K128 ubiquitination constrains RAS activity by expanding its binding interface with GAP proteins.

The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.

TIPRL1 and its ATM-dependent phosphorylation promote radiotherapy resistance in head and neck cancer.

PURPOSE: TIPRL1 (target of rapamycin signaling pathway regulator-like 1) is a known interactor and inhibitor of protein phosphatases PP2A, PP4 and PP6 - all pleiotropic modulators of the DNA Damage Response (DDR). Here, we investigated the role of TIPRL1 in the radiotherapy (RT) response of Head and Neck Squamous Cell Carcinoma (HNSCC). METHODS: TIPRL1 mRNA (cBioportal) and protein expression (immunohistochemistry) in HNSCC samples were linked with clinical patient data. TIPRL1-depleted HNSCC cells were generated by CRISPR/Cas9 editing, and effects on colony growth, micronuclei formation (microscopy), cell cycle (flow cytometry), DDR signaling (immunoblots) and proteome (mass spectrometry) following RT were assessed. Mass spectrometry was used for TIPRL1 phosphorylation and interactomics analysis in irradiated cells. RESULTS: TIPRL1 expression was increased in tumor versus non-tumor tissue, with high tumoral TIPRL1 expression associating with lower locoregional control and decreased survival of RT-treated patients. TIPRL1 deletion in HNSCC cells resulted in increased RT sensitivity, a faster but prolonged cell cycle arrest, increased micronuclei formation and an altered proteome-wide DDR. Upon irradiation, ATM phosphorylates TIPRL1 at Ser265. A non-phospho Ser265Ala mutant could not rescue the increased radiosensitivity phenotype of TIPRL1-depleted cells. While binding to PP2A-like phosphatases was confirmed, DNA-dependent protein kinase (DNA-PKcs), RAD51 recombinase and nucleosomal histones were identified as novel TIPRL1 interactors. Histone binding, although stimulated by RT, was adversely affected by TIPRL1 Ser265 phosphorylation. CONCLUSIONS: Our findings underscore a clinically relevant role for TIPRL1 and its ATM-dependent phosphorylation in RT resistance through modulation of the DDR, highlighting its potential as a new HNSCC predictive marker and therapeutic target.

Spatial Mechano-Signaling Regulation of GTPases through Non-Degradative Ubiquitination.

Blood flow produces shear stress exerted on the endothelial layer of the vessels. Spatial characterization of the endothelial proteome is required to uncover the mechanisms of endothelial activation by shear stress, as blood flow varies in the vasculature. An integrative ubiquitinome and proteome analysis of shear-stressed endothelial cells demonstrated that the non-degradative ubiquitination of several GTPases is regulated by mechano-signaling. Spatial analysis reveals increased ubiquitination of the small GTPase RAP1 in the descending aorta, a region exposed to laminar shear stress. The ubiquitin ligase WWP2 is identified as a novel regulator of RAP1 ubiquitination during shear stress response. Non-degradative ubiquitination fine-tunes the function of GTPases by modifying their interacting network. Specifically, WWP2-mediated RAP1 ubiquitination at lysine 31 switches the balance from the RAP1/ Talin 1 (TLN1) toward RAP1/ Afadin (AFDN) or RAP1/ RAS Interacting Protein 1 (RASIP1) complex formation, which is essential to suppress shear stress-induced reactive oxygen species (ROS) production and maintain endothelial barrier integrity. Increased ROS production in endothelial cells in the descending aorta of endothelial-specific Wwp2-knockout mice leads to increased levels of oxidized lipids and inflammation. These results highlight the importance of the spatially regulated non-degradative ubiquitination of GTPases in endothelial mechano-activation.

The deubiquitinase OTUB1 governs lung cancer cell fitness by modulating proteostasis of OXPHOS proteins.

