Overexpression Of MSI2 Research Articles

Musashi-2 induces cardiac hypertrophy and heart failure by inducing mitochondrial dysfunction through destabilizing cluh and smyd1 mRNA

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This work is funded by Ramalingaswami Re-entry Fellowship (BT/RLF/re-entry/14/2019) from the Department of Biotechnology, Government of India Background Regulation of RNA stability and translation by RNA-binding proteins (RBPs) is a crucial process altering gene expression. Musashi family of RBPs comprising Msi1 and Msi2 are known to control RNA stability and translation with a well-characterized role in the hematopoietic system, gut and cancer. However, the functions of Musashi in the heart is not explored. Purpose Despite the expression of MSI2 in the heart, its role remains largely unknown. In our study, we endeavor to understand the cardiac function of MSI2. Methods RNA-seq analysis, adeno-associated virus (AAV), lentivirus, echocardiography, histopathology, proteomics, electron microscopy, luciferase assay, ROS measurement, TMRE red staining, seahorse assay, MSI2 pooldown, and H3K4me3 ChIP assay were used to elucidate the function of Msi2 in cardiomyocytes. Results Among the two Musashi members, the heart expresses only Msi2. We confirmed the presence of MSI2 in the adult mouse, rat heart, and neonatal rat cardiomyocytes. Furthermore, Msi2 is significantly enriched in the heart cardiomyocytee fraction. Next, using RNA-seq data and isoform-specific PCR primers, we identified Msi2 isoforms 1, 4 and 5, and two novel putative isoforms labeled as Msi2 6 and 7 to be expressed in the heart. Overexpression of Msi2 isoforms led to cardiac hypertrophy in cultured cardiomyocytes. Additionally, Msi2 exhibited a significant increase in a pressure-overload model of cardiac hypertrophy. We selected isoforms 4 and 7 to validate the hypertrophic effects due to their unique alternative splicing patterns. AAV9-mediated overexpression of Msi2 isoforms 4 and 7 in murine hearts led to cardiac hypertrophy, dilation, heart failure,and eventually early death, confirming a pathological function for Msi2. Using global proteomics, gene ontology, transmission electron microscopy, seahorse, and transmembrane potential measurement assys, increased MSI2 was found to cause mitochondrial dysfunction in the heart. Mechanistically, we identified Cluh and Smyd1 as direct downstream target of Msi2. Overexpression of Cluh and Smyd1 inhbited Msi2-induced cardiac mitochondrial dysfunction. Collectively, we show that Msi2 inducs hypertrophy, mitochondrial dysfunction, and heart failure. Conclusion We here show that Msi2 has a pro-hypertrophic function in cardiomyocytes leading to heart failure and death in mice. AAV9 mediated overexpression of Msi2 promoted degradation of Cluh and Smyd1 and thus led to mitochondrial dysfunction. Overexpression of Cluh and Smyd1 inhibits the pro-hypertrophic and mitochondrial dysfunction induced by Msi2.

The RNA-binding protein Msi2 regulates autophagy during myogenic differentiation.

Skeletal muscle development is a highly ordered process orchestrated transcriptionally by the myogenic regulatory factors. However, the downstream molecular mechanisms of myogenic regulatory factor functions in myogenesis are not fully understood. Here, we identified the RNA-binding protein Musashi2 (Msi2) as a myogenin target gene and a post-transcriptional regulator of myoblast differentiation. Msi2 knockdown in murine myoblasts blocked differentiation without affecting the expression of MyoD or myogenin. Msi2 overexpression was also sufficient to promote myoblast differentiation and myocyte fusion. Msi2 loss attenuated autophagosome formation via down-regulation of the autophagic protein MAPL1LC3/ATG8 (LC3) at the early phase of myoblast differentiation. Moreover, forced activation of autophagy effectively suppressed the differentiation defects incurred by Msi2 loss. Consistent with its functions in myoblasts in vitro, mice deficient for Msi2 exhibited smaller limb skeletal muscles, poorer exercise performance, and muscle fiber-type switching in vivo. Collectively, our study demonstrates that Msi2 is a novel regulator of mammalian myogenesis and establishes a new functional link between muscular development and autophagy regulation.

