Musashi-1 Research Articles

OR26-5 Musashi-2 Regulatory Interactions Promote Adrenal Steroidogenesis

Abstract The adrenal gland is the site of steroid hormone synthesis. These hormones regulate many physiological processes including metabolism, blood pressure and volume, and sexual characteristic development. While signaling pathways, steroidogenic enzymes, and transcription factors have all been well characterized, little is known about the contribution of RNA-binding proteins (RBPs) in regulating steroidogenesis. We recently demonstrated that regulated RNA decay controls the timing of the steroidogenic gene expression response and identified 9 RBPs that regulate aldosterone production in an immortalized human cell line model for steroidogenesis, H295R. One of the RBPs we identified was Musashi-2 (MSI2), which is required for development of other steroidogenic tissues such as ovary and testis in mice. We found that MSI2 mRNA is enriched in human adrenal glands relative to other tissues, positively correlates in expression with mRNAs encoding steroidogenic enzymes, and abundantly expressed in H295R cells. Staining of normal human adrenal gland sections demonstrated that MSI2 protein is expressed in all three layers of the human adrenal cortex. Taken together, we hypothesized that MSI2 promotes steroidogenesis through regulating expression of target mRNAs encoding. Using the selective MSI2 inhibitor Ro-08-2750 (Ro) we found that aldosterone and cortisol production is decreased in a dosage dependent manner in H295R cells, which is consistent with siRNA knockdown of MSI2. We performed RNA-immunoprecipitation and sequencing and identified 1428 mRNA targets of MSI2 in H295R cells. A target of interest was Stearoyl-CoA Desaturase (SCD), which functions to convert saturated fatty acids to unsaturated fatty acids that promote formation of lipid droplets. Cholesterol esters in lipid droplets are a source of cholesterol used to produce all steroid hormones. Ro treatment of H295R cells resulted in a suppression of SCD mRNA and protein induction. Furthermore, inhibition of SCD with A939572 caused a dosage dependent decrease in aldosterone production. These data suggest that potentiation of stimulation dependent SCD induction is one mechanism by which MSI2 promotes steroidogenesis. It will be important to determine if and how MSI2 promotes steroidogenesis in primary human adrenal cells and mouse models. Since it is possible to deliver oligonucleotides to the adrenal cortex, disruption of MSI2-SCD mRNA regulatory interactions may open new therapeutic avenues for modulate aldosterone production. Presentation: Monday, June 13, 2022 12:00 p.m. - 12:15 p.m.

PMON29 A Comparative Analysis of Musashi-Dependent Control of Mouse and Human Pituitary mRNA Translation

Abstract Specialized cell lineages within the anterior pituitary produce specific hormones which, in turn, regulate diverse biological processes ranging from growth, metabolism, and reproduction to stress. Interestingly, these specialized cell lineages exhibit a high degree of cellular plasticity, readily altering their differentiation state to produce different hormones in response to organismal demand. Underlying molecular mechanisms controlling cellular plasticity in the pituitary are not fully defined but appear to involve post-transcriptional regulation exerted by the Musashi family of sequence-specific RNA binding proteins. Musashi is a bifunctional regulator of target mRNAs and can exert either repression or activation of translation in a context-dependent manner. Our recent work has implicated a role for Musashi-dependent repression of key pituitary mRNAs in the mouse, including a lineage-specific transcription factor (Pou1f1), the gonadotropin releasing hormone receptor (Gnrhr), and two anterior pituitary hormones (Prl and Tshb). In this study, we look to determine the potential relevance of Musashi-dependent mRNA translational control to the human pituitary. Reflecting our findings in the adult mouse, a re-analysis of published human fetal pituitary single cell RNA sequencing data revealed Musashi1 (MSI1) and Musashi2 (MSI2) expression in both stem/progenitor cell populations as well as in all hormone-producing cell lineages of the anterior pituitary. We further report that the human homologs of identified Musashi-target mRNAs in the mouse pituitary also possess Musashi binding elements (MBEs) in their 3' untranslated regions (UTRs). In the case of the human Pou1f1 3' UTR, we were able to verify Musashi-dependent mRNA translational repression in reporter assays as seen with the murine Pou1f1 3' UTR. Interestingly, a germline Pou1f1 mutation identified in human patients, disrupts a consensus MBE in the human Pou1f1 3' UTR and abolishes Musashi-dependent repression. We developed a bioinformatic pipeline to characterize pathogenic human mutations catalogued in the Geno2MP database for disruption or creation of consensus MBEs in pituitary-specific mRNA 3' UTRs. Our findings suggest an evolutionarily conserved role for Musashi in the post-transcriptional regulation of pituitary mRNAs in both mice and humans and identify several human mutations which may perturb Musashi regulation of human pituitary function. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Musashi-1 and miR-147 Precursor Interaction Mediates Synergistic Oncogenicity Induced by Co-Infection of Two Avian Retroviruses.