Aerobic glycolysis is a hallmark of cancer development, but this dogma has been challenged by reports showing a key role of oxidative phosphorylation (OXPHOS) in cancer cell survival. It has been proposed that increased levels of intramitochondrial proteins in cancer cells are associated with high OXPHOS activity and increased sensitivity to OXPHOS inhibitors. However, the molecular mechanisms leading to the high expression of OXPHOS proteins in cancer cells remain unknown. Multiple proteomics studies have detected the ubiquitination of intramitochondrial proteins, suggesting the contribution of the ubiquitin system to the proteostatic regulation of OXPHOS proteins. Here, we identified the ubiquitin hydrolase OTUB1 as a regulator of the mitochondrial metabolic machinery essential for lung cancer cell survival. Mitochondria-localized OTUB1 modulates respiration by inhibiting K48-linked ubiquitination and turnover of OXPHOS proteins. An increase in OTUB1 expression is commonly observed in one-third of non-small-cell lung carcinomas and is associated with high OXPHOS signatures. Moreover, OTUB1 expression highly correlates with the sensitivity of lung cancer cells to mitochondrial inhibitors.

Breast tumors interfere with endothelial TRAIL at the premetastatic niche to promote cancer cell seeding.

Endothelial cells (ECs) grant access of disseminated cancer cells to distant organs. However, the molecular players regulating the activation of quiescent ECs at the premetastatic niche (PMN) remain elusive. Here, we find that ECs at the PMN coexpress tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its cognate death receptor 5 (DR5). Unexpectedly, endothelial TRAIL interacts intracellularly with DR5 to prevent its signaling and preserve a quiescent vascular phenotype. In absence of endothelial TRAIL, DR5 activation induces EC death and nuclear factor κB/p38-dependent EC stickiness, compromising vascular integrity and promoting myeloid cell infiltration, breast cancer cell adhesion, and metastasis. Consistently, both down-regulation of endothelial TRAIL at the PMN by proangiogenic tumor-secreted factors and the presence of the endogenous TRAIL inhibitors decoy receptor 1 (DcR1) and DcR2 favor metastasis. This study discloses an intracrine mechanism whereby TRAIL blocks DR5 signaling in quiescent endothelia, acting as gatekeeper of the vascular barrier that is corrupted by the tumor during cancer cell dissemination.

The regulation of the protein interaction network by monoubiquitination.

The conjugation of a single ubiquitin or monoubiquitination acts as a versatile signal that can have both degradative and non-degradative functions. The latter is of particular interest as emerging evidence indicates that ubiquitin-driven alterations of the protein interaction landscape play a key role in multiple signaling pathways. Whereas early studies were focused on how monoubiquitination alters the interactions of proteins containing ubiquitin-binding domains, more recent reports demonstrate that ubiquitin conjugation can also affect the binding mode by changing the surface of the ubiquitinated substrate. Furthermore, monoubiquitination modulates the interactions with other macromolecules, such as DNA or lipids, underscoring the diverse role of monoubiquitination in cellular processes. In this review, we discussed how monoubiquitination achieves its function by modulating the interaction landscape.

Loss-of-Function Mutations in TRAF7 and KLF4 Cooperatively Activate RAS-Like GTPase Signaling and Promote Meningioma Development.

Meningiomas are the most common benign brain tumors. Mutations of the E3 ubiquitin ligase TRAF7 occur in 25% of meningiomas and commonly cooccur with mutations in KLF4, yet the functional link between TRAF7 and KLF4 mutations remains unclear. By generating an in vitro meningioma model derived from primary meningeal cells, we elucidated the cooperative interactions that promote meningioma development. By integrating TRAF7-driven ubiquitinome and proteome alterations in meningeal cells and the TRAF7 interactome, we identified TRAF7 as a proteostatic regulator of RAS-related small GTPases. Meningioma-associated TRAF7 mutations disrupted either its catalytic activity or its interaction with RAS GTPases. TRAF7 loss in meningeal cells altered actin dynamics and promoted anchorage-independent growth by inducing CDC42 and RAS signaling. TRAF deficiency-driven activation of the RAS/MAPK pathway promoted KLF4-dependent transcription that led to upregulation of the tumor-suppressive Semaphorin pathway, a negative regulator of small GTPases. KLF4 loss of function disrupted this negative feedback loop and enhanced mutant TRAF7-mediated cell transformation. Overall, this study provides new mechanistic insights into meningioma development, which could lead to novel treatment strategies. SIGNIFICANCE: The intricate molecular cross-talk between the ubiquitin ligase TRAF7 and the transcription factor KLF4 provides a first step toward the identification of new therapies for patients with meningioma.

