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A Fusion Transcription Factor-Driven Cancer Progresses to a Fusion-Independent Relapse Via Constitutive Activation of a Downstream Transcriptional Target

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A Fusion Transcription Factor-Driven Cancer Progresses to a Fusion-Independent Relapse Via Constitutive Activation of a Downstream Transcriptional Target Author Manuscript Published OnlineFirst on February 15, 2021; DOI: 10.1158/0008-5472.CAN-20-1613 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. A fusion transcription factor-driven cancer progresses to a fusion-independent relapse via constitutive activation of a downstream transcriptional target Salah Boudjadi1, Puspa Raj Pandey1, Bishwanath Chatterjee1, Thanh Hung Nguyen1, Wenyue Sun1, Frederic G. Barr1* 1Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, MD. Running Title Fusion-independent recurrence from a fusion-driven cancer Keywords Targeted therapy, FGF8, PAX3-FOXO1, tumor recurrence Conflict of interest statement The authors declare no potential conflicts of interest. *Corresponding Author: Frederic G. Barr, MD PhD, Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, 10 Center Drive, Room 2S235D, Bethesda, MD 20892-1500. Phone: 301-480-7176; Fax: 301-480-0611; E-mail: [email protected]. 1 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 15, 2021; DOI: 10.1158/0008-5472.CAN-20-1613 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Targeted monotherapies usually fail due to development of resistance by a subgroup of cells that evolve into recurrent tumors. Alveolar rhabdomyosarcoma is an aggressive myogenic soft tissue cancer that is associated with a characteristic PAX3-FOXO1 gene fusion encoding a novel fusion transcription factor. In our myoblast model of PAX3- FOXO1-induced rhabdomyosarcoma, de-induction of PAX3-FOXO1 simulates a targeted therapy that antagonizes the fusion oncoprotein. This simulated therapy results initially in regression of the primary tumors, but PAX3-FOXO1-independent recurrent tumors eventually form after a delay. We report here that upregulation of the fibroblast growth factor FGF8, a direct transcriptional target of PAX3-FOXO1, is a mechanism responsible for PAX3-FOXO1-independent tumor recurrence. As a transcriptional target of PAX3- FOXO1, FGF8 promoted oncogenic activity in PAX3-FOXO1-expressing primary tumors that developed in the myoblast system. In the recurrent tumors forming after PAX3- FOXO1 de-induction, FGF8 expression was necessary and sufficient to induce PAX3- FOXO1-independent tumor growth through an autocrine mechanism. FGF8 was also expressed in human PAX3-FOXO1-expressing rhabdomyosarcoma cell lines and contributed to proliferation and transformation. In a human rhabdomyosarcoma cell line with reduced PAX3-FOXO1 expression, FGF8 upregulation rescued oncogenicity and simulated recurrence after PAX3-FOXO1-targeted therapy. We propose that deregulated expression of a PAX3-FOXO1 transcriptional target can generate resistance to therapy directed against this oncogenic transcription factor and postulate that this resistance mechanism may ultimately be countered by therapeutic approaches that antagonize the corresponding downstream pathways. Statement of significance In a model of cancer initiated by a fusion transcription factor, constitutive activation of a downstream transcriptional target leads to fusion oncoprotein-independent recurrences, thereby highlighting a novel progression mechanism and therapeutic target. 2 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 15, 2021; DOI: 10.1158/0008-5472.CAN-20-1613 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric soft tissue sarcoma that occurs frequently in adolescents and young adults and is characterized by early metastasis, poor response to chemotherapy, and frequent relapses after chemotherapy (1). This cancer usually occurs within skeletal muscle and is hypothesized to develop from skeletal muscle progenitors (2). ARMS is distinguished by a recurrent 2;13 chromosome translocation involving the PAX3 and FOXO1 genes and less frequently by a 1;13 translocation involving the PAX7 and FOXO1 genes. These translocations generate PAX3-FOXO1 (P3F) or PAX7-FOXO1 fusion genes (3,4), which encode novel chimeric transcription factors. We previously demonstrated that expression of P3F, along with MYCN, induces transforming and tumorigenic activity in human myoblasts, and that P3F plays a crucial role in regulating the proliferation, survival and differentiation of these cells (5). These properties make P3F an interesting therapeutic target in the treatment of ARMS, particularly given the fact that conventional