Synergism between avian leukosis virus subgroup J (ALV-J) and reticuloendotheliosis virus (REV) has been reported frequently in co-infected chicken flocks. Although significant progress has been made in understanding the tumorigenesis mechanisms of ALV and REV, how these two simple oncogenic retroviruses induce synergistic oncogenicity remains unclear. In this study, we found that ALV-J and REV synergistically promoted mutual replication, suppressed cellular senescence, and activated epithelial-mesenchymal transition (EMT) in vitro. Mechanistically, structural proteins from ALV-J and REV synergistically activated the expression of Musashi-1(MSI1), which directly targeted pri-miR-147 through its RNA binding site. This inhibited the maturation of miR-147, which relieved the inhibition of NF-κB/KIAA1199/EGFR signaling, thereby suppressing cellular senescence and activating EMT. We revealed a synergistic oncogenicity mechanism induced by ALV-J and REV in vitro. The elucidation of the synergistic oncogenicity of these two simple retroviruses could help in understanding the mechanism of tumorigenesis in ALV-J and REV co-infection and help identify promising molecular targets and key obstacles for the joint control of ALV-J and REV and the development of clinical technologies.

TP53 mutations and RNA-binding protein MUSASHI-2 drive resistance to PRMT5-targeted therapy in B-cell lymphoma

To identify drivers of sensitivity and resistance to Protein Arginine Methyltransferase 5 (PRMT5) inhibition, we perform a genome-wide CRISPR/Cas9 screen. We identify TP53 and RNA-binding protein MUSASHI2 (MSI2) as the top-ranked sensitizer and driver of resistance to specific PRMT5i, GSK-591, respectively. TP53 deletion and TP53R248W mutation are biomarkers of resistance to GSK-591. PRMT5 expression correlates with MSI2 expression in lymphoma patients. MSI2 depletion and pharmacological inhibition using Ro 08-2750 (Ro) both synergize with GSK-591 to reduce cell growth. Ro reduces MSI2 binding to its global targets and dual treatment of Ro and PRMT5 inhibitors result in synergistic gene expression changes including cell cycle, P53 and MYC signatures. Dual MSI2 and PRMT5 inhibition further blocks c-MYC and BCL-2 translation. BCL-2 depletion or inhibition with venetoclax synergizes with a PRMT5 inhibitor by inducing reduced cell growth and apoptosis. Thus, we propose a therapeutic strategy in lymphoma that combines PRMT5 with MSI2 or BCL-2 inhibition.

Theoretical studies on RNA recognition by Musashi 1 RNA-binding protein

The Musashi (MSI) family of RNA-binding proteins, comprising the two homologs Musashi-1 (MSI1) and Musashi-2 (MSI2), typically regulates translation and is involved in cell proliferation and tumorigenesis. MSI proteins contain two ribonucleoprotein-like RNA-binding domains, RBD1 and RBD2, that bind single-stranded RNA motifs with a central UAG trinucleotide with high affinity and specificity. The finding that MSI also promotes the replication of Zika virus, a neurotropic Flavivirus, has triggered further investigations of the biochemical principles behind MSI–RNA interactions. However, a detailed molecular understanding of the specificity of MSI RBD1/2 interaction with RNA is still missing. Here, we performed computational studies of MSI1–RNA association complexes, investigating different RNA pentamer motifs using molecular dynamics simulations with binding free energy calculations based on the solvated interaction energy method. Simulations with Alphafold2 suggest that predicted MSI protein structures are highly similar to experimentally determined structures. The binding free energies show that two out of four RNA pentamers exhibit a considerably higher binding affinity to MSI1 RBD1 and RBD2, respectively. The obtained structural information on MSI1 RBD1 and RBD2 will be useful for a detailed functional and mechanistic understanding of this type of RNA–protein interactions.

