Thomas W. Bebee

Mouse models of SMA: tools for disease characterization and therapeutic development

Mouse models of human disease are an important tool for studying disease mechanism and manifestation in a way that is physiologically relevant. Spinal muscular atrophy (SMA) is a neurodegenerative disease that is caused by deletion or mutation of the survival motor neuron gene (SMN1). The SMA disease is present in a spectrum of disease severities ranging from infant mortality, in the most severe cases, to minor motor impairment, in the mildest cases. The variability of disease severity inversely correlates with the copy number, and thus expression of a second, partially functional survival motor neuron gene, SMN2. Correspondingly, a plethora of mouse models has been developed to mimic these different types of SMA. These models express a range of SMN protein levels and extensively cover the severe and mild types of SMA, with neurological and physiological manifestation of disease supporting the relevance of these models. The SMA models provide a strong background for studying SMA and have already shown to be useful in pre-clinical therapeutic studies. The purpose of this review is to succinctly summarize the genetic and disease characteristic of the SMA mouse models and to highlight their use for therapeutic testing.

Splicing regulation of the survival motor neuron genes and implications for treatment of spinal muscular atrophy.

Proximal spinal muscular atrophy (SMA) is a neuromuscular disease caused by low levels of the survival motor neuron (SMN) protein. The reduced SMN levels are due to loss of the survival motor neuron-1 (SMN1) gene. Humans carry a nearly identical SMN2 gene that generates a truncated protein, due to a C to T nucleotide alteration in exon 7 that leads to inefficient RNA splicing of exon 7. This exclusion of SMN exon 7 is central to the onset of the SMA disease, however, this offers a unique therapeutic intervention in which corrective splicing of the SMN2 gene would restore SMN function. Exon 7 splicing is regulated by a number of exonic and intronic splicing regulatory sequences and trans-factors that bind them. A better understanding of the way SMN pre-mRNA is spliced has lead to the development of targeted therapies aimed at correcting SMN2 splicing. As therapeutics targeted toward correction of SMN2 splicing continue to be developed available SMA mouse models can be utilized in validating their potential in disease treatment.

A humanized Smn gene containing the SMN2 nucleotide alteration in exon 7 mimics SMN2 splicing and the SMA disease phenotype

Proximal spinal muscular atrophy (SMA) is a neurodegenerative disease caused by low levels of the survival motor neuron (SMN) protein. In humans, SMN1 and SMN2 encode the SMN protein. In SMA patients, the SMN1 gene is lost and the remaining SMN2 gene only partially compensates. Mediated by a C>T nucleotide transition in SMN2, the inefficient recognition of exon 7 by the splicing machinery results in low levels of SMN. Because the SMN2 gene is capable of expressing SMN protein, correction of SMN2 splicing is an attractive therapeutic option. Although current mouse models of SMA characterized by Smn knock-out alleles in combination with SMN2 transgenes adequately model the disease phenotype, their complex genetics and short lifespan have hindered the development and testing of therapies aimed at SMN2 splicing correction. Here we show that the mouse and human minigenes are regulated similarly by conserved elements within in exon 7 and its downstream intron. Importantly, the C>T mutation is sufficient to induce exon 7 skipping in the mouse minigene as in the human SMN2. When the mouse Smn gene was humanized to carry the C>T mutation, keeping it under the control of the endogenous promoter, and in the natural genomic context, the resulting mice exhibit exon 7 skipping and mild adult onset SMA characterized by muscle weakness, decreased activity and an alteration of the muscle fibers size. This Smn C>T mouse represents a new model for an adult onset form of SMA (type III/IV) also know as the Kugelberg-Welander disease.

Conserved sequences in the final intron of MDM2 are essential for the regulation of alternative splicing of MDM2 in response to stress

Alternative splicing plays a fundamental role in generating proteome diversity and is critical in regulation of eukaryotic gene expression. It is estimated that 50% of disease-causing mutations alter splicing efficiency and/or patterns of splicing. An alternatively spliced form of murine double-minute 2, MDM2-ALT1, is associated with pediatric rhabdomyosarcoma (RMS) at high frequency in primary human tumors and RMS cell lines. We have identified that this isoform can be induced in response to specific types of stress (UV and cisplatin). However, the mechanism of alternative splicing of MDM2 in human cancer is unknown. Using UV and cisplatin to model alternative splicing of the MDM2 gene, we have developed a damage-inducible in vitro splicing system. This system employs an MDM2 minigene that mimics the damage-induced alternative splicing observed in vivo. Using this in vitro splicing system, we have shown that conserved intronic sequences in intron 11 of MDM2 are required for normal splicing. Furthermore, we showed that these intronic elements are also required for the regulated damage-induced alternative splicing of MDM2. The use of this novel damage-inducible system will allow for the systematic identification of regulatory elements and factors involved in the splicing regulation of the MDM2 gene in response to stress. This study has implications for identification of novel intervention points for development of future therapeutics for rhabdomyosarcoma.

