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.

Generation of induced pluripotent stem cell line (RCMGi012-A) from fibroblasts of patient with mucopolysaccharidosis type VI.

Skin fibroblasts obtained from a 5-year-old girl with genetically proven (two heterozygous mutations in ARSB gene) and clinically manifested mucopolysaccharidosis type VI were successfully transformed into induced pluripotent stem cells by using Sendai virus-based reprogramming vectors including the four Yamanaka factors namely SOX2, OCT3/4, KLF4, and c-MYC. These iPSCs expressed pluripotency markers, had a normal karyotype and the potential to differentiate into three germ layers in spontaneous differentiation assay. The line may be used for cell differentiation and pharmacological investigations, and also may provide a model for development of a personalized treatment including drug screening and genome editing.

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.

Monte Carlo Computer Simulations of Spin-Transfer Torque.

This article performs computer simulations of the change in magnetization in the ferromagnetic film when polarized electric current passes through it. The model examines multilayer structures from ferromagnetic and nonmagnetic films. A sandwich system comprises two ferromagnetic layers separated by a nonmagnetic gasket. Ferromagnetic films have different magnetic susceptibility. The first ferromagnetic film is magnetically hard and acts as a fixed layer. The second ferromagnetic film is magnetically soft, with a switched direction of magnetization. The current direction is perpendicular to the film plane (CPP geometry). Spin transfer is carried out by electrons that polarize in the first ferromagnetic film and transmit spin to the second ferromagnetic film. We use the Ising model to describe the magnetic properties of the system and the Metropolis algorithm to form the thermodynamic states of the spin system. Simulations are performed at temperatures below the Curie points for both materials. The result of computer simulation is the dependence of magnetization in the magnetically soft film on the current strength in the system. Calculations show that there is a critical value of the current at which the magnetization sign of the controlled film changes. The magnetization versus current plot is stepwise. The change in the magnetization sign is due to an increase in the polarization of the electron gas. The plot of electron gas polarization versus current is also stepwise.

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.

Enhanced magnetic modulation of surface plasmon polaritons on hyperbolic metasurfaces.

In this Letter we demonstrate a fundamentally new, to the best of our knowledge, concept to enhance the magnetic modulation of the surface plasmon polaritons (SPPs) by using hybrid magneto-plasmonic structures consisting of hyperbolic plasmonic metasurfaces and magnetic dielectric substrates. Our results show that the magnetic modulation of SPPs in the proposed structures can be an order of magnitude stronger than in the hybrid metal-ferromagnet multilayer structures conventionally used in active magneto-plasmonics. We believe that this effect will allow for the further miniaturization of magneto-plasmonic devices.

Magnetic Properties of 2D Nanowire Arrays: Computer Simulations.

The paper considers a nanowires 2D array located in the nodes of a square lattice. Computer simulations use the Heisenberg model and Metropolis algorithm. The array consists of small nanowires that are monodomain. The exchange interaction orders the spins within a single nanowire. Dipole-dipole forces act between neighboring nanowires. The shape of an individual nanowire affects its magnetic anisotropy. Computer simulations examine the phase transition temperature and magnetization behavior of the system. The type of magnetic moments ordering in the array of nanowires depends on the orientation of their long axis. We consider two types of systems. The nanowires' long axes are oriented perpendicular to the plane of their location in the first case. A dipole-dipole interaction results in first-type superantiferromagnetic ordering of the nanowires' magnetic moments for such orientation. The nanowires' long axes are oriented in the plane of the system in the second case. Dipole-dipole interaction results in second-type superantiferromagnetic ordering in such systems. The dependence of the phase transition temperature on the dipole-dipole interaction intensity is investigated.

Functional investigation of SCN1A deep-intronic variants activating poison exons inclusion.

Dravet syndrome is a devastating epileptic syndrome characterized by intractable epilepsy with an early age of onset, regression of developmental milestones, ataxia, and motor deficits. Loss-of-function pathogenic variants in the SCN1A gene are found in the majority of patients with Dravet syndrome; however, a significant number of patients remain undiagnosed even after comprehensive genetic testing. Previously, it was shown that intronic elements in the SCN1A gene called poison exons can incorporate into SCN1A mRNA, leading to haploinsufficiency and potentially causing Dravet syndrome. Here, we developed a splicing reporter assay for all described poison exons of the SCN1A gene and validated it using previously reported and artificially introduced variants. Overall, we tested 18 deep-intronic single nucleotide variants and one complex allele in the SCN1A gene. Our approach is capable of evaluating the effect of both variants affecting cis-regulatory sequences and splice-site variants, with the potential to functionally annotate every possible variant within these elements. Moreover, using antisense-modified uridine-rich U7 small nuclear RNAs, we were able to block poison exon incorporation in mutant constructs, an approach that could be used as a promising therapeutic intervention in Dravet syndrome patients with deep-intronic variants.

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.

Leigh Syndrome: Spectrum of Molecular Defects and Clinical Features in Russia.

