Pilot Study of High-Dose Pemetrexed in Patients with Progressive Chordoma.

PURPOSE: Chordomas are ultrarare tumors of the axial spine and skull-base without approved systemic therapy. Most chordomas have negative expression of thymidylate synthase (TS), suggesting a potential for responding to the antifolate agent pemetrexed, which inhibits TS and other enzymes involved in nucleotide biosynthesis. We evaluated the therapeutic activity and safety of high-dose pemetrexed in progressive chordoma. PATIENTS AND METHODS: Adult patients with previously treated, progressive chordoma participated in an open-label, single-institution, single-arm, pilot clinical trial of intravenous pemetrexed 900 mg/m2 every 3 weeks and supportive medications of folic acid, vitamin B12, and dexamethasone. The primary endpoint was objective response rate according to RECIST v1.1. Secondary endpoints included adverse events, progression-free survival (PFS), tumor molecular profiles, and alterations in tissue and blood-based biomarkers. RESULTS: Fifteen patients were enrolled and the median number of doses administered was 15 (range, 4-31). One patient discontinued treatment due to psychosocial issues after four cycles and one contracted COVID-19 after 13 cycles. Of the 14 response-evaluable patients, 2 (14%) achieved a partial response and 10 (71%) demonstrated stable disease. Median PFS was 10.5 months (95% confidence interval: 9 months-undetermined) and 6-month PFS was 67%. Adverse events were expected and relatively mild, with one grade 3 creatinine increased, and one each of grade 3 and 4 lymphopenia. No grade 5 adverse events, unexpected toxicities, or dose-limiting toxicities were observed. Several patients reported clinical improvement in disease-related symptoms. CONCLUSIONS: High-dose pemetrexed appears tolerable and shows objective antitumor activity in patients with chordoma. Phase II studies of high-dose pemetrexed are warranted.

Diagnostic miRNA Signatures in Paired Tumor, Plasma, and Urine Specimens From Renal Cell Carcinoma Patients.

BACKGROUND: The incidence of patients diagnosed with renal cell carcinoma (RCC) is increasing. There are no approved biofluid biomarkers for routine diagnosis of RCC patients. This retrospective study aims to identify cell-free microRNA (cfmiR) signatures in urine samples that can be utilized as biomarkers for early diagnosis of sporadic RCC patients. METHODS: Tissue, plasma, and urine samples (n = 221) from 56 sporadic RCC patients and respective normal healthy donors were profiled for 2083 microRNAs (miRs) using the next-generation sequencing-based HTG EdgeSeq miR Whole Transcriptome Assay. DESeq2 (FC |1.2|, false discovery rate <0.05) was performed to identify differentially expressed miRs. Data from RCC tissue samples of The Cancer Genome Atlas database were used for miR validation. RESULTS: We found a 10-miR signature that distinguished RCC tissues from remote normal kidney tissue or benign kidney lesion samples. Additionally, we identified subtype-specific miRs (miR-122-5p, miR-210-3p, and miR-21-3p) and miRs specific for all RCC subtypes (miR-106b-3p, miR-629-5p, and miR-885-5p). We observed that miR-155-5p was associated with tumor size. Using The Cancer Genome Atlas data sets, we validated the miRs found in RCC tissue samples. In plasma or urine analysis, we found cfmiRs that were consistently and significantly upregulated in RCC tissue samples. A 15-cfmiR signature was proposed in urine samples of RCC patients, of which miR-1275 was consistently upregulated in tissue, plasma, and urine samples. CONCLUSIONS: This integrative study found diagnostic miRs/cfmiRs for RCC patients, which were validated using The Cancer Genome Atlas data sets. Distinctive cfmiR signatures found in urine may have clinical utility for the diagnosis of RCC.

MiR-181a targets STING to drive PARP inhibitor resistance in BRCA- mutated triple-negative breast cancer and ovarian cancer.