Msi2 enhances muscle dysfunction in a myotonic dystrophy type 1 mouse model

BackgroundMyotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the 3′ untranslated region of the DM1 protein kinase gene. Characteristic degenerative muscle symptoms include myotonia, atrophy, and weakness. We previously proposed an Musashi homolog 2 (MSI2)>miR-7>autophagy axis whereby MSI2 overexpression repressed miR-7 biogenesis that subsequently de-repressed muscle catabolism through excessive autophagy. Because the DM1 HSALR mouse model expressing expanded CUG repeats shows weak muscle-wasting phenotypes, we hypothesized that MSI2 overexpression was sufficient to promote muscle dysfunction in vivo. MethodsBy means of recombinant AAV murine MSI2 was overexpressed in neonates HSALR mice skeletal muscle to induce DM1-like phenotypes. ResultsSustained overexpression of the murine MSI2 protein in HSALR neonates induced autophagic flux and expression of critical autophagy proteins, increased central nuclei and reduced myofibers area, and weakened muscle strength. Importantly, these changes were independent of MBNL1, MBNL2, and Celf1 protein levels, which remained unchanged upon Msi2 overexpression. ConclusionsGlobally, molecular, histological, and functional data from these experiments in the HSALR mouse model confirms the pathological role of MSI2 expression levels as an atrophy-associated component that impacts the characteristic muscle dysfunction symptoms in DM1 patients.

Glioma glycolipid metabolism: MSI2-SNORD12B-FIP1L1-ZBTB4 feedback loop as a potential treatment target.

Abnormal energy metabolism, including enhanced aerobic glycolysis and lipid synthesis, is a well‐established feature of glioblastoma (GBM) cells. Thus, targeting the cellular glycolipid metabolism can be a feasible therapeutic strategy for GBM. This study aimed to evaluate the roles of MSI2, SNORD12B, and ZBTB4 in regulating the glycolipid metabolism and proliferation of GBM cells. MSI2 and SNORD12B expression was significantly upregulated and ZBTB4 expression was significantly low in GBM tissues and cells. Knockdown of MSI2 or SNORD12B or overexpression of ZBTB4 inhibited GBM cell glycolipid metabolism and proliferation. MSI2 may improve SNORD12B expression by increasing its stability. Importantly, SNORD12B increased utilization of the ZBTB4 mRNA transcript distal polyadenylation signal in alternative polyadenylation processing by competitively combining with FIP1L1, which decreased ZBTB4 expression because of the increased proportion of the 3′ untranslated region long transcript. ZBTB4 transcriptionally suppressed the expression of HK2 and ACLY by binding directly to the promoter regions. Additionally, ZBTB4 bound the MSI promoter region to transcriptionally suppress MSI2 expression, thereby forming an MSI2/SNORD12B/FIP1L1/ZBTB4 feedback loop to regulate the glycolipid metabolism and proliferation of GBM cells. In conclusion, MSI2 increased the stability of SNORD12B, which regulated ZBTB4 alternative polyadenylation processing by competitively binding to FIP1L1. Thus, the MSI2/SNORD12B/FIP1L1/ZBTB4 positive feedback loop plays a crucial role in regulating the glycolipid metabolism of GBM cells and provides a potential drug target for glioma treatment.

Abstract 955: Musashi-2 regulates EGFR expression in NSCLC, cell proliferation and survival, response to EGFR inhibitors in EGFR-mutant NSCLC

Abstract Musashi-2 (MSI2) is an RNA-binding protein that regulates mRNA translation. We recently established that MSI2 is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression and drives NSCLC metastasis, in part based on activity supporting a TGF-beta/SMAD3/claudin signaling cascade. Here, reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+;P53R172HΔG/+ murine NSCLC cell lines identified a significant upregulation of HER3 (ERBB3) upon MSI2 depletion. Negative MSI2-dependent regulation of ERBB3 protein was confirmed in multiple murine and human NSCLC models, based on analysis of MSI2 depletion or overexpression. Further, MSI2 positively regulated expression of the epidermal growth factor receptor (EGFR) protein in the same models. Comparing EGFR and KRAS driven models, we found that MSI2 depletion significantly impairs cell proliferation and clonogenicity only in EGFR-mutant NSCLC cell lines. Using RNA immunoprecipitation analysis coupled with qPCR, we show that MSI2 directly binds to EGFR and, to a lesser extent, to HER3 mRNA. MSI2 mRNA binding was correlating with the presence of predicted MSI2 binding sites in corresponding mRNAs. Taken together, this data suggest direct regulation of EGFR protein expression by MSI2 at post transcriptional level. NSCLC lung tissue microarray immunohistochemistry analysis revealed that MSI2 total positivity by H-score, 2+/3+ and 3+ positivity correlated with EGFR 3+ staining. Finally, EGFR inhibitors erlotinib and afatinib synergized with MSI2 depletion in EGFR mutant models, suggesting that therapeutic targeting of MSI2 could be of clinical value, especially in EGFR-mutant lung cancer. In vivo experiments of erlotinib treatments of wild type or MSI2 depleted tumors are ongoing and these results will be presented. Citation Format: Alexander Kudinov, Petr Makhov, Bulat Faezov, Igor Bychkov, Alexander Deneka, Emmanuelle Nicolas, Kathy Q Q. Caid, Rohan Brebione, Eleanor Avrilf, Anna S. Nikonova, Ilya G. Serebriiskii, Hossein Borghaei, Erica A. Golemis, Yanis Boumber. Musashi-2 regulates EGFR expression in NSCLC, cell proliferation and survival, response to EGFR inhibitors in EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 955.