Abstract 874: MSI2 regulates VEGFR2 signaling and tumor progression in NSCLC

Abstract 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-Msi2tm1.1Cjl/J (M2) mice with B6/129S/Sv-Krastm3Tyj/J;Trp53tm1Brn/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.

Integrative genome-wide analysis reveals EIF3A as a key downstream regulator of translational repressor protein Musashi 2 (MSI2).

Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate decisions in normal and cancer stem cells. MSI2 appears to repress translation by binding to 3′ untranslated regions (3′UTRs) of mRNA, but the identity of functional targets remains unknown. Here, we used individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) to identify direct RNA binding partners of MSI2 and integrated these data with polysome profiling to obtain insights into MSI2 function. iCLIP revealed specific MSI2 binding to thousands of mRNAs largely in 3′UTRs, but translational differences were restricted to a small fraction of these transcripts, indicating that MSI2 regulation is not triggered by simple binding. Instead, the functional targets identified here were bound at higher density and contain more ‘UAG’ motifs compared to targets bound nonproductively. To further distinguish direct and indirect targets, MSI2 was acutely depleted. Surprisingly, only 50 transcripts were found to undergo translational induction on acute loss. Using complementary approaches, we determined eukaryotic translation initiation factor 3A (EIF3A) to be an immediate, direct target. We propose that MSI2 downregulation of EIF3A amplifies these effects on translation. Our results also underscore the challenges in defining functional targets of RBPs since mere binding does not imply a discernible functional interaction.

Inhibition of Musashi-1 enhances chemotherapeutic sensitivity in gastric cancer patient-derived xenografts.

Musashi-1 (MSI1), a neural RNA-binding protein, is considered a gastric and intestinal stem cell marker. Although the function of MSI1 in gastric cancer has attracted increasing interest, it is not known whether MSI1 can be used as a biomarker to monitor gastric cancer development and response to treatment. Here, the role of MSI1 in the chemotherapeutic sensitivity of gastric cancer was investigated. Patients with high MSI1 levels had poor outcomes, implicating the gene in the development and progression of the disease. We overexpressed and silenced MSI1 in the human gastric cancer cell lines MKN45 and HGC27, finding that knockdown reduced proliferation, invasion, and migration, while promoting apoptosis. A patient-derived xenograft gastric cancer model was constructed in which mice received chemical drugs, si-MSI1, or a drug-si-MSI1 combination. It was found that blocking MSI1 expression reduced gastric cancer drug tolerance. The combination treatment with si-MSI1 was superior to 5F-dUMP and cisplatin, either separately or in combination, indicating that including si-MSI1 was better than drug therapy alone. Transcriptome sequencing analysis showed that MSI1 altered cell cycle regulation and growth signal transduction, including that of blood vessel epicardial substance (BVES). These results suggest that MSI1 reduces the tolerance of gastric cancer to chemical drugs through modulation of MSI1/BVES signaling.

Small Molecule Palmatine Targeting Musashi-2 in Colorectal Cancer.

Musashi-2 (MSI2) is an evolutionally conserved RNA-binding protein and recently considered as an attractive therapeutic target in a wide spectrum of malignancies. However, MSI2-engaged mRNAs are not well profiled, and no MSI2-dependent antagonist is available so far. In the study, we created MSI2 knockout cancer cells and demonstrated that MSI2 is required for the survival of colorectal cancer HCT116 cells but not non-small cell lung cancer A549 cells. In addition, the global profiling of the transcriptome and proteomics of MSI2 knockout colorectal cells revealed 38 candidate MSI2-targeted genes. In a loss–rescue screening, palmatine was identified as a functional MSI2 antagonist inhibiting the MSI2-dependent growth of colorectal cancer cells. Finally, we confirmed that palmatine is directly bound to MSI2 at its C-terminal. Our findings not only indicated MSI2 as a promising therapeutic target of colorectal cancer but also provided a small molecule palmatine as a direct and functional MSI2 antagonist for cancer therapy.

Strategic Decoy Peptides Interfere with MSI1/AGO2 Interaction to Elicit Tumor Suppression Effects.

