Msi1 mRNA Research Articles

502 Spy1 as a Novel Cell Cycle Target Against Medulloblastoma

INTRODUCTION: The current treatment strategies for medulloblastoma (MB) are highly aggressive, cytotoxic, and ultimately unsuccessful. One recent therapeutic approach is the use of synthetic cyclin-dependent kinase inhibitors (CKIs). Speedy (Spy1) is a cyclin-like protein that primes its complexes with CDKs insensitive to natural CKIs and promotes uncontrolled cell proliferation by overriding the safety cell cycle checkpoints. Spy1 has been implicated in the expansion of stem-like populations of therapy-resistant tumour initiating cells (TICs) in neural systems. METHODS: We established an in vitro neurosphere platform as well as in vivo high-throughput Zebrafish (ZF) xenograft model using ONS-76 and DAOY MB cell lines derived from three patients. Fluorescent Activated Cell Sorting (FACS) was used to enrich for specific CD133+/- or CD15 +/- populations of MB. Spy1 levels were manipulated using lentivirus to obtain overexpression or knockdown of Spy1 protein levels in the derived TICs as well as heterogenous cell populations. RESULTS: We found that downregulation of Spy1 levels in the employed cell lines resulted in a decrease in neurospheres formation efficiency as well as significantly lower tumour burden in Zebrafish when compared to control (p<0.001). Spy1 overexpression in CD133 or CD15 negative MB populations resulted in the upregulation of Msi1 mRNA levels. Finally, in vivo treatment of ZF xenografts with R-roscovitine, a synthetic CKI, demonstrated declined migration of Spy1 KD MB cells in comparison to control cells (p<0.05). CONCLUSION: This study shows that Spy1 plays a role in the regulation of stemness and aggressive characteristics of TICs in medulloblastoma. The assessed effect on CKI treatment efficacy on the tumours in vivo demonstrated that downregulation of Spy1 may sensitize, at least a subset of MB, to therapy. This work may contribute toward optimizing the design of CKIs and their use in combination therapy for patients with MB.

Assessment of MUSASHI 1 and MUSASHI 2 expression in spermatozoa and testicular tissue.

MUSASHI (MSI) family plays the main role in the spermatogenesis process. The purpose of this study was the assessment of sperm MSI1 and MSI2, and sperm functional tests in infertile men (n=30) with varicocele and fertile men (n=30). Furthermore, MSI1 and MSI2 proteins were assessed in testicular tissue of azoospermic men (n=9) as well as epididymal spermatozoa and testis of mice. Expression of MSI1 and MSI2 was assessed at RNA and protein levels in human spermatozoa. Sperm concentration and motility were significantly lower, while abnormal sperm morphology, lipid peroxidation, DNA fragmentation and protamine deficiency were significantly higher in men with varicocele compared to fertile individuals. Any significant difference was not observed in the expression of MSI1 and MSI2 mRNA between the two groups. Unlike MSI1 protein that was not detectable in humans, the relative expression of MSI2 protein was similar in varicocele and fertile individuals. The expression level of both Msi1 and Msi2 proteins was also observable in mouse spermatozoa. No significant relationship was observed between sperm functional parameters with expression of these genes. The data of this study demonstrated that although MSI1 and MSI2 play important roles during spermatogenesis, their relative expression in spermatozoa was not affected by varicocele.

The pyriproxyfen metabolite, 4′–OH–PPF, disrupts thyroid hormone signaling in neural stem cells, modifying neurodevelopmental genes affected by ZIKA virus infection.

North-Eastern Brazil saw intensive application of the insecticide pyriproxyfen (PPF) during the microcephaly outbreak caused by the Zika virus (ZIKV). ZIKV requires the neural RNA-binding protein Musashi-1 to replicate. Thyroid hormone (TH) represses MSI1. PPF is a suspected TH disruptor. We hypothesized that co-exposure to the main metabolite of PPF, 4′–OH–PPF, could exacerbate ZIKV effects through increased MSI1 expression. Exposing an in vivo reporter model, Xenopus laevis, to 4′–OH–PPF decreased TH signaling and increased msi1 mRNA and protein, confirming TH-antagonistic properties. Next, we investigated the metabolite's effects on mouse subventricular zone-derived neural stem cells (NSCs). Exposure to 4′–OH–PPF dose-dependently reduced neuroprogenitor proliferation and dysregulated genes implicated in neurogliogenesis. The highest dose induced Msi1 mRNA and protein, increasing cell apoptosis and the ratio of neurons to glial cells. Given these effects of the metabolite alone, we considered if combined infection with ZIKV worsened neurogenic events. Only at the fourth and last day of incubation did co-exposure of 4′–OH–PPF and ZIKV decrease viral replication, but viral RNA copies stayed within the same order of magnitude. Intracellular RNA content of NSCs was decreased in the combined presence of 4′–OH–PPF and ZIKV, suggesting a synergistic block of transcriptional machinery. Seven out of 12 tested key genes in TH signaling and neuroglial commitment were dysregulated by co-exposure, of which four were unaltered when exposed to 4′–OH–PPF alone. We conclude that 4′–OH–PPF is an active TH-antagonist, altering NSC processes known to underlie correct cortical development. A combination of the TH-disrupting metabolite and ZIKV could aggravate the microcephaly phenotype.

