Trisomy 8 Defines a Distinct Subtype of Myeloproliferative Neoplasms Driven by the MYC-Alarmin Axis.

Despite advances in understanding the genetic abnormalities in myeloproliferative neoplasms (MPN) and the development of JAK2 inhibitors, there is an urgent need to devise new treatment strategies, particularly for patients with triple-negative (TN) myelofibrosis (MF) who lack mutations in the JAK2 kinase pathway and have very poor clinical outcomes. Here we report that MYC copy number gain and increased MYC expression frequently occur in TN-MF and that MYC-directed activation of S100A9, an alarmin protein that plays pivotal roles in inflammation and innate immunity, is necessary and sufficient to drive development and progression of MF. Notably, the MYC-S100A9 circuit provokes a complex network of inflammatory signaling that involves numerous hematopoietic cell types in the bone marrow microenvironment. Accordingly, genetic ablation of S100A9 or treatment with small molecules targeting the MYC-S100A9 pathway effectively ameliorates MF phenotypes, highlighting the MYC-alarmin axis as a novel therapeutic vulnerability for this subgroup of MPNs. Significance: This study establishes that MYC expression is increased in TN-MPNs via trisomy 8, that a MYC-S100A9 circuit manifest in these cases is sufficient to provoke myelofibrosis and inflammation in diverse hematopoietic cell types in the BM niche, and that the MYC-S100A9 circuit is targetable in TN-MPNs.

DOG1 as a novel antibody-drug conjugate target for the treatment of multiple gastrointestinal tumors and liver metastasis.

Discovered On Gastrointestinal stromal tumors protein 1 (DOG1), a major calcium-activated chloride channel, has been used as a common diagnostic marker for gastrointestinal stromal tumors. However, the therapeutic application of DOG1 was not well defined. Here, we aim to investigate its potential as a therapeutic target for an antibody-drug conjugate (ADC) in various cancers of the alimentary tract and metastasis. The DOG1 expression profile was determined among TCGA samples and tissue microarrays. High levels of DOG1 expression were ubiquitously observed in multiple cancer samples from the alimentary tract determined by TCGA samples and tissue microarrays. Circulating tumor cells isolated from metastatic colon cancer patients were also positive for DOG1 expression. The mechanisms of anti-DOG1 antibody were investigated by dual-luciferase reporter assay. The anti-DOG1 antibody could inhibit proliferation and metastasis via p53 signaling in limited cancer cell lines. The anti-DOG1 antibody was conjugated with a microtubule inhibitor DM4, to construct a new anti-DOG1-DM4-ADC to strengthen its activity. The anti-DOG1-DM4-ADC showed cytotoxicity at the nanomolar level in vitro. In the murine xenograft tumor models, treatment of anti-DOG1-DM4-ADC achieved a significant tumor growth inhibition rate. Our study indicates that anti-DOG1-DM4-ADC may be promising therapeutic molecules for DOG1-positive alimentary tract tumors and may be effective in inhibiting recurrence after curative resection of liver metastases of colorectal origin.

SPOP and OTUD7A Control EWS-FLI1 Protein Stability to Govern Ewing Sarcoma Growth.

Chromosomal translocation results in development of an Ewing sarcoma breakpoint region 1-Friend leukemia integration 1 (EWS-FLI1) fusion oncogene in the majority of Ewing sarcoma. The persistent dependence of the tumor for this oncoprotein points to EWS-FLI1 as an ideal drug target. Although EWS-FLI1 transcriptional targets and binding partners are evaluated, the mechanisms regulating EWS-FLI1 protein stability remain elusive. Speckle-type POZ protein (SPOP) and OTU domain-containing protein 7A (OTUD7A) are identified as the bona fide E3 ligase and deubiquitinase, respectively, that control EWS-FLI1 protein turnover in Ewing sarcoma. Casein kinase 1-mediated phosphorylation of the VTSSS degron in the FLI1 domain enhances SPOP activity to degrade EWS-FLI1. Opposing this process, OTUD7A deubiquitinates and stabilizes EWS-FLI1. Depletion of OTUD7A in Ewing sarcoma cell lines reduces EWS-FLI1 protein abundance and impedes Ewing sarcoma growth in vitro and in mice. Performing an artificial-intelligence-based virtual drug screen of a 4-million small molecule library, 7Ai is identified as a potential OTUD7A catalytic inhibitor. 7Ai reduces EWS-FLI1 protein levels and decreases Ewing sarcoma growth in vitro and in a xenograft mouse model. This study supports the therapeutic targeting of OTUD7A as a novel strategy for Ewing sarcoma bearing EWS-FLI1 and related fusions, and may also be applicable to other cancers dependent on aberrant FLI1 expression.