MSI2-TGF-β/TGF-β R1/SMAD3 positive feedback regulation in glioblastoma.

Glioblastoma is the most malignant glioma tumors with inevitable relapse and resistance to chemotherapy; however, the mechanisms driving chemoresistance remain to be fully elucidated. This study is to explore the molecular and cellular mechanisms involving in the chemoresistance of glioblastoma. The expression of musashi (MSI) RNA-binding protein in the tumor tissues and cells of glioblastoma was measured. The effects of MSI2 in epithelial-to-mesenchymal transition (EMT), resistance to temozolomide (TMZ), tumor cell invasion, migration, and proliferation and associated signaling were evaluated. High MSI2 expression was observed in the glioblastoma tissues. Silencing or overexpression of MSI2 significantly affected tumor cells invasion, migration, and proliferation. Silencing of MSI2 expression significantly inhibited O6-methylguanine-DNA methyltransferase (MGMT) expression and tumor growth, and reversed resistance to TMZ in xenograft tumor models. MSI2 expression regulated EMT through activating the transcription factors Snail and the TGFβ R1/SMAD3 signaling. Our study demonstrated a positive feedback loop of MSI2-TGFβ/SMAD3 signaling which activates the EMT and MGMT which may contribute to chemoresistance in glioblastoma. This study also highlights that MSI2 could be a new target for the therapy of glioblastoma.

Abstract 4452: Musashi-2 regulates EGFR/HER3 expression in NSCLC, cell proliferation and response to EGFR inhibitors in EGFR-mutant NSCLC

Abstract Musashi-2 (MSI2) is an RNA-binding protein that regulates mRNA translation. We recently established that MSI2 is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression and drives NSCLC metastasis, in part based on activity supporting a TGF-beta/SMAD3/claudin signaling cascade. Here, reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+;P53R172HΔG/+ murine NSCLC cell lines identified a significant ~2.7-fold upregulation of HER3 (ERBB3) upon MSI2 depletion. Negative MSI2-dependent regulation of ERBB3 protein was confirmed in multiple NSCLC models, based on analysis of MSI2 depletion or overexpression. Further, MSI2 positively regulated expression of the epidermal growth factor receptor (EGFR) protein in the same models. Comparing EGFR and KRAS driven models, we found that MSI2 depletion significantly impairs cell proliferation only in EGFR-mutant NSCLC cell lines. Using RNA immunoprecipitation analysis coupled with qPCR, we show that MSI2 directly binds to EGFR and, to a lesser extent, to HER3 mRNA. MSI2 mRNA binding was approximately correlating with the presence of predicted MSI2 binding sites in corresponding mRNAs. NSCLC lung tissue microarray analysis revealed that MSI2 total positivity by H-score, 2+/3+ and 3+ positivity correlated with EGFR 3+ staining. Finally, EGFR inhibitors erlotinib and afatinib synergized with MSI2 depletion in EGFR mutant models, suggesting that therapeutic targeting of MSI2 could be of clinical value, especially in EGFR-mutant lung cancer. Citation Format: Peter Makhov, Alexander Kudinov, Alexander Deneka, Brian L. Egleston, Emmanuelle Nicolas, Kathy Q. Cai, Rohan Brebion, Eleanor Avril, Mark Hitrik, Anna S. Nikonova, Ilya G. Serebriiskii, Vladimir Khazak, Hossein Borghaei, Erica A. Golemis, Yanis Boumber. Musashi-2 regulates EGFR/HER3 expression in NSCLC, cell proliferation and response to EGFR inhibitors in EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4452.

PLAG1 and USF2 Co-regulate Expression of Musashi-2 in Human Hematopoietic Stem and Progenitor Cells.

SummaryMSI2, which is expressed predominantly in hematopoietic stem and progenitor cells (HSPCs), enforces HSPC expansion when overexpressed and is upregulated in myeloid leukemias, indicating its regulated transcription is critical to balanced self-renewal and leukemia restraint. Despite this, little is understood of the factors that enforce appropriate physiological levels of MSI2 in the blood system. Here, we define a promoter region that reports on endogenous expression of MSI2 and identify USF2 and PLAG1 as transcription factors whose promoter binding drives reporter activity. We show that these factors co-regulate, and are required for, efficient transactivation of endogenous MSI2. Coincident overexpression of USF2 and PLAG1 in primitive cord blood cells enhanced MSI2 transcription and yielded cellular phenotypes, including expansion of CD34+ cells in vitro, consistent with that achieved by direct MSI2 overexpression. Global chromatin immunoprecipitation sequencing analyses confirm a preferential co-binding of PLAG1 and USF2 at the promoter of MSI2, as well as regulatory regions corresponding to genes with roles in HSPC homeostasis. PLAG1 and USF2 cooperation is thus an important contributor to stem cell-specific expression of MSI2 and HSPC-specific transcriptional circuitry.

