Mi-2ß promotes immune evasion in melanoma by activating EZH2 methylation.

Recent development of new immune checkpoint inhibitors has been particularly successfully in cancer treatment, but still the majority patients fail to benefit. Converting resistant tumors to immunotherapy sensitive will provide a significant improvement in patient outcome. Here we identify Mi-2ß as a key melanoma-intrinsic effector regulating the adaptive anti-tumor immune response. Studies in genetically engineered mouse melanoma models indicate that loss of Mi-2ß rescues the immune response to immunotherapy in vivo. Mechanistically, ATAC-seq analysis shows that Mi-2ß controls the accessibility of IFN-γ-stimulated genes (ISGs). Mi-2ß binds to EZH2 and promotes K510 methylation of EZH2, subsequently activating the trimethylation of H3K27 to inhibit the transcription of ISGs. Finally, we develop an Mi-2ß-targeted inhibitor, Z36-MP5, which reduces Mi-2ß ATPase activity and reactivates ISG transcription. Consequently, Z36-MP5 induces a response to immune checkpoint inhibitors in otherwise resistant melanoma models. Our work provides a potential therapeutic strategy to convert immunotherapy resistant melanomas to sensitive ones.

Dysregulation of PD-L1 by UFMylation imparts tumor immune evasion and identified as a potential therapeutic target.

Immunotherapy of PD-L1/PD-1 blockage elicited impressive clinical benefits for cancer treatment. However, the relative low response and therapy resistance highlight the need to better understand the molecular regulation of PD-L1 in tumors. Here, we report that PD-L1 is a target of UFMylation. UFMylation of PD-L1 destabilizes PD-L1 by synergizing its ubiquitination. Inhibition of PD-L1 UFMylation via silencing of UFL1 or Ubiquitin-fold modifier 1 (UFM1), or the defective UFMylation of PD-L1, stabilizes the PD-L1 in multiple human and murine cancer cells, and undermines antitumor immunity in vitro and mice, respectively. Clinically, UFL1 expression was decreased in multiple cancers and lower expression of UFL1 negatively correlated with the response of anti-PD1 therapy in melanoma patients. Moreover, we identified a covalent inhibitor of UFSP2 that promoted the UFMylation activity and contributed to the combination therapy with PD-1 blockade. Our findings identified a previously unrecognized regulator of PD-L1 and highlighted UFMylation as a potential therapeutic target.

Transcriptional Regulation of Endogenous Retroviruses and Their Misregulation in Human Diseases.

Endogenous retroviruses (ERVs), deriving from exogenous retroviral infections of germ line cells occurred millions of years ago, represent ~8% of human genome. Most ERVs are highly inactivated because of the accumulation of mutations, insertions, deletions, and/or truncations. However, it is becoming increasingly apparent that ERVs influence host biology through genetic and epigenetic mechanisms under particular physiological and pathological conditions, which provide both beneficial and deleterious effects for the host. For instance, certain ERVs expression is essential for human embryonic development. Whereas abnormal activation of ERVs was found to be involved in numbers of human diseases, such as cancer and neurodegenerative diseases. Therefore, understanding the mechanisms of regulation of ERVs would provide insights into the role of ERVs in health and diseases. Here, we provide an overview of mechanisms of transcriptional regulation of ERVs and their dysregulation in human diseases.

Ufl1 deficiency causes skin pigmentation by up-regulation of Endothelin-1.

Ufmylation (UFM1 modification) is a newly identified ubiquitin-like modification system involved in numerous cellular processes. However, the regulatory mechanisms and biological functions of this modification remain mostly unknown. We have recently reported that Ufmylation family genes have frequent somatic copy number alterations in human cancer including melanoma, suggesting involvement of Ufmylation in skin function and disease. UFL1 is the only known Ufmylation E3-like ligase. In this study, we generated the skin-specific Ufl1 knockout mice and show that ablation of Ufl1 caused epidermal thickening, pigmentation and shortened life span. RNA-Seq analysis indicated that Ufl1 deletion resulted in upregulation of the genes involved in melanin biosynthesis. Mechanistically, we found that Endothelin-1 (ET-1) is a novel substrate of Ufmylation and this modification regulates ET-1 stability, and thereby deletion of Ufl1 upregulates the expression and secretion of ET-1, which in turn results in up-regulation of genes in melanin biosynthesis and skin pigmentation. Our findings establish the role of Ufl1 in skin pigmentation through Ufmylation modification of ET-1 and provide opportunities for therapeutic intervention of skin diseases.