Downregulation of SOX2-OT Prevents Hepatocellular Carcinoma Progression Through miR-143-3p/MSI2.

ObjectiveLncRNA SOX2-OT is involved in a variety of cancers. This study explored the effect of lncRNA SOX2-OT on hepatocellular carcinoma (HCC) cells.MethodsSOX2-OT expressions were detected in HCC tissues and normal tissues, normal cells, and HCC cells. The relationship between SOX2-OT and prognosis was analyzed by TCGA. After SOX2-OT expression was inhibited using siRNA, HCC cell malignant behaviors were evaluated. The subcellular localization of SOX2-OT in HCC cells was predicted and analyzed. The binding relationships among SOX2-OT, miR-143-3p, and MSI2 were analyzed by bioinformatics website, dual-luciferase assay, and RNA pull-down assay. The effect of miR-143-3p and MSI2 on the regulation of SOX2-OT on biological behaviors of HCC cells was confirmed by functional rescue experiments. The effect of SOX2-OT on the tumorigenicity of HCC was evaluated by subcutaneous tumorigenesis in nude mice.ResultsSOX2-OT was highly expressed in HCC cells and tissues. The prognosis was poor in HCC patients with high SOX2-OT expression. Downregulating SOX2-OT inhibited HCC cell malignant behaviors. SOX2-OT bound to miR-143-3p to promote MSI2 expression. Downregulating miR-143-3p or upregulating MSI2 averted the role of si-SOX2-OT in HCC cells. Nude mouse subcutaneous tumorigenesis showed that SOX2-OT downregulation decreased the tumorigenicity of HCC, and affected the levels of miR-143-3p and MSI2 mRNA in tumor tissues.ConclusionSOX2-OT inhibited the targeted inhibition of miR-143-3p on MSI2 through competitively binding to miR-143-3p, thus promoting MSI2 expression and proliferation, invasion, and migration of HCC cells.

MiR-598 Represses Cell Migration and Invasion of Non-Small-Cell Lung Cancer by Inhibiting MSI2

Non-small-cell lung cancer (NSCLC) is one of the most frequent solid tumors and regarded as a significant threat to individual health around the world. MicroRNAs (miRs) are recognized as critical governors of gene expression during carcinogenesis, while their clinical significance and mechanism in NSCLC occurrence and development are required for further investigation. In this report, we characterized the functional role of miR-598 and its regulation mechanism in NSCLC. The expression level of miR-598 in NSCLC tissues and cell lines was detected by qRT-PCR. A549 cells were transiently transfected with miR-598 mimics or miR-598 inhibitors. Scratch assay and Transwell assay were used to detect cell transfection, migration, and invasion. Possible binding sites of miR-598 in MSI2 mRNA were predicted by bioinformatics and validated by dual-luciferase reporter gene system. The ability of migration and invasion was examined on cells transfected with MSI2 alone or cotransfected A549 cells with miR-598. The expression of miR-598 in NSCLC tissues was significantly lower than that in adjacent tissues, and the expression of miR-598 in NSCLC cell lines (A549, H1650, and H1299) was also significantly lower than that of normal lung epithelial cell line BEAS-2B. A549 cells were significantly inhibited in migration and invasion after transfection with miR-598 mimics, while miR-598 inhibitors were significantly enhanced in migration and invasion. MSI2 was a direct target gene of miR-598. MSI2 can promote the migration and invasion of A549 cells, but the ability to promote cell migration and invasion was reversed when miR-598 was introduced. In conclusion, miR-598 inhibits the migration and invasion of NSCLC by downregulating the target gene MSI2.

Control of the Anterior Pituitary Cell Lineage Regulator POU1F1 by the Stem Cell Determinant Musashi.