Increased Musashi-2 and Decreased NUMB Protein Levels Observed in Human Colorectal Cancer are reverted to Normal Levels by ATRA-Induced Cell Differentiation.

Background:Musashi stem cell (SC) proteins (MSI-1 & MSI-2) are known to become over expressed during colorectal tumorigenesis in humans and mice. MSI-1 overexpression induces tumorigenesis through Notch activation via inactivation of NUMB. Previous studies also show that MSI-2 overexpression in mice induces intestinal tumorigenesis but the mechanism is independent of NUMB. However, whether the MSI-2/NUMB pathway contributes to colorectal cancer (CRC) development in humans is still undetermined.Methods:We evaluated expression of MSI-2 and NUMB proteins in matched normal and CRC patient samples, as well as in human CRC cell lines. We also determined whether induction of cellular differentiation by all-trans retinoic acid (ATRA) influences MSI-2 and NUMB expression.Results:Analysis of matched patient tissue samples and CRC cell lines showed that MSI-2 protein expression is significantly increased and NUMB expression is decreased in CRCs compared to the normal colonic tissue. Immunostaining of normal and adenomatous colonic epithelium revealed that MSI-1+ andMSI-2+ SCs reside in the SC niche and they become overpopulated during colon tumorigenesis. Moreover, promoting cellular differentiation by ATRA reduces MSI-2 protein levels, while increasing NUMB protein levels in human CRC cell lines.Conclusions:MSI-2/NUMB protein expression is altered during colon tumorigenesis, and indicates that MSI-2/NUMB signaling in human colonic stem cells is closely linked to normal colonic epithelial homeostasis.Implications:The ability to normalize MSI-2/NUMB signaling by inducing differentiation of cancer SCs suggests a novel therapeutic approach for CRC treatment.

Msi2 plays a carcinogenic role in esophageal squamous cell carcinoma via regulation of the Wnt/β-catenin and Hedgehog signaling pathways

Msi2 has been widely reported to be upregulated and strongly associated with fast progress and poor prognosis in many cancers. However, the expression and role of Msi2 in esophageal squamous cell carcinoma (ESCC) remain unknown. In this study, we found that Msi2 was upregulated in ESCC clinical samples, and was significantly associated with tumor size, differentiation status, and lymph node metastasis in ESCC patients. Multivariate Cox regression analysis showed that Msi2 was an independent predictor for disease-free survival (DFS) and overall survival (OS). Moreover, knockdown of Msi2 impaired ESCC cell proliferation, epithelial-mesenchymal transition (EMT) and migration, while overexpression of Msi2 promoted ESCC cell proliferation, EMT and migration in vitro. Animal experiments also confirmed that Msi2 promoted ESCC cell proliferation in vivo. Mechanistically, Msi2 promoted ESCC cell proliferation, EMT and migration via regulation of the Wnt/β-catenin and Hedgehog (Hh) signaling pathways. Taken together, our study suggested that Msi2 could serve as a candidate for diagnosis and prognosis and as a potential therapeutic target in ESCC.

Abstract 2542: MSI2 binds LncRNAs and promotes self-renewal and oncogenesis through MYC expression

Abstract Backgrounds and Aims: RNA-binding protein MSI2 is elevated in several cancers and is linked to poor prognosis. Here, we sought to elucidate the role of MSI2 in regulating the expression of proto-oncogenes or tumor suppressor genes in hepatocellular carcinoma (HCC). Methods: We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs) and subsequent validation by qPCR analysis. Results: Among the MSI2-bound RNAs, MYC mRNA and long noncoding RNA (LncRNA) miR22 host gene (MIR22HG) and MARAT1 were identified by RIP-qPCR analysis. MSI2-MARAT1 binding dereppresses polycomb reppressor to recruit E2F1 in MYC promoter regions. It is known that the 5’-untranslated region of MYC contains an internal ribosome entry sequence (IRES) which allows IRES-dependent translation initiation along with canonical cap-dependent mechanisms. Our data show that MSI2 acts as an IRES trans-acting factors for MYC mRNA translation. MSI2 does not have any effect on the steady state level of MYC mRNA but significantly increases MYC protein levels, indicating that MSI2 regulates MYC at the post-transcriptional level. Further, we observed that MSI2 reduced the level of mature miR22 derived from MIR22HG processing. We showed that miR22 suppresses the expression of MYC thorough a 3’-UTR dependent process. Overexpression of MSI2 promotes TIC self-renewal and tumor initiation property in xenograft tumor mouse models, whereas silencing MSI2 reduces this occurrence. Conclusion: we demonstrate that MSI2 promotes liver tumorigenesis by maintenance of MYC expression through dereppression of polycomb repressor and an IRES-dependent translation mechanism and possibly by inhibition of miR22 processing. Thus, MSI2 may be a useful prognostic factor for HCC and MSI2 targeted therapy and thus may be beneficial in the treatment of HCC patients. Citation Format: Hifzur Siddique, Padmani Narayan, Vasu Punj, Douglas E. Feldman, Keigo Machida. MSI2 binds LncRNAs and promotes self-renewal and oncogenesis through MYC expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2542. doi:10.1158/1538-7445.AM2017-2542