Human UFSP1 translated from an upstream near-cognate initiation codon functions as an active UFM1-specific protease.

Ubiquitin-fold modifier 1 (UFM1) is a recently identified ubiquitin-like posttranslational modification with important biological functions. However, the regulatory mechanisms governing UFM1 modification of target proteins (UFMylation) and the cellular processes controlled by UFMylation remain largely unknown. It has been previously shown that a UFM1-specific protease (UFSP2) mediates the maturation of the UFM1 precursor and drives the de-UFMylation reaction. Furthermore, it has long been thought that UFSP1, an ortholog of UFSP2, is inactive in many organisms, including human, because it lacks an apparent protease domain when translated from the canonical start codon (445AUG). Here, we demonstrate using the combination of site-directed mutagenesis, CRISPR/Cas9-mediated genome editing, and mass spectrometry approaches that translation of human UFSP1 initiates from an upstream near-cognate codon, 217CUG, via eukaryotic translation initiation factor eIF2A-mediated translational initiation rather than from the annotated 445AUG, revealing the presence of a catalytic protease domain containing a Cys active site. Moreover, we show that both UFSP1 and UFSP2 mediate maturation of UFM1 and de-UFMylation of target proteins. This study demonstrates that human UFSP1 functions as an active UFM1-specific protease, thus contributing to our understanding of the UFMylation/de-UFMylation process.

TERT activates endogenous retroviruses to promote an immunosuppressive tumour microenvironment.

Telomerase plays a pivotal role in tumorigenesis by both telomere-dependent and telomere-independent activities, although the underlying mechanisms are not completely understood. Using single-sample gene set enrichment analysis (ssGSEA) across 9,264 tumour samples, we observe that expression of telomerase reverse transcriptase (TERT) is closely associated with immunosuppressive signatures. We demonstrate that TERT can activate a subclass of endogenous retroviruses (ERVs) independent of its telomerase activity to form double-stranded RNAs (dsRNAs), which are sensed by the RIG-1/MDA5-MAVS signalling pathway and trigger interferon signalling in cancer cells. Furthermore, we show that TERT-induced ERV/interferon signalling stimulates the expression of chemokines, including CXCL10, which induces the infiltration of suppressive T-cell populations with increased percentage of CD4+ and FOXP3+ cells. These data reveal an unanticipated role for telomerase as a transcriptional activator of ERVs and provide strong evidence that TERT-mediated ERV/interferon signalling contributes to immune suppression in tumours.

Human endogenous retroviruses in development and disease.

Human endogenous retroviruses (HERVs) represent ∼8% of human genome, deriving from exogenous retroviral infections of germ line cells occurred millions of years ago and being inherited by the offspring in a Mendelian fashion. Most of HERVs are nonprotein-coding because of the accumulation of mutations, insertions, deletions, and/or truncations. It has been long thought that HERVs were "junk DNA". However, it is now known that HERVs are involved in various biological processes through encoding proteins, acting as promoters/enhancers, or lncRNAs to affect human health and disease. In this review, we summarized recent findings about HERVs, with implications in embryonic development, pluripotency, cancer, aging, and neurodegenerative diseases.

Neuroprotective effects of salidroside on ageing hippocampal neurons and naturally ageing mice via the PI3K/Akt/TERT pathway.

