Loss of LCMT1 and biased protein phosphatase 2A heterotrimerization drive prostate cancer progression and therapy resistance.

Loss of the tumor suppressive activity of the protein phosphatase 2A (PP2A) is associated with cancer, but the underlying molecular mechanisms are unclear. PP2A holoenzyme comprises a heterodimeric core, a scaffolding A subunit and a catalytic C subunit, and one of over 20 distinct substrate-directing regulatory B subunits. Methylation of the C subunit regulates PP2A heterotrimerization, affecting B subunit binding and substrate specificity. Here, we report that the leucine carboxy methyltransferase (LCMT1), which methylates the L309 residue of the C subunit, acts as a suppressor of androgen receptor (AR) addicted prostate cancer (PCa). Decreased methyl-PP2A-C levels in prostate tumors is associated with biochemical recurrence and metastasis. Silencing LCMT1 increases AR activity and promotes castration-resistant prostate cancer growth. LCMT1-dependent methyl-sensitive AB56αCme heterotrimers target AR and its critical coactivator MED1 for dephosphorylation, resulting in the eviction of the AR-MED1 complex from chromatin and loss of target gene expression. Mechanistically, LCMT1 is regulated by S6K1-mediated phosphorylation-induced degradation requiring the ß-TRCP, leading to acquired resistance to anti-androgens. Finally, feedforward stabilization of LCMT1 by small molecule activator of phosphatase (SMAP) results in attenuation of AR-signaling and tumor growth inhibition in anti-androgen refractory PCa. These findings highlight methyl-PP2A-C as a prognostic marker and that the loss of LCMT1 is a major determinant in AR-addicted PCa, suggesting therapeutic potential for AR degraders or PP2A modulators in prostate cancer treatment.

p53 restoration in small cell lung cancer identifies a latent cyclophilin-dependent necrosis mechanism.

The p53 tumor suppressor regulates multiple context-dependent tumor suppressive programs. Although p53 is mutated in ~90% of small cell lung cancer (SCLC) tumors, how p53 mediates tumor suppression in this context is unknown. Here, using a mouse model of SCLC in which endogenous p53 expression can be conditionally and temporally regulated, we show that SCLC tumors maintain a requirement for p53 inactivation. However, we identify tumor subtype heterogeneity between SCLC tumors such that p53 reactivation induces senescence in a subset of tumors, while in others, p53 induces necrosis. We pinpoint cyclophilins as critical determinants of a p53-induced transcriptional program that is specific to SCLC tumors and cell lines poised to undergo p53-mediated necrosis. Importantly, inhibition of cyclophilin isomerase activity, or genetic ablation of specific cyclophilin genes, suppresses p53-mediated necrosis by limiting p53 transcriptional output without impacting p53 chromatin binding. Our study demonstrates that intertumoral heterogeneity in SCLC influences the biological response to p53 restoration, describes a cyclophilin-dependent mechanism of p53-regulated cell death, and uncovers putative mechanisms for the treatment of this most-recalcitrant tumor type.

DLBCL-associated NOTCH2 mutations escape ubiquitin-dependent degradation and promote chemoresistance.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Up to 40% of patients with DLBCL display refractory disease or relapse after standard chemotherapy treatment (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone [R-CHOP]), leading to significant morbidity and mortality. The molecular mechanisms of chemoresistance in DLBCL remain incompletely understood. Using a cullin-really interesting new gene (RING) ligase-based CRISPR-Cas9 library, we identify that inactivation of the E3 ubiquitin ligase KLHL6 promotes DLBCL chemoresistance. Furthermore, proteomic approaches helped identify KLHL6 as a novel master regulator of plasma membrane-associated NOTCH2 via proteasome-dependent degradation. In CHOP-resistant DLBCL tumors, mutations of NOTCH2 result in a protein that escapes the mechanism of ubiquitin-dependent proteolysis, leading to protein stabilization and activation of the oncogenic RAS signaling pathway. Targeting CHOP-resistant DLBCL tumors with the phase 3 clinical trial molecules nirogacestat, a selective γ-secretase inhibitor, and ipatasertib, a pan-AKT inhibitor, synergistically promotes DLBCL destruction. These findings establish the rationale for therapeutic strategies aimed at targeting the oncogenic pathway activated in KLHL6- or NOTCH2-mutated DLBCL.

Disease-associated KBTBD4 mutations in medulloblastoma elicit neomorphic ubiquitylation activity to promote CoREST degradation.