Simple SummaryPeptide drugs that can specifically target undesirable protein–protein interactions that lead to oncogenic developments have emerged as the next era of future medicine for cancers. To combat GBM tumor progression, our study offers an alternative therapeutic strategy via targeting the protein–protein interaction between MSI1 and AGO2 with synthetic peptides identified from the C-terminus of MSI1 in peptide arrays. Our present data revealed for the first time that peptidic disruption to the MSI1/AGO2 complex known for promoting cancer stemness and progression could lead to encouraging therapeutic efficacy at both in vitro and in vivo levels. The significantly suppressed tumor growth and prolonged survival rates in PDX tumor models by decoy peptides evidently provided a new rationale for stratifying patients with MSI1/AGO2-targeted therapeutics.Peptide drugs that target protein–protein interactions have attracted mounting research efforts towards clinical developments over the past decades. Increasing reports have indicated that expression of Musashi 1 (MSI1) is tightly correlated to high grade of cancers as well as enrichment of cancer stem cells. Treatment failure in malignant tumors glioblastoma multiform (GBM) had also been correlated to CSC-regulating properties of MSI1. It is thus imperative to develop new therapeutics that could effectively improve current regimens used in clinics. MSI1 and AGO2 are two emerging oncogenic molecules that both contribute to GBM tumorigenesis through mRNA regulation of targets involved in apoptosis and cell cycle. In this study, we designed peptide arrays covering the C-terminus of MSI1 and identified two peptides (Pep#11 and Pep#26) that could specifically interfere with the binding with AGO2. Our Biacore analyses ascertained binding between the identified peptides and AGO2. Recombinant reporter system Gaussian luciferase and fluorescent bioconjugate techniques were employed to determine biological functions and pharmacokinetic characteristics of these two peptides. Our data suggested that Pep#11 and Pep#26 could function as decoy peptides by mimicking the interaction function of MSI1 with its binding partner AGO2 in vitro and in vivo. Further experiments using GMB animal models corroborated the ability of Pep#11 and Pep#26 in disrupting MSI1/AGO2 interaction and consequently anti-tumorigenicity and prolonged survival rates. These striking therapeutic efficacies orchestrated by the synthetic peptides were attributed to the decoy function to C-terminal MSI1, especially in malignant brain tumors and glioblastoma.

LncRNA LINC00942 promotes chemoresistance in gastric cancer by suppressing MSI2 degradation to enhance c-Myc mRNA stability.

BackgroundChemoresistance to cisplatin (DDP) remains a major challenge in advanced gastric cancer (GC) treatment. Although accumulating evidence suggests an association between dysregulation of long non‐coding RNAs (lncRNAs) and chemoresistance, the regulatory functions and complexities of lncRNAs in modulating DDP‐based chemotherapy in GC remain under‐investigated. This study was designed to explore the critical chemoresistance‐related lncRNAs in GC and identify novel therapeutic targets for patients with chemoresistant GC.MethodsChemoresistance‐related lncRNAs were identified through microarray and verified through a quantitative real‐time polymerase chain reaction (qRT‐PCR). Proteins bound by lncRNAs were identified through a human proteome array and validated through RNA immunoprecipitation (RIP) and RNA pull‐down assays. Co‐immunoprecipitation and ubiquitination assays were performed to explore the molecular mechanisms of the Musashi2 (MSI2) post‐modification. The effects of LINC00942 (LNC942) and MSI2 on DDP‐based chemotherapy were investigated through MTS, apoptosis assays and xenograft tumour formation in vivo.ResultsLNC942 was found to be up‐regulated in chemoresistant GC cells, and its high expression was positively correlated with the poor prognosis of patients with GC. Functional studies indicated that LNC942 confers chemoresistance to GC cells by impairing apoptosis and inducing stemness. Mechanically, LNC942 up‐regulated the MSI2 expression by preventing its interaction with SCFβ‐TRCP E3 ubiquitin ligase, eventually inhibiting ubiquitination. Then, LNC942 stabilized c‐Myc mRNA in an N6‐methyladenosine (m6A)‐dependent manner. As a potential m6A recognition protein, MSI2 stabilized c‐Myc mRNA with m6A modifications. Moreover, inhibition of the LNC942‐MSI2‐c‐Myc axis was found to restore chemosensitivity both in vitro and in vivo.ConclusionsThese results uncover a chemoresistant accelerating function of LNC942 in GC, and disrupting the LNC942‐MSI2‐c‐Myc axis could be a novel therapeutic strategy for GC patients undergoing chemoresistance.

Mechanism of RNA recognition by a Musashi RNA-binding protein.

