genes associated with Rhabdomyosarcoma
studies of Rhabdomyosarcoma
Galindo, K.A., Endicott, T.R., Avirneni-Vadlamudi, U. and Galindo, R.L. (2014). A rapid one-generation genetic screen in a Drosophila model to capture rhabdomyosarcoma effectors and therapeutic targets. G3 (Bethesda) 5: 205-217. PubMed ID: PubMed ID: 25491943
With this platform, new PAX-FOXO1 pathogenesis underpinnings were probed. Though very similar in molecular structure, PAX3-FOXO1− and PAX7-FOXO1−positive RMS demonstrate differing clinical behaviors, as PAX3-FOXO1 tumors are more common and notoriously aggressive. Consequently, PAX3-FOXO1 is the PAX-FOXO1 fusion most commonly investigated in vertebrate models. This study focuses on PAX7-FOXO1 in the Drosophila system, which demonstrates phenotypes that are better penetrant and experimentally tractable due to the fact that human PAX7 demonstrates slightly greater sequence identity to fly PAX3/7 than does human PAX3. Additionally, as no other animal models of PAX7-FOXO1 presently exist, the fly PAX7-FOXO1 model also conveniently serves as a complement to vertebrate PAX3-FOXO1 models (Galindo, 2014).
The extent to which observations from the PAX7-FOXO1 fly model would impact the clinically more aggressive PAX3-FOXO1 RMS subtype, as well as PAX-FOXO1-negative (embryonal) RMS, is unknown. Notably, previous studies show that genetic modifiers identified from the Drosophila system impact PAX3-FOXO1 RMS oncogenesis and tumorigenesis. Furthermore, unpublished studies suggest that fly PAX7-FOXO1 genetic modifiers are similarly involved in Embryonal RMS. These findings provide marked validation for the applicability and value of this genetic fly system to human RMS (Galindo, 2014).
Interestingly, though PAX7-FOXO1 induces expression of the late myogenic differentiation marker MHC, PAX-FOXO1 RMS myoblasts in culture and in vivo demonstrate only partial differentiation with little-to-no MHC expression. In considering this discrepancy, it should be first noted that PAX-FOXO1 is a relatively weak driver of RMS in culture and in vivo and requires additional/sequential genetic aberrations to induce oncogenic transformation. Thus, secondary mutations might be necessary to force the strength of RMS myoblast differentiation-arrest seen in human RMS tumors; by contrast, the PAX7-FOXO1 model of this study differs in that the system is free of any additional background mutations. Second, earlier studies show that expression of PAX3-FOXO1 in mouse embryonic cultured cells induces the formation of MHC-positive myocytes and myotube, similar to the Drosophila system in this study as the da-Gal4/UAS-PAX7-FOXO1 expression system targets undifferentiated embryonic primordia. Uncovering of the genetic/molecular sequence of RMS pathogenesis and the cell(s) origin will shed further insight into the underlying mechanisms that account for the myoblast differentiation arrest phenotypes seen in RMS in vivo (Galindo, 2014).
The differentiation and fusion of myoblasts into postmitotic, syncytial muscle requires that the bHLH myogenic regulatory factors (MRFs: Myf5, Mrf4, MyoD, and Myogenin) interact with E-proteins, which drive and regulate critical aspects of myogenic fate determination. The MRFs subsequently interact with the MEF2 transcription factors that, although lacking intrinsic myogenic activity, cooperate with the MRFs to synergistically activate muscle-specific genes and the downstream myogenic terminal differentiation program. Vertebrates possess four MEF2 family member genes (-A, -B, -C, -D), which demonstrate complex overlapping spatial and temporal expression patterns in embryonic and adult tissues, with greatest expression levels seen in striated muscle and brain. Because of genetic redundancy and overlapping expression patterns of the MEF2 genes, interrogating individual MEF2 gene activity in mammals is experimentally challenging, with loss-of-function mutation studies revealing only limited insights into MEF2 gene function in tissues in which the MEF2 genes do not overlap/compensate. Conveniently, flies possess only one Mef2 gene (D-Mef2) and serve as an excellent model system to delineate MEF2’s critical role in myogenesis. The study speculates that the lack of Mef2 redundancy in flies provides a marked experimental advantage in isolating D-Mef2 as a PAX7-FOXO1 effector. Similarly, the identification of mam was also likely facilitated by the fact that flies possess one mam gene, whereas mammals contain three mam orthologs. Thus, the study proposes that the comparative lack of genetic compensation/redundancy is an attractive advantage to Drosophila as a disease model system (Galindo, 2014).