Medulloblastoma is the most common malignant brain tumour in children. Genomic studies have identified distinct disease subgroups: wnt/wingless (WNT), sonic hedgehog (SHH), and non-WNT/non-SHH, comprising group 3 and group 4. Alterations in WNT and SHH signalling form the pathogenetic basis for their subgroups, whereas those for non-WNT/non-SHH tumours remain largely elusive. Recent analyses have revealed recurrent in-frame insertions in the E3 ubiquitin ligase adaptor Kelch Repeat and BTB Domain Containing 4 (KBTBD4) in cases of group 3/4 medulloblastoma. Critically, group 3/4 tumours with KBTBD4 mutations typically lack other gene-specific alterations, such as MYC amplification, indicating KBTBD4 insertion mutations as the primary genetic driver. Delineating the role of KBTBD4 mutations thus offers significant opportunities to understand tumour pathogenesis and to exploit the underpinning mechanisms therapeutically. Here, we show a novel mechanism in cancer pathogenesis whereby indel mutations in KBTBD4 drive its recognition of neo-substrates for degradation. We observe that KBTBD4 mutants promote the recruitment and ubiquitylation of the REST Corepressor (CoREST), which forms a complex to modulate chromatin accessibility and transcriptional programmes. The degradation of CoREST promoted by KBTBD4 mutation diverts epigenetic programmes inducing significant alterations in transcription to promote increased stemness of cancer cells. Transcriptional analysis of >200 human group 3 and 4 medulloblastomas by RNA-seq, highlights the presence of CoREST and stem-like signatures in tumours with KBTBD4 mutations, which extend to a further sub-set of non-mutant tumours, suggesting CoREST alterations as a novel pathogenetic mechanism of wide relevance in groups 3 and 4. Our findings uncover KBTBD4 mutation as a novel driver of epigenetic reprogramming in non-WNT/non-SHH medulloblastoma, establish a novel mode of tumorigenesis through gain-of-function mutations in ubiquitin ligases (neo-substrate recruitment) and identify both mutant KBTBD4 and CoREST complexes as new druggable targets for improved tumour-specific therapies.

CRL4AMBRA1 is a master regulator of D-type cyclins.

D-type cyclins are central regulators of the cell division cycle and are among the most frequently deregulated therapeutic targets in human cancer1, but the mechanisms that regulate their turnover are still being debated2,3. Here, by combining biochemical and genetics studies in somatic cells, we identify CRL4AMBRA1 (also known as CRL4DCAF3) as the ubiquitin ligase that targets all three D-type cyclins for degradation. During development, loss of Ambra1 induces the accumulation of D-type cyclins and retinoblastoma (RB) hyperphosphorylation and hyperproliferation, and results in defects of the nervous system that are reduced by treating pregnant mice with the FDA-approved CDK4 and CDK6 (CDK4/6) inhibitor abemaciclib. Moreover, AMBRA1 acts as a tumour suppressor in mouse models and low AMBRA1 mRNA levels are predictive of poor survival in cancer patients. Cancer hotspot mutations in D-type cyclins abrogate their binding to AMBRA1 and induce their stabilization. Finally, a whole-genome, CRISPR-Cas9 screen identified AMBRA1 as a regulator of the response to CDK4/6 inhibition. Loss of AMBRA1 reduces sensitivity to CDK4/6 inhibitors by promoting the formation of complexes of D-type cyclins with CDK2. Collectively, our results reveal the molecular mechanism that controls the stability of D-type cyclins during cell-cycle progression, in development and in human cancer, and implicate AMBRA1 as a critical regulator of the RB pathway.

FBXW7 Triggers Degradation of KMT2D to Favor Growth of Diffuse Large B-cell Lymphoma Cells.

Mature B-cell neoplasms are the fifth most common neoplasm. Due to significant heterogeneity at the clinical and genetic levels, current therapies for these cancers fail to provide long-term cures. The clinical success of proteasome inhibition for the treatment of multiple myeloma and B-cell lymphomas has made the ubiquitin pathway an important emerging therapeutic target. In this study, we assessed the role of the E3 ligase FBXW7 in mature B-cell neoplasms. FBXW7 targeted the frequently inactivated tumor suppressor KMT2D for protein degradation, subsequently regulating gene expression signatures related to oxidative phosphorylation (OxPhos). Loss of FBXW7 inhibited diffuse large B-cell lymphoma cell growth and further sensitized cells to OxPhos inhibition. These data elucidate a novel mechanism of regulation of KMT2D levels by the ubiquitin pathway and uncover a role of FBXW7 in regulating oxidative phosphorylation in B-cell malignancies. SIGNIFICANCE: These findings characterize FBXW7 as a prosurvival factor in B-cell lymphoma via degradation of the chromatin modifier KMT2D.