The Musashi RNA-binding proteins (RBPs) regulate translation of target mRNAs and maintenance of cell stemness and tumorigenesis. Musashi-1 (MSI1), long considered as an intestinal and neural stem cell marker, has been more recently found to be over expressed in many cancers. It has served as an important drug target for treating acute myeloid leukemia and solid tumors such as ovarian, colorectal and bladder cancer. One of the reported binding targets of MSI1 is Numb, a negative regulator of the Notch signaling. However, the dynamic mechanism of Numb RNA binding to MSI1 remains unknown, largely hindering effective drug design targeting this critical interaction. Here, we have performed extensive all-atom microsecond-timescale simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which successfully captured multiple times of spontaneous and highly accurate binding of the Numb RNA from bulk solvent to the MSI1 protein target site. GaMD simulations revealed that Numb RNA binding to MSI1 involved largely induced fit in both the RNA and protein. The simulations also identified important low-energy intermediate conformational states during RNA binding, in which Numb interacted mainly with the β2-β3 loop and C terminus of MSI1. The mechanistic understanding of RNA binding obtained from our GaMD simulations is expected to facilitate rational structure-based drug design targeting MSI1 and other RBPs.

TP53 Mutations and RNA Binding Protein Musashi 2 Drive Resistance to PRMT5-Targeted Therapy in B-Cell Lymphoma

PRMT5 catalyzes the arginine symmetric demethylation of histone and non-histone proteins. PRMT5 is overexpressed in lymphoma and plays a key role in lymphomagenesis through the repression of several tumor suppressors, including P53, while promoting the expression of lymphoma-drivers, such as c-MYC.Currently, three PRMT5 inhibitors are being clinically evaluated in B-cell lymphoma patients, however, the mechanism of action of these molecules remain poorly understood. Thus, to identify the mechanism of action of the potent selective PRMT5 inhibitor, GSK3203591 (GSK-591), we performed a genome-wide CRISPR/Cas9 KO screen in the human mantle cell line, Z-138 and a validation CRISPR /Cas9 screen in the DLBCL cell line, OCI-LY19. Our genome-wide CRISPR screen identified 316 sensitizing genes and 89 genes involved in resistance. The top sensitizing gene was TP53, which validated our screens, as it is a well characterized target of PRMT5. Furthermore, we found that TP53 deletion and the hot spot TP53 R248Wmutation are biomarkers of resistance to GSK-591.RNA-binding protein MUSASHI 2 (MSI2) was identified as the top-ranked driver of resistance to GSK-591. MSI2 plays a key role in hematopoietic stem cells activation, myeloid leukemia and CLL, however, its function in lymphoma remains unknown. MSI2 is overexpressed in DLBCL patients (n=96). Relapsed MCL and DLBCL primary samples express high levels of both PRMT5 and MSI2. Furthermore, MSI2 depletion decreased cell viability and sensitized lymphoma cells to PRMT5 inhibitor in vitro and vivo. In contrast, overexpression of MSI2 reverted sensitivity to GSK-591 in lymphoma cells. Consistent with our genetic studies, inhibition of MSI2 using Ro 08-2750 (Ro) (Minuesa et al. Nat Commun. 2019), conferred sensitivity to GSK-591, and the combination therapy was synergistic inducing cytotoxicity.In order to identify specific downstream targets of MSI2 that may contribute to resistance to PRMT5 inhibition in lymphoma, we performed MSI2-HyperTRIBE, a recently discovered technology that allows mapping the MSI2 targeting network (Nguyen et al. Nat Commun. 2020). Using MSI2-HyperTRIBE, we were able to identify MSI2 targets in lymphoma cells. Moreover, we found that Ro treatment significantly blocked MSI2 binding activity, while GSK-591 had no effect on MSI2 targets.To uncover the mechanism involved in the synergy of PRMT5 and MSI2 inhibitors, we performed RNA-sequencing of Z-138 cells treated with GSK-591, Ro or the combination. Gene set enrichment analysis (GSEA) demonstrated a loss in the c-MYC pathway upon drug combination.MSI2 RNA-IP assays showed that MSI2 binds c-MYC and this interaction is disrupted by the combination of GSK-591 and Ro. We observed that the combination of MSI2 and PRMT5 inhibitors does not affect c-MYC mRNA levels but, rather, controls translation. Interestingly, in the human B cell line P493-6 that express a conditional, tetracycline-regulated c-MYC, the depletion of c-MYC significantly increased the anti-proliferative activity of GSK-591 and Ro alone or in combination.To further investigate whether there are other functionally relevant downstream targets of MSI2 promoting resistance to PRMT5 inhibition, we overlapped the differentially expressed genes upon treatment with GSK-591 and Ro (RNA-Seq), the Ro-direct targets in lymphoma (MSI2-HyperTRIBE) and the resistance genes to PRMT5 inhibition (whole-genome CRISPR screen) and we identified the anti-apoptotic protein BCL-2 as a common target of the PRMT5-MSI2 axis. We established first that the combination of GSK-591 and Ro blocked the binding of MSI2 to BCL-2 mRNA. We also found that the drug combination significantly decreased BCL-2 mRNA and protein abundance. Furthermore, Z-138 and OCI-LY19 BCL-2 KO cells are more sensitive to GSK-591 and Ro alone or in combination. Using venetoclax, we found that BCL-2 inhibition enhanced GSK-591 activity in vitro and in vivo. Thus, these data confirmed that BCL-2 is a key driver of resistance to PRMT5 inhibition.Overall, our study uncovered a novel oncogenic axis, PRMT5/MSI2/c-MYC/BCL-2 that drives resistance to PRMT5-targeted therapy in lymphoma. We demonstrated that TP53 LOF and MSI2 expression could be used as biomarkers for patient stratification. Moreover, we proposed two novel drug combination strategies, with venetoclax or a MSI2 inhibitor, to be considered in further clinical studies with PRMT5 inhibitors. DisclosuresBarbash: GlaxoSmithKline: Current Employment, Current equity holder in publicly-traded company. Batlevi: Viatris: Current holder of individual stocks in a privately-held company; Juno/Celgene: Consultancy; Pfizer: Current holder of individual stocks in a privately-held company; Bayer: Research Funding; ADC Therapeutics: Consultancy; TG Therapeutics: Consultancy; Medscape: Honoraria; BMS: Current holder of individual stocks in a privately-held company; Memorial Sloan Kettering Cancer Center: Current Employment; Kite Pharma: Consultancy; Life Sciences: Consultancy; Seattle Genetics: Consultancy; Karyopharm: Consultancy; Regeneron: Current holder of individual stocks in a privately-held company; Moderna: Current holder of individual stocks in a privately-held company; TouchIME: Honoraria; GLG Pharma: Consultancy; Dava Oncology: Honoraria; Xynomic: Research Funding; Roche/Genentech: Research Funding; Novartis: Research Funding; Epizyme: Research Funding; Janssen: Research Funding; Autolus: Research Funding. Younes: AZ: Current Employment, Other: Senior Vice President, Global Head of Haematology (Early and Late Stage) Oncology R&D at AstraZeneca.