The adipokine leptin regulates energy homeostasis through ubiquitously expressed leptin receptors. Leptin has a number of major signaling targets in the brain, including cells of the anterior pituitary (AP). We have previously reported that mice lacking leptin receptors in AP somatotropes display growth hormone (GH) deficiency, metabolic dysfunction, and adult-onset obesity. Among other targets, leptin signaling promotes increased levels of the pituitary transcription factor POU1F1, which in turn regulates the specification of somatotrope, lactotrope, and thyrotrope cell lineages within the AP. Leptin's mechanism of action on somatotropes is sex dependent, with females demonstrating posttranscriptional control of Pou1f1 messenger RNA (mRNA) translation. Here, we report that the stem cell marker and mRNA translational control protein, Musashi1, exerts repression of the Pou1f1 mRNA. In female somatotropes, Msi1 mRNA and protein levels are increased in the mouse model that lacks leptin signaling (Gh-CRE Lepr-null), coincident with lack of POU1f1 protein, despite normal levels of Pou1f1 mRNA. Single-cell RNA sequencing of pituitary cells from control female animals indicates that both Msi1 and Pou1f1 mRNAs are expressed in Gh-expressing somatotropes, and immunocytochemistry confirms that Musashi1 protein is present in the somatotrope cell population. We demonstrate that Musashi interacts directly with the Pou1f1 mRNA 3' untranslated region and exerts translational repression of a Pou1f1 mRNA translation reporter in a leptin-sensitive manner. Musashi immunoprecipitation from whole pituitary reveals coassociated Pou1f1 mRNA. These findings suggest a mechanism in which leptin stimulation is required to reverse Musashi-mediated Pou1f1 mRNA translational control to coordinate AP somatotrope function with metabolic status.

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.

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.

Musashi-2 is a prognostic marker for the survival of patients with cervical cancer.

Cervical cancer is one of the most common gynecological malignancies. Mousasi 2 (Msi2) is a RNA-binding protein that regulates various key cellular functions and has emerged as a crucial regulator of cancer development. However, the clinical significance and biological functions of Msi2 in cervical cancer remain unknown. The current study assessed the expression of Msi2 mRNA using reverse transcription-quantitative polymerase chain reaction. Furthermore, the expression of Msi2 was examined in 162 cervical cancer samples using immunohistochemistry and the association between Msi2 expression and patient clinicopathological features was analyzed. The overall survival (OS) and progression-free survival (PFS) of patients were estimated using the Kaplan-Meier method and Cox regression analysis was performed to investigate the clinicopathological significance of Msi2 expression. In vitro migration and invasion assays were performed in Sinha and Caskie cells. The results demonstrated that, compared with normal cervical tissues, the expression of Msi2 was increased in cervical cancer tissues. The expression of Msi2 was significantly correlated with International Federation of Gynaecology and Obstetrics (FIGO) stage (P=0.049) and lymph node metastasis (P=0.036). Furthermore, patients with higher Msi2 expression exhibited significantly poorer OS (P=0.013) and PFS (P=0.006) than patients with low Msi2 expression. Notably, high Msi2 expression was correlated with poorer OS in patients with a FIGO stage ≤I (P=0.015), a smaller tumor size (P=0.043) and grade 3 tumor (P=0.002). High Msi2 expression was also correlated with a poorer PFS in patients with a FIGO stage ≤I (P=0.016) and grade 3 tumor (P=0.001). Multivariate analysis suggested that Msi2 expression was an independent prognostic marker of the OS (P=0.027) and PFS (P=0.013) of patients with cervical cancer. Furthermore, Msi2 knockdown significantly (P<0.05) inhibited the invasion and migration of cervical cancer cells. The results of the current study demonstrate that Msi2 may act as a prognostic biomarker in patients with cervical cancer. Targeting Msi2 may therefore offer a promising therapeutic strategy for the treatment of patients with cervical cancer.

Association of Gnrhr mRNA With the Stem Cell Determinant Musashi: A Mechanism for Leptin-Mediated Modulation of GnRHR Expression.

The cyclic expression of pituitary gonadotropin-releasing hormone receptors (GnRHRs) may be an important checkpoint for leptin regulatory signals. Gonadotrope Lepr-null mice have reduced GnRHR levels, suggesting these receptors may be leptin targets. To determine if leptin stimulated GnRHR directly, primary pituitary cultures or pieces were exposed to 1 to 100 nM leptin. Leptin increased GnRHR protein levels and the percentages of gonadotropes that bound biotinylated analogs of gonadotropin-releasing hormone (bio-GnRH) but had no effect on Gnrhr messenger RNA (mRNA). An in silico analysis revealed three consensus Musashi (MSI) binding elements (MBEs) for this translational control protein in the 3' untranslated region (UTR) of Gnrhr mRNA. Several experiments determined that these Gnrhr mRNA MBE were active: (1) RNA electrophoretic mobility shift assay analyses showed that MSI1 specifically bound Gnrhr mRNA 3'-UTR; (2) RNA immunoprecipitation of pituitary fractions with MSI1 antibody pulled down a complex enriched in endogenous MSI protein and endogenous Gnrhr mRNA; and (3) fluorescence reporter assays showed that MSI1 repressed translation of the reporter coupled to the Gnrhr 3'-UTR. In vitro, leptin stimulation of pituitary pieces reduced Msi1 mRNA in female pituitaries, and leptin stimulation of pituitary cultures reduced MSI1 proteins selectively in gonadotropes identified by binding to bio-GnRH. These findings show that leptin's direct stimulatory actions on gonadotrope GnRHR correlate with a direct inhibition of expression of the posttranscriptional regulator MSI1. We also show MSI1 interaction with the 3'-UTR of Gnrhr mRNA. These findings now open the door to future studies of leptin-modulated posttranscriptional pathways.