The study suggests that further interrogation of MEF2 in RMS will open new avenues for RMS chemotherapy, which for high-risk disease has not improved for decades. For example, since MEF2 activity is tightly governed by class IIa histone deacetylases, histone deacetylase inhibitors are now ripe for preclinical testing as new anti-RMS agents. Additionally, it was found that the MEF2 cofactor Mastermind, which interacts with MEF2C and mediates crosstalk between Notch signals during myogenic differentiation, similarly influences PAX-FOXO1 pathogenicity in flies. Interestingly, Mastermind-specific, cell-permeable peptide inhibitors have been shown to block the progression of T-cell acute lymphoblastic leukemia in mice in vivo and thus are also new agents available for RMS preclinical testing. Further characterization of MEF2 in RMS cell and mouse models will continue to refine both our understanding and the potential targeting of MEF2 activity in RMS (Galindo, 2014).
In conclusion, the study postulates that: 1) The Drosophila PAX7-FOXO1 model is uniquely configured for the quick uncovering of new RMS genetic effectors with one simple genetic screening cross; 2) a putative PAX-FOXO1-to-MEF2/MASTERMIND axis underlies A-RMS; and 3) Drosophila conditional expression models are an efficient and powerful gene discovery platform for the rapid dissection of human disease (Galindo, 2014).
Crose, L.E., Galindo, K.A., Kephart, J.G., Chen, C., Fitamant, J., Bardeesy, N., Bentley, R.C., Galindo, R.L., Chi, J.T. and Linardic, C.M. (2014). Alveolar rhabdomyosarcoma-associated PAX3-FOXO1 promotes tumorigenesis via Hippo pathway suppression. J Clin Invest 124: 285-296. PubMed ID: 24334454
Using this Drosophila model, the expression data for transcriptional changes promoted by PAX7-FOXO1 was inspected. (Human PAX7 demonstrates a slightly higher sequence identity with Drosophila PAX3/7 than does human PAX3 and is used in this study.) It was found that dRASSF mRNA overexpresses 2.6-fold in PAX7-FOXO1 larval muscle, suggesting that similarly to the human myoblast model, this Drosophila model of aRMS shows upregulated RASSF. Next, it was examined whether dRASSF promotes PAX-FOXO1 phenotypes by testing the effect of Drosophila mutants that alter dRASSF levels or function. A Drosophila chromosomal deletion, Df(3R)Exel6193, that dominantly suppresses PAX7-FOXO1 semilethality was used. Interestingly, Df(3R)Exel6193 removed segments 94D3-94E4 on chromosome 3, which includes dRASSF. It was hypothesized that heterozygous deletion of dRASSF might account for Df(3R)Exel6193-mediated PAX7-FOXO1 suppression and that, consistent with PAX3-FOXO1 activation of RASSF4 in mammalian myoblasts, dRASSF might act as a PAX3/7-FOXO1 downstream target and effector. Next, two publicly available transposable-element insertion loss-of-function dRASSF alleles, DG30608 and A531, were also tested for suppression of PAX7-FOXO1. The DG30608 element inserts into the 5′UTR, which does not alter DRASSF protein structure but instead presumably alters dRASSF expression. The A531 insertion disrupts exon 5, resulting in a truncated dRASSF lacking the C-terminal SARAH domain, which mediates RASSF-MST1 physical association. Both dRASSF loss-of-function alleles were found to ameliorate PAX-FOXO1 pathogenicity, with A531 acting as the stronger suppressor. These data show that dRASSF and RASSF4 promote PAX-FOXO1 phenotypes in both Drosophila and mammalian models of aRMS (Crose, 2014).
Although the progrowth function of RASSF4 in aRMS found in this study may be distinct from the presumed canonical role of RASSFs in cancer, it does resolve a conflict between RASSF function in mammals versus Drosophila. While there are 10 RASSF genes in human cells, the Drosophila genome contains only two orthologs, known as dRASSF and dRASSF8. Of these genes, dRASSF is most similar to the C-terminal RASSF1-6, and most similar to RASSF4 within the Ras-association and SARAH domains. In contrast to the majority of mammalian RASSFs, dRASSF promotes cell proliferation and inhibits Hippo pathway activation through recruitment of phosphatases or competition with the SAV1 ortholog Salvador. While RASSF4 and DRASSF can inhibit Hippo signaling, rescue experiments for the RASSF4-knockdown phenotype with dRASSF expression were not successful in this study. Therefore, RASSF4 is likely not a true ortholog of dRASSF. However, the observations in this study on the effects of RASSF4 on aRMS may reflect one evolutionarily conserved role of RASSFs in cell proliferation (Crose, 2014).
