The most frequent focal alterations in human retinoblastoma are mutations in the tumor-suppressor gene retinoblastoma (RB) and amplification of the oncogene MYCN. Whether MYCN overexpression drives retinoblastoma has not been assessed in model systems. Here, we have shown that Rb inactivation collaborates strongly with MYCN overexpression and leads to retinoblastoma in mice. Overexpression of human MYCN in the context of Rb inactivation increased the expression of MYC-, E2F-, and ribosome-related gene sets, promoted excessive proliferation, and led to retinoblastoma with anaplastic changes. We then modeled responses to MYCN-directed therapy by suppressing MYCN expression in MYCN-driven retinoblastomas. Initially, MYCN suppression led to proliferation arrest and partial tumor regression with loss of anaplasia. However, over time, retinoblastomas reemerged, typically without reactivation of human MYCN or amplification of murine Mycn. A subset of returning retinoblastomas showed genomic amplification of a Mycn target gene encoding the miR cluster miR-17~92, while most retinoblastomas reemerged without clear genetic alterations in either Mycn or known Mycn targets. This Rb/MYCN model recapitulates key genetic driver alterations seen in human retinoblastoma and reveals the emergence of MYCN independence in an initially MYCN-driven tumor.
Authors
Nan Wu, Deshui Jia, Breanna Bates, Ryan Basom, Charles G. Eberhart, David MacPherson
(A) Strategy to overexpress MYCN in Rb or Rb/p107-deficient retina. Retina-specific Pax6 α enhancer Cre (Pax6 α Cre) drives the expression of rtTA in the developing retina. The TRE-MYCN allele allowed for DOX-dependent expression of MYCN and luciferase in the retina. Image shows an in vivo assessment of bioluminescence in a tumor-bearing animal. (B) Kaplan-Meier curve shows the time to externally evident retinoblastoma in Rb/p107 (Rb/p107 DKO) compared with Rb/p107/TET-MYCN (Rb/p107 DKO MYCN) mice. The time to tumor presence in the first eye (unilateral curve) and second eye (bilateral curve) is shown. (C) H&E-stained eye from an Rb/p107/TET-MYCN mouse at P12, showing extensive retinoblastoma. The inset image is of tumor cells (original magnification, ×100) and shows Homer Wright rosettes typical of human and mouse retinoblastomas. O.N., optic nerve. (D) Kaplan-Meier curve showing the time to unilateral and then bilateral retinoblastoma formation in Rb/TET-MYCN (Rb KO MYCN) mice. (E) H&E staining of retinoblastoma-containing eye from an Rb/TET-MYCN model (inset: original magnification, ×100). Black scale bar: 500 μm; white scale bar: 25 μm.