Correlation Between Meis1 and Msi1 in Esophageal Squamous Cell Carcinoma.

Homeobox (HOX) transcription factors are critical regulators of cell fate, stem cell functions, and gastrointestinal development. They require three-amino acid loop extension (TALE) homeodomain proteins such as Meis1 to enhance their transcriptional efficiencies. There are complicated associations between different signaling pathways such as the Wnt and NOTCH and tumor progression. It has been investigated that GSK-3 as an important component of the Wnt pathway facilitates the expression of HOX target genes. Therefore, in the present study, we assessed the probable correlation between Wnt, NOTCH, and HOX genes in esophageal squamous cell carcinoma (ESCC) progression and metastasis through the correlational study between the Msi1 as an important activator for both of the NOTCH and Wnt pathways and Meis1. Levels of Meis1 and Msi1 messenger RNA (mRNA) expression in 51 ESCC patients were compared to the normal tissues using real-time polymerase chain reaction. Only 3 out of 51 (5.9 %) cases had Meis1/Msi1 overexpression and also 3/51 (5.9 %) cases had Meis1/Msi1 underexpression. There was a significant correlation between the Msi1 and Mesi1 mRNA expression (p = 0.037). All of the Msi1/Meis1 underexpressed tumors were poorly differentiated (p = 0.003). Meis1 under/Msi1 overexpressed cases also were in T3 tumor depth of invasion (p = 0.019). And there was a significant correlation between the Msi1/Meis1 underexpression and gender (p = 0.045). Our results show that Meis1 may have a positive feedback with Msi1 during the ESCC progression.

Role of Msi1 and PYGO2 in esophageal squamous cell carcinoma depth of invasion.

Deregulation of developmental signaling pathways such as Wnt/b-catenin and NOTCH are commonly observed in different cancers. A normal wnt pathway is essential for development and tissue homeostasis to preserve a normal balance between the differentiation and proliferation. PYGO2 is the main transcription factor of wnt pathway, while Msi1 is one of the wnt inhibitors. In this study we assessed the correlation between Msi1 and PYGO2 mRNA expression using Real time polymerase chain reaction in 48 esophageal squamous cell carcinoma (ESCC) patients. Although, there was not any significant correlation between the levels of Msi1 and PYGO2 mRNA expression, we observed a significant correlation between the Msi1 and PYGO2 overexpressed cases and depth of tumor invasion (p=0.05). In conclusion, despite the role of these markers in tumor depth of invasion there is not any feedback between Msi1 and PYGO2 gene expression in ESCC.

Role of Msi1 and MAML1 in Regulation of Notch Signaling Pathway in Patients with Esophageal Squamous Cell Carcinoma.

Developmental pathways such as Wnt and Notch are involved in different cellular functions from the cell cycle regulation to self-renewal. Therefore, aberrations in these pathways may cause tumorigenesis. Msi1 has a critical regulatory role for the Wnt and Notch pathways. In the present study, we have assessed the probable correlation between the Msi1 and MAML1 in esophageal squamous cell carcinoma (ESCC) progression and metastasis. Levels of Msi1 and MAML1 mRNA expression in 51 ESCC patients were compared to the normal tissues using real-time polymerase chain reaction (PCR). Nine out of 51 (17.6 %) cases had Msi1/MAML1 overexpression, and there was a significant correlation between such overexpressed cases and tumor location (p = 0.013). We showed that there is not any direct correlation and feedback between the Msi1 and MAML1 in ESCC patients.

Identification and validation of novel small molecule disruptors of HuR-mRNA interaction.