PPP2R4 dysfunction promotes KRAS-mutant lung adenocarcinoma development and mediates opposite responses to MEK and mTOR inhibition.

KRAS-mutant lung adenocarcinomas represent the largest molecular subgroup of non-small cell lung cancers (NSCLC) and are notorious for their dismal survival perspectives. To gain more insights in etiology and therapeutic response, we focused on the tumor suppressor Protein Phosphatase 2A (PP2A) as a player in KRAS oncogenic signaling. We report that the PP2A activator PTPA (encoded by PPP2R4) is commonly affected in NSCLC by heterozygous loss and low-frequent loss-of-function mutation, and this is specifically associated with poorer overall survival of KRAS-mutant lung adenocarcinoma patients. Reduced or mutant PPP2R4 expression in A549 cells increased anchorage-independent growth in vitro and xenograft growth in vivo, correlating with increased Ki67 and c-MYC expression. Moreover, KrasG12D-induced lung tumorigenesis was significantly accelerated in Ppp2r4 gene trapped mice as compared to Ppp2r4 wild-type. A confined kinase inhibitor screen revealed that PPP2R4-depletion induced resistance against selumetinib (MEK inhibitor), but unexpectedly sensitized cells for temsirolimus (mTOR inhibitor), in vitro and in vivo. Our findings underscore a clinically relevant role for PTPA loss-of-function in KRAS-mutant NSCLC etiology and kinase inhibitor response.

The mitotic checkpoint is a targetable vulnerability of carboplatin-resistant triple negative breast cancers.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, lacking effective therapy. Many TNBCs show remarkable response to carboplatin-based chemotherapy, but often develop resistance over time. With increasing use of carboplatin in the clinic, there is a pressing need to identify vulnerabilities of carboplatin-resistant tumors. In this study, we generated carboplatin-resistant TNBC MDA-MB-468 cell line and patient derived TNBC xenograft models. Mass spectrometry-based proteome profiling demonstrated that carboplatin resistance in TNBC is linked to drastic metabolism rewiring and upregulation of anti-oxidative response that supports cell replication by maintaining low levels of DNA damage in the presence of carboplatin. Carboplatin-resistant cells also exhibited dysregulation of the mitotic checkpoint. A kinome shRNA screen revealed that carboplatin-resistant cells are vulnerable to the depletion of the mitotic checkpoint regulators, whereas the checkpoint kinases CHEK1 and WEE1 are indispensable for the survival of carboplatin-resistant cells in the presence of carboplatin. We confirmed that pharmacological inhibition of CHEK1 by prexasertib in the presence of carboplatin is well tolerated by mice and suppresses the growth of carboplatin-resistant TNBC xenografts. Thus, abrogation of the mitotic checkpoint by CHEK1 inhibition re-sensitizes carboplatin-resistant TNBCs to carboplatin and represents a potential strategy for the treatment of carboplatin-resistant TNBCs.

Loss of 9p21 Regulatory Hub Promotes Kidney Cancer Progression by Upregulating HOXB13.