Leigh syndrome (LS), also known as infantile subacute necrotizing encephalopathy, is the most frequent mitochondrial disorder in children. Recently, more than 80 genes have been associated with LS, which greatly complicates the diagnosis. In this article, we present clinical and molecular findings of 219 patients with LS and give the detailed description of three cases with rare findings in nuclear genes MORC2, NARS2 and VPS13D, demonstrating wide genetic heterogeneity of this mitochondrial disease. The most common cause of LS in Russian patients are pathogenic variants in the SURF1 gene (44.3% of patients). The most frequent pathogenic variant is c.845_846delCT (66.0% of mutant alleles; 128/192), which is also widespread in Eastern Europe. Five main LS genes, SURF1, SCO2, MT-ATP6, MT-ND5 and PDHA1, account for 70% of all LS cases in the Russian Federation. Using next generation sequencing (NGS) technique, we were able to detect pathogenic variants in other nuclear genes: NDUFV1, NDUFS2, NDUFS8, NDUFAF5, NDUFAF6, NDUFA10, SUCLG1, GFM2, COX10, PMPCB, NARS2, PDHB and SLC19A3, including two genes previously associated with Leigh-like phenotypes-MORC2 and VPS13D. We found 49 previously undescribed nucleotide variants, including two deep intronic variants which affect splicing.

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.

Functional Characterization of Two Novel Intron 4 SERPING1 Gene Splice Site Pathogenic Variants in Families with Hereditary Angioedema.

Variants that affect splice sites comprise 14.3% of all pathogenic variants in the SERPING1 gene; more than half of them are located outside the canonical sites. To make a clinical decision concerning patients with such variants, it is essential to know the exact way in which the effect of the variant would be realized. The optimal approach to determine the consequences is considered to be mRNA analysis. In the current study, we present the results of functional analysis of two previously non-described variants in the SERPING1 gene (NM_000062.3) affecting intron 4: c.686-1G>A and c.685+4dup, which were detected in members of two Russian families with autosomal dominant inheritance of angioedema type 1. Analysis of the patients' mRNA (extracted from whole blood) showed that the SERPING1(NM_000062.3):c.685+4dup variant leads to the loss of the donor splice site and the activation of the cryptic site in exon 4: r.710_745del (p.Gly217_Pro228del), while the SERPING1(NM_000062.3):c.686-1G>A variant leads to the skipping of exon 5: r.746_949del (p.Asp229_Ser296del).

A Study of Magnetic Properties in a 2D Ferromagnetic Nanolattice through Computer Simulation.

This article investigated the magnetic properties of a 2D nanolattice through computer modeling. A square antidote nanolattice in thin films was considered. For our computer simulation, we used the Heisenberg model. Ferromagnetic phase transition was studied for lattices with pores of various sizes. We determined the Curie temperature based on the finite-dimensional scaling theory. Using Wolf's algorithm, we simulated the behavior of the system. The dependence of the phase transition temperature on the density of spins was found to be power. Using Metropolis' algorithm, we calculated a hysteresis loop for an antidote lattice film. The hysteresis loop narrowed as the pore sizes increased. The dependence of coercive force on the size of the nanolattice obeyed the logarithmic law.

Expanding the genetic spectrum of the pyruvate carboxylase deficiency with novel missense, deep intronic and structural variants.

Introduction: Pathogenic variants in the pyruvate carboxylase (PC) gene cause a wide spectrum of recessive phenotypes, ranging from the early-onset fatal encephalopathy to the adult-onset benign form. Results: Patient 1 is a 6 y.o. boy with ataxia, hypoglycemia and episodes of lactic acidosis. WGS revealed the novel heterozygous missense variant c.1372A > G (p.Asn458Asp) in the PC gene. Additional analysis revealed discordant reads mapped to chromosomes 11 and 1, so a reciprocal translocation disrupted the PC gene was suspected. The translocation was validated via FISH-analysis and Sanger sequencing of its boundaries.Patient 2 is a 13 y.o. girl with psychomotor delay, episodes of lactic acidosis and ketonuria. WES revealed the novel homozygous intronic variant c.1983-116C > T. The PC's mRNA analysis demonstrated the exonization of several intron 16 sequences and some residual amount of WT mRNA isoform.Two other patients had more severe course of the disease. Their genotype represents missense variants in compound heterozygous and homozygous state (c.1876C > T (p.Arg626Trp), c.2606G > C (p.Gly869Ala), c.2435C > A (p.Ala812Asp). Conclusion: In patients with metabolic crises, lactic acidosis and hypoglycemia analysis of PC gene is recommended. WGS with deep bioinformatic analysis should be taken into consideration when none or the only one pathogenic variant in the PC gene is found.

Processed pseudogene insertion in GLB1 causes Morquio B disease by altering intronic splicing regulatory landscape.

Morquio B disease (MBD) is an ultra-rare lysosomal storage disease, which represents the relatively mild form of GLB1-associated disorders. In this article, we present the unique case of "pure" MBD associated with an insertion of the mobile genetic element from the class of retrotransposons. Using whole-genome sequencing (WGS), we identified an integration of the processed pseudogene NPM1 deep in the intron 5 of GLB1. The patient's mRNA analysis and the detailed functional analysis revealed the underlying molecular genetic mechanism of pathogenesis, which is an alteration of the GLB1 normal splicing. By co-expression of minigenes and antisense splice-modulating oligonucleotides (ASMOs), we demonstrated that pseudogene-derived splicing regulatory motifs contributed to an activation of the cryptic exon located 36 bp upstream of the integration site. Blocking the cryptic exon with ASMOs incorporated in the modified U7 small nuclear RNA (modU7snRNA) almost completely restored the wild-type splicing in the model cell line, that could be further extended toward the personalized genetic therapy. To our knowledge, this is the second reported case of the processed pseudogene insertion for monogenic disorders. Our data emphasizes the unique role of WGS in identification of such rare and probably underrepresented in literature types of disease-associated genetic variants.