Roles of Ubiquitination and SUMOylation in DNA Damage Response.

Ubiquitin and ubiquitin-like modifiers, such as SUMO, exert distinct physiological functions by conjugating to protein substrates. Ubiquitination or SUMOylation of protein substrates determine the fate of modified proteins, including proteasomal degradation, cellular re-localization, alternations in binding partners and serving as a protein-binding platform, in a ubiquitin or SUMO linkage-dependent manner. DNA damage occurs constantly in living organisms but is also repaired by distinct tightly controlled mechanisms including homologous recombination, non-homologous end joining, inter-strand crosslink repair, nucleotide excision repair and base excision repair. On sensing damaged DNA, a ubiquitination/SUMOylation landscape is established to recruit DNA damage repair factors. Meanwhile, misloaded and mission-completed repair factors will be turned over by ubiquitin or SUMO modifications as well. These ubiquitination and SUMOylation events are tightly controlled by both E3 ubiquitin/SUMO ligases and deubiquitinases/deSUMOylases. In this review, we will summarize identified ubiquitin and SUMO-related modifications and their function in distinct DNA damage repair pathways, and provide evidence for responsible E3 ligases, deubiquitinases, SUMOylases and deSUMOylases in these processes. Given that genome instability leads to human disorders including cancer, understanding detailed molecular mechanisms for ubiquitin and SUMO-related regulations in DNA damage response may provide novel insights into therapeutic modalities to treat human diseases associated with deregulated DNA damage response.

BRD4 Promotes Gastric Cancer Progression and Metastasis through Acetylation-Dependent Stabilization of Snail.

Cancer metastasis, a leading cause of death in patients, is associated with aberrant expression of epigenetic modifiers, yet it remains poorly defined how epigenetic readers drive metastatic growth and whether epigenetic readers are targetable to control metastasis. Here, we report that bromodomain-containing protein 4 (BRD4), a histone acetylation reader and emerging anticancer therapeutic target, promotes progression and metastasis of gastric cancer. The abundance of BRD4 in human gastric cancer tissues correlated with shortened metastasis-free gastric cancer patient survival. Consistently, BRD4 maintained invasiveness of cancer cells in vitro and their dissemination at distal organs in vivo. Surprisingly, BRD4 function in this context was independent of its putative transcriptional targets such as MYC or BCL2, but rather through stabilization of Snail at posttranslational levels. In an acetylation-dependent manner, BRD4 recognized acetylated lysine 146 (K146) and K187 on Snail to prevent Snail recognition by its E3 ubiquitin ligases FBXL14 and ß-Trcp1, thereby inhibiting Snail polyubiquitination and proteasomal degradation. Accordingly, genome-wide transcriptome analyses identified that BRD4 and Snail regulate a partially shared metastatic gene signature in gastric cancer cells. These findings reveal a noncanonical posttranscriptional regulatory function of BRD4 in maintaining cancer growth and dissemination, with immediate translational implications for treating gastric metastatic malignancies with clinically available bromodomain inhibitors. SIGNIFICANCE: These findings reveal a novel posttranscriptional regulatory function of the epigenetic reader BRD4 in cancer metastasis via stabilizing Snail, with immediate translational implication for treating metastatic malignancies with clinically available bromodomain inhibitors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/19/4869/F1.large.jpg.

Structural characterization and immune regulation of a novel polysaccharide from Maerkang Lactarius deliciosus Gray.

The present study investigated the structural characterization and immune regulation of a novel polysaccharide from Maerkang Lactarius deliciosus Gray. Chemical methods, high performance gel permeation chromatography, fourier transform infrared spectroscopy, nuclear magnetic resonance spectrum and gas chromatography­mass spectrometry were used to characterize the polysaccharide structure. The immunomodulatory abilities of the Maerkang L. deliciosus Gray polysaccharide (LDG­M) were also investigated. LDG­M was primarily composed of ß­D­glucose and α­D­lyxose with the ratio of 2:1. The possible structure of LDG­M had a backbone of 1,6­linked­ß­D­glucose and 1,4,6­linked­ß­D­glucose, with branches primarily composed of one (1→4)­linked­α­D­lyxose residue. The immunoregulatory activity results demonstrated that LDG­M promoted the proliferation and phagocytosis of macrophages, and induced cytokine release. LDG­M also promoted the proliferation of B cells by affecting the G0/G1, S and G2/M phases. The present study introduced LDG­M as a valuable source with unique immunoregulatory properties.