Loss of ELF5-FBXW7 stabilizes IFNGR1 to promote the growth and metastasis of triple-negative breast cancer through interferon-γ signalling.

Triple-negative breast cancer (TNBC) is characterized by a high degree of immune infiltrate in the tumour microenvironment, which may influence the fate of TNBC cells. We reveal that loss of the tumour suppressive transcription factor Elf5 in TNBC cells activates intrinsic interferon-γ (IFN-γ) signalling, promoting tumour progression and metastasis. Mechanistically, we find that loss of the Elf5-regulated ubiquitin ligase FBXW7 ensures stabilization of its putative protein substrate IFN-γ receptor 1 (IFNGR1) at the protein level in TNBC. Elf5low tumours show enhanced IFN-γ signalling accompanied by an increase of immunosuppressive neutrophils within the tumour microenvironment and increased programmed death ligand 1 expression. Inactivation of either programmed death ligand 1 or IFNGR1 elicited a robust anti-tumour and/or anti-metastatic effect. A positive correlation between ELF5 and FBXW7 expression and a negative correlation between ELF5, FBXW7 and IFNGR1 expression in the tumours of patients with TNBC strongly suggest that this signalling axis could be exploited for patient stratification and immunotherapeutic treatment strategies for Elf5low patients with TNBC.

Targeting the E3 ubiquitin ligases DCAF15 and cereblon for cancer therapy.

Small molecule-mediated inhibition of protein function is the rational behind therapeutic efficacy of the majority clinically used drugs. In order for a drug to achieve pharmacologically relevant inhibition, efficient target engagement at high selectivity and specificity is necessary to obtain the desired therapeutic effect minimizing offtarget outcomes. Majority of small molecules approaches developed so far have failed in their attempt to reach clinical efficacy because of low selectivity and low specificity in achieving close to 100 % target inhibition. Recently, approaches that directly control cellular protein levels have opened the potential to accomplish a high grade of efficacy not imaginable with traditional small-molecule inhibitors. Research in this area has just started opening avenues to effectively degrade a cellular target of choice and will soon impact clinical efficacy.

Disruption of the menin-MLL interaction triggers menin protein degradation via ubiquitin-proteasome pathway.

Menin, a protein encoded by the MEN1 gene, suppresses cancers associated with multiple endocrine neoplasia type 1 (MEN1), but promotes the development of a subset of leukemia induced by mixed lineage leukemia (MLL)-derived fusion proteins (MLL-FPs). The crystal structure of menin indicates that it acts as a scaffold protein to bind the N-terminus of MLL via a central pocket. Small molecule menin-MLL inhibitors (MIs) bind the menin pocket to disrupt the menin/MLL interaction, resulting in suppression of MLL-FP-transformed acute myeoloid leukemia (AML). It is thought that MIs suppress the MLL-FP-induced leukemia by blocking the menin/MLL interaction and menin/MLL-induced HOX gene transcription. However, it is not clear whether MIs also affect other aspects of menin biology beyond disruption of the menin/MLL interaction. Here we show for the first time that MIs reduced menin protein levels and decreased the half-life of menin protein but have no effect on mRNA level in MLL-FP-expressing leukemia cells, and proteasome or E1 ligase inhibitor rescued the MI-induced menin degradation. Notably, the MI-induced reduction of H3K4m3 and HOXA9 expression was rescued with a proteasome inhibitor that blocks MI-induced menin protein degradation. Mechanistically, MIs promote the interaction of menin with Hsp70-associated ubiquitin ligase CHIP, resulting in increased menin ubiquitination, leading to increased menin degradation. Together, these findings uncover a novel mechanism whereby small molecule MIs increase menin degradation by triggering the Hsp70/CHIP-mediated ubiquitin-proteasome pathway that ultimately leads to the reduction in HOXA9 gene expression and leukemia suppression.

The E3 ubiquitin ligase SPOP controls resolution of systemic inflammation by triggering MYD88 degradation.

