. Simvastatin, which degrades the mutant form of p53, also degraded RNF128 Iso1 protein in BE cells and slowed growth of EAC xenograft tumors in mice. We found that isoform 2 of RNF128 is decreased in BE cells, resulting in increased levels of mutant p53, whereas isoform 1 of RNF128 is increased in BE cells, further promoting the stabilization of mutant p53.

Notch and the pre-TCR coordinate thymocyte proliferation by induction of the SCF subunits Fbxl1 and Fbxl12.

Proliferation is tightly regulated during T cell development and is limited to immature CD4−CD8− thymocytes. The major proliferative event is initiated at the ‘β-selection’ stage following successful rearrangement of Tcrβ and is triggered by and dependent on concurrent signaling by Notch and the pre-TCR; however, it is unclear how these signals cooperate to promote cell proliferation. Here we found that β-selection-associated proliferation required the combined activity of two SCF ubiquitin ligase complexes that included as substrate recognition subunits the F-box proteins Fbxl1 or Fbxl12. Both SCF complexes targeted the cyclin-dependent kinase inhibitor Cdkn1b for ubiquitinylaton and degradation. We found that Notch signals induced the transcription of Fbxl1 whereas pre-TCR signals induced the transcription of Fbxl12. Thus, concurrent Notch and pre-TCR signaling induced the expression of two genes, Fbxl1 and Fbxl12, whose products functioned identically but additively to promote degradation of Cdkn1b, cell cycle progression, and proliferation of β-selected thymocytes.

FBXW11 VARIANTS DECREASED HSCS QUIESCENCE AND MYELOID-BIASED DIFFERENTIATION IN VIVO

Hematopoietic stem cells (HSCs) maintain their balance of self-renewal and differentiation under the regulation of sophisticated signal transduction networks. However, the intrinsic molecular mechanism that regulates this transformative property remains elusive. Fbxw11, as a constituent of the SCF (Skp1-Cul1-F box) ubiquitin ligase complex, can target for degradation several important transcription factors. So, Fbxw11 may play a pivotal role in many aspects of hematopoiesis through regulating various signal transduction pathways. In this study, we found that the expression of Fbxw11were lower in long term hematopoietic stem cell (LT‐HSCs), and higher in hematopoietic stem and progenitor cells (LSK) and various hematopoietic progenitor cells compared with BM cells. Competitive repopulation assays were performed and we observed that Fbxw11-overexpressed HSCs impaired their ability to repopulation. Colony-forming cell assay (CFC) showed that the number of CFU-GEMM, CFU-GM, CFU-G and the total number of CFU were lower in LSK with overexpression of Fbxw11 variants when compared with LSK control. Hoechst 33342 and Ki67 staining assay showed that the percentage of LSK cells in G0 phase was decreased when Fbxw11 overexpression. Fbxw11-overexpressed HSCs expressed lower level of self-renewal associated transcription factors, such as HoxA5, Hoxbl3, Myc, Sox2 and Tim1, suggesting that Fbxw11 decreased the self-renewal ability of HSCs. Fbxw11 also led to a progressive enlargement of myeloid population and reduce of lymphocyte population. Fbxw11-overexpressed HSCs expressed higher level of myeloid differentiation associated Csf1r, CSF2r and Runx1. Our results, therefore, demonstrate a critical and unique role for Fbxw11 in regulating self-renewal of HSCs and myeloid differentiation.

Cyclin F Controls Cell-Cycle Transcriptional Outputs by Directing the Degradation of the Three Activator E2Fs.

E2F1, E2F2, and E2F3A, the three activators of the E2F family of transcription factors, are key regulators of the G1/S transition, promoting transcription of hundreds of genes critical for cell-cycle progression. We found that during late S and in G2, the degradation of all three activator E2Fs is controlled by cyclinF, the substrate receptor of 1 of 69 human SCF ubiquitin ligase complexes. E2F1, E2F2, and E2F3A interact with the cyclin box of cyclin F via their conserved N-terminal cyclin binding motifs. In the short term, E2F mutants unable to bind cyclin F remain stable throughout the cell cycle, induce unscheduled transcription in G2 and mitosis, and promote faster entry into the next S phase. However, in the long term, they impair cell fitness. We propose that by restricting E2F activity to the S phase, cyclinF controls one of the main and most critical transcriptional engines of the cell cycle.