Structure-activity relationship and synthetic methodologies of α-santonin derivatives with diverse bioactivities: A mini-review.

α-Santonin, a sesquiterpene lactone isolated from Artemisia Santonica, possesses diverse bioactivities including antioxidant, anti-inflammation, immunosuppressive, anti-roundworm, anti-malaria, etc. However, its bioactivities are not satisfactory and need to be further optimized. Thus, many α-santonin derivatives were synthesized on the basis of rings A, B and C for the discovery of new analogues with prominent bioactivities. Herein, we reviewed and discussed the related synthetic methodologies, diverse bioactivities and structure-activity relationships (SAR) of α-santonin derivatives.

MERTK mediated novel site Akt phosphorylation alleviates SAV1 suppression.

Akt plays indispensable roles in cell proliferation, survival and metabolism. Mechanisms underlying posttranslational modification-mediated Akt activation have been extensively studied yet the Akt interactome is less understood. Here, we report that SAV1, a Hippo signaling component, inhibits Akt, a function independent of its role in Hippo signaling. Binding to a proline-tyrosine motif in the Akt-PH domain, SAV1 suppresses Akt activation by blocking Akt's movement to plasma membrane. We further identify cancer-associated SAV1 mutations with impaired ability to bind Akt, leading to Akt hyperactivation. We also determine that MERTK phosphorylates Akt1-Y26, releasing SAV1 binding and allowing Akt responsiveness to canonical PI-3K pathway activation. This work provides a mechanism underlying MERTK-mediated Akt activation and survival signaling in kidney cancer. Akt activation drives oncogenesis and therapeutic resistance; this mechanism of Akt regulation by MERTK/SAV1 provides yet another complexity in an extensively studied pathway, and may yield prognostic information and therapeutic targets.

Control of mTOR signaling by ubiquitin.

The evolutionarily conserved mTOR signaling pathway plays essential roles in cell growth, proliferation, metabolism and responses to cellular stresses. Hyperactivation of the mTOR signaling is observed in virtually all solid tumors and has been an attractive drug target. In addition to changes at genetic levels, aberrant activation of the mTOR signaling is also a result from dysregulated posttranslational modifications on key pathway members, such as phosphorylation that has been extensively studied. Emerging evidence also supports a critical role for ubiquitin-mediated modifications in dynamically regulating the mTOR signaling pathway, while a comprehensive review for relevant studies is missing. In this review, we will summarize characterized ubiquitination events on major mTOR signaling components, their modifying E3 ubiquitin ligases, deubiquitinases and corresponding pathophysiological functions. We will also reveal methodologies that have been used to identify E3 ligases or DUBs to facilitate the search for yet-to-be discovered ubiquitin-mediated regulatory mechanisms in mTOR signaling. We hope that our review and perspectives provide rationales and strategies to target ubiquitination for inhibiting mTOR signaling to treat human diseases.

K63-linked polyubiquitin chains bind to DNA to facilitate DNA damage repair.

Polyubiquitylation is canonically viewed as a posttranslational modification that governs protein stability or protein-protein interactions, in which distinct polyubiquitin linkages ultimately determine the fate of modified protein(s). We explored whether polyubiquitin chains have any nonprotein-related function. Using in vitro pull-down assays with synthetic materials, we found that polyubiquitin chains with the Lys63 (K63) linkage bound to DNA through a motif we called the "DNA-interacting patch" (DIP), which is composed of the adjacent residues Thr9, Lys11, and Glu34 Upon DNA damage, the binding of K63-linked polyubiquitin chains to DNA enhanced the recruitment of repair factors through their interaction with an Ile44 patch in ubiquitin to facilitate DNA repair. Furthermore, experimental or cancer patient-derived mutations within the DIP impaired the DNA binding capacity of ubiquitin and subsequently attenuated K63-linked polyubiquitin chain accumulation at sites of DNA damage, thereby resulting in defective DNA repair and increased cellular sensitivity to DNA-damaging agents. Our results therefore highlight a critical physiological role for K63-linked polyubiquitin chains in binding to DNA to facilitate DNA damage repair.

Erratum: Genomic variants in mouse model induced by azoxymethane and dextran sodium sulfate improperly mimic human colorectal cancer.

