PAX3–FOXO1 is essential for tumour initiation and maintenance but not recurrence in a human myoblast model of rhabdomyosarcoma

Abstract

The PAX3-FOXO1 fusion gene is generated by a 2;13 chromosomal translocation and is a characteristic feature of an aggressive subset of rhabdomyosarcoma (RMS). To dissect the mechanism of oncogene action during RMS tumourigenesis and progression, doxycycline-inducible PAX3-FOXO1 and constitutive MYCN expression constructs were introduced into immortalised human myoblasts. Though myoblasts expressing PAX3-FOXO1 or MYCN alone were not transformed in focus formation assays, combined PAX3-FOXO1 and MYCN expression resulted in transformation. Following intramuscular injection into immunodeficient mice, myoblasts expressing PAX3-FOXO1 and MYCN formed rapidly growing RMS tumours whereas myoblasts expressing only PAX3-FOXO1 formed tumours after a longer latency period. Doxycycline withdrawal in myoblasts expressing inducible PAX3-FOXO1 and constitutive MYCN following tumour formation in vivo or focus formation in vitro resulted in tumour regression or smaller foci associated with myogenic differentiation and cell death. Following regression, most tumours recurred in the absence of doxycycline. Analysis of recurrent tumours revealed a subset without PAX3-FOXO1 expression, and cell lines derived from these recurrent tumours demonstrated transformation in the absence of doxycycline. The doxycycline-independent oncogenicity in these recurrent tumour-derived lines persisted even after PAX3-FOXO1 was inactivated by a CRISPR-Cas9 editing strategy. Whereas cell lines derived from primary tumours were dependent on PAX3-FOXO1 and differentiated following doxycycline withdrawal, recurrent tumour-derived cells without PAX3-FOXO1 expression did not differentiate under these conditions. These findings indicate that PAX3-FOXO1 collaborates with MYCN during early RMS tumourigenesis to dysregulate proliferation and inhibit myogenic differentiation and cell death. Although most cells in the primary tumours are dependent on PAX3-FOXO1, recurrent tumours can develop by a PAX3-FOXO1-independent mechanism, in which rare cells are postulated to acquire secondary transforming events that were activated or selected by initial PAX3-FOXO1 expression.

Deciphering the roles of FOXO1 in human neoplasms

“…They discovered that FILNC1 represses c‐Myc‐mediated energy metabolism and inhibits renal tumor development . Additionally, studies have also demonstrated that FOXO1 has antitumor actions in spindle cell lipoma, rhabdomyosarcomas and Kaposi's sarcoma (Table ).…”

Early detection of the PAX3‐FOXO1 fusion gene in circulating tumor‐derived DNA in a case of alveolar rhabdomyosarcoma

“…One important pitfall of using the tumor‐specific fusion gene as a biomarker is the possibility of the tumor progressing to a fusion‐independent state due to the acquisition of other genetic abnormalities after therapeutic interventions are performed for the primary tumor. The actual progression to a PAX3‐FOXO1 ‐independent state during the recurrence of the tumor has been reported recently in a mouse model . Therefore, fusion‐specific assays may become problematic in the later stages of tumor progression in some cases, particularly in settings involving treatment with targeted agents that may enhance clonal selection inside the tumors.…”

“…FOXO1 (exon 2) 0 6 3 0 5 3 0 4 3 0 3 3 0 2 3 0 1 3 0 0 3 0 9 2 0 8 2 0 7 2 0 6 2 0 5 2 during the recurrence of the tumor has been reported recently in a mouse model. 22 Therefore, fusion-specific assays may become problematic in the later stages of tumor progression in some cases, particularly in settings involving treatment with targeted agents that may enhance clonal selection inside the tumors.…”

Fusion gene addiction: can tumours be forced to give up the habit?

“…This and previous functional work show cell death and differentiation results from ablation of the fusion protein in tumour‐derived cells and have led to the view that the fusion protein is an Achilles heel of ARMS. The work carried out by Pandey et al is the first to test the reliance of these tumours on the fusion gene in vivo beyond the stage of tumour initiation. They mimicked a targeting strategy by reducing levels of the fusion protein in an established tumour model and discovered that despite a dramatic initial response, tumours recurred that in some cases lacked PAX3–FOXO1 expression.…”

“…Model of alveolar rhabdomyosarcoma demonstrating that PAX3–FOXO1‐independent recurrences can arise from PAX3–FOXO1‐expressing primary tumours. Pandey et al have shown that tumours and in vitro transformed clones arise when MYCN is constitutively expressed and PAX3–FOXO1 is induced in immortalized human myoblasts in an orthotopic xenograft model ( in vivo ). These regress when PAX3–FOXO1 expression is switched off; however, they recur in two ways: firstly, when cells re‐express the fusion gene (PAX3–FOXO1‐dependent); and, secondly, via a PAX3–FOXO1‐independent mechanism.…”