Studies have found that salidroside, isolated from Rhodiola rosea L, has various pharmacological activities, but there have been no studies on the effects of salidroside on brain hippocampal senescence. The purpose of this study was to investigate the mechanistic role of salidroside in hippocampal neuron senescence and injury. In this study, long-term cultured primary rat hippocampal neurons and naturally aged C57 mice were treated with salidroside. The results showed that salidroside increased the viability and MAP2 expression, reduced ß-galactosidase (ß-gal) levels of rat primary hippocampal neurons. Salidroside also improved cognition dysfunction in ageing mice and alleviated neuronal degeneration in the ageing mice CA1 region. Moreover, salidroside decreased the levels of oxidative stress and p21, p16 protein expressions of hippocampal neurons and ageing mice. Salidroside promoted telomerase reverse transcriptase (TERT) protein expression via the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pathway. In conclusion, our findings suggest that salidroside has the potential to be used as a therapeutic strategy for anti-ageing and ageing-related disease treatment.

Polymerase I and transcript release factor transgenic mice show impaired function of hematopoietic stem cells.

The age-dependent decline in stem cell function plays a critical role in aging, although the molecular mechanisms remain unclear. PTRF/Cavin-1 is an essential component in the biogenesis and function of caveolae, which regulates cell proliferation, endocytosis, signal transduction and senescence. This study aimed to analyze the role of PTRF in hematopoietic stem cells (HSCs) senescence using PTRF transgenic mice. Flow cytometry was used to detect the frequency of immune cells and hematopoietic stem/progenitor cells (HSCs and HPCs). The results showed than the HSC compartment was significantly expanded in the bone marrow of PTRF transgenic mice compared to age-matched wild-type (WT) mice, and exhibited the senescent phenotype characterized by G1 cell cycle arrest, increased SA-ß-Gal activity and high levels of reactive oxygen species (ROS). The PTRF-overexpressing HSCs also showed significantly lower self-renewal and ability to reconstitute hematopoiesis in vitro and in vivo. Real-time PCR was performed to analyze the expression levels of senescence-related genes. PTRF induced HSCs senescence via the ROS-p38-p16 and caveolin-1-p53-p21 pathways. Furthermore, the PTRF+cav-1-/- mice showed similar HSCs function as WT mice, indicating that PTRF induces senescence in HSCs partly through caveolin-1. Thus PTRF impaired HSCs aging partly via caveolin-1.

UFMylation maintains tumour suppressor p53 stability by antagonizing its ubiquitination.

p53 is the most intensively studied tumour suppressor1. The regulation of p53 homeostasis is essential for its tumour-suppressive function2,3. Although p53 is regulated by an array of post-translational modifications, both during normal homeostasis and in stress-induced responses2-4, how p53 maintains its homeostasis remains unclear. UFMylation is a recently identified ubiquitin-like modification with essential biological functions5-7. Deficiency in this modification leads to embryonic lethality in mice and disease in humans8-12. Here, we report that p53 can be covalently modified by UFM1 and that this modification stabilizes p53 by antagonizing its ubiquitination and proteasome degradation. Mechanistically, UFL1, the UFM1 ligase6, competes with MDM2 to bind to p53 for its stabilization. Depletion of UFL1 or DDRGK1, the critical regulator of UFMylation6,13, decreases p53 stability and in turn promotes cell growth and tumour formation in vivo. Clinically, UFL1 and DDRGK1 expression are downregulated and positively correlated with levels of p53 in a high percentage of renal cell carcinomas. Our results identify UFMylation as a crucial post-translational modification for maintenance of p53 stability and tumour-suppressive function, and point to UFMylation as a promising therapeutic target in cancer.

Implications of telomerase reverse transcriptase in tumor metastasis.

Metastasis is the main culprit of the great majority of cancerrelated deaths. However, the complicated process of the invasion-metastasis cascade remains the least understood aspect of cancer biology. Telomerase plays a pivotal role in bypassing cellular senescence and sustaining the cancer progression by maintaining telomere homeostasis and genomic integrity. Telomerase reverse transcriptase (TERT) exerts a series of fundamental functions that are independent of its enzymatic cellular activity, including proliferation, inflammation, epithelia-mesenchymal transition (EMT), angiogenesis, DNA repair, and gene expression. Accumulating evidence indicates that TERT may facilitate most steps of the invasion-metastasis cascade. In this review, we summarize important advances that have revealed some of the mechanisms by which TERT facilitates tumor metastasis, providing an update on the non-canonical functions of telomerase beyond telomere maintaining. [BMB Reports 2020; 53(9): 458-465].