Avirneni-Vadlamudi, U., Galindo, K.A., Endicott, T.R., Paulson, V., Cameron, S. and Galindo, R.L. (2012). Drosophila and mammalian models uncover a role for the myoblast fusion gene TANC1 in rhabdomyosarcoma. J Clin Invest 122: 403-407. PubMed ID: 22182840
Of the 2 alternative transcripts expressed from the rols locus, only one of which is expressed in myoblasts; expression of the second is restricted to endodermal/ectodermal precursors. In this study, 2 rols homozygous-lethal, P-element insertion loss-of-function alleles, P1027 and P1729, were tested for suppression of PAX7-FOXO1. Of these 2 alleles, only the P1729 insertion disrupts expression of the myoblast rols transcript (myoblast expression of rols is unperturbed in P1027); accordingly, only the rolsP1729 allele suppresses PAX7-FOXO1–induced lethality and muscle pathogenicity (Avirneni-Vadlamudi, 2012).
To investigate whether rols acts as a downstream PAX-FOXO1 target, the daughterless-Gal4 transgene was used to drive ubiquitous embryonic expression of UAS-PAX7-FOXO1 and probed for Rols misexpression. Since native Rols expression initiates at embryonic stage 11, the study focused only on embryos stage 10 or earlier. Diffuse expression of PAX7-FOXO1 and Rols is observed in blastoderm (stage 4–5) embryos, which consist of uncommitted precursor cells, and expression persists in all examined cells — including nonmyogenic ectodermal and endodermal cells — of gastrulated (stage 9–10) embryos. Taken together, these Drosophila studies reveal that rols acts as a PAX7-FOXO1 downstream target gene, direct or indirect, and as a bona fide genetic effector (Avirneni-Vadlamudi, 2012).
RMS model systems conveniently promote insights into not only neoplasia, but also muscle development. Although ultrastructural studies suggest that myoblast fusion biology is conserved, few of the Drosophila fusigenic genes have been identified as essential in mammals, and none of these are from the founder subfamily. As the name implies, founder myoblasts are seminal to Drosophila myogenesis, uniquely dictating the location and physiology of each individual muscle. With rols and Tanc1, it was shown that founder gene function is conserved in mammals and, furthermore, participates in human disease. How founder gene activity influences other forms of neuromuscular disease now becomes an intriguing issue (Avirneni-Vadlamudi, 2012).
Genetic screening in a Drosophila model and loss-of-function/gain-of-function studies in mammalian platforms have collaboratively uncovered a PAX-FOXO1-to-TANC1 neoplasia axis, a finding that the study suggests to be novel. Results from this study also argue that the relationship between myogenesis transcription factor (e.g., MyoD) signaling and myoblast fusion genes is intricate. In the presence of altered fusion potential, both Drosophila and mammalian myoblasts transition to differentiated myocytes, which suggests that later aspects of myogenesis signaling must uncouple from the TANC1 fusigenic pathway. Yet correcting PAX-FOXO1–mediated overexpression of rols/TANC1 rescues PAX-FOXO1–induced differentiation and arrest. These results intimate that correction of the TANC1 fusigenic axis feeds back to and rescues PAX-FOXO1–mediated misregulation of myogenic signaling, raising fascinating questions regarding the mechanisms by which this occurs. The observation in this study that PAX3-FOXO1 protein levels remain unchanged in TANC1-silenced cells argues that rescue does not originate from decreased expression of PAX3-FOXO1 from the PAX3 promoter. Thus, the study speculates that rescue occurs epistatically downstream of PAX3-FOXO1 (Avirneni-Vadlamudi, 2012).