MSI2 expression in adrenocortical carcinoma: Association with unfavorable prognosis and correlation with steroid and immune-related pathways.

Adrenocortical carcinoma (ACC) is a rare, but highly aggressive cancer of the adrenal cortex with a generally poor prognosis. Despite being rare, completely resected ACCs present a high risk of recurrence. Musashi-2 (MSI2) has recently been recognized as a potential prognostic biomarker and therapeutic target in many cancers. However, no studies have evaluated the clinical significance of MSI2 expression in ACC. Here, we addressed MSI2 expression and its association with ACC prognosis and clinicopathological parameters. MSI2 expression was analyzed in TCGA, GSE12368, GSE33371, and GSE49278 ACC datasets; and its correlation with other genes and immune cell infiltration were investigated by using the R2: Genomics Analysis and Visualization Platform and TIMER databases, respectively. Enrichment analysis was performed with the DAVID Functional Annotation Tool. Kaplan-Meier curves, log-rank tests, and Cox regression analyses were used to explore the prognostic role of MSI2 in ACC. Our findings demonstrated the potential value of MSI2 overexpression as an independent predictor of poor prognosis in patients with completely resected ACC (hazard ratio 6.715, 95% confidence interval 1.266 - 35.620, p =.025). In addition, MSI2 overexpression was associated with characteristics of unfavorable prognosis, such as cortisol excess (p = .002), recurrence (p =.003), and death (p =.015); positively correlated with genes related to steroid biosynthesis (p < .05); and negatively correlated with immune-related pathways (p < .05). Our findings demonstrate that MSI2 has value as a prognostic marker for completely resected ACC and reinforce the investigation of its role as a possible therapeutic target for patients with ACC.

