Iuliia Topchu

Introduction. Histone methyltransferase NSD1 is often inactivated in head and neck squamous cell carcinoma. Loss of NSD1 is associated with improved prognosis in HPV (human papilloma virus) negative cancer. However, data on the signaling pathways regulated by NSD1 are limited. Aim. Validation and systemic assessment of reproducibility of the NSD1-Akt/mTORC1 axis based on integrative analysis of open transcriptomic and proteomic data from cell lines and samples of head and neck squamous cell carcinoma. Materials and methods. Systemic analysis was performed of the connection between NSD1 and Akt/mTORC1 signaling pathway using open transcriptomic and proteomic data (panel of squamous cell carcinoma cell lines on the DepMap portal and clinical cohorts from the The Cancer Genome Atlas (TCGA)). Pathway activity was evaluated using ssGSEA/GSEA (single-sample Gene Set Enrichment Analysis), proteomic activity – using protein expression data obtained with Reverse Phase Protein Array (RPPA), statistical significance – using standard correlation and nonparametric tests. Results. In the cell line panel, elevated NSD1 expression correlated with stronger PI3K/AKT/mTOR transcription signature and increased phosphorylation of the key pathway components according to RPPA data. Similar correlation was observed in patient tumor samples. Data on isogenic cell lines with NSD1 knockout demonstrated decreased expression of mTORC1 target genes. Conclusion. Reproducible correlation between NSD1 level and Akt/mTORC1 signaling pathway activity in head and neck cancer was established. However, additional confirmation of the NSD1 role in regulation of the Akt/mTORC1 pathways requires in vitro and in vivo investigation of expression and phosphorylation of protein components of this cascade, as well as functional experiments to evaluate proliferation, survival and drug sensitivity of head and neck cancer cells. Clinical significance of the established correlation in the context of disease progression requires further verification.

Lung cancer is one of the most common types of cancer worldwide. Non-small cell lung cancer (NSCLC), typically caused by KRAS and TP53 driver mutations, represents the majority of all new lung cancer diagnoses. Overexpression of the RNA-binding protein (RBP) Musashi-2 (MSI2) has been associated with NSCLC progression. To investigate the role of MSI2 in NSCLC development, we compared the tumorigenesis in mice with lung-specific Kras -activating mutation and Trp53 deletion, with and without Msi2 deletion (KPM2 versus KP mice). KPM2 mice showed decreased lung tumorigenesis in comparison with KP mice. In addition, KPM2 lung tumors showed evidence of decreased proliferation, but increased DNA damage, marked by increased levels of phH2AX (S139) and phCHK1 (S345), but decreased total and activated ATM. Using cell lines from KP and KPM2 tumors, and human NSCLC cell lines, we found that MSI2 directly binds ATM mRNA and regulates its translation. MSI2 depletion impaired DNA damage response (DDR) signaling and sensitized human and murine NSCLC cells to treatment with PARP inhibitors in vitro and in vivo. Taken together, we conclude that MSI2 supports NSCLC tumorigenesis, in part, by supporting repair of DNA damage by controlling expression of DDR proteins. These results suggest that targeting MSI2 may be a promising strategy for lung cancers treated with DNA-damaging agents.