HuR, an RNA binding protein, binds to adenine- and uridine-rich elements (ARE) in the 3'-untranslated region (UTR) of target mRNAs, regulating their stability and translation. HuR is highly abundant in many types of cancer, and it promotes tumorigenesis by interacting with cancer-associated mRNAs, which encode proteins that are implicated in different tumor processes including cell proliferation, cell survival, angiogenesis, invasion, and metastasis. Drugs that disrupt the stabilizing effect of HuR upon mRNA targets could have dramatic effects on inhibiting cancer growth and persistence. In order to identify small molecules that directly disrupt the HuR-ARE interaction, we established a fluorescence polarization (FP) assay optimized for high throughput screening (HTS) using HuR protein and an ARE oligo from Musashi RNA-binding protein 1 (Msi1) mRNA, a HuR target. Following the performance of an HTS of ∼6000 compounds, we discovered a cluster of potential disruptors, which were then validated by AlphaLISA (Amplified Luminescent Proximity Homogeneous Assay), surface plasmon resonance (SPR), ribonucleoprotein immunoprecipitation (RNP IP) assay, and luciferase reporter functional studies. These compounds disrupted HuR-ARE interactions at the nanomolar level and blocked HuR function by competitive binding to HuR. These results support future studies toward chemical probes for a HuR function study and possibly a novel therapy for HuR-overexpressing cancers.

MSI2 and TGFβR1 Expression Levels in CML Patients and Hematopoietic Cell Lines

Musashi 2 (also known as MSI2), a mRNA binding protein is reported to control critical stem cell fate decisions by binding to the 3’untranslated region of target mRNAs, thereby inhibiting translation. MSI2 is preferentially expressed in hematopoietic tissue, in particular early myeloid progenitors. Moreover, investigators suggest upregulated MSI2 disrupts regulatory pathway/s leading to hematopoietic stem cell proliferation, impaired myeloid differentiation and worse clinical prognosis in CML and AML (Kharas et al. Nat Med. 2010; 16:903; Ito et al. Nature. 2010; 466:765). Indeed we have confirmed increased MSI2 levels in CML patients in blast crisis (BC) compared with those in chronic phase (CP), irrespective of lymphoid or myeloid transformation. Furthermore, we have shown MSI2 and BCR-ABL1 expressions correlate. Here we report data implying MSI2 functions viaTGFβ1 signalling pathway.We retrospectively studied 54 CML cDNA samples from 34 patients (M:15 ; F:19) in CP with median age 55.5 years (range 12-74). Apart from 3 patients treated with interferon+AraC, the remainder were prescribed Imatinib mesylate. Of the 54 samples 29 were collected at diagnosis (Dx) and 25 were obtained at 3 months post therapy (3M). For 20 of the patients, samples collected at Dx and 3M were available. Eight of the patients failed to achieve major molecular response (MMR) within 12 months post therapy. In addition to patient samples we included 19 normal cDNA controls from healthy blood donors (M:10 ; F:9), with 41 years median age (range 20-61). We also included 20 cDNA samples derived from hematopoietic cell lines (lymphoma: 7; AML: 3; CML:6 ; essential thrombocythemia: 1 ; hyperesoinophilic syndrome: 1 ; Acute lymphoblastic leukemia: 1). The cDNA was synthesized using reverse transcriptase and random hexamers. All the samples were subjected to quantitative real time PCR using TaqMan assay to quantify MSI2, TGFRβ1 and GUSβmRNA levels. Furthermore, we subjected protein isolated from the cells lines to Western blot analysis to assess TGFβR1 expression.MSI2 mRNA median levels in patient samples were significantly decreased compared with the NC group at Dx and at 3M, p=0.002 and p=0.013, respectively. But we found no significant difference in MSI2 mRNA levels at Dx nor at 3M, between those who achieved major molecular response and those who did not within 12 months of starting therapy, p=0.215 and p=1.871, respectively. Also we observed no significant difference between the patient samples and the NC group TGFβR1 transcript numbers at Dx nor at 3M, p=0.057 and p=0.097, respectively. Equally we observed no significant difference in TGFβR1 levels, either at Dx or 3M, between those who did and failed to achieve MMR. But we found strong to moderate correlation between MSI2 and TGFβR1 expression when we compared the Dx and 3M data as determined by Spearman correlation coefficient, r=0.6975 and 0.5715, respectively. More importantly, we observed 9 of the cell lines, with increased (>6.7%) or detectable MSI2 mRNA expression, of which 6 express BCR-ABL1 (BV173, Lama87, K562, KCL22, KU812 and SupB15), the TGFβR1 protein was considerably decreased. The BCR-ABL1 positive cell line Meg01 was the one exception with increased MSI2 mRNA levels (7.1%) and clearly detectable TGFβR1 protein. Conversely, of the 10 BCR-ABL1 negative cell lines with decreased or undetectable MSI2 transcripts the TGFβR1 protein was clearly detectable in 8. Of these 7 were lymphoid cell lines (L-248, KM-H2, L-1236, HDLM-2, BJAB, U-266, U-2932) and one was essential thrombocythemia, SET2. The 2 exceptions were AML cell lines OCI-AML-2 and OCI-AML-3 with considerably decreased MSI2mRNA levels and markedly decreased TGFβR1 protein expression.We suggest MSI2 expression is significantly decreased in CML patients in CP because the early progenitors expressing it are masked by large bulk of mature myeloid cells in these patients. Therefore to assess the prognostic value of MSI2 levels at diagnosis it should be quantified in CD34+ enriched samples at diagnosis. More importantly, finding cell lines expressing MSI2, with the exception of 3 cell lines (OCI-AML-2, OCI-AML-3 and Meg01) had decreased TGFβR1 protein is consistent with its role as a post transcription regulator. These findings combined with the previous data showing MSI2 mRNA levels are increased in CML BC support the notion that it functions via TGFβR1 signaling pathway to influence CML transformation. Disclosuresle Coutre:Novartis: Honoraria.