Cooperation of Musashi-2, Numb, MDM2, and P53 in drug resistance and malignant biology of pancreatic cancer.

Our earlier work showed that Musashi (MSI)-2 promoted the development of pancreatic cancer (PC) by down-regulating Numb, which prevented murine double-minute (MDM)-2-mediated p53 ubiquitin degradation. Thus, we investigate the relationship among MSI2, Numb, MDM2, and p53 in PC in vitro and invivo, an association that has not been reported to our knowledge. MSI2 had no relationship with mutant p53 (mtp53) and wild-type p53 (wtp53) in normal PC cells. However, in response to gemcitabine or cisplatin treatment, MSI2 silencing simultaneously down-regulated MDM2 and up-regulated Numb and wtp53 protein levels. Moreover, these 4 endogenous proteins can be coimmunoprecipitated as a quaternary complex. Numb small interfering RNA (siRNA) reversed the MSI2 silencing-induced p53 increase. During treatment with chemical agents, MSI2 silencing decreased drug resistance and cell motility in vitro and inhibited tumor growth in vivo, all of which were significantly reversed by p53 siRNA. MSI2 was also negatively associated with Numb and positively associated with MDM2 expression in tissue. Overexpression of MSI2, MDM2, and mtp53 and weak expression of Numb were closely associated with aggressive clinicopathologic characteristics and poor prognosis for patients with PC. MSI2 negatively regulates wtp53 protein by up-regulating MDM2 and down-regulating Numb after treatment with chemical agents. MSI2 promotes drug resistance and malignant biology of PC in a p53-dependent manner.-Sheng, W., Dong, M., Chen, C., Wang, Z., Li, Y., Wang, K., Li, Y., Zhou, J. Cooperation of Musashi-2, Numb, MDM2, and P53 in drug resistance and malignant biology of pancreatic cancer.

Musashi-2 promotes migration and invasion in bladder cancer via activation of the JAK2/STAT3 pathway

Musashi-2 (Msi2) is considered to have a crucial role in regulating various key cellular functions. However, the clinical significance and biological role of Msi2 in bladder cancer remains unknown. We examined the expression of Msi2 in bladder cancer cell lines in 167 clinical samples and the biological role of Msi2 in bladder cancer cells. Western blotting was used to investigate the possible mechanism of Msi2-induced migration and invasion in bladder cancer. Msi2 was significantly upregulated in bladder cancer cells and tissues compared with normal bladder urothelial cells and tissues. Immunohistochemical analysis revealed high expression of Msi2 in 57 of 167 (34.1%) bladder cancer specimens. Statistical analysis showed a significant correlation of Msi2 expression with advanced clinical stage, lymph node metastasis, and poor prognosis. Overexpression and ablation of Msi2 promoted and inhibited, respectively, the migration and invasion of bladder cancer cells. Furthermore, we found that Msi2 activated the JAK2/STAT3 pathway and promoted expression of genes downstream of JAK2/STAT3 in bladder cancer. This study demonstrates that Msi2 can induce bladder cancer cell migration and invasion by activating the JAK2/STAT3 pathway, and that Msi2 may be a valuable prognostic biomarker for bladder cancer patients.

MSI2 is required for maintaining activated myelodysplastic syndrome stem cells.

Myelodysplastic syndromes (MDS) are driven by complex genetic and epigenetic alterations. The MSI2 RNA-binding protein has been demonstrated to have a role in acute myeloid leukaemia and stem cell function, but its role in MDS is unknown. Here, we demonstrate that elevated MSI2 expression correlates with poor survival in MDS. Conditional deletion of Msi2 in a mouse model of MDS results in a rapid loss of MDS haematopoietic stem and progenitor cells (HSPCs) and reverses the clinical features of MDS. Inversely, inducible overexpression of MSI2 drives myeloid disease progression. The MDS HSPCs remain dependent on MSI2 expression after disease initiation. Furthermore, MSI2 expression expands and maintains a more activated (G1) MDS HSPC. Gene expression profiling of HSPCs from the MSI2 MDS mice identifies a signature that correlates with poor survival in MDS patients. Overall, we identify a role for MSI2 in MDS representing a therapeutic target in this disease.