The response to systemic infection and injury requires the rapid adaptation of hematopoietic stem cells (HSCs), which proliferate and divert their differentiation toward the myeloid lineage. Significant interest has emerged in understanding the signals that trigger the emergency hematopoietic program. However, the mechanisms that halt this response of HSCs, which is critical to restore homeostasis, remain unknown. Here we reveal that the E3 ubiquitin ligase Speckle-type BTB-POZ protein (SPOP) restrains the inflammatory activation of HSCs. In the absence of Spop, systemic inflammation proceeded in an unresolved manner, and the sustained response in the HSCs resulted in a lethal phenotype reminiscent of hyper-inflammatory syndrome or sepsis. Our proteomic studies decipher that SPOP restricted inflammation by ubiquitinating the innate signal transducer myeloid differentiation primary response protein 88 (MYD88). These findings unearth an HSC-intrinsic post-translational mechanism that is essential for reestablishing homeostasis after emergency hematopoiesis.

Copper chaperone ATOX1 is required for MAPK signaling and growth in BRAF mutation-positive melanoma.

Copper (Cu) is a tightly regulated micronutrient that functions as a structural or catalytic cofactor for specific proteins essential for a diverse array of biological processes. While the study of the extremely rare genetic diseases, Menkes and Wilson, has highlighted the requirement for proper Cu acquisition and elimination in biological systems for cellular growth and proliferation, the importance of dedicated Cu transport systems, like the Cu chaperones ATOX1 and CCS, in the pathophysiology of cancer is not well defined. We found that ATOX1 was significantly overexpressed in human blood, breast, and skin cancer samples, while CCS was significantly altered in human brain, liver, ovarian, and prostate cancer when compared to normal tissue. Further analysis of genetic expression data in Cancer Cell Line Encyclopedia (CCLE) revealed that ATOX1 is highly expressed in melanoma cell lines over other cancer cell lines. We previously found that Cu is required for BRAFV600E-driven MAPK signaling and melanomagenesis. Here we show that genetic loss of ATOX1 decreased BRAFV600E-dependent growth and signaling in human melanoma cell lines. Pharmacological inhibition of ATOX1 with a small molecule, DCAC50, decreased the phosphorylation of ERK1/2 and reduced the growth of BRAF mutation-positive melanoma cell lines in a dose-dependent manner. Taken together, these results suggest that targeting the Cu chaperone ATOX1 as a novel therapeutic angle in BRAFV600E-driven melanomas.

ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of ßTrCP.

The Hedgehog (Hh) pathway is essential for embryonic development and tissue homeostasis. Aberrant Hh signaling may occur in a wide range of human cancers, such as medulloblastoma, the most common brain malignancy in childhood. Here, we identify endoplasmic reticulum aminopeptidase 1 (ERAP1), a key regulator of innate and adaptive antitumor immune responses, as a previously unknown player in the Hh signaling pathway. We demonstrate that ERAP1 binds the deubiquitylase enzyme USP47, displaces the USP47-associated ßTrCP, the substrate-receptor subunit of the SCFßTrCP ubiquitin ligase, and promotes ßTrCP degradation. These events result in the modulation of Gli transcription factors, the final effectors of the Hh pathway, and the enhancement of Hh activity. Remarkably, genetic or pharmacological inhibition of ERAP1 suppresses Hh-dependent tumor growth in vitro and in vivo. Our findings unveil an unexpected role for ERAP1 in cancer and indicate ERAP1 as a promising therapeutic target for Hh-driven tumors.

E3 ubiquitin ligases in B-cell malignancies.

Ubiquitylation is a post-translational modification (PTM) that controls various cellular signaling pathways. It is orchestrated by a three-step enzymatic cascade know as the ubiquitin proteasome system (UPS). E3 ligases dictate the specificity to the substrates, primarily leading to proteasome-dependent degradation. Deregulation of the UPS components by various mechanisms contributes to the pathogenesis of cancer. This review focuses on E3 ligase-substrates pairings that are implicated in B-cell malignancies. Understanding the molecular mechanism of specific E3 ubiquitin ligases will present potential opportunities for the development of targeted therapeutic approaches.

RUNX proteins desensitize multiple myeloma to lenalidomide via protecting IKZFs from degradation.

Ikaros family zinc finger protein 1 and 3 (IKZF1 and IKZF3) are transcription factors that promote multiple myeloma (MM) proliferation. The immunomodulatory imide drug (IMiD) lenalidomide promotes myeloma cell death via Cereblon (CRBN)-dependent ubiquitylation and proteasome-dependent degradation of IKZF1 and IKZF3. Although IMiDs have been used as first-line drugs for MM, the overall survival of refractory MM patients remains poor and demands the identification of novel agents to potentiate the therapeutic effect of IMiDs. Using an unbiased screen based on mass spectrometry, we identified the Runt-related transcription factor 1 and 3 (RUNX1 and RUNX3) as interactors of IKZF1 and IKZF3. Interaction with RUNX1 and RUNX3 inhibits CRBN-dependent binding, ubiquitylation, and degradation of IKZF1 and IKZF3 upon lenalidomide treatment. Inhibition of RUNXs, via genetic ablation or a small molecule (AI-10-104), results in sensitization of myeloma cell lines and primary tumors to lenalidomide. Thus, RUNX inhibition represents a valuable therapeutic opportunity to potentiate IMiDs therapy for the treatment of multiple myeloma.