BACKGROUND: Poly (ADP-ribose) polymerase inhibitors (PARPi) are approved for the treatment of BRCA-mutated breast cancer (BC), including triple-negative BC (TNBC) and ovarian cancer (OvCa). A key challenge is to identify the factors associated with PARPi resistance; although, previous studies suggest that platinum-based agents and PARPi share similar resistance mechanisms. METHODS: Olaparib-resistant (OlaR) cell lines were analyzed using HTG EdgeSeq miRNA Whole Transcriptomic Analysis (WTA). Functional assays were performed in three BRCA-mutated TNBC cell lines. In-silico analysis were performed using multiple databases including The Cancer Genome Atlas, the Genotype-Tissue Expression, The Cancer Cell Line Encyclopedia, Genomics of Drug Sensitivity in Cancer, and Gene Omnibus Expression. RESULTS: High miR-181a levels were identified in OlaR TNBC cell lines (p = 0.001) as well as in tumor tissues from TNBC patients (p = 0.001). We hypothesized that miR-181a downregulates the stimulator of interferon genes (STING) and the downstream proinflammatory cytokines to mediate PARPi resistance. BRCA1 mutated TNBC cell lines with miR-181a-overexpression were more resistant to olaparib and showed downregulation in STING and the downstream genes controlled by STING. Extracellular vesicles derived from PARPi-resistant TNBC cell lines horizontally transferred miR-181a to parental cells which conferred PARPi-resistance and targeted STING. In clinical settings, STING levels were positively correlated with interferon gamma (IFNG) response scores (p = 0.01). In addition, low IFNG response scores were associated with worse response to neoadjuvant treatment including PARPi for high-risk HER2 negative BC patients (p = 0.001). OlaR TNBC cell lines showed resistance to platinum-based drugs. OvCa cell lines resistant to platinum showed resistance to olaparib. Knockout of miR-181a significantly improved olaparib sensitivity in OvCa cell lines (p = 0.001). CONCLUSION: miR-181a is a key factor controlling the STING pathway and driving PARPi and platinum-based drug resistance in TNBC and OvCa. The miR-181a-STING axis can be used as a potential marker for predicting PARPi responses in TNBC and OvCa tumors.

Multi-omic profiling reveals discrepant immunogenic properties and a unique tumor microenvironment among melanoma brain metastases.

Melanoma brain metastases (MBM) are clinically challenging to treat and exhibit variable responses to immune checkpoint therapies. Prior research suggests that MBM exhibit poor tumor immune responses and are enriched in oxidative phosphorylation. Here, we report results from a multi-omic analysis of a large, real-world melanoma cohort. MBM exhibited lower interferon-gamma (IFNγ) scores and T cell-inflamed scores compared to primary cutaneous melanoma (PCM) or extracranial metastases (ECM), which was independent of tumor mutational burden. Among MBM, there were fewer computationally inferred immune cell infiltrates, which correlated with lower TNF and IL12B mRNA levels. Ingenuity pathway analysis (IPA) revealed suppression of inflammatory responses and dendritic cell maturation pathways. MBM also demonstrated a higher frequency of pathogenic PTEN mutations and angiogenic signaling. Oxidative phosphorylation (OXPHOS) was enriched in MBM and negatively correlated with NK cell and B cell-associated transcriptomic signatures. Modulating metabolic or angiogenic pathways in MBM may improve responses to immunotherapy in this difficult-to-treat patient subset.

Spatial profiling of cancer-associated fibroblasts of sporadic early onset colon cancer microenvironment.