MSI2 Is Required for Maintaining the Activated Myelodysplastic Syndrome Stem Cell

+ The first four authors contributed equally to this project.Myelodysplastic syndromes (MDS) are a group of blood cell disorders, characterized by ineffective hematopoiesis and severe cytopenias, which often transform to acute leukemia. MDS is also considered to be a clonal stem cell disease driven by alterations that are both genetic and epigenetic. However, it remains unclear how stem cell function is dysregulated and what factors drive these alterations in MDS HSCs. MSI2 is an important RNA-binding protein in normal HSC maintenance and can promote aggressive myeloid leukemia. Our preliminary data indicate that MSI2 expression is increased in high-risk MDS compared to low-risk MDS and correlates with poor survival. In order to model the role of MSI2 in MDS, we utilized the NUP98-HOXD13 transgenic (NHD13) model, which recapitulates many salient features of MDS including, leukopenia, severe anemia, erythroid dysplasia and leukemic transformation. Despite the lethal MDS or AML disease found in primary NHD13 animals, bone marrow cells transplanted into congenic mice generate a non-lethal MDS that rarely transform. Depletion of Msi2 utilizing a conditional knockout (NHD13-Msi2f/f -MX1-Cre) reversed the MDS phenotype and after one month the diseased HSPCs were eliminated. Conversely, we found that tetracycline inducible MSI2 overexpression in the context of the NHD13 transgene (NHD13/MSI2 mice) resulted in a worse MDS disease and a fully penetrant and lethal transformation to an AML, which was further accelerated during serial transplantation. AML arising in NHD13/MSI2 mice remained dependent on sustained MSI2 overexpression as mice removed from doxycycline demonstrated improved survival. Most interestingly, MSI2 overexpression expanded and maintained a more activated (G1) MDS hematopoietic stem and progenitor compartment (HSPC) in NHD13 cells. Gene expression profiling of the LSKs (Lineagelo, c-Kit+, Sca1+) before disease progression identified 891 significant genes, of which 137 genes were up-regulated (log2 fold change > 0) and 754 genes were down-regulated (log2 fold change <= 0). Furthermore, Gene Set Enrichment Analysis (GSEA) demonstrated a more progenitor like gene expression signature, enrichment in an NRAS activated signature, and a reduced quiescent phenotype. Unsupervised hierarchical clustering of the NHD13/MSI2 LSK gene signature in MDS patients resulted in four distinct clusters. Clusters segregated MSI2 high expressing MDS patients and this "MSI2 high cluster" predicted poor survival. In summary, our findings suggest that MSI2 plays a critical functional role in the maintenance of the hematopoietic stem and progenitor compartment in MDS and highlights it as a novel therapeutic target in this disease. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4968: Msi2 directly linked to Hedgehog and Wnt signaling in non-triple negative breast cancer

Abstract Introduction: Previously, we have shown that a gain on 17q22.24.2 was a predictor of invasiveness when found in duct carcinoma in situ (DCIS) and of nodal metastasis when found in invasive duct carcinoma (IDC). Within this amplicon, we have identified a gene that is a likely driver of breast tumorigenesis and progression: Musashi homolog 2 (Msi2). Msi2 has a functional role in neural stem cell maintenance and regulation of hematopoietic stem cells. Previously, Msi2 has been implicated in Notch and Hedgehog signaling in leukemia, and here we link Msi2 to an increase in Wnt activation. Methodology: To understand the expression of Msi2 in patient tissue, tissue microarrays (TMAs) containing 232 breast cancers were analyzed by immunohistochemistry. MDA-MB-231 and MCF7 cells expressing Msi2-GFP and GFP clones were plated on transwells to examine cell migration and invasiveness. Proliferation was analyzed by the MTS assay. TOP-FOP and Gli1 Luciferase assays were used to confirm activation of Hedgehog and Wnt signaling. Western blots were used to assess total beta-catenin levels in the cell lines. Analysis of publicly available expression microarray databases and RT-PCR of the over-expression cell lines validated the proposed mechanism of action. Results: Tissue microarrays confirmed that Allred scores for Msi2 significantly correlated with grade, size stage, and non-triple negative receptor status. Over-expression of Msi2, in MDA-MB-231 (triple negative) and MCF7 (luminal cell type with epithelial morphology) cell lines, caused an increase in proliferation, migration and invasion. The knockdowns demonstrated the reverse effect. As determined by the TOP-FOP luciferase assay and RT-PCR, Msi2 leads to increased signaling of Hedgehog and Wnt. Furthermore, Msi2-mediated activation of Wnt occurs independently of total beta-catenin levels. Microarray analysis of breast cancer cell lines and patients' tumors supports this mechanism of action for Msi2 in non-triple negative cell lines (as assessed by Pearson's correlation coefficient of pathway interactions). Conclusion: When overexpressed in a cancerous cell system in vitro, Msi2 levels correlate with increased migratory and invasive capabilities. Furthermore, its implication in Notch, Hedgehog, and Wnt Signaling, make Msi2 a likely driver of the 17q22-24.2 amplicon in breast cancer. This study furthers our understanding of Msi2, and hints at the possibility that Msi2 plays a more significant role in cellular events and tumorigenesis than previously thought. Citation Format: Moustafa Abdalla, Nisha Kanwar, Yanglong Zhu, Ranju Nair, Bruce J. Youngson, Naomi A. Miller, Tianyu Liu, Susan J. Done. Msi2 directly linked to Hedgehog and Wnt signaling in non-triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4968. doi:10.1158/1538-7445.AM2014-4968