treatment, combining chemotherapy, surgery and radiotherapy, is often ineffective, with a 5-year overall survival less than 50%. Though efforts to develop effective P3F-directed targeted therapy are ongoing, it is well known that resistance to a single therapeutic agent often develops and thus may ultimately occur in monotherapies targeting the P3F oncoprotein. We propose that a model of P3F targeted therapy will be useful in the investigation of such resistance mechanisms. To simulate this targeted therapy, we designed a doxycycline-inducible P3F (iP3F) expression construct to control the timing and level of P3F expression in vitro and in vivo (6). We observed that human Dbt myoblasts engineered with constitutive MYCN and iP3F expression constructs (Dbt/MYCN/iP3F) rapidly form primary rhabdomyosarcoma (RMS) tumors when injected into mice fed a doxycycline-containing diet. When doxycycline induction is stopped to turn off P3F expression, tumors initially regress, and then recurrent tumors without P3F expression form after a variable delay. These findings suggest that a subset of tumor cells is able to conserve their tumorigenicity by developing or maintaining mechanisms that lead to clonal expansion and formation of P3F- independent recurrent tumors. We hypothesize that P3F is required for initial tumor development but is not needed in later stages of tumorigenesis due to the accumulation of additional oncogenic changes. In this study, we analyzed the expression profile of primary and recurrent tumor-derived (TD) cells and found that fibroblast growth factor 8 (FGF8), a transcriptional target of P3F, often remains upregulated in recurrent TD cells. Furthermore, assays of oncogenic activity reveal that FGF8 is required for proliferation, transformation, migration and invasion of these recurrent TD cells. In addition, FGF8 is also necessary for the oncogenic transformation of primary TD cells as well as human ARMS tumor cells, and FGF8 overexpression in human ARMS cells is sufficient to maintain their oncogenicity 3 Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2021 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 15, 2021; DOI: 10.1158/0008-5472.CAN-20-1613 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. when P3F expression is repressed. Our findings provide biological information regarding the consequences of therapeutically targeting oncogenic fusion proteins and may enable development of combined approaches to effectively treat these aggressive cancers. Materials and Methods Cell culture Engineered derivatives of a Duchenne muscular dystrophy myoblast cell line (Dbt) and human RMS cell lines were cultured as described previously (5,7). The source of the cell line is as follow: Dbt, Dr D. Trono; RH30 American Type Culture Collection; Rh28 Dr. B. Emanuel; Rh5 Dr. J. Khan; CW9019 Dr. J. Biegel; Rh41 Dr. C. Linardic; MP4 Dr. T. Cripe. Verification of cell line identity was performed using the short tandem repeat genotyping analysis with the AmpFLSTR profiler plus polymerase chain reaction amplification kit (Applied Biosystems, Foster City, CA). Cell lines were periodically checked with a PCR- based mycoplasma detection kit (ATCC, # 30-1012K, Manassas, VA) to rule out the possibility of mycoplasma contamination. Transfection and lentiviral transduction Dbt cells were previously transduced either with a constitutive MYCN expression construct (Dbt/MYCN) or with MYCN and an inducible P3F expression construct (Dbt/MYCN/iP3F) (5,6). The pCMV6-Entry vector expressing FGF8b cDNA or the empty vector EV (Origene, Rockville, MD) was stably transfected using lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA). CRISPR/Cas9 targeting of FGF8 was performed with single-guide RNAs cloned into the lentiCRISPR v2-eGFP (GenScript, Piscataway NJ) and compared with the control non-targeting vector lentiCRISPR v2- eGFP (CR-control). Dbt/MYCN/iP3F cells were transduced with a green fluorescent protein (GFP) expression construct as previously described (6). Lentiviral particles were packaged using HEK293T cells with the human immunodeficiency virus-based pPACK- H1 Packaging Plasmid Mix (System Biosciences, Mountain View, CA). RNA extraction and gene expression profiling Total RNA was extracted using the AllPrep Kit (Qiagen, Germantown, MD). Synthesized cDNA was hybridized to the GeneChip™ Human Transcriptome Array 2.0 (#902162, Affymetrix, Santa Clara, CA). Generated CEL files were analyzed using Partek® Flow® software, version 8.0.19.111© (Partek, St. Louis, MO), using the standard recommendations provided by the software. Gene expression changes with >1.5- or <- 1.5-fold alterations and
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