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Genomic variants in mouse model induced by azoxymethane and dextran sodium sulfate improperly mimic human colorectal cancer.

Mouse model induced by azoxymethane (AOM) and dextran sodium sulfate (DSS) is generally accepted as an ideal object to study on the carcinogenesis mechanisms of human colorectal cancer (CRC). The genomic responses to the AOM/DSS treatment in mouse that possibly lead to elucidation of CRC pathological mechanism are still poorly understood. For the first time, we investigated the cancer genome landscape of AOM/DSS mouse model by exome sequencing, to testify its molecular faithfulness to human CRC. Of 14 neoplastic samples, 7575 somatic variants were identified, which resulted in 2507 mutant genes and exhibited a large diversity in both colorectal aberrant crypt foci (ACF) and tumors even those tissues that were gained from the similar morphology or same treatment period. Cross-species comparison of the somatic variants demonstrated the totally different patterns of variable sites, mutant genes and perturbed pathways between mouse and human CRC. We therefore come to a conclusion that the tumorigenesis at genomic level in AOM/DSS model may not be properly comparable with that in human CRC, and the molecular mechanism elicited from this animal model should be carefully evaluated.

Lowering Endogenous Cathepsin D Abundance Results in Reactive Oxygen Species Accumulation and Cell Senescence.

Cathepsin D is reportedly to be closely associated with tumor development, migration, and invasion, but its pathological mechanism is not fully elucidated. We aimed to evaluate phenotypic changes and molecular events in response to cathepsin D knockdown. Lowering endogenous cathepsin D abundance (CR) induced senescence in HeLa cells, leading to reduced rate of cell proliferation and impaired tumorigenesis in a mouse model. Quantitative proteomics revealed that compared with control cells (EV), the abundances of several typical lysosomal proteases were decreased in the lysosomal fraction in CR cells. We further showed that cathepsin D knockdown caused increased permeability of lysosomal membrane and reactive oxygen species accumulation in CR cells, and the scavenging of reactive oxygen species by antioxidant was able to rescue cell senescence. Despite the increased reactive oxygen species, the proteomic data suggested a global reduction of redox-related proteins in CR cells. Subsequent analysis indicated that the transcriptional activity of nuclear factor erythroid-related factor 2 (Nrf2), which regulates the expression of groups of antioxidant enzymes, was down-regulated by cathepsin D knockdown. Importantly, Nrf2 overexpression significantly reduced cell senescence. Although transient oxidative stress promoted the accumulation of Nrf2 in the nucleus, we showed that the Nrf2 protein exited nucleus if oxidative stress persisted. In addition, when cathepsin D was transiently knocked down, the cathepsin-related events followed a sequential order, including lysosomal leakage during the early stage, followed by oxidative stress augmentation, and ultimately Nrf2 down-regulation and senescence. Our results suggest the roles of cathepsin D in cancer cells in maintaining lysosomal integrity, redox balance, and Nrf2 activity, thus promoting tumorigenesis. The MS Data are available via ProteomeXchange with identifier PXD002844.

Isoliquiritigenin prevents the progression of psoriasis-like symptoms by inhibiting NF-κB and proinflammatory cytokines.

UNLABELLED: Isoliquiritigenin (ISL) is an important flavonoid component of licorice and has been reported to possess anti-inflammatory and antioxidant properties, but its exact mechanism of action remains poorly understood. Previously, we demonstrated that ISL could suppress IL-6 expression in multiple myeloma. Here, we further characterized the anti-inflammatory effects of ISL in several psoriasis models, including the keratin 14/vascular endothelial growth factor (VEGF) transgenic mouse, the imiquimod (IMQ)-induced psoriasis-like mouse, and the human keratinocytes HaCaT and NHEK in vitro. We found that ISL ameliorated the inflammatory process in psoriasis models but not in their respective controls. Moreover, the anti-inflammatory effects of ISL were attributed to the suppression of nuclear factor-κB (NF-κB) activity, which consequently resulted in the reduction of pro-inflammation cytokines IL-6 and IL-8 expression. In conclusion, ISL exhibited anti-inflammatory effects in psoriasis models, by downregulating IL-6 and IL-8 via suppression of NF-κB activity, suggesting that ISL might serve as a potential candidate for treatment of psoriasis and other autoimmune inflammatory diseases. KEY MESSAGE: ISL could ameliorate the inflammatory process of psoriasis. ISL could suppress NF-κB and subsequent production of a series of pro-inflammatory cytokines. Dual-inhibitory activity against IL-6 and IL-8 of ISL is implemented via inhibiting NF-κB. ISL exerts no inhibitory effects on normal human keratinocytes or wild-type Balb/c mice, implying its low toxicity and safety.