Head and neck squamous cell carcinoma (HNSCC) ranks among the most prevalent global cancers. Despite advancements in treatments, the five-year survival rate remains at approximately 66%. The histone methyltransferase NSD1, known for its role in catalyzing histone H3 lysine 36 di-methylation (H3K36me²), emerges as a potential oncogenic factor in HNSCC. Our study, employing Reverse Phase Protein Array (RPPA) analysis and subsequent validation, reveals that PIP4K2B is a key downstream target of NSD1. Notably, PIP4K2B depletion in HNSCC induces downregulation of the mTOR pathway, resulting in diminished cell growth in vitro. Our investigation highlights a direct, positive regulatory role of NSD1 on PIP4K2B gene transcription through an H3K36me2-dependent mechanism. Importantly, the impact of PIP4K2B appears to be context-dependent, with overexpression rescuing cell growth in laryngeal HNSCC cells but not in tongue/hypopharynx cells. In conclusion, our findings implicate PIP4K2B as a novel NSD1-dependent protein in HNSCC, suggesting its potential significance for laryngeal cancer cell survival. This insight contributes to our understanding of the molecular landscape in HNSCC and establishes PIP4KB as a promising target for drug development.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Despite advances in therapeutic management and immunotherapy, the 5-year survival rate for head and neck cancer remains at ~66% of all diagnosed cases. A better definition of drivers of HPV-negative HNSCC that are targetable points of tumor vulnerability could lead to significant clinical advances. NSD1 is a histone methyltransferase that catalyzes histone H3 lysine 36 di-methylation (H3K36me ² ); mutations inactivating NSD1 have been linked to improved outcomes in HNSCC. In this study, we show that NSD1 induces H3K36me ² levels in HNSCC and that the depletion of NSD1 reduces HNSCC of cell growth in vitro and in vivo. We also find that NSD1 strongly promotes activation of the Akt/mTORC1 signaling pathway. NSD1 depletion in HNSCC induces an autophagic gene program activation, causes accumulation of the p62 and LC3B-II proteins, and decreases the autophagic signaling protein ULK1 at both protein and mRNA levels. Reflecting these signaling defects, the knockdown of NSD1 disrupts autophagic flux in HNSCC cells. Taken together, these data identify positive regulation of Akt/mTORC1 signaling and autophagy as novel NSD1 functions in HNSCC, suggesting that NSD1 may be of value as a therapeutic target in this cancer.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Despite advances in therapeutic management and immunotherapy, the five-year survival rate for head and neck cancer remains at ∼66% of all diagnosed cases. A better definition of drivers of HPV-negative HNSCC that are targetable points of tumor vulnerability could lead to significant clinical advances. NSD1 is a histone methyltransferase which catalyzes histone H3 lysine 36 di-methylation (H3K36me ² ); mutations inactivating NSD1 have been linked to improved outcomes in HNSCC. In this study, we show that NSD1 induces H3K36me ² levels in HNSCC, and that the depletion of NSD1 reduces HNSCC of cell growth in vitro and in vivo . We also find that NSD1 strongly promotes activation of the Akt/mTORC1 signaling pathway. NSD1 depletion in HNSCC induces an autophagic gene program activation, causes accumulation of the p62 and LC3B-II proteins, and decreases the autophagic signaling protein ULK1 at both protein and mRNA levels. Reflecting these signaling defects, knockdown of NSD1 disrupts autophagic flux in HNSCC cells. Taken together, these data identify positive regulation of Akt/mTORC1 signaling and autophagy as novel NSD1 functions in HNSCC, suggesting that NSD1 may be of value as a therapeutic target in this cancer.

Introduction. The histone methylation regulates gene expression and plays a role in genomic stability participating in DNA repair. Dimethylation of histone 3 lysine 36 (H3K36me²) is an important histone modification which is responsible for gene expression activation. H3K36me² is a product of methyltransferase activity of NSD1, NSD2, NSD3, and ASH1L proteins. NSD1 mutations are known to often occur in head and neck squamous carcinoma. The presence of NSD1 mutations highly correlates with increased survival, especially for patients with laryngeal cancer. The aim of this study was an in vitro investigation of the role of NSD1 in the cell proliferation of laryngeal squamous cell cancer and non-small lung cancer cells, as well as a study of the effect of disruption of the NSD1 gene expression on cisplatin treatment response.Material and Methods. Using TCGA, correlation analysis was performed to compare NSD1 wild type and mutant patient survival. NSD1 knockout cell lines models of laryngeal and non-small cell lung cancer were developed using the CRISPR/ Cas9 system. The effect of NSD1 knockout on H3K36me2 level was evaluated by western blot. Proliferation and IC50 of cisplatin in control and knockout cells were studied as well.Results. It was demonstrated that NSD1 knockout decreased the H3K36me² level and cell proliferation in laryngeal squamous cell cancer cells and increased the sensitivity of head and neck cancer cells to cisplatin treatment, while there was no effect of NSD1 knockout in a non-small cell lung cancer cell line.Conclusion. Based on the data obtained, it can be concluded that the NSD1 protein is a potential target for inhibitor development following in vitro and in vivo testing in head-neck squamous cell carcinoma models. More studies are needed for better understanding of the regulation of tumor cell growth by NSD1.

Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the diagnoses of lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, which are expressed on both endothelial and tumor cells, are one of the key proteins contributing to cancer development, and are involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer, which suggests that VEGFR2 protein is strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human lung adenocarcinoma cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR, which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting a direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human lung adenocarcinoma samples. We conclude that the MSI2/VEGFR2 axis contributes to lung adenocarcinoma progression and is worth further investigations and therapeutic targeting.