The incidence of sporadic early-onset colon cancer (EOCC) has increased worldwide. The molecular mechanisms in the tumor and the tumor microenvironment (TME) in EOCC are not fully understood. The aim of this study is to unravel unique spatial transcriptomic and proteomic profiles in tumor epithelial cells and cancer-associated fibroblasts (CAFs). Here, we divide the sporadic colon cancer tissue samples with transcriptomic data into patients diagnosed with EOCC (<50 yrs) and late-onset colon cancer (LOCC, ≥50 yrs) and then, analyze the data using CIBERSORTx deconvolution software. EOCC tumors are more enriched in CAFs with fibroblast associated protein positive expression (FAP(+)) than LOCC tumors. EOCC patients with higher FAP mRNA levels in CAFs have shorter OS (Log-rank test, p < 0.029). Spatial transcriptomic analysis of 112 areas of interest, using NanoString GeoMx digital spatial profiling, demonstrate that FAP(+) CAFs at the EOCC tumor invasive margin show a significant upregulation of WNT signaling and higher mRNA/protein levels of fibroblast growth factor 20 (FGF20). Tumor epithelial cells at tumor invasive margin of EOCC tumors neighboring FAP(+) CAFs show significantly higher mRNA/protein levels of fibroblast growth factor receptor (FGFR2) and PI3K/Akt signaling activation. NichNET analysis show a potential interaction between FGF20 and FGFFR2. The role of FGF20 in activating FGFR2/pFGFR2 and AKT/pAKT was validated in-vitro. In conclusion, we identify a unique FAP(+) CAF population that showed WNT signaling upregulation and increased FGF20 levels; while neighbor tumor cells show the upregulation/activation of FGFR2-PI3K/Akt signaling at the tumor invasive margin of EOCC tumors.

Inhibition of Microsomal Prostaglandin E2 Synthase Reduces Collagen Deposition in Melanoma Tumors and May Improve Immunotherapy Efficacy by Reducing T-cell Exhaustion.

The arachidonic acid pathway participates in immunosuppression in various types of cancer. Our previous observation detailed that microsomal prostaglandin E2 synthase 1 (mPGES-1), an enzyme downstream of cyclooxygenase 2 (COX-2), limited antitumor immunity in melanoma; in addition, genetic depletion of mPGES-1 specifically enhanced immune checkpoint blockade therapy. The current study set out to distinguish the roles of mPGES-1 from those of COX-2 in tumor immunity and determine the potential of mPGES-1 inhibitors for reinforcing immunotherapy in melanoma. Genetic deletion of mPGES-1 showed different profiles of prostaglandin metabolites from that of COX-2 deletion. In our syngeneic mouse model, mPGES-1-deficient cells exhibited similar tumorigenicity to that of COX-2-deficient cells, despite a lower ability to suppress PGE2 synthesis by mPGES-1 depletion, indicating the presence of factors other than PGE2 that are likely to regulate tumor immunity. RNA-sequencing analysis revealed that mPGES-1 depletion reduced the expressions of collagen-related genes, which have been found to be associated with immunosuppressive signatures. In our mouse model, collagen was reduced in mPGES-1-deficient tumors, and phenotypic analysis of tumor-infiltrating lymphocytes indicated that mPGES-1-deficient tumors had fewer TIM3+ exhausted CD8+ T cells compared with COX-2-deficient tumors. CAY10678, an mPGES-1 inhibitor, was equivalent to celecoxib, a selective COX-2 inhibitor, in reinforcing anti-PD-1 treatment. Our study indicates that mPGES-1 inhibitors represent a promising adjuvant for immunotherapies in melanoma by reducing collagen deposition and T-cell exhaustion. Significance: Collagen is a predominant component of the extracellular matrix that may influence the tumor immune microenvironment for cancer progression. We present here that mPGES-1 has specific roles in regulating tumor immunity, associated with several collagen-related genes and propose that pharmacologic inhibition of mPGES-1 may hold therapeutic promise for improving immune checkpoint-based therapies.

The Vicious Cycle of Melanoma-Microglia Crosstalk: Inter-Melanoma Variations in the Brain-Metastasis-Promoting IL-6/JAK/STAT3 Signaling Pathway.