MSI2 and HOXA9 Expression Is Significantly Increased in Patients with Primary Myelofibrosis

Musashi 2 (also known as MSI2), a mRNA binding protein, is reported to control critical stem cell fate decisions by binding to the 3’untranslated region of target mRNAs, thereby inhibiting translation. MSI2 is preferentially expressed in hematopoietic tissue and in particular in early myeloid progenitors. Furthermore, investigators have shown MSI2 expression is upregulated by HOXA9 resulting in dysfunction of the regulatory pathway, leading to hematopoietic stem cell proliferation, impaired myeloid differentiation and is associated with worse prognosis in chronic myeloid leukemia (CML) and acute myeloid leukemia (Kharas, et al. Nat Med 2010; 16:903; Ito, et al. Nature 2010; 466:765). We verified MSI2 levels are significantly increased in CML patients in blast crisis compared with those in chronic phase, irrespective of lymphoid or myeloid transformation (Kaeda, et al. Blood. 2011;118;supplement). Given these observations we sought to assess MSI2 and HOXA9 expression in BCR-ABL1negative myeloproliferative neoplasm (MPN) patients.We retrospectively quantified MSI2 and HOXA9 expression in 38 samples of which 22 were from highly heterogeneous MPN patients, all of whom are alive. These 22 MPN patients were classified as polycythemia vera (PV) n= 5; essential thrombocythemia (ET) n=3; primary myelofibrosis (PMF) n=11 and unclassified MPN (U-MPN) n=3, based on clinical hematologic and molecular parameters in accordance with the World Health Organization 2008 classification. The MPN patients demographic features were (M: 13; F:9) with median age of 69.5 years (range 53-88). Of the 22 patients 17 had the V617F allele, in one it was undetectable and 4 were not tested. The remaining cDNA samples were unselected normal controls (NC) from healthy blood donors (M: 6; F:10) with median age of 41 years (range 34-61). MSI2 and HOXA9 mRNA levels were quantified by quantitative real time PCR (Q-PCR) and normalized to GUSβendogenous control gene, expression levels. Patient and control data were subjected to Mann-Whitney unpaired test two-tailed anaylsis using Graphpad Prism version 6.04 software.MSI2 and HOXA9 expression was detectable in all the samples by Q-PCR. However, HOXA9 transcript numbers, median 0.115% (range 0.020-2.360) were significantly increased (p=0.0123) in MPN patients when compared with the NC Group, median 0.060% (range 0.030-0.120). Conversely, median MSI2 expression level 0.715% (range 0.370-2.030) in MPN patients, was significantly decreased, p<0.0001 compared to that observed in the NC Group, median 2.817% (range 1.445-7.533). But both MSI2 and HOXA9 were significantly increased, p<0.0001, when comparing NC Group (n=16) with the PMF patients (n=11). Of these 11 PMF patients 6 had the V617F allele, in one it was undetectable and 4 were not screened. Interestingly, we noted a difference between PV and PMF data with respect to HOXA9 and MSI2 expression. Specifically, the PMF patients’ HOXA9 median [n=11; median age 62 years (range 53-81)], 0.790% (range 0.03-2.36); was significantly higher, (p=0.0021), than the PV patients’ [n=5; median age 77 years (range 69-88)], 0.030% (range 0.10-0.40). By contrast no significant difference, p=0.0504, was detected when comparing MSI2expression between patients with PV and PMF. The sample size was too small to reliably evaluate the other MPN groups.Our data are consistent with the notion that MSI2 plays a significant role in the biology of myeloid malignancies. While PV and ET patient numbers in this study were too small for evaluation, we did note finding increased HOXA9 expression among PMF subjects is consistent with reports that up to 30% of PMF patients harbor ASXL1 mutations, which are reported to lead to increased HOXA9 expression. Furthermore, the ASXL1 mutations are associated with worse prognosis, such that stem cell transplant (SCT) is recommended for patients harboring these mutations. In this study 2 of the 11 PMF patients had SCT and were among three patients with the highest HOXA9 levels determined. Given the findings it would be interesting to screen the PMF patients for ASXL1 mutations. Equally, finding MSI2 and HOXA9 increased expression in PMF patients is supportive of the later regulating the former. However, we observed no correlation between MSI2 and HOXA9 levels. An expanded study is required to assess these preliminary data implying HOXA9 expression is a prognostic marker in PMF patients. Disclosuresle Coutre:Novartis: Honoraria.