Elongin and the Elongin A ubiquitin ligase complex in transcription and the response to DNA damage

Lesions induced by a variety of DNA damaging agents can block RNA polymerase II (Pol II) elongation. Mechanisms that reactivate or remove Pol II that has become stalled or arrested at such lesions are critical for resumption of gene expression after DNA damage repair and, in addition, may contribute to efficiency of the DNA repair process by enhancing access of the damage repair machinery to the lesion. Following exposure of cells to UV irradiation or other DNA damaging agents, Pol II is ubiquitinated in a transcription‐coupled reaction and degraded by the proteasome, and it has been proposed that ubiquitination and degradation of Pol II stalled at DNA lesions may contribute to transcription‐coupled DNA repair and/or to reactivation of transcription after repair of the damaged DNA.Elongin is a multisubunit complex that was first identified and purified as an activity that stimulates the rate at which Pol II elongates nascent transcripts in vitro. Elongin is composed of 3 subunits, Elongin A (ELOA), the ubiquitin‐like protein Elongin B (ELOB), and Elongin C (ELOC), which is similar to the SCF ubiquitin ligase subunit Skp1. Elongin A performs dual functions in cells as a component of RNA polymerase II (Pol II) transcription elongation factor Elongin and as the substrate recognition subunit of a Cullin‐RING (CRL) E3 ubiquitin ligase that has been shown to target Pol II stalled at sites of DNA damage. Here we investigate the mechanism(s) governing conversion of the Elongin complex from its elongation factor to its ubiquitin ligase form. We find that assembly of the Elongin A ubiquitin ligase is a tightly regulated process. In unstressed cells, the majority of Elongin A is found as part of Pol II elongation factor Elongin. Assembly of Elongin A into the ubiquitin ligase is strongly induced by genotoxic stress, by transcriptional stresses that lead to accumulation of stalled Pol II, and by other stimuli, including endoplasmic reticulum and nutrient stress and retinoic acid signaling, that activate Elongin A‐dependent transcription. We will describe ongoing studies to define signal transduction pathways that lead to assembly and activation of the Elongin A‐ubiquitin ligase as well as experiments addressing the possibility that the Elongin A ubiquitin ligase plays a more direct role in Elongin A‐dependent transcription activation than previously anticipated.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PAM forms an atypical SCF ubiquitin ligase complex that ubiquitinates and degrades NMNAT2

PHR (PAM/Highwire/RPM‐1) proteins are conserved RING E3 ubiquitin ligases that function in developmental processes, such as axon termination and synapse formation, as well as axon degeneration. At present, our understanding of how PHR proteins form ubiquitin ligase complexes remains incomplete. Although genetic studies indicate NMNAT2 is an important mediator of PHR protein function in axon degeneration, it remains unknown how PHR proteins inhibit NMNAT2. Here, we decipher the biochemical basis for how the human PHR protein PAM, also called MYCBP2, forms a noncanonical Skp/Cullin/F box (SCF) complex that contains the F‐box protein FBXO45 and SKP1 but lacks CUL1. We show FBXO45 does not simply function in substrate recognition but is important for assembly of the PAM/FBXO45/SKP1 complex. Interestingly, we demonstrate a novel role for SKP1 as an auxiliary component of the target recognition module that enhances binding of FBXO45 to NMNAT2. Finally, we provide biochemical evidence that PAM polyubiquitinates NMNAT2 and regulates NMNAT2 protein stability and degradation by the proteasome.Support or Funding InformationNational Institutes of Health Grant R01 NS072129This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Local Chain Dynamics of Intrinsically Disordered Sic1 Protein Inferred from Fluorescence Anisotropy Decay Measurements

As part of the cell cycle of the budding yeast Saccharomyces cerevisiae, the cyclin-dependent kinase inhibitor Sic1 is an intrinsically disordered protein (IDP) that is degraded by the Cell division control protein 4 (Cdc4) substrate of the SCF ubiquitin ligase complex upon multi-site phosphorylation. More specifically, the binding affinity of Sic1 to the WD40 binding domain of Cdc4 increases nonlinearly with the number of phosphate groups added to the Sic1 sequence. Previous studies have shown that the overall compaction of Sic1 is not altered significantly upon phosphorylation and/or binding. In fact, the Sic1 protein remains disordered upon binding and it is thought to form a “fuzzy” complex with Cdc4. The internal chain dynamics of the disordered region of Sic1 were examined by fluorescence anisotropy decay (FAD) measurements using site-specific labelling with Alexa Fluor-488 at six different positions along the chain. Anisotropy decays for each labelled construct were fit with two rotational correlation times (∼0.3 ns and ∼2 ns), much faster than the tumbling rate of the whole protein. Conversely, these data provide a map of the dye/linker rotational freedom and the intrinsic backbone torsional mobility of separate regions in the Sic1 sequence. Differential quenching as measured by the fluorescence lifetime points to local/distant contacts between some of the labelling sites and the only aromatic residue in Sic1 (Y14). Additionally, the backbone segmental dynamics are due to dihedral angle fluctuations, and they were compared with a Ramachandran plot obtained from the (p)Sic1 conformations available in the ENSEMBLE database. Going forward, chain dynamics data obtained from fluorescence anisotropy decays can be incorporated as an additional (local) constraint in ENSEMBLE to complement existing constraints from NMR (local) and SAXS (global).