Previous studies from our lab demonstrated that the crosstalk between brain-metastasizing melanoma cells and microglia, the macrophage-like cells of the central nervous system, fuels progression to metastasis. In the present study, an in-depth investigation of melanoma-microglia interactions elucidated a pro-metastatic molecular mechanism that drives a vicious melanoma-brain-metastasis cycle. We employed RNA-Sequencing, HTG miRNA whole transcriptome assay, and reverse phase protein arrays (RPPA) to analyze the impact of melanoma-microglia interactions on sustainability and progression of four different human brain-metastasizing melanoma cell lines. Microglia cells exposed to melanoma-derived IL-6 exhibited upregulated levels of STAT3 phosphorylation and SOCS3 expression, which, in turn, promoted melanoma cell viability and metastatic potential. IL-6/STAT3 pathway inhibitors diminished the pro-metastatic functions of microglia and reduced melanoma progression. SOCS3 overexpression in microglia cells evoked microglial support in melanoma brain metastasis by increasing melanoma cell migration and proliferation. Different melanomas exhibited heterogeneity in their microglia-activating capacity as well as in their response to microglia-derived signals. In spite of this reality and based on the results of the present study, we concluded that the activation of the IL-6/STAT3/SOCS3 pathway in microglia is a major mechanism by which reciprocal melanoma-microglia signaling engineers the interacting microglia to reinforce the progression of melanoma brain metastasis. This mechanism may operate differently in different melanomas.

Genomic Amplification of UBQLN4 Is a Prognostic and Treatment Resistance Factor.

Ubiquilin-4 (UBQLN4) is a proteasomal shuttle factor that directly binds to ubiquitylated proteins and delivers its cargo to the 26S proteasome for degradation. We previously showed that upregulated UBQLN4 determines the DNA damage response (DDR) through the degradation of MRE11A. However, the regulatory mechanism at DNA level, transcriptionally and post-transcriptional levels that control UBQLN4 mRNA levels remains unknown. In this study, we screened 32 solid tumor types and validated our findings by immunohistochemistry analysis. UBQLN4 is upregulated at both mRNA and protein levels and the most significant values were observed in liver, breast, ovarian, lung, and esophageal cancers. Patients with high UBQLN4 mRNA levels had significantly poor prognoses in 20 of 32 cancer types. DNA amplification was identified as the main mechanism promoting UBQLN4 upregulation in multiple cancers, even in the early phases of tumor development. Using CRISPR screen datasets, UBQLN4 was identified as a common essential gene for tumor cell viability in 81.1% (860/1,060) of the solid tumor derived cell lines. Ovarian cancer cell lines with high UBQLN4 mRNA levels were platinum-based chemotherapy resistant, while they were more sensitive to poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi). Our findings highlight the utilities of UBQLN4 as a significant pan-cancer theranostic factor and a precision oncology biomarker for DDR-related drug resistance.

Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy.

The hypoxic tumor microenvironment has been implicated in immune escape, but the underlying mechanism remains elusive. Using an in vitro culture system modeling human T cell dysfunction and exhaustion in triple-negative breast cancer (TNBC), we find that hypoxia suppresses immune effector gene expression, including in T and NK cells, resulting in immune effector cell dysfunction and resistance to immunotherapy. We demonstrate that hypoxia-induced factor 1α (HIF1α) interaction with HDAC1 and concurrent PRC2 dependency causes chromatin remolding resulting in epigenetic suppression of effector genes and subsequent immune dysfunction. Targeting HIF1α and the associated epigenetic machinery can reverse the immune effector dysfunction and overcome resistance to PD-1 blockade, as demonstrated both in vitro and in vivo using syngeneic and humanized mice models. These findings identify a HIF1α-mediated epigenetic mechanism in immune dysfunction and provide a potential strategy to overcome immune resistance in TNBC.

Multi-modal molecular programs regulate melanoma cell state.