Musashi-2 contributes to myotonic dystrophy muscle dysfunction by promoting excessive autophagy through miR-7 biogenesis repression

Skeletal muscle symptoms strongly contribute to mortality of myotonic dystrophy type 1 (DM1) patients. DM1 is a neuromuscular genetic disease caused by CTG repeat expansions that, upon transcription, sequester the Muscleblind-like family of proteins and dysregulate alternative splicing of hundreds of genes. However, mis-splicing does not satisfactorily explain muscle atrophy and wasting, and several other contributing factors have been suggested, including hyperactivated autophagy leading to excessive catabolism. MicroRNA (miR)-7 has been demonstrated to be necessary and sufficient to repress the autophagy pathway in cell models of the disease, but the origin of its low levels in DM1 was unknown. We have found that the RNA-binding protein Musashi-2 (MSI2) is upregulated in patient-derived myoblasts and biopsy samples. Because it has been previously reported that MSI2 controls miR-7 biogenesis, we tested the hypothesis that excessive MSI2 was repressing miR-7 maturation. Using gene-silencing strategies (small interfering RNAs [siRNAs] and gapmers) and the small molecule MSI2-inhibitor Ro 08-2750, we demonstrate that reducing MSI2 levels or activity boosts miR-7 expression, represses excessive autophagy, and downregulates atrophy-related genes of the UPS system. We also detect a significant upregulation of MBNL1 upon MSI2 silencing. Taken together, we propose MSI2 as a new therapeutic target to treat muscle dysfunction in DM1.

MUSASHI-2 confers resistance to third-generation EGFR-tyrosine kinase inhibitor osimertinib in lung adenocarcinoma.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKIs) are effective in patients with non–small‐cell lung cancer (NSCLC) harboring EGFR mutations. However, due to acquired resistance to EGFR‐TKIs, even patients on third‐generation osimertinib have a poor prognosis. Resistance mechanisms are still not fully understood. Here, we demonstrate that the increased expression of MUSASHI‐2 (MSI2), an RNA‐binding protein, is a novel mechanism for resistance to EGFR‐TKIs. We found that after a long‐term exposure to gefitinib, the first‐generation EGFR‐TKI lung cancer cells harboring the EGFR‐TKI‐sensitive mutations became resistant to both gefitinib and osimertinib. Although other mutations in EGFR were not found, expression levels of Nanog, a stemness core protein, and activities of aldehyde dehydrogenase (ALDH) were increased, suggesting that cancer stem‐like properties were increased. Transcriptome analysis revealed that MSI2 was among the stemness‐related genes highly upregulated in EGFR‐TKI‐resistant cells. Knockdown of MSI2 reduced cancer stem‐like properties, including the expression levels of Nanog, a core stemness factor. We demonstrated that knockdown of MSI2 restored sensitivity to osimertinib or gefitinib in EGFR‐TKI‐resistant cells to levels similar to those of parental cells in vitro. An RNA immunoprecipitation (RIP) assay revealed that antibodies against MSI2 were bound to Nanog mRNA, suggesting that MSI2 increases Nanog expression by binding to Nanog mRNA. Moreover, overexpression of MSI2 or Nanog conferred resistance to osimertinib or gefitinib in parental cells. Finally, MSI2 knockdown greatly increased the sensitivity to osimertinib in vivo. Collectively, our findings provide proof of principle that targeting the MSI2‐Nanog axis in combination with EGFR‐TKIs would effectively prevent the emergence of acquired resistance.

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

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. 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. 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 TGFβ1, E-cadherin, and ZO1. 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.

Overview of the molecular mechanisms of migration and invasion in glioblastoma multiforme.

Glioblastoma (GBM) is one of the most devastating cancers, with an approximate median survival of only 16 months. Although some new insights into the fantastic heterogeneity of this kind of brain tumor have been revealed in recent studies, all subclasses of GBM still demonstrate highly aggressive invasion properties to the surrounding parenchyma. This behavior has become the main obstruction to current curative therapies as invasive GBM cells migrate away from these foci after surgical therapies. Therefore, this review aimed to provide a relatively comprehensive study of GBM invasion mechanisms, which contains an intricate network of interactions and signaling pathways with the extracellular matrix (ECM). Among these related molecules, TGF-β, the ECM, Akt, and microRNAs are most significant in terms of cellular procedures related to GBM motility and invasion. Moreover, we also review data indicating that Musashi-1 (MSI1), a neural RNA-binding protein (RBP), regulates GBM motility and invasion, maintains stem cell populations in GBM, and promotes drug-resistant GBM phenotypes by stimulating necessary oncogenic signaling pathways through binding and regulating mRNA stability. Importantly, these necessary oncogenic signaling pathways have a close connection with TGF-β, ECM, and Akt. Thus, it appears promising to find MSI-specific inhibitors or RNA interference-based treatments to prevent the actions of these molecules despite using RBPs, which are known as hard therapeutic targets. In summary, this review aims to provide a better understanding of these signaling pathways to help in developing novel therapeutic approaches with better outcomes in preclinical studies.