Developmental Expression of Musashi-1 and Musashi-2 RNA-Binding Proteins During Spermatogenesis: Analysis of the Deleterious Effects of Dysregulated Expression1

Spermatogenesis is a complex developmental process whereby diploid spermatogenic stem cells become haploid and undergo a series of morphological changes to produce physically mature spermatozoa. Crucial to this process are a number of RNA-binding proteins, responsible for the posttranscriptional control of essential mRNAs and particularly pertinent to the two periods of inactive transcription that occur in spermatogenesis. One such group of RNA-binding proteins is the Musashi family, specifically Musashi-1 (MSI1) and Musashi-2 (MSI2), which act as key translational regulators in various stem cell populations and have been linked with the induction of tumorigenesis. In the present study, we examined the differential expression of mammalian MSI1 and MSI2 during germ cell development in the mouse testis. MSI1 was found to be predominately localized in mitotic gonocytes and spermatogonia, whereas MSI2 was detected in meiotic spermatocytes and differentiating spermatids. Extensive examination of the function of Musashi in spermatogenesis was achieved through the use of two transgenic mouse models with germ cell-specific overexpression of full-length isoforms of Msi1 or Msi2. These models demonstrated that aberrant expression of either Msi1 or Msi2 has deleterious effects on normal spermatogenesis, with Msi2 overexpression resulting in male sterility. Studies undertaken on human testicular seminoma tumors provide further insights into the relevance of MSI1 and MSI2 overexpression as diagnostic markers to human stem cell cancers. Overall this study provides further evidence for the unique functions that RNA-binding protein isoforms occupy within spermatogenesis, and introduces the potential manipulation of the Musashi family proteins to elucidate the mechanisms of posttranscriptional gene expression during germ cell development.