Lung cancer remains the second most commonly diagnosed cancer worldwide and the leading cause of cancer-related mortality. The mapping of genomic alterations and their role in lung-cancer progression has been followed by the development of new therapeutic options. Several novel drugs, such as targeted therapy and immunotherapy, have significantly improved outcomes. However, many patients with lung cancer do not benefit from existing therapies or develop progressive disease, leading to increased morbidity and mortality despite initial responses to treatment. Alterations in DNA-damage repair (DDR) genes represent a cancer hallmark that impairs a cell’s ability to prevent deleterious mutation accumulation and repair. These alterations have recently emerged as a therapeutic target in breast, ovarian, prostate, and pancreatic cancers. The role of DDR alterations remains largely unknown in lung cancer. Nevertheless, recent research efforts have highlighted a potential role of some DDR alterations as predictive biomarkers of response to treatment. Despite the failure of PARP inhibitors (main class of DDR targeting agents) to improve outcomes in lung cancer patients, there is some evidence suggesting a role of PARP inhibitors and other DDR targeting agents in benefiting a distinct subset of lung cancer patients. In this review, we will discuss the existing literature on DDR alterations and homologous recombination deficiency (HRD) state as predictive biomarkers and therapeutic targets in both non-small cell lung and small cell lung cancer.

Lung cancer holds second place in incidence and first place in mortality among all cancers, both in men and women, with metastasis contributing to the vast majority of deaths. Recently, expression of vascular endothelial growth factor receptor-2 (VEGFR2) on both endothelial and tumor cells was demonstrated as one of the key mechanisms contributing to growth of non-small cell lung cancer (NSCLC), the most common subtype of lung cancer. VEGFR2 dependent signaling is critical both for tumor autocrine functions related to NSCLC migration, and in paracrine control of tumor angiogenesis through action on tumor-proximal endothelial cells, suggesting a potential mechanism for growth control. Several drugs targeting VEGFR2 signaling pathway demonstrated their clinical efficacy in NSCLC setting, including VEGFR2 and VEGF-A inhibitors, VEGF-A/PIGF trap. Recently our group demonstrated that RNA-binding protein Musashi-2 (MSI2) is driving progression and metastasis of NSCLC. MSI2 regulates mRNA translation of multiple targets, its expression is upregulated in the metastasis-competent murine and human lung cancer cell lines and is progressively elevated in lung cancer samples. The goal of this study is to define the role of MSI2 in sustaining VEGFR2 signaling during NSCLC progression. Using reverse RNA immunoprecipitation (RIP) qPCR analysis, we demonstrate that MSI2 directly binds VEGFR2 mRNA. Reverse protein phase array (RPPA) of several NSCLC cell line models with expression or knockdown of MSI2 and subsequent direct validation showed that MSI2 strongly and positively regulates expression of VEGFR2. Subsequent mechanistical analysis revealed that MSI2 knockdown leads to decreased VEGFR2 protein and mRNA levels expression in several NSCLC cell lines in vitro, resulting in increased apoptosis via inhibition of VEGFR2 signaling pathway. To better characterize MSI2 biology, we’ve crossed the B6/129S4-Msi2^tm1.1Cjl/J (M2) mice with B6/129S/Sv-Kras^tm3Tyj/J;Trp53^tm1Brn/J (KP) mice and generated novel B6/129S/Sv-KP;Msi2^-/- (KPM2) mouse model. Induction of tumorigenesis in KP mice using Cre adenovirus leads to development of lung adenocarcinoma. Induction of lung-specific Cre resulted in tumorigenesis in both KP and KPM2 mice. Interestingly, we observed significant decrease in both total lung tumor number and total lung tumor burden in KPM2 compared to KP mice. Next, we established three novel KP and KPM2 derived cell lines from mouse lung tumors and found that KPM2 cell lines demonstrate reduced proliferation capacity and VEGFR2 mRNA level relative to KP cell lines. Additional studies to understand an impact of MSI2 deletion in KP cell lines are ongoing. Taken together, MSI2 is a promising target for NSCLC treatment, as this protein regulates VEGFR2 signaling and apoptosis in cell models and contributes to KP tumorigenesis in vivo. Citation Format: Igor Bychkov, Alexander Deneka, Iuliia Topchu, Petr Makhov, Alexander Kudinov, Anna Nikonova, John Karanicolas, Erica Golemis, Christopher Lengner, Hossein Borghaei, Jyoti Patel, Yanis Boumber. MSI2 regulates VEGFR2 signaling and tumor progression in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 874.