Loss of chromosome 9p21 is observed in one-thirds of clear-cell renal cell carcinoma (ccRCC) and is associated with poorer patient survival. Unexpectedly, 9p21 LOH does not lead to decreased expression of the 9p21 tumor suppressor genes, CDKN2A and CDKN2B, suggesting alternative mechanisms of 9p-mediated tumorigenesis. Concordantly, CRISPR-mediated 9p21 deletion promotes growth of immortalized human embryonic kidney epithelial cells independently of the CDKN2A/B pathway inactivation. The 9p21 locus has a highly accessible chromatin structure, suggesting that 9p21 loss might contribute to kidney cancer progression by dysregulating genes distal to the 9p21 locus. We identified several 9p21 regulatory hubs by assessing which of the 9p21-interacting genes are dysregulated in 9p21-deleted kidney cells and ccRCCs. By focusing on the analysis of the homeobox gene 13 (HOXB13) locus, we found that 9p21 loss relieves the HOXB13 locus, decreasing HOXB13 methylation and promoting its expression. Upregulation of HOXB13 facilitates cell growth and is associated with poorer survival of patients with ccRCC. IMPLICATIONS: The results of our study propose a novel tumor suppressive mechanism on the basis of coordinated expression of physically associated genes, providing a better understanding of the role of chromosomal deletions in cancer.

Cracking the Monoubiquitin Code of Genetic Diseases.

Ubiquitination is a versatile and dynamic post-translational modification in which single ubiquitin molecules or polyubiquitin chains are attached to target proteins, giving rise to mono- or poly-ubiquitination, respectively. The majority of research in the ubiquitin field focused on degradative polyubiquitination, whereas more recent studies uncovered the role of single ubiquitin modification in important physiological processes. Monoubiquitination can modulate the stability, subcellular localization, binding properties, and activity of the target proteins. Understanding the function of monoubiquitination in normal physiology and pathology has important therapeutic implications, as alterations in the monoubiquitin pathway are found in a broad range of genetic diseases. This review highlights a link between monoubiquitin signaling and the pathogenesis of genetic disorders.

The Mystery of Rap1 Suppression of Oncogenic Ras.

Decades ago, Rap1, a small GTPase very similar to Ras, was observed to suppress oncogenic Ras phenotype, reverting its transformation. The proposed reason, persisting since, has been competition between Ras and Rap1 for a common target. Yet, none was found. There was also Rap1's puzzling suppression of Raf-1 versus activation of BRAF. Reemerging interest in Rap1 envisages capturing its Ras suppression action by inhibitors. Here, we review the literature and resolve the enigma. In vivo oncogenic Ras exists in isoform-distinct nanoclusters. The presence of Rap1 within the nanoclusters reduces the number of the clustered oncogenic Ras molecules, thus suppressing Raf-1 activation and mitogen-activated protein kinase (MAPK) signaling. Nanoclustering suggests that Rap1 suppression is Ras isoform dependent. Altogether, a potent Rap1-like inhibitor appears unlikely.

The Noonan Syndrome Gene Lztr1 Controls Cardiovascular Function by Regulating Vesicular Trafficking.

RATIONALE: Noonan syndrome (NS) is one of the most frequent genetic disorders. Bleeding problems are among the most common, yet poorly defined complications associated with NS. A lack of consensus on the management of bleeding complications in patients with NS indicates an urgent need for new therapeutic approaches. OBJECTIVE: Bleeding disorders have recently been described in patients with NS harboring mutations of LZTR1 (leucine zipper-like transcription regulator 1), an adaptor for CUL3 (CULLIN3) ubiquitin ligase complex. Here, we assessed the pathobiology of LZTR1-mediated bleeding disorders. METHODS AND RESULTS: Whole-body and vascular specific knockout of Lztr1 results in perinatal lethality due to cardiovascular dysfunction. Lztr1 deletion in blood vessels of adult mice leads to abnormal vascular leakage. We found that defective adherent and tight junctions in Lztr1-depleted endothelial cells are caused by dysregulation of vesicular trafficking. LZTR1 affects the dynamics of fusion and fission of recycling endosomes by controlling ubiquitination of the ESCRT-III (endosomal sorting complex required for transport III) component CHMP1B (charged multivesicular protein 1B), whereas NS-associated LZTR1 mutations diminish CHMP1B ubiquitination. LZTR1-mediated dysregulation of CHMP1B ubiquitination triggers endosomal accumulation and subsequent activation of VEGFR2 (vascular endothelial growth factor receptor 2) and decreases blood levels of soluble VEGFR2 in Lztr1 haploinsufficient mice. Inhibition of VEGFR2 activity by cediranib rescues vascular abnormalities observed in Lztr1 knockout mice Conclusions: Lztr1 deletion phenotypically overlaps with bleeding diathesis observed in patients with NS. ELISA screening of soluble VEGFR2 in the blood of LZTR1-mutated patients with NS may predict both the severity of NS phenotypes and potential responders to anti-VEGF therapy. VEGFR inhibitors could be beneficial for the treatment of bleeding disorders in patients with NS.

