Musashi-2 (MSI2) regulation of DNA damage response in lung cancer.

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.

PIP4K2B Protein Regulation by NSD1 in HPV-Negative Head and Neck Squamous Cell Carcinoma.

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.

NSD1 supports cell growth and regulates autophagy in HPV-negative head and neck squamous cell carcinoma.

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 (H3K36me2); mutations inactivating NSD1 have been linked to improved outcomes in HNSCC. In this study, we show that NSD1 induces H3K36me2 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.

NSD1 supports cell growth and regulates autophagy in HPV-negative head and neck squamous cell carcinoma.

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 (H3K36me2); mutations inactivating NSD1 have been linked to improved outcomes in HNSCC. In this study, we show that NSD1 induces H3K36me2 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.

Musashi-2 (MSI2) regulation of DNA damage response in lung cancer.

Lung cancer is one of the most common types of cancers 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 (KP versus KPM2 mice). KPM2 mice showed decreased lung tumorigenesis in comparison with KP mice what supports published data. In addition, using cell lines from KP and KPM2 tumors, and human NSCLC cell lines, we found that MSI2 directly binds ATM/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 lung tumorigenesis, in part, by direct positive regulation of ATM protein expression and DDR. This adds the knowledge of MSI2 function in lung cancer development. Targeting MSI2 may be a promising strategy to treat lung cancer. Significance: This study shows the novel role of Musashi-2 as regulator of ATM expression and DDR in lung cancer.

Regulation of VEGFR2 and AKT Signaling by Musashi-2 in Lung Cancer.

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.

Regulation of VEGFR2 and AKT signaling by Musashi-2 in lung cancer.

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 lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, expressed on both endothelial and tumor cells which is one of the key proteins contributing to cancer development and 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 nominated VEGFR2 protein as strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human NSCLC 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 direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human NSCLC samples. We conclude that MSI2/VEGFR2 axis contributes to NSCLC progression and is worth further investigations and therapeutic targeting.

Rationally designed inhibitors of the Musashi protein-RNA interaction by hotspot mimicry.

RNA-binding proteins (RBPs) are key post-transcriptional regulators of gene expression, and thus underlie many important biological processes. Here, we developed a strategy that entails extracting a "hotspot pharmacophore" from the structure of a protein-RNA complex, to create a template for designing small-molecule inhibitors and for exploring the selectivity of the resulting inhibitors. We demonstrate this approach by designing inhibitors of Musashi proteins MSI1 and MSI2, key regulators of mRNA stability and translation that are upregulated in many cancers. We report this novel series of MSI1/MSI2 inhibitors is specific and active in biochemical, biophysical, and cellular assays. This study extends the paradigm of "hotspots" from protein-protein complexes to protein-RNA complexes, supports the "druggability" of RNA-binding protein surfaces, and represents one of the first rationally-designed inhibitors of non-enzymatic RNA-binding proteins. Owing to its simplicity and generality, we anticipate that this approach may also be used to develop inhibitors of many other RNA-binding proteins; we also consider the prospects of identifying potential off-target interactions by searching for other RBPs that recognize their cognate RNAs using similar interaction geometries. Beyond inhibitors, we also expect that compounds designed using this approach can serve as warheads for new PROTACs that selectively degrade RNA-binding proteins.

Rationally designed inhibitors of the Musashi protein-RNA interaction by hotspot mimicry.