The Splicing Factor FUBP1 Is Required for the Efficient Splicing of Oncogene MDM2 Pre-mRNA

Background: MDM2 is alternatively spliced into shorter isoforms under DNA damage and in several cancers through unknown mechanisms. Results: FUBP1 inactivation decreases splicing efficiency of an MDM2 minigene and causes exon skipping of endogenous MDM2 under normal conditions. Its overexpression attenuates damage-induced skipping of MDM2 exons. Conclusion: FUBP1 positively regulates efficient MDM2 splicing. Significance: This work uncovers an important mechanism regulating splicing efficiency of the oncogene MDM2. Alternative splicing of the oncogene MDM2 is a phenomenon that occurs in cells in response to genotoxic stress and is also a hallmark of several cancer types with important implications in carcinogenesis. However, the mechanisms regulating this splicing event remain unclear. Previously, we uncovered the importance of intron 11 in MDM2 that affects the splicing of a damage-responsive MDM2 minigene. Here, we have identified discrete cis regulatory elements within intron 11 and report the binding of FUBP1 (Far Upstream element-Binding Protein 1) to these elements and the role it plays in MDM2 splicing. Best known for its oncogenic role as a transcription factor in the context of c-MYC, FUBP1 was recently described as a splicing regulator with splicing repressive functions. In the case of MDM2, we describe FUBP1 as a positive splicing regulatory factor. We observed that blocking the function of FUBP1 in in vitro splicing reactions caused a decrease in splicing efficiency of the introns of the MDM2 minigene. Moreover, knockdown of FUBP1 in cells induced the formation of MDM2-ALT1, a stress-induced splice variant of MDM2, even under normal conditions. These results indicate that FUBP1 is also a strong positive splicing regulator that facilitates efficient splicing of the MDM2 pre-mRNA by binding its introns. These findings are the first report describing the regulation of alternative splicing of MDM2 mediated by the oncogenic factor FUBP1.

Stress-Induced Alternative Splice Forms of MDM2 and MDMX Modulate the p53-Pathway in Distinct Ways

MDM2 and MDMX are the chief negative regulators of the tumor-suppressor protein p53 and are essential for maintaining homeostasis within the cell. In response to genotoxic stress and also in several cancer types, MDM2 and MDMX are alternatively spliced. The splice variants MDM2-ALT1 and MDMX-ALT2 lack the p53-binding domain and are incapable of negatively regulating p53. However, they retain the RING domain that facilitates dimerization of the full-length MDM proteins. Concordantly, MDM2-ALT1 has been shown to lead to the stabilization of p53 through its interaction with and inactivation of full-length MDM2. The impact of MDM2-ALT1 expression on the p53 pathway and the nature of its interaction with MDMX remain unclear. Also, the role of the architecturally similar MDMX-ALT2 and its influence of the MDM2-MDMX-p53 axis are yet to be elucidated. We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2. Additionally, we demonstrate that MDMX-ALT2 is able to dimerize with both full-length MDMX and MDM2 and that the expression of MDM2-ALT1 and MDMX-ALT2 leads to the upregulation of p53 protein, and also of its downstream target p21. Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21. Finally we present evidence that MDM2-ALT1 and MDMX-ALT2 expression can activate subtly distinct subsets of p53-transcriptional targets implying that these splice variants can modulate the p53 tumor suppressor pathway in unique ways. In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response.

A novel mouse model of rhabdomyosarcoma underscores the dichotomy of MDM2-ALT1 function in vivo

Alternative splicing of the oncogene murine double minute 2 (MDM2) is induced in response to genotoxic stress. MDM2-ALT1, the major splice variant generated, is known to activate the p53 pathway and impede full-length MDM2’s negative regulation of p53. Despite this perceptible tumor-suppressive role, MDM2-ALT1 is also associated with several cancers. Furthermore, expression of MDM2-ALT1 has been observed in aggressive metastatic disease in pediatric rhabdomyosarcoma (RMS), irrespective of histological subtype. Therefore, we generated a transgenic MDM2-ALT1 mouse model that would allow us to investigate the effects of this splice variant on the progression of tumorigenesis. Here we show that when MDM2-ALT1 is ubiquitously expressed in p53 null mice it leads to increased incidence of spindle cell sarcomas, including RMS. Our data provide evidence that constitutive MDM2-ALT1 expression is itself an oncogenic lesion that aggravates the tumorigenesis induced by p53 loss. On the contrary, when MDM2-ALT1 is expressed solely in B-cells in the presence of homozygous wild-type p53 it leads to significantly increased lymphomagenesis (56%) when compared with control mice (27%). However, this phenotype is observable only at later stages in life (⩾18 months). Moreover, flow cytometric analyses for B-cell markers revealed an MDM2-ALT1-associated decrease in the B-cell population of the spleens of these animals. Our data suggest that the B-cell loss is p53 dependent and is a response mounted to persistent MDM2-ALT1 expression in a wild-type p53 background. Overall, our findings highlight the importance of an MDM2 splice variant as a critical modifier of both p53-dependent and -independent tumorigenesis, underscoring the complexity of MDM2 posttranscriptional regulation in cancer. Furthermore, MDM2-ALT1-expressing p53 null mice represent a novel mouse model of fusion-negative RMS.