KLHL6 is a tumor suppressor gene in diffuse large B-cell lymphoma.

The ubiquitin proteasome system (UPS) plays a critical function in cellular homeostasis. The misregulation of UPS is often found in human diseases, including cancer. Kelch-like protein 6 (KLHL6) is an E3 ligase gene mutated in diffused large B-cell lymphoma (DLBCL). This review discusses the function of KLHL6 as a cullin3-RING ligase and how cancer-associated mutations disrupt the interaction with the cullin3, resulting in the loss of KLHL6 function. Furthermore, the mRNA decay factor Roquin2 is discussed as the first bona fide substrate of KLHL6 in the context of B-cell receptor activation and B-cell lymphoma. Importantly, the tumor-suppressing mechanism of KLHL6 via the degradation of Roquin2 and the mRNA decay in the context of the NF-κB pathway is summarized.

PTPN14 regulates Roquin2 stability by tyrosine dephosphorylation.

Protein phosphorylation regulates a variety of cellular signaling pathways and fundamental mechanisms in cells. In this paper, we demonstrate that the mRNA decay factor Roquin2 is phosphorylated at tyrosine residue in position 691 in vivo. This phosphorylation disrupts the interaction with KLHL6, the E3 ligase for Roquin2. Furthermore, we establish that the tyrosine phosphatase PTPN14 specifically interacts with Roquin2 through its phosphatase domain and dephosphorylates Roquin2 tyrosine 691. Overexpression of PTPN14 promotes Roquin2 degradation in a KLHL6-dependant manner by promoting interaction with KLHL6. Collectively, our findings reveal that PTPN14 negatively regulates the protein stability of Roquin2, thereby adding a new layer of regulation to the KLHL6-Roquin2 axis.

Loss of KLHL6 promotes diffuse large B-cell lymphoma growth and survival by stabilizing the mRNA decay factor roquin2.

Kelch-like protein 6 (KLHL6) is an uncharacterized gene mutated in diffuse large B-cell lymphoma (DLBCL). Here we report that KLHL6 assembles with cullin3 to form a functional cullin-RING ubiquitin ligase. Mutations in KLHL6 inhibit its ligase activity by disrupting the interaction with cullin3. Loss of KLHL6 favours DLBCL growth and survival both in vitro and in xenograft models. We further established that the mRNA decay factor roquin2 is a substrate of KLHL6. Degradation of roquin2 is dependent on B-cell receptor activation, and requires the integrity of the Tyr691 residue in roquin2 that is essential for its interaction with KLHL6. A non-degradable roquin2(Y691F) mutant requires its RNA-binding ability to phenocopy the effect of KLHL6 loss. Stabilization of roquin2 promotes mRNA decay of the tumour suppressor and NF-κB pathway inhibitor, tumour necrosis factor-α-inducible gene 3. Collectively, our findings uncover the tumour suppressing mechanism of KLHL6.

KMT2D regulates p63 target enhancers to coordinate epithelial homeostasis.

Epithelial tissues rely on a highly coordinated balance between self-renewal, proliferation, and differentiation, disruption of which may drive carcinogenesis. The epigenetic regulator KMT2D (MLL4) is one of the most frequently mutated genes in all cancers, particularly epithelial cancers, yet its normal function in these tissues is unknown. Here, we identify a novel role for KMT2D in coordinating this fine balance, as depletion of KMT2D from undifferentiated epidermal keratinocytes results in reduced proliferation, premature spurious activation of terminal differentiation genes, and disorganized epidermal stratification. Genome-wide, KMT2D interacts with p63 and is enriched at its target enhancers. Depletion of KMT2D results in a broad loss of enhancer histone modifications H3 Lys 4 (H3K4) monomethylation (H3K4me1) and H3K27 acetylation (H3K27ac) as well as reduced expression of p63 target genes, including key genes involved in epithelial development and adhesion. Together, these results reveal a critical role for KMT2D in the control of epithelial enhancers and p63 target gene expression, including the requirement of KMT2D for the maintenance of epithelial progenitor gene expression and the coordination of proper terminal differentiation.