Expression of musashi-2 gene in leukemia stem cells from acute myeloid leukemia patients

This study was aimed to detect the expression of Musashi-2 (Msi2) in acute myeloid leukemia (AML) and investigate the relationship between Msi2 and other clinical parameters, especially CD34. A total RNA was extracted from bone marrow of newly diagnosed AML patietns. The Msi2 mRNA expression in newly diagnosed AML patients was detected with real-time fluorescence quantitative RT-PCR. The expression level of CD34 in above-menthioned patients was detected by flow cytometry (FCM). The relationship between the expression of Msi2 mRNA and clinical outcome in AML patients was analysed. The results showed that (1)the expression of Msi2 mRNA in newly diagnosed AML patients was much higher than that in healthy volunteers (P < 0.05) , especially in M1, M4 and M5 patients; (2)the expression level of Msi2 did not correlate with age, sex, white blood cell count of peripheral blood, AML1/ETO and PML/RARa fusion gene (P > 0.05); (3) Msi2 expression level in patients with CD34(+) cells was significantly higher than that in patients with CD34(-) cells (P < 0.05). It is concluded that the Msi2 mRNA expresses in leukamia stem cells, the high expression of Msi2 mRNA has been found in newly diagnosed AML patients, especially in M1, M4 and M5 patients, the high expression also has been observed in patients with CD34(+).

NOTCH3 Signaling Regulates MUSASHI-1 Expression in Metastatic Colorectal Cancer Cells

MUSASHI-1 (MSI-1) is a well-established stem cell marker in both normal and malignant colon cells and it acts by positively regulating the NOTCH pathway through inactivation of NUMB, a NOTCH signaling repressor. To date, the mechanisms of regulation of MSI-1 levels remain largely unknown. Here, we investigated the regulation of MSI-1 by NOTCH signaling in colorectal cancer cell lines and in primary cultures of colorectal cancer metastases. Stimulation by the NOTCH ligand DLL4 was associated with an increase of MSI-1 mRNA and protein levels, and this phenomenon was prevented by the addition of an antibody neutralizing NOTCH2/3 but not NOTCH1. Moreover, forced expression of activated NOTCH3 increased MSI-1 levels, whereas silencing of NOTCH3 by short hairpin RNA reduced MSI-1 levels in both colorectal cancer cells and CRC tumor xenografts. Consistent with these findings, enforced NOTCH3 expression or stimulation by DLL4 increased levels of activated NOTCH1 in colorectal cell lines. Finally, treatment of colorectal cancer cells with anti-NOTCH2/3 antibody increased NUMB protein while significantly reducing formation of tumor cell spheroids. This novel feed-forward circuit involving DLL4, NOTCH3, MSI-1, NUMB, and NOTCH1 may be relevant for regulation of NOTCH signaling in physiologic processes as well as in tumor development. With regard to therapeutic implications, NOTCH3-specific drugs could represent a valuable strategy to limit NOTCH signaling in the context of colorectal cancers overexpressing this receptor.

Hematopoietic Cell Lines and Patient Samples Show a Correlation Between Upregulated MSI2 and BCR-ABL Expression