Melanoma cells display distinct intrinsic phenotypic states. Here, we seek to characterize the molecular regulation of these states using multi-omic analyses of whole exome, transcriptome, microRNA, long non-coding RNA and DNA methylation data together with reverse-phase protein array data on a panel of 68 highly annotated early passage melanoma cell lines. We demonstrate that clearly defined cancer cell intrinsic transcriptomic programs are maintained in melanoma cells ex vivo and remain highly conserved within melanoma tumors, are associated with distinct immune features within tumors, and differentially correlate with checkpoint inhibitor and adoptive T cell therapy efficacy. Through integrative analyses we demonstrate highly complex multi-omic regulation of melanoma cell intrinsic programs that provide key insights into the molecular maintenance of phenotypic states. These findings have implications for cancer biology and the identification of new therapeutic strategies. Further, these deeply characterized cell lines will serve as an invaluable resource for future research in the field.

Urine Cell-Free MicroRNAs in Localized Prostate Cancer Patients.

Prostate cancer (PCa) is the most common cancer in men. Prostate-specific antigen screening is recommended for the detection of PCa. However, its specificity is limited. Thus, there is a need to find more reliable biomarkers that allow non-invasive screening for early-stage PCa. This study aims to explore urine microRNAs (miRs) as diagnostic biomarkers for PCa. We assessed cell-free miR (cfmiR) profiles of urine and plasma samples from pre- and post-operative PCa patients (n = 11) and normal healthy donors (16 urine and 24 plasma) using HTG EdgeSeq miRNA Whole Transcriptome Assay based on next-generation sequencing. Furthermore, tumor-related miRs were detected in formalin-fixed paraffin-embedded tumor tissues obtained from patients with localized PCa. Specific cfmiRs signatures were found in urine samples of localized PCa patients using differential expression analysis. Forty-two cfmiRs that were detected were common to urine, plasma, and tumor samples. These urine cfmiRs may have potential utility in diagnosing early-stage PCa and complementing or improving currently available PCa screening assays. Future studies may validate the findings.

Interplay between soluble CD74 and macrophage-migration inhibitory factor drives tumor growth and influences patient survival in melanoma.

Soluble forms of receptors play distinctive roles in modulating signal-transduction pathways. Soluble CD74 (sCD74) has been identified in sera of inflammatory diseases and implicated in their pathophysiology; however, few relevant data are available in the context of cancer. Here we assessed the composition and production mechanisms, as well as the clinical significance and biological properties, of sCD74 in melanoma. Serum sCD74 levels were significantly elevated in advanced melanoma patients compared with normal healthy donors, and the high ratio of sCD74 to macrophage-migration inhibitory factor (MIF) conferred significant predictive value for prolonged survival in these patients (p = 0.0035). Secretion of sCD74 was observed primarily in melanoma cell lines as well as a THP-1 line of macrophages from monocytes and primary macrophages, especially in response to interferon-γ (IFN-γ). A predominant form that showed clinical relevance was the 25-KDa sCD74, which originated from the 33-KDa isoform of CD74. The release of this sCD74 was regulated by either a disintegrin and metalloproteinase-mediated cell-surface cleavage or cysteine-protease-mediated lysosomal cleavage, depending on cell types. Both recombinant and THP-1 macrophage-released endogenous sCD74 suppressed melanoma cell growth and induced apoptosis under IFN-γ stimulatory conditions via inhibiting the MIF/CD74/AKT-survival pathway. Our findings demonstrate that the interplay between sCD74 and MIF regulates tumor progression and determines patient outcomes in advanced melanoma.

Recent Developments of Circulating Tumor Cell Analysis for Monitoring Cutaneous Melanoma Patients.