Human telomerase reverse transcriptase is a novel target of Hippo-YAP pathway.

Telomerase plays a pivotal role in tumorigenesis by maintaining telomere homeostasis, a hallmark of cancer. However, the mechanisms by which telomerase is reactivated or upregulated during tumorigenesis remain incompletely understood. Here, we report that the Hippo pathway effector Yes-associated protein (YAP) regulates the expression of human telomerase reverse transcriptase (hTERT). Ectopic expression or physiological activation of YAP increases hTERT expression, whereas knockdown of YAP decreases the expression of hTERT. YAP binds to the hTERT promoter through interaction with the TEA domain family transcription factors and activates hTERT transcription. Furthermore, sustained YAP hyperactivation promotes telomerase activity and extends telomere length, with increased hTERT expression. In addition, we show that hTERT expression is positively correlated with YAP activation in human liver cancer tissues. Together, our results demonstrate that YAP promotes hTERT expression, which could contribute to tumor progression.

Role of telomerase in the tumour microenvironment.

Telomeres are specialized genomic structures that protect chromosomal ends to maintain genomic stability. Telomeric length is primarily regulated by the telomerase complex, essentially consisting of an RNA template (TERC), an enzymatic subunit (telomerase reverse transcriptase, TERT). In humans, telomerase activity is repressed during embryonic differentiation and is absent in most somatic cells. However, it is upregulated or reactivated in 80%-90% of the primary tumours in humans. The human TERT (hTERT) plays a pivotal role in cellular immortality and tumourigenesis. However, the molecular mechanisms of telomerase functioning in cancer have not been fully understood beyond the telomere maintenance. Several research groups, including ours, have demonstrated that hTERT possesses vital functions independent of its telomere maintenance, including angiogenesis, inflammation, cancer cell stemness, and epithelial-mesenchymal transformation (EMT). All these telomere-independent activities of hTERT may contribute to the regulation of the dynamics and homeostasis of the tumour microenvironment (TME), thereby promoting tumour growth and development. Cancer progression and metastasis largely depend upon the interactions between cancer cells and their microenvironment. In this review, the involvement of TERT in the tumour microenvironment and the underlying implications in cancer therapeutics have been summarized.

MRE11 UFMylation promotes ATM activation.

A proper DNA damage response (DDR) is essential to maintain genome integrity and prevent tumorigenesis. DNA double-strand breaks (DSBs) are the most toxic DNA lesion and their repair is orchestrated by the ATM kinase. ATM is activated via the MRE11-RAD50-NBS1 (MRN) complex along with its autophosphorylation at S1981 and acetylation at K3106. Activated ATM rapidly phosphorylates a vast number of substrates in local chromatin, providing a scaffold for the assembly of higher-order complexes that can repair damaged DNA. While reversible ubiquitination has an important role in the DSB response, modification of the newly identified ubiquitin-like protein ubiquitin-fold modifier 1 and the function of UFMylation in the DDR is largely unknown. Here, we found that MRE11 is UFMylated on K282 and this UFMylation is required for the MRN complex formation under unperturbed conditions and DSB-induced optimal ATM activation, homologous recombination-mediated repair and genome integrity. A pathogenic mutation MRE11(G285C) identified in uterine endometrioid carcinoma exhibited a similar cellular phenotype as the UFMylation-defective mutant MRE11(K282R). Taken together, MRE11 UFMylation promotes ATM activation, DSB repair and genome stability, and potentially serves as a therapeutic target.

Author Correction: PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

In this Letter, two relevant references were omitted; see the accompanying Amendment for details. The original Letter has not been corrected.

HuR regulates telomerase activity through TERC methylation.