RNA-binding proteins (RBPs) are key post-transcriptional regulators of gene expression, and thus underlie many important biological processes. Here, we developed a strategy that entails extracting a "hotspot pharmacophore" from the structure of a protein-RNA complex, to create a template for designing small-molecule inhibitors and for exploring the selectivity of the resulting inhibitors. We demonstrate this approach by designing inhibitors of Musashi proteins MSI1 and MSI2, key regulators of mRNA stability and translation that are upregulated in many cancers. We report this novel series of MSI1/MSI2 inhibitors is specific and active in biochemical, biophysical, and cellular assays. This study extends the paradigm of "hotspots" from protein-protein complexes to protein-RNA complexes, supports the "druggability" of RNA-binding protein surfaces, and represents one of the first rationally-designed inhibitors of non-enzymatic RNA-binding proteins. Owing to its simplicity and generality, we anticipate that this approach may also be used to develop inhibitors of many other RNA-binding proteins; we also consider the prospects of identifying potential off-target interactions by searching for other RBPs that recognize their cognate RNAs using similar interaction geometries. Beyond inhibitors, we also expect that compounds designed using this approach can serve as warheads for new PROTACs that selectively degrade RNA-binding proteins.

The role of NSD1, NSD2, and NSD3 histone methyltransferases in solid tumors.

NSD1, NSD2, and NSD3 constitute the nuclear receptor-binding SET Domain (NSD) family of histone 3 lysine 36 (H3K36) methyltransferases. These structurally similar enzymes mono- and di-methylate H3K36, which contribute to the maintenance of chromatin integrity and regulate the expression of genes that control cell division, apoptosis, DNA repair, and epithelial-mesenchymal transition (EMT). Aberrant expression or mutation of members of the NSD family is associated with developmental defects and the occurrence of some types of cancer. In this review, we discuss the effect of alterations in NSDs on cancer patient's prognosis and response to treatment. We summarize the current understanding of the biological functions of NSD proteins, focusing on their activities and the role in the formation and progression in solid tumors biology, as well as how it depends on tumor etiologies. This review also discusses ongoing efforts to develop NSD inhibitors as a promising new class of cancer therapeutic agents.

Prognostic role and biologic features of Musashi-2 expression in colon polyps and during colorectal cancer progression.

BACKGROUND: The RNA-binding protein Musashi-2 (MSI2) controls the translation of proteins that support stem cell identity and lineage determination and is associated with progression in some cancers. We assessed MSI2 as potential clinical biomarker in colorectal cancer (CRC) and tubulovillous adenoma (TA) of colon mucosa. METHODS: We assessed 125 patients, of whom 20 had polyps of the colon (TAs), and 105 had CRC. Among 105 patients with CRC, 45 had stages I-III; among metastatic CRC (mCRC) patients, 31 had synchronous and 29 metachronous liver metastases. We used immunohistochemistry to measure MSI2 expression in matching specimens of normal tissue versus TAs, primary CRC tumors, and metastases, correlating expression to clinical outcomes. We analyzed the biological effects of depleting MSI2 expression in human CRC cells. RESULTS: MSI2 expression was significantly elevated in polyps versus primary tissue, and further significantly elevated in primary tumors and metastases. MSI2 expression correlated with decreased progression free survival (PFS) and overall survival (OS), higher tumor grade, and right-side localization (p = 0.004) of tumors. In metastases, high MSI2 expression correlated with E-cadherin expression. Knockdown of MSI2 in CRC cells suppressed proliferation, survival and clonogenic capacity, and decreased expression of TGFß1, E-cadherin, and ZO1. CONCLUSION: Elevated expression of MSI2 is associated with pre-cancerous TAs in the colonic mucosa, suggesting it is an early event in transformation. MSI2 expression is further elevated during CRC progression, and associated with poor prognosis. Depletion of MSI2 reduces CRC cell growth. These data imply a causative role of MSI2 overexpression at multiple stages of CRC formation and progression.

Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC).