Circulating tumor cells (CTCs) have been studied using multiple technical approaches for interrogating various cancers, as they allow for the real-time assessment of tumor progression, disease recurrence, treatment response, and tumor molecular profiling without the need for a tumor tissue biopsy. Here, we will review studies from the last 15 years on the assessment of CTCs in cutaneous melanoma patients in relation to different clinical outcomes. The focus will be on CTC detection in blood samples obtained from cutaneous melanoma patients of different clinical stages and treatments utilizing multiple platforms. Assessment of multiple molecular melanoma-associated antigen (MAA) markers by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was the most common assay allowing for the improvement of assay sensitivity, tumor heterogeneity, and to predict patient outcomes. Multicenter studies demonstrate the utility of CTC assays reducing the bias observed in single- center trials. The recent development of CTC enrichment platforms has provided reproducible methods. CTC assessment enables both multiple mRNAs and DNAs genomic aberration profiling. CTC provides specific important translational information on tumor progression, prediction of treatment response, and survival outcomes for cutaneous melanoma patients. The molecular studies on melanoma CTCs have provided and may set standards for other solid tumor CTC analyses.

Cell-free plasma microRNAs that identify patients with glioblastoma.

Glioblastoma (GBM) is still one of the most commonly diagnosed advanced stage primary brain tumors. Current treatments for patients with primary GBM (pGBM) are often not effective and a significant proportion of the patients with pGBM recur. The effective treatment options for recurrent GBM (rGBM) are limited and survival outcomes are poor. This retrospective multicenter pilot study aims to determine potential cell-free microRNAs (cfmiRs) that identify patients with pGBM and rGBM tumors. 2,083 miRs were assessed using the HTG miRNA whole transcriptome assay (WTA). CfmiRs detection was compared in pre-operative plasma samples from patients with pGBM (n = 32) and rGBM (n = 13) to control plasma samples from normal healthy donors (n = 73). 265 cfmiRs were found differentially expressed in plasma samples from pGBM patients compared to normal healthy donors (FDR < 0.05). Of those 193 miRs were also detected in pGBM tumor tissues (n = 15). Additionally, we found 179 cfmiRs differentially expressed in rGBM, of which 68 cfmiRs were commonly differentially expressed in pGBM. Using Random Forest algorithm, specific cfmiR classifiers were found in the plasma of pGBM, rGBM, and both pGBM and rGBM combined. Two common cfmiR classifiers, miR-3180-3p and miR-5739, were found in all the comparisons. In receiving operating characteristic (ROC) curves analysis for rGBM miR-3180-3p showed a specificity of 87.7% and a sensitivity of 100% (AUC = 98.5%); while miR-5739 had a specificity of 79.5% and sensitivity of 92.3% (AUC = 90.2%). This study demonstrated that plasma samples from pGBM and rGBM patients have specific miR signatures. CfmiR-3180-3p and cfmiR-5739 have potential utility in diagnosing patients with pGBM and rGBM tumors using a minimally invasive blood assay.

Current status of gastrointestinal tract cancer brain metastasis and the use of blood-based cancer biomarker biopsy.

Brain metastasis (BM) frequently occurs in patients with cutaneous melanoma, lung, and breast cancer; although, BM rarely arises from cancers of the gastrointestinal tract (GIT). The reported incidence of GIT cancer BM is less than 4%. In the last few years, effective systemic therapy has prolonged the survival of GIT patients and consequently, the incidence of developing BM is rising. Therefore, the epidemiology and biology of BM arising from GIT cancer requires a more comprehensive understanding. In spite of the development of new therapeutic agents for patients with metastatic GIT cancers, survival for patients with BM still remains poor, with a median survival after diagnosis of less than 4 months. Limited evidence suggests that early detection of isolated intra-cranial lesions will enable surgical resection plus systemic and/or radiation therapy, which may lead to an increase in overall survival. Novel diagnostic methods such as blood-based biomarker biopsies may play a crucial role in the early detection of BM. Circulating tumor cells and circulating cell-free nucleic acids are known to serve as blood biomarkers for early detection and treatment response monitoring of multiple cancers. Blood biopsy may improve early diagnosis and treatment monitoring of GIT cancers BM, thus prolonging patients' survivals.