Abstract LB-489: MDM2 alternative splicing: fine-tuning the p53 damage response

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Cell survival is dependent upon precise control of the activity of the tumor suppressor p53. p53 is a transcription factor and is known to control a number of biological processes including cell cycle arrest and apoptosis by transactivating downstream genes. Several proteins are reported to affect p53 activity but the murine double-minute protein, MDM2, and a closely related family member, MDM4, are two of the most critical regulators of p53. In previous work, we have identified alternative splicing of both the MDM2 and MDM4 genes in response to UV damage implicating alternative splicing in the cells normal surveillance mechanism. However, the role of the splice variants in the DNA damage pathway remains elusive. To determine the role the alternative splice forms play in the p53 damage response, overexpression of the UV-induced isoforms, MDM2-ALT1 and MDM4-ALT2, was performed. Both MDM2-ALT1 and MDM4-ALT2 are capable of negatively regulating the full-length MDM2 and MDM4 full-length proteins thereby increasing p53 levels. Interestingly, each splice variant activates a distinct and unique subset of p53 target genes. An additional type of cellular stress, hypoxia, induces alternative splicing of MDM2 as well, but generates a different splice form known as MDM2-ALT2. This splice form activates a third unique profile of p53 target genes. Hence, the splicing status of the MDM2 and MDM4 genes is capable of dictating unique p53 programs. We are also currently investigating p53-independent functions for the damage-induced splice variants to better understand their role in the damage response and tumorigenesis. This work elucidates regulated splicing as a novel mechanism by which cellular injury can control the activity of p53 within the cell. By producing specific alternatively spliced variants of both MDM2 and MDM4, yet another layer of p53 regulation is initiated in order to fine-tune the cells response to damage. Because MDM2 and MDM4 alternative splice variants are characteristic of numerous cancer types, this work indicates that the status of MDM2 and MDM4 splicing could be a consideration in determining treatment options for cancers for which p53 activation is a therapeutic strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-489. doi:1538-7445.AM2012-LB-489

Modeling Spinal Muscular Atrophy in Mouse: A Disease of Splicing, Stability, and Timing

Proximal spinal muscular atrophy (SMA) is a progressive neurodegenerative disease associated with the loss of alpha motor neurons in the lumbar spinal cord. The loss of these motor neurons leads to the progressive atrophy of the associated proximal muscles, eventual respiratory distress, and death. SMA occurs in about 1 in 6,000 to 10,000 live births and is a leading genetic cause of infant mortality (Burnett et al. 2009). SMA is subdivided into several groupings based on disease onset, severity, and outcome. The most severe form of SMA is referred to as type 0, or embryonic SMA, wherein patients are born with severe muscle atrophy and have expected lifespans of less than 6 months. The most common type of SMA is type I, which is characterized by disease onset within 6 months and mortality by age 2. Children with SMA type I often fail to sit unaided or walk, have difficulty breathing, and often require respiratory assistance. SMA type II children experience disease onset between 6 to 18 months and will not gain the ability to walk. Two forms of SMA are characterized by disease onset later in life and have unaltered life expectancies: juvenile and adult onset SMA, or type III and IV, respectively. Juvenile onset SMA type III is distinguished by disease onset after the age of 2, but there is variability of onset that can extend to early teen years. Type III children are often able to walk, and the ability to remain ambulatory throughout life can further subdivide this juvenile onset SMA. The least severe form of SMA is adult onset SMA type IV. This form of SMA does not emerge until the adult years and often presents with difficulty in performing previously attainable activities, such as climbing stairs (Zerres et al. 1995; Zerres et al. 1997; Zerres et al. 1997). In SMA disease progression, the inability to achieve normal motor function is often the primary indication of disease. The level of motor function decline in SMA disease progression can be measured by the loss of functional motor units in SMA patients. In severe forms of SMA, there is a rapid loss of motor neurons innervating proximal muscles. This measure of reduced motor units can be observed by evaluating the Compound Motor Action Potential (CMAP) and Motor Unit Number Estimation (MUNE). The total number of innervated motor units is reduced in SMA disease as measured by these two tests and is correlated with age, severity of disease, and disease progression (Swoboda et al. 2005). The loss of motor neurons early in the onset of disease argues for the significance of motor neurons in disease, although additional components of the disease may provide for the