MYCN induces cell-specific tumorigenic growth in RB1-proficient human retinal organoid and chicken retina models of retinoblastoma

Abstract

Retinoblastoma is a rare, intraocular paediatric cancer that originates in the neural retina and is most frequently caused by bi-allelic loss of RB1 gene function. Other oncogenic mutations, such as amplification and increased expression of the MYCN gene, have been found even with proficient RB1 function. In this study, we investigated whether MYCN over-expression can drive carcinogenesis independently of RB1 loss-of-function mutations. The aim was to elucidate the events that result in carcinogenesis and identify the cancer cell-of-origin. We used the chicken retina, a well-established model for studying retinal neurogenesis, and established human embryonic stem cell-derived retinal organoids as model systems. We over-expressed MYCN by electroporation of piggyBac genome-integrating expression vectors. We found that over-expression of MYCN induced tumorigenic growth with high frequency in RB1-proficient chicken retinas and human organoids. In both systems, the tumorigenic cells expressed markers for undifferentiated cone photoreceptor/horizontal cell progenitors. The over-expression resulted in metastatic retinoblastoma within 7–9 weeks in chicken. Cells expressing MYCN could be grown in vitro and, when orthotopically injected, formed tumours that infiltrated the sclera and optic nerve and expressed markers for cone progenitors. Investigation of the tumour cell phenotype determined that the potential for neoplastic growth was embryonic stage-dependent and featured a cell-specific resistance to apoptosis in the cone/horizontal cell lineage, but not in ganglion or amacrine cells. We conclude that MYCN over-expression is sufficient to drive tumorigenesis and that a cell-specific resistance to apoptosis in the cone/horizontal cell lineage mediates the cancer phenotype.

Mutation spectrum of retinoblastoma patients in Vietnam

“…This controversy has been pointed out recently by Gallie's research group as they also encountered RB patients bearing not Rb1 mutations, in which they detected high copy numbers of the MYCN gene, which is a member of MYC transcription factors (Rushlow et al, 2013). More recent research was conducted and revealed more about the mechanism and interactions between the over‐expression of MYCN and retinoblastoma (Blixt et al, 2022; Zugbi et al, 2020). Despite a low population of patients with this new subtype of retinoblastoma, MYCN amplification has been shown to cope with much lower sensitivity with standard chemotherapy of RB, hence, highly dangerous as no specific clinical guidelines to aid or salvage patient's vision (Zugbi et al, 2020).…”

“…T A B L E 2 (Continued) and interactions between the over-expression of MYCN and retinoblastoma (Blixt et al, 2022;Zugbi et al, 2020). Despite a low population of patients with this new subtype of retinoblastoma, MYCN amplification has been shown to cope with much lower sensitivity with standard chemotherapy of RB, hence, highly dangerous as no specific clinical guidelines to aid or salvage patient's vision (Zugbi et al, 2020).…”

A flexible electrode Array for genetic transfection of different layers of the retina by electroporation

“…To overcome the cargo limitation of AAV or other similar viral vectors, many efforts have been paid to test the applicability of non-viral retinal gene delivery, including ion electroosmosis, 11 particle bombardment, 12 electroporation (EP), 13 magnetic transfection, 14 photoporation 15 and acoustic perforation. 16 In all non-viral delivery methods, EP has emerged as one of the most likely alternatives to AAV [17][18][19][20][21] because of its high transfection efficiency 22,23 and nontoxicity. 24 Electroporation, in which high-intensity electric fields are applied to destabilize cell membranes to facilitate transmembrane particle transport, 25 has been demonstrated to be capable of delivering DNA plasmids into the retina.…”

“…To overcome the cargo limitation of AAV or other similar viral vectors, many efforts have been paid to test the applicability of non-viral retinal gene delivery, including ion electroosmosis, 11 particle bombardment, 12 electroporation (EP), 13 magnetic transfection, 14 photoporation 15 and acoustic perforation. 16 In all non-viral delivery methods, EP has emerged as one of the most likely alternatives to AAV 17–21 because of its high transfection efficiency 22,23 and nontoxicity. 24…”

Inhibition of high level E2F in a RB1 proficient MYCN overexpressing chicken retinoblastoma model normalizes neoplastic behaviour

“…The MYCN A amplified RB1 -proficient retinoblastoma tumours also have distinct molecular signatures from RB1 -deficient tumours [ 5 ]. Tumorigenic growth can be induced with high frequency when MYCN is over-expressed in RB1 -proficient human retinal organoids as well as in embryonic chicken retina in vivo [ 7 ]. In both systems, the tumorigenic cells are anaplastic and express markers for active proliferation and undifferentiated cone photoreceptors (cPR) [ 7 ].…”