Interleukin enhancer-binding factor 2 promotes cell proliferation and DNA damage response in metastatic melanoma.

BACKGROUND: 1q21.3 amplification, which is frequently observed in metastatic melanoma, is associated with cancer progression. Interleukin enhancer-binding factor 2 (ILF2) is located in the 1q21.3 amplified region, but its functional role or contribution to tumour aggressiveness in cutaneous melanoma is unknown. METHODS: In silico analyses were performed using the TCGA SKCM dataset with clinical annotations and three melanoma microarray cohorts from the GEO datasets. RNA in situ hybridisation and immunohistochemistry were utilised to validate the gene expression in melanoma tissues. Four stable melanoma cell lines were established for in vitro ILF2 functional characterisation. RESULTS: Our results showed that the ILF2 copy number variation (CNV) is positively correlated with ILF2 mRNA expression (r = 0.68, p < .0001). Additionally, ILF2 expression is significantly increased with melanoma progression (p < .0001), and significantly associated with poor overall survival for metastatic melanoma patients (p = .026). The overexpression of ILF2 (ILF2-OV) promotes proliferation in metastatic melanoma cells, whereas ILF2 knockdown decreases proliferation by blocking the cell cycle. Mechanistically, we demonstrated the interaction between ILF2 and the splicing factor U2AF2, whose knockdown reverses the proliferation effects mediated by ILF2-OV. Stage IIIB-C melanoma patients with high ILF2-U2AF2 expression showed significantly shorter overall survival (p = .024). Enhanced ILF2/U2AF2 expression promotes a more efficient DNA-damage repair by increasing RAD50 and ATM mRNA expression. Paradoxically, metastatic melanoma cells with ILF2-OV were more sensitive to ATM inhibitors. CONCLUSION: Our study uncovered that ILF2 amplification of the 1q21.3 chromosome is associated with melanoma progression and triggers a functional downstream pathway in metastatic melanoma promoting drug resistance.

Acetylated DNMT1 Downregulation and Related Regulatory Factors Influence Metastatic Melanoma Patients Survival.

The role of post-translational modifications (PTM) of the key epigenetic factor DNMT1 protein has not been well explored in cutaneous metastatic melanoma progression. The acetylated DNMT1 (ac-DNMT1) protein level was assessed using an anti-acetylated lysine antibody in a clinically annotated melanoma patient tumor specimen cohort. In this study, we showed that surgically resected tumors have significantly higher DNMT1 protein expression in metastatic melanoma (stage III metastasis n = 17, p = 0.0009; stage IV metastasis n = 164, p = 0.003) compared to normal organ tissues (n = 19). Additionally, reduced ac-DNMT1 protein levels were associated with melanoma progression. There was a significant inverse correlation between ac-DNMT1 and DNMT1 protein levels in stage IV metastatic melanoma (r = -0.18, p = 0.02, n = 164). Additionally, ac-DNMT1 protein levels were also significantly positively correlated with TIP60 (r = 0.6, p < 0.0001) and USP7 (r = 0.74, p < 0.0001) protein levels in stage IV metastatic melanoma (n = 164). Protein analysis in metastatic melanoma tumor tissues showed that with high ac-DNMT1 (p = 0.006, n = 59), or concurrent high ac-DNMT1 with low DNMT1 (p = 0.05, n = 27), or high TIP60 (p = 0.007, n = 41), or high USP7 (p = 0.01, n = 48) consistently showed better 4-year melanoma-specific survival (MSS). Multivariate Cox proportional hazard analysis showed that ac-DNMT1 level is a significant independent factor associated with MSS (HR, 0.994; 95% confidential interval (CI), 0.990-0.998; p = 0.002). These results demonstrated that low ac-DNMT1 levels may represent an important regulatory factor in controlling metastatic melanoma progression and a promising factor for stratifying aggressive stage IV metastasis.