The Role of MSI2 Expression Levels on Outcome of MDS and AML Patients

Abstract 1734Musashi (MSI) represents an evolutionarily conserved family of RNA-binding proteins. MSI2 is the predominant form in hematopoetic stem cells (HSC). Recently, overexpression of MSI2 in a mouse model was found to increase cell cycle progression and to cooperate with BCR-ABL1 to induce aggressive leukemia. The aim of this study was to analyze the prognostic importance of MSI2 expression in myelodysplastic syndrome (MDS) and in acute myeloid leukemia (AML). Diagnostic bone marrow or peripheral blood samples were analyzed from 454 adult patients (aged 17–60 years) with de novo (n=406) or secondary AML (n=48) with French-American-British (FAB] classification M0-M2, or M4-M7, who were entered into the multicenter treatment trials AML SHG 0199 or AML SHG 0295. Additionally, we analyzed a cohort of 148 patients (aged 36–92 years) with MDS with low to high IPSS scores. Real-time reverse-transcriptase-polymerase chain reaction (RT-PCR) was performed using patient-derived cDNA and ABL as an endogenous control. MSI-2 expression levels were quantified using the TaqMan Gene Expression Assay (Applied Biosystems, Assay ID MSI2: Hs01592567_m1). To define the MSI2 expression status patient cohorts were divided into four quartiles (Q) according to levels of MSI2/ABL expression and subsequently dichotomized into two groups including the three lower Qs (Q1, Q2, and Q3) and the upper Q (Q4) of MSI2/ABL values. Patients of the quartile with highest MSI2 expression levels were classified as high MSI2 expressing patients.In MDS, MSI2 high versus low expression was independent of transfusions (P=.55), cytogenetic risk (P=.6), and ASXL1 mutations (P=.99), but was associated with a higher blast percentage (p=.034). In AML, higher MSI2 expression correlated with increased peripheral blood blasts (P=.036), and inversely correlated with the favourable cytogenetic risk group defined by inv(16) and t(8;21) according to Medical Research Council criteria (P<.001). Additionally, patients with higher MSI2 expression were likely to be FLT3-ITD positive (P<.001), NPM1 (P<.001), DNMT3A (P=.003), NRAS (P=.018) and CEBPA double mutated (P=.043). No association was found with other molecular markers including IDH1, IDH2 and WT1 mutations.Next, we evaluated the effect of MSI2 expression levels on patient outcome in MDS and AML. In MDS, high MSI2 expression predicted an increased likelihood of and more rapid progression to AML (HR 2.95, 95%CI 1.55–5.64, P=.001), but MSI2 expression levels did not affect overall survival (OS, HR 1.34, 95%CI 0.81–2.23, P=.25). In multivariate analysis when adjusting for karyotype, transfusion dependence, percentage of bone marrow blasts, ASXL1 frameshift mutation status and IDH1 mutation status, high MSI2 expression remained an independent prognostic factor for AML transformation in MDS (HR 2.33, 95%CI 1.16–4.67, P=.018).In AML, OS was shorter in AML patients with higher MSI2 expression as compared to low expression (HR 1.48, 95%CI 1.13–1.95, P=.005). However, relapse free survival (RFS) and complete remission (CR) rates were not influenced by high versus low MSI2 expression (RFS, HR 1.27, 95%CI.92–1.76, P=.15; CR, P=.39). In multivariate analysis, when considering MSI2 high versus low expression, DNMT3A mutations, age, platelet count, secondary versus de novo AML, cytogenetic risk group, NPM1/FLT3-ITD high vs. low risk group, and MLL5 expression levels, MSI2 expression did not remain an independent prognostic marker for OS (HR 1.31, 95%CI.98–1.76, P=.07). In patients with cytogenetically normal AML (CN-AML), MSI2 also presented a negative prognostic marker for OS (HR 1.59, 95%CI 1.08–2.33, P=.019) but did not influence RFS (HR 1.10, 95%CI 0.69–1.76, P=.68) and CR (P=.39). In multivariate analyses for CN-AML, when considering age, platelet count, DNMT3A mutations, NPM1FLT3-ITD high vs. low risk group, CEBPA mutation status and WT1 SNPrs16754, MSI2 did not independently predict OS (HR 144, 95%CI 0.94–2.19, P=.096). In summary, our data suggest that MSI2 expression might contribute to the transformation from MDS to AML. Whether MSI2 expression is dysregulated through other associated prognostic markers, or whether it is an independent event in AML pathogenesis remains to be determined. However, it may represent a valuable therapeutic target both in MDS and AML patients. Disclosures:Ottmann:Novartis Corporation: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding. Hofmann:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Musashi expression in β-cells coordinates insulin expression, apoptosis and proliferation in response to endoplasmic reticulum stress in diabetes

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic β-cells. In other systems, Musashi genes regulate cell fate via Notch signaling, which we recently showed regulates β-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms, as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of β-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1, while it decreased Ins1 and Ins2 expression, revealing a molecular link between ER stress and β-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1, stimulate proliferation, inhibit apoptosis and reduce insulin expression, whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together, these results demonstrate overlapping, but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and β-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory β-cell proliferation and the loss of β-cell gene expression seen in specific phases of the progression to type 2 diabetes.

Roles for MSI2 and PROX1 in hematopoietic stem cell activity

The MSI2 and PROX1 proteins are increasingly recognized for their critical roles in the biology of primitive hematopoietic cells and for their potential contributions to leukemic pathogenesis. Here we summarize the studies that have shed light on the hematopoietic-specific roles of MSI2 and PROX1 and give an overview of the molecular mechanisms underlying their function. In addition to a likely role in cell cycle restraint, the hematopoietic stem cell agonist MSI2 is essential for the maintenance of primitive cell fate through ensuring appropriate balance between self-renewal and differentiation. Overexpression of Msi2 can contribute to the progression of murine myeloid leukemia and in the human setting is associated with poor prognosis. Regulatory control imposed by MSI2 may be achieved partly through regulation of the Notch signaling pathway. Prox1 behaves in an opposing manner to Msi2, resulting in elevated stem cell numbers when depleted. It has a potential role in cell cycle control and may act at the level of primitive hematopoietic stem and progenitor cells as it does in other systems by directly promoting commitment and differentiation. PROX1 functions as a tumor suppressor in numerous tissue types and has been found mutated in hematopoietic cell lines and primary blood malignancies. Deciphering the molecular mechanisms through which MSI2 and PROX1 affect primitive hematopoietic cell fate will provide insight into the regulation of normal hematopoiesis and facilitate better understanding of the leukemic transformation process. This will be directly applicable to the development of effective regenerative therapies and targeted leukemia treatments.