Increased expression of Musashi 2 (also known as MSI2), a mRNA binding protein, is reported to trigger progression of chronic myeloid leukemia (CML) from chronic phase (CP) to blast crisis (BC), which is frequently fatal. The data imply that MSI2 is upregulated by HOXA9 leading to disruption of the critical hematopoietic stem cell (HSC) fate decisions via the NUMB-NOTCH signaling pathway. These events lead to HSC proliferation, impaired myeloid differentiation and are associated with worse prognosis in CML and acute myeloid leukemia (Kharas, et al. Nat Med 2010; 16:903; Ito, et al. Nature 2010; 466:765). Previously, we confirmed increased MSI2 levels in CML patients in blast crisis (BC) compared with those in CP, irrespective of lymphoid or myeloid transformation (Kaeda, et al. Blood. 2011;118;supplement). To further elucidate this regulatory pathway we assessed whether HOXA9 expression correlated with increased MSI2mRNA levels and upregulation of NOTCH.Because of the rarity of BC samples and finding MSI2 increased in lymphoid and myeloid BC CML patients, we studied lymphoid [n=12 (Lymphoma:8; Myeloma:3; ALL:1)] and myeloid [n=12 (AML:4; CML:6; HES:1; ET:1)] cells lines. We also included 29 BCR-ABL positive (e1a2: 18; e13a2: 6; e14a2: 5) diagnostic samples from acute lymphoblastic leukaemia (ALL) patients (M:18; F:11); median age 50 years (range: 26-74). We quantified MSI2 and HOXA9 mRNA transcripts by quantitative PCR and expressed the data as % ratio of the control gene, GUSβ.Western blotting of cell line protein extracts was performed to assess expression of NOTCH 1, 2, 3 receptors and delta like ligand 3.We found MSI2 [median: 7.10% (range:0.06-38.71)] and HOXA9 [median: 2.40% (range:0.00-51.13)] expression was generally restricted to myeloid cell lines. Of the 12 lymphoid cell lines, MSI2 was only detectable in SUPB15 with 8.44%. The latter is a B-lymphocyte ALL cell line, known to express BCR-ABL e1a2 transcript. Similarly, HOXA9 [median: 0.00 (range:0.00-27.0%)] expression ranged from undetectable to 0.08%, among this group, apart from HD-MY-Z with 27.0%. In contrast, HOXA9 [median: 0.02% (range:0.01-2.18)] and MSI2 [median: 3.58% (range: 1.24-22.38)] transcripts were detectable in all 29 BCR-ABL positive ALL patients, irrespective of the transcript type expressed. Among the ALL samples 7 (24%) had increased MSI2 levels, i.e. >6.7% (Kaeda, et al. Blood. 2011;118;supplement) and of these 6 expressed e1a2 transcript and the other e13a2. In summary, upregulated MSI2 expression was observed in 17 (32.0%) of the 53 samples screened. But only 4 [KG1, EOL1, HEL and MEG-01 (all myeloid cell lines)] of the 17 also had increased HOXA9 levels. Remarkably, 5 (62%) of the 8 myeloid cell lines with increased MSI2 are known to express BCR-ABL. NOTCH1 receptor was detectable in all the lymphoid cell lines. But, NOTCH expression was highly variable in myeloid cell lines. Overall, an upregulated MSI2 mRNA expression was not reflected in the NOTCH receptor levels nor in the HOXA9levels.Our observations are consistent with MSI2 being limited to myeloid linage. However, in contrast to earlier reports our data imply that MSI2 functions via a pathway other than NOTCH signaling and is not regulated by HOXA9 alone. But the cell lines and ALL patients' data provide further evidence of correlation between MSI2 and BCR-ABL expression, suggesting they interact, directly or indirectly, to arrest cell differentiation and trigger BC. These findings, together with our reported data, show increased MSI2 levels to be an important biomarker of poor prognosis and are likely to have an impact in optimizing clinical management. It also represents a potential novel therapeutic target, especially for the BCR-ABL positive stem cells resistant to tyrosine kinase inhibitors. Disclosures:Kaeda:Novartis: Research Funding. le Coutre:Novartis: Research Funding.

Musashi1 as a potential therapeutic target and diagnostic marker for lung cancer

Lung cancer remains one of the leading causes of cancer-related deaths worldwide with a 5-year survival rate of less than 20%. One approach to improving survival is the identification of biomarkers to detect early stage disease. In this study, we investigated the potential of the stem cell and progenitor cell marker, Musashi1 (Msi1), as a diagnostic marker and potential therapeutic target for lung cancer. Functional studies in A549 bronchioalveolar carcinoma and NCI-H520 squamous cell carcinoma cells revealed that Msi1 was enriched in spheroid cultures of tumor cells and in the CD133+ cell population. Downregulation of Msi1 by lentivirus-mediated expression of an Msi1 shRNA reduced spheroid colony proliferation. Growth inhibition was associated with reduced nuclear localization of β-catenin and inhibition of the processing of intracellular Notch. In primary lung cancer, Msi1 protein expression was elevated in 86% of 202 tissue microarray specimens, and Msi1 mRNA was increased in 80% of 118 bronchoscopic biopsies, including metastatic disease, but was rarely detected in adjacent normal lung tissue and in non-malignant diseased tissue. Msi1 was expressed in a diffuse pattern in most tumor subtypes, except in squamous cell carcinomas, where it appeared in a focal pattern in 50% of specimens. Thus, Msi1 is a sensitive and specific diagnostic marker for all lung cancer subtypes.