Galindo, R.L., Allport, J.A. and Olson, E.N. (2006). A Drosophila model of the rhabdomyosarcoma initiator PAX7-FKHR. Proc Natl Acad Sci U S A 103: 13439-13444. PubMed ID: 16938866
Despite intensive study, the tissue of origin for ARMS has been puzzling. Because skeletal muscle is irreversibly postmitotic and syncytial, the origin has long been hypothesized to be a muscle precursor cell or stem-like cell. Yet, transgenic expression of PAX3-FKHR in muscle precursor cells or muscle-specific satellite stem cells demonstrates no evidence of tumorigenesis, whereas expression of PAX3-FKHR in terminal differentiating myofibers causes rhabdomyosarcomagenesis. Because no evidence exists, either in cell culture or in vivo, that PAX-FKHR can induce individual cells to form de novo from synytial tissue, questions persist regarding whether the mouse model has undetectably generated tumors from an unknown cell. Because most PAX3-FKHR mice in the ARMS model do not grow tumors, this study predicted that exhaustively surveying mouse muscle for focal or subtle cellular changes would be difficult. In contrast, it was postulated that Drosophila would provide a practical approach for this type of study, given the amenability of the organism to rapid, thorough, serial examination of living muscle. Additionally, because fly muscle contains no known mechanism for repair (including satellite cells), it was predicted that muscle dysmorphology would not be obfuscated by physiologic regeneration. With this approach, this study was able to document evidence of cells generating de novo from syncytial tissue. These results show that PAX-FKHR can specify this process and that cells can form from differentiated muscle in vivo. Thus, in a complementary fashion, the ARMS mouse and PAX7-FKHR fly strongly argue that muscle can serve as a RMS tissue of origin (Galindo, 2006).
How might PAX-FKHR cause muscle “dedifferentiation?” Because both wild-type PAX and PAX-FKHR demonstrate activity in fly muscle and, therefore, presumably share gene targets and cofactors, wild-type PAX can be used for providing mechanistic insights. Specifically, wild-type PAX performs at least two distinct developmental functions: (i) PAX proteins regulate early organogenesis and lineage determination; and (ii) PAX functions late in lineage development to repress the terminal differentiation of tissue-specific precursors. For example, in melanocyte development, PAX3 inhibits the terminal differentiation of maturing melanoblasts, thereby maintaining a population of partially differentiated precursors available for damage response. PAX7 likewise inhibits late steps in the terminal differentiation of muscle satellite cells, where PAX7 and myogenin expression are mutually exclusive, and overexpression of PAX7 interferes with MyoD-dependent transcriptional activation and down-regulates MyoD expression. PAX5 has also been characterized as a negative modulator of plasma cell terminal differentiation. This study proposes that PAX-FKHR, upon chromosomal rearrangement and misexpression, reacquires its inhibitory activity on terminal differentiation and interferes with the integrity of differentiated myofibers (Galindo, 2006).
For ARMS tumorigenesis to originate from postmitotic muscle, newly generated PAX-FKHR cells would need to re-enter the cell cycle to proliferate. The fact that PAX3-FKHR in the ARMS mouse model requires loss of cell-cycle regulation for appreciable levels of tumorigenesis argues that PAX3-FKHR alone is not sufficient to cause cell-cycle reentry. Consistent with this observation, this study did not find evidence of cell-cycle reentry in myofiber nuclei or individual GFP-positive myogenic cells in either PAX7-FKHR or PAX7-FKHR, RasV12 animals after staining with phosphohistone H3 antibody. These results are similar to earlier findings that activated Ras does not promote proliferation of either mammalian or fly myogenic precursor cells. Genetic gain-of-function and loss-of-function ras experiments in this study suggest, nonetheless, that Ras signaling is involved in PAX7-FKHR-mediated dedifferentiation of muscle. In this regard, it is noteworthy that constitutively activated Ras strongly interferes with the terminal differentiation of mammalian myoblasts, whereas ras gain-of-function or loss-of-function alters the differentiation of Drosophila founder cell myoblasts, the patterning pioneers of fly muscle. Therefore, it is possible that the interaction observed between PAX7-FKHR and Ras results not from proliferation but from the fact that both molecules functionally perturb muscle differentiation (Galindo, 2006).
Outcomes for advanced ARMS remain dismal despite intensive therapy, underscoring the need to characterize the pathogenetic mechanisms underlying tumorigenesis. As presented in this study, the PAX7-FKHR Drosophila phenotypes are sensitive to both genetic suppression and enhancement. Therefore, this PAX-FKHR model is well situated for unbiased genetic screens. This approach, unexplored with regards to PAX-FKHR, should allow for the isolation of previously unknown PAX-FKHR gene targets and cofactors. Uncovering these molecular entities should represent a starting point for the conceptual development of therapeutics to target these interactions and poison ARMS (Galindo, 2006).
Gonzalez, C. (2013). Drosophila melanogaster: a model and a tool to investigate malignancy and identify new therapeutics. Nat Rev Cancer 13: 172-183. PubMed ID: 23388617
daughterless homologs, myogenesis and rhabdomyosarcoma
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Date revised: 20 June 2015
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