Musashi-1-A Stemness RBP for Cancer Therapy?

Simple SummaryMusashi–1 (MSI1) is an RNA–binding protein that promotes stemness properties. It was initially discovered as a regulator of neuronal development and oocyte maturation in flies and frogs. Due to its specific expression pattern with high levels during development and in a variety of cancers, MSI1 evolved as an interesting target for cancer therapy. In cancer cells, the protein mainly promotes an undifferentiated state enhancing cancer growth and therapy resistance. In this review, we summarize previous findings from development of other organisms, outline MSI1′s expression and function in different cancer entities and highlight the development of MSI1–directed inhibitors.The RNA–binding protein Musashi–1 (MSI1) promotes stemness during development and cancer. By controlling target mRNA turnover and translation, MSI1 is implicated in the regulation of cancer hallmarks such as cell cycle or Notch signaling. Thereby, the protein enhanced cancer growth and therapy resistance to standard regimes. Due to its specific expression pattern and diverse functions, MSI1 represents an interesting target for cancer therapy in the future. In this review we summarize previous findings on MSI1′s implications in developmental processes of other organisms. We revisit MSI1′s expression in a set of solid cancers, describe mechanistic details and implications in MSI1 associated cancer hallmark pathways and highlight current research in drug development identifying the first MSI1–directed inhibitors with anti–tumor activity.

Musashi-1 Regulates MIF1-Mediated M2 Macrophage Polarization in Promoting Glioblastoma Progression

Simple SummaryGlioblastoma (GBM) is the most lethal type of brain cancer. It is well known that the malignancy of cancers is dependent not only on the oncogenic properties of the tumor cells, but also on the composition of the tumor microenvironment, which includes macrophages of the immune system. The prevalence of M2 type macrophages usually promotes tumor progression as opposed to tumor-suppressing function of M1 type macrophages. In our previous studies, we identified Musashi-1 (MSI1) RNA-binding protein as a principal oncogenic factor in GBM. In this study, in a pursuit of finding secreted factors that may alter tumor microenvironment in GBM, we identified MIF1 cytokine to be positively regulated by MSI1. Moreover, we found that MSI1-mediated MIF1 secretion promotes differentiation of macrophages into pro-oncogenic M2 phenotype. The oncogenic role of MSI1/MIF1/M2 macrophage regulatory axis was also confirmed in GBM mouse models, which makes it a promising target for novel drug discovery.Glioblastoma (GBM) is the most malignant brain tumor which is characterized by high proliferation and migration capacity. The poor survival rate has been attributed to limitations of the current standard therapies. The search for novel biological targets that can effectively hamper tumor progression remains extremely challenging. Previous studies indicated that tumor-associated macrophages (TAMs) are the abundant elements in the tumor microenvironment that are closely implicated in glioma progression and tumor pathogenesis. M2 type TAMs are immunosuppressive and promote GBM proliferation. RNA-binding protein Musashi-1 (MSI1) has recently been identified as a marker of neural stem/progenitor cells, and its high expression has been shown to correlate with the growth of GBM. Nevertheless, the relationship between MSI1 and TAMs in GBM is still unknown. Thus, in our present study, we aimed to investigate the molecular interplay between MSI1 and TAMs in contributing to GBM tumorigenesis. Our data revealed that the secretion of macrophage inhibitory factor 1 (MIF1) is significantly upregulated by MSI1 overexpression in vitro. Importantly, M2 surface markers of THP-1-derived macrophages were induced by recombinant MIF1 and reduced by using MIF1 inhibitor (S,R)-3-(4-hHydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (ISO-1). Furthermore, GBM tumor model data suggested that the tumor growth, MIF1 expression and M2 macrophage population were significantly downregulated when MSI1 expression was silenced in vivo. Collectively, our findings identified a novel role of MSI1 in the secretion of MIF1 and the consequent polarization of macrophages into the M2 phenotype in promoting GBM tumor progression.