Stress-induced lncRNA LASTR fosters cancer cell fitness by regulating the activity of the U4/U6 recycling factor SART3.

Dysregulated splicing is a common event in cancer even in the absence of mutations in the core splicing machinery. The aberrant long non-coding transcriptome constitutes an uncharacterized level of regulation of post-transcriptional events in cancer. Here, we found that the stress-induced long non-coding RNA (lncRNA), LINC02657 or LASTR (lncRNA associated with SART3 regulation of splicing), is upregulated in hypoxic breast cancer and is essential for the growth of LASTR-positive triple-negative breast tumors. LASTR is upregulated in several types of epithelial cancers due to the activation of the stress-induced JNK/c-JUN pathway. Using a mass-spectrometry based approach, we identified the RNA-splicing factor SART3 as a LASTR-interacting partner. We found that LASTR promotes splicing efficiency by controlling SART3 association with the U4 and U6 small nuclear ribonucleoproteins (snRNP) during spliceosome recycling. Intron retention induced by LASTR depletion downregulates expression of essential genes, ultimately decreasing the fitness of cancer cells.

Co-regulation of the antagonistic RepoMan:Aurora-B pair in proliferating cells.

Chromosome segregation during mitosis is antagonistically regulated by the Aurora-B kinase and RepoMan (recruits PP1 onto mitotic chromatin at anaphase)-associated phosphatases PP1/PP2A. Aurora B is overexpressed in many cancers but, surprisingly, this only rarely causes lethal aneuploidy. Here we show that RepoMan abundance is regulated by the same mechanisms that control Aurora B, including FOXM1-regulated expression and proteasomal degradation following ubiquitination by APC/C-CDH1 or SCFFBXW7. The deregulation of these mechanisms can account for the balanced co-overexpression of Aurora B and RepoMan in many cancers, which limits chromosome segregation errors. In addition, Aurora B and RepoMan independently promote cancer cell proliferation by reducing checkpoint--induced cell-cycle arrest during interphase. The co-up-regulation of RepoMan and Aurora B in tumors is inversely correlated with patient survival, underscoring its potential importance for tumor progression. Finally, we demonstrate that high RepoMan levels sensitize cancer cells to Aurora-B inhibitors. Hence, the co-up-regulation of RepoMan and Aurora B is associated with tumor aggressiveness but also exposes a vulnerable target for therapeutic intervention.

Radiosensitization approaches for HPV-positive and HPV-negative head and neck squamous carcinomas.

Radiotherapy is one of the most used treatment approaches for head and neck squamous cell carcinoma (HNSCC). Targeted inhibition of DNA repair machinery has the potential to improve treatment response by tailoring treatment to cancer cells lacking specific DNA repair pathways. Human papillomavirus (HPV)-negative and HPV-positive HNSCCs respond differently to radiotherapy treatment, suggesting that different approaches of DNA repair inhibition should be employed for these HNSCC groups. Here, we searched for optimal radiosensitization approaches for HPV-positive and HPV-negative HNSCCs by performing a targeted CRISPR-Cas9 screen. We found that inhibition of base excision repair resulted in a better radiotherapy response in HPV-positive HNSCC, which is correlated with upregulation of genes involved in base excision repair. In contrast, inhibition of nonhomologous end-joining and mismatch repair showed strong effects in both HNSCC groups. We validated the screen results by combining radiotherapy with targeted inhibition of DNA repair in several preclinical models including primary and recurrent patient-derived HNSCC xenografts. These findings underline the importance of stratifying HNSCC patients for combination treatments.