Discovery of potential colorectal cancer serum biomarkers through quantitative proteomics on the colonic tissue interstitial fluids from the AOM-DSS mouse model.

Quantitative proteomic analysis was performed using iTRAQ to discover colorectal cancer (CRC)-related proteins in tissue interstitial fluids (TIFs). A typical inflammation-related CRC mouse model was generated using azoxymethane-dextran sodium sulfate (AOM-DSS), and TIFs were collected from these mice in four stages during CRC development. Using stringent criteria, a total of 144 proteins displayed changes in their abundances during tumor growth, including 45 that consecutively increased, 17 that consecutively decreased and 82 that changed irregularly. Of these 144 proteins, 24 of the consecutively changed proteins were measured using MRM in individual TIF samples, and 18 were verified. Twelve proteins verified to be consecutively increased in TIFs were examined using MRM to evaluate changes in their abundance in individual mouse serum samples. The abundances of leucine-rich alpha-2-glycoprotein 1 (LRG1), tubulin beta-5 chain (TUBB5) and immunoglobulin J chain (IGJ) were significantly higher in CRC mice than in control mice. Using clinical samples and MRM, we further verified that LRG1 and TUBB5 are potential CRC serum biomarkers. These data demonstrate that coupling dynamic TIF proteomics with targeted serum proteomics in an animal model is a promising avenue for pursuing the discovery of tumor serum biomarkers. BIOLOGICAL SIGNIFICANCE: Colorectal cancer (CRC) is one of the most dangerous diseases worldwide. However, few of CRC biomarkers possess satisfied specificity and sensitivity in clinical practices. Exploration of more CRC biomarkers, especially in serum, is an urgent and also a time-consuming campaign in the CRC study. Our study demonstrates that quantitatively evaluating the phase-dependent proteins in colonic tissue interstitial fluids from AOM-DSS mice is a feasible and effective way for exploration of the CRC-related proteins and the potential serum biomarkers. We identified two proteins, LRG1 and TUBB5, which may be practicable in human clinical samples as CRC serum biomarkers. To sum up, this study provides a novel angle to explore the critical factors in tumorigenesis and a new pipeline for potential serum biomarker discovery and verification.

A Comprehensive Proteomics Analysis Reveals a Secretory Path- and Status-Dependent Signature of Exosomes Released from Tumor-Associated Macrophages.

Exosomes are 30-120 nm-sized membrane vesicles of endocytic origin that are released into the extracellular environment and play roles in cell-cell communication. Tumor-associated macrophages (TAMs) are important constituents of the tumor microenvironment; thus, it is critical to study the features and complex biological functions of TAM-derived exosomes. Here, we constructed a TAM cell model from a mouse macrophage cell line, Ana-1, and performed comparative proteomics on exosomes, exosome-free media, and cells between TAMs and Ana-1. Proteomic analysis between exosome and exosome-free fractions indicated that the functions of exosome dominant proteins were mainly enriched in RNA processing and proteolysis. TAM status dramatically affected the abundances of 20S proteasome subunits and ribosomal proteins in their exosomes. The 20S proteasome activity assay strongly indicated that TAM exosomes possessed higher proteolytic activity. In addition, Ana-1- and TAM-derived exosomes have different RNA profiles, which may result from differential RNA processing proteins. Taken together, our comprehensive proteomics study provides novel views for understanding the complicated roles of macrophage-derived exosomes in the tumor microenvironment.

BAG2 is a target of the c-Myc gene and is involved in cellular senescence via the p21(CIP1) pathway.

Suppression of c-Myc is likely to induce cellular senescence in many tumors with unclear mechanisms. A proteomics survey indicated that high levels of BCL2-associated athanogene 2 (BAG2) were found in response to c-Myc repression in TRE293 cells. This observation led to the investigation into the role of BAG2 in c-Myc-induced senescence. The association of the c-Myc/SP1 complex with the BAG2 promoter verified the role of c-Myc/SP1 in regulating BAG2 transcription. Furthermore, high levels of BAG2 were found to induce p21(CIP1)-dependent senescence and subsequent carcinogenetic arrest, suggesting its possible role as an indirect activator of the p21(CIP1) pathway.