Small molecules that selectively inhibit growth of MYCN^ARB1^+/+ retinoblastoma cells

Abstract

Purpose: Retinoblastoma is the most common pediatric ocular cancer, traditionally thought to be always caused by loss of both alleles of the RB1 tumor suppressor gene. However, recent work showed that amplification of the MYCN oncogene can also spur the development of retinoblastoma in a fraction of unilateral patients with normal RB1 alleles. Here, our aim was to identify small molecules from a library of known bioactive compounds that show selective growth inhibition of a MYCN