Telomerase consists of the catalytic protein TERT and the RNA TERC. Mutations in TERC are linked to human diseases, but the underlying mechanisms are poorly understood. Here we report that the RNA-binding protein HuR associates with TERC and promotes the assembly of the TERC/TERT complex by facilitating TERC C106 methylation. Dyskeratosis congenita (DC)-related TERC U100A mutation impair the association of HuR with TERC, thereby reducing C106 methylation. Two other TERC mutations linked to aplastic anemia and autosomal dominant DC, G107U, and GC107/108AG, likewise disrupt methylation at C106. Loss-of-HuR binding and hence lower TERC methylation leads to decreased telomerase activity and telomere shortening. Furthermore, HuR deficiency or mutation of mTERC HuR binding or methylation sites impair the renewal of mouse hematopoietic stem cells, recapitulating the bone marrow failure seen in DC. Collectively, our findings reveal a novel function of HuR, linking HuR to telomerase function and TERC-associated DC.

Inhibitor of the human telomerase reverse trancriptase (hTERT) gene promoter induces cell apoptosis via a mitochondrial-dependent pathway.

Telomerase is aberrantly expressed in many cancers and plays an important role in the development of cellular immortality and oncogenesis, which makes it a potential cancer therapeutic target for drug discovery. Here, we constructed a firefly luciferase reporter driven by the human telomerase reverse trancriptase (hTERT) gene promoter to screen for inhibitory compounds. Compound 5c was discovered and shown to significantly inhibit the promoter activity of hTERT gene. Furthermore, five analogs of compound 5c were synthesized, and compound 8b was shown to be a more potent inhibitor of hTERT gene promoter activity and subsequent expression of hTERT mRNA and protein. The viability of HeLa cells was inhibited by a knockdown of hTERT gene expression, and the same effect was also observed by treating with compound 8b. Moreover, our results indicated that compound 8b induced apoptosis of HeLa cells, and activated caspase-9 and caspase-3 enzymes. Taken together, these results suggested that compound 8b down-regulates the expression of hTERT and induces mitochondrial-dependent apoptosis.

PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas.

Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi and MEKi) therapies have markedly improved the clinical outcomes of patients with metastatic melanoma. Unfortunately, the efficacy of these treatments is often countered by the acquisition of drug resistance. Here we investigated the molecular mechanisms that underlie acquired resistance to BRAFi and to the combined therapy. Consistent with previous studies, we show that resistance to BRAFi is mediated by ERK pathway reactivation. Resistance to the combined therapy, however, is mediated by mechanisms independent of reactivation of ERK in many resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in cells with acquired drug resistance and have a pivotal role in mediating resistance. Our screening, using a reverse-phase protein array, revealed distinct mechanisms by which PAKs mediate resistance to BRAFi and the combined therapy. In BRAFi-resistant cells, PAKs phosphorylate CRAF and MEK to reactivate ERK. In cells that are resistant to the combined therapy, PAKs regulate JNK and ß-catenin phosphorylation and mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide insights into the molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.

Src Inhibits the Hippo Tumor Suppressor Pathway through Tyrosine Phosphorylation of Lats1.

The Hippo pathway regulates cell proliferation, apoptosis, and stem cell self-renewal, and its inactivation in animal models causes organ enlargement followed by tumorigenesis. Hippo pathway deregulation occurs in many human cancers, but the underlying mechanisms are not fully understood. Here, we report tyrosine phosphorylation of the Hippo pathway tumor suppressor LATS1 as a mechanism underlying its regulation by cell adhesion. A tyrosine kinase library screen identified Src as the kinase to directly phosphorylate LATS1 on multiple residues, causing attenuated Mob kinase activator binding and structural alteration of the substrate-binding pocket in the kinase domain. Cell matrix adhesion activated the Hippo pathway effector transcription coactivator YAP partially through Src-mediated phosphorylation and inhibition of LATS1. Aberrant Src activation abolished the tumor suppressor activity of LATS1 and induced tumorigenesis in a YAP-dependent manner. Protein levels of Src in human breast cancer tissues correlated with accumulation of active YAP dephosphorylated on the LATS1 target site. These findings reveal tyrosine phosphorylation of LATS1 by Src as a novel mechanism of Hippo pathway regulation by cell adhesion and suggest Src activation as an underlying reason for YAP deregulation in tumorigenesis. Cancer Res; 77(18); 4868-80. ©2017 AACR.