Non-small cell lung cancer (NSCLC) has limited treatment options. Expression of the RNA-binding protein (RBP) Musashi-2 (MSI2) is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression, and drives NSCLC metastasis. We evaluated the mechanism of MSI2 action in NSCLC to gain therapeutically useful insights. Reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+; Trp53R172HΔG/+ NSCLC cell lines identified EGFR as a MSI2-regulated protein. MSI2 control of EGFR expression and activity in an NSCLC cell line panel was studied using RT-PCR, Western blots, and RNA immunoprecipitation. Functional consequences of MSI2 depletion were explored for cell growth and response to EGFR-targeting drugs, in vitro and in vivo. Expression relationships were validated using human tissue microarrays. MSI2 depletion significantly reduced EGFR protein expression, phosphorylation, or both. Comparison of protein and mRNA expression indicated a post-transcriptional activity of MSI2 in control of steady state levels of EGFR. RNA immunoprecipitation analysis demonstrated that MSI2 directly binds to EGFR mRNA, and sequence analysis predicted MSI2 binding sites in the murine and human EGFR mRNAs. MSI2 depletion selectively impaired cell proliferation in NSCLC cell lines with activating mutations of EGFR (EGFRmut). Further, depletion of MSI2 in combination with EGFR inhibitors such as erlotinib, afatinib, and osimertinib selectively reduced the growth of EGFRmut NSCLC cells and xenografts. EGFR and MSI2 were significantly co-expressed in EGFRmut human NSCLCs. These results define MSI2 as a direct regulator of EGFR protein expression, and suggest inhibition of MSI2 could be of clinical value in EGFRmut NSCLC.

Alkaptonuria in Russia: mutational spectrum and novel variants.

Alkaptonuria is a rare genetic disease caused by mutations in HGD gene. Here we report the results of genetic and biochemical analysis of 49 Russian patients with alkaptonuria. One of the common variants c.481G > A; p.(Gly161Arg) comprising 72.4% of identified alleles was found in 45 of 49 patients in our cohort, which is probably the highest frequency of this variant worldwide. 9 novel variants were found: 6 missense, 2 splicing and 1 loss of start-codon. For missense variants we performed bioinformatic analysis, protein 3D-modeling and molecular dynamics simulations, which strongly suggest their pathogenic effect. For the rare synonymous variant c.753C > T; p.(Gly251Gly), which was found in 3 cases and predicted to activate cryptic splice site, we performed the detailed functional analysis on patient's cDNA and minigene assay and confirmed its pathogenicity.

Effect of BN Nanoparticles Loaded with Doxorubicin on Tumor Cells with Multiple Drug Resistance.

Herein we study the effect of doxorubicin-loaded BN nanoparticles (DOX-BNNPs) on cell lines that differ in the multidrug resistance (MDR), namely KB-3-1 and MDR KB-8-5 cervical carcinoma lines, and K562 and MDR i-S9 leukemia lines. We aim at revealing the possible differences in the cytotoxic effect of free DOX and DOX-BNNP nanoconjugates on these types of cells. The spectrophotometric measurements have demonstrated that the maximum amount of DOX in the DOX-BNNPs is obtained after saturation in alkaline solution (pH 8.4), indicating the high efficiency of BNNPs saturation with DOX. DOX release from DOX-BNNPs is a pH-dependent and DOX is more effectively released in acid medium (pH 4.0-5.0). Confocal laser scanning microscopy has shown that the DOX-BNNPs are internalized by neoplastic cells using endocytic pathway and distributed in cell cytoplasm near the nucleus. The cytotoxic studies have demonstrated a higher sensitivity of the leukemia lines to DOX-BNNPs compared with the carcinoma lines: IC50(DOX-BNNPs) is 1.13, 4.68, 0.025, and 0.14 µg/mL for the KB-3-1, MDR KB-8-5, K562, and MDR i-S9 cell lines, respectively. To uncover the mechanism of cytotoxic effect of nanocarriers on MDR cells, DOX distribution in both the nucleus and cytoplasm has been studied. The results indicate that the DOX-BNNP nanoconjugates significantly change the dynamics of DOX accumulation in the nuclei of both KB-3-1 and KB-8-5 cells. Unlike free DOX, the utilization of DOX-BNNPs nanoconjugates allows for maintaining a high and stable level of DOX in the nucleus of MDR KB-8-5 cells.