hnRNPM protects against the dsRNA-mediated interferon response by repressing LINE-associated cryptic splicing.

RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.

Dysregulation and Epigenetic Reprogramming of NRF2 Signaling Axis Promote Acquisition of Cisplatin Resistance and Metastasis in Head and Neck Squamous Cell Carcinoma.

PURPOSE: Cisplatin (CDDP)-based chemotherapy is a first-line treatment for patients with advanced head and neck squamous cell carcinomas (HNSCC), despite a high rate of treatment failures, acquired resistance, and subsequent aggressive behavior. The purpose of this study was to study the mechanism of CDDP resistance and metastasis in HNSCC. We investigated the role of NRF2 pathway activation as a driven event for tumor progression and metastasis of HNSCC. EXPERIMENTAL DESIGN: Human HNSCC cell lines that are highly resistant to CDDP were generated. Clonogenic survival assays and a mouse model of oral cancer were used to examine the impact of NRF2 activation in vitro and in vivo on CDDP sensitivity and development of metastasis. Western blotting, immunostaining, whole-exome sequencing, single-cell transcriptomic and epigenomic profiling platforms were performed to dissect clonal evolution and molecular mechanisms. RESULTS: Implantation of CDDP-resistant HNSCC cells into the tongues of nude mice resulted in a very high rate of distant metastases. The CDDP-resistant cells had significantly higher expression of NRF2 pathway genes in the presence of newly acquired KEAP1 mutations, or via epigenomic activation of target genes. Knockdown of NRF2 or restoration of the wild-type KEAP1 genes resensitized resistant cells to CDDP and decreased distant metastasis (DM). Finally, treatment with inhibitor of glutaminase-1, a NRF2 target gene, alleviated CDDP resistance. CONCLUSIONS: CDDP resistance and development of DM are associated with dysregulated and epigenetically reprogrammed KEAP1-NRF2 signaling pathway. A strategy targeting KEAP1/NRF2 pathway or glutamine metabolism deserves further clinical investigation in patients with CDDP-resistant head and neck tumors.

Combined Inhibition of Rad51 and Wee1 Enhances Cell Killing in HNSCC Through Induction of Apoptosis Associated With Excessive DNA Damage and Replication Stress.

Despite advances in surgery, chemotherapy, and radiation, there are limited treatment options for advanced head and neck squamous cell carcinoma (HNSCC) and survival remains very poor. Therefore, effective therapies are desperately needed. Recently, selective exploitation of DNA damage and replication stress responses has become a novel approach for cancer treatment. Wee1 kinase and Rad51 recombinase are two proteins involved in regulating replication stress and homologous recombination repair in cancer cells. In this study, we investigated the combined effect of Rad51 inhibitor (B02) and Wee1 inhibitor (AZD1775) in vitro and in vivo in various HNSCC cell lines. Clonogenic survival assays demonstrated that B02 synergized with AZD1775 in vitro in all HNSCC cell lines tested. The synergy between these drugs was associated with forced CDK1 activation and reduced Chk1 phosphorylation leading to induction of excessive DNA damage and replication stress, culminating in aberrant mitosis and apoptosis. Our results showed that elevated Rad51 mRNA expression correlated with worse survival in HNSCC patients with HPV-positive tumors. The combination of B02 and AZD1775 significantly inhibited tumor growth in vivo in mice bearing HPV-positive HNSCC tumors as compared to HPV-negative HNSCC. This differential sensitivity appears to be linked to HPV-positive tumors having more in vivo endogenous replication stress owing to transformation by E6 and E7 oncogenes. Furthermore, addition of B02 radiosensitized the HPV-negative HNSCC tumors in vitro and in vivo In conclusion, our data implicate that a novel rational combination with Rad51 and Wee1 inhibitors holds promise as synthetic lethal therapy, particularly in high-risk HPV-positive HNSCC.

Clinical Translation of Scarless 0.33-mm Core Microbiopsy for Molecular Evaluation of Human Skin.

BACKGROUND: Skin scarring can occur after punch biopsies, prohibiting their routine utilization, especially in the central face. OBJECTIVES: This paper describes a scarless, 0.33-mm-diameter skin microbiopsy for molecular analysis of skin. METHODS: This is was single-center, randomized, prospective study with 15 patients receiving no biopsy or biopsy on the left or right nasolabial fold. Six blinded raters assessed participant photos at baseline, 1 month, and 3 months post biopsy to evaluate for a visualized scar. Patient and Observer Scar Assessment Scale was completed. Additionally, biopsies from various skin regions of body along with arm skin after treatment with a single Erbium-YAG laser were processed for molecular analysis. RESULTS: No patients exhibited scar formation based on evaluation of photographs and patient feedback. There was no mark at the biopsy site 7 days post-procedure. Optical coherence tomography showed a complete closing of the biopsy-punch wound 48 hours post-biopsy. One month post-biopsy, photography reviewers were unable to identify a scar, on average, 90% of the time at 3-month follow-up. Microbiopsies from various anatomical regions were successfully extracted for histology, electron microscopy, and gene expression analysis. Selected skin rejuvenation markers in the biopsies from Erbium-YAG-treated forearm skin resulted in significant gene upregulation in extracellular matrix molecules at 1 month posttreatment compared with untreated skin. CONCLUSIONS: A core microbiopsy of 0.33 mm can be extracted reproducibly for histological, ultrastructural, and gene expression analysis without scarring. This allows repeated sampling for assessment of skin treatments and diseases, including aesthetics and wound-healing progress.

Utility of Adipocyte Fractions in Fat Grafting in an Athymic Rat Model.

BACKGROUND: Multiple processing and handling methods of autologous fat yield to variations in graft retention and viability, which results in unpredictable clinical outcomes. OBJECTIVES: This study aims to understand the skin effects of fat graft preparations that contain a varying ratio of free-lipid and stem-cell-bearing stromal vascular fractions (SVF). METHODS: Lipoaspirates from consenting patients were processed into emulsified fat and then SVF and adipocyte fractions (free-lipid). SVF enriched with 0%, 5%, and 15% free-lipid were grafted along the dorsum of athymic rats. The xenografts were collected 45 days after grafting and then prepped for immunostaining. RESULTS: Xenografts resulted in viable tissue mass under the panniculus carnosus of rats as confirmed with human specific markers. A low percentage of human cells was also detected in the lower reticular dermis. Although grafts with SVF formed adipocytes of normal architecture, grafts formed with free-lipid alone resulted in large lipid vacuoles in varying sizes. Among graft preparations, SVF with 10% free-lipid resulted in much-developed adipocyte architecture with collagen and elastin. Compared with SVF alone grafts, SVF with free-lipid had higher CD44 expression, suggesting a localized immune response of adipocytes. CONCLUSIONS: Current studies suggest that SVF enriched with approximately 10% free-lipid provides the best conditions for fat graft differentiation into viable fat tissue formation as well as collagen and elastin production to provide mechanical support for overlaying skin in an athymic rat model. Additionally, application of this therapeutic modality in a simple clinical setting may offer a practical way to concentrate SVF with free-lipid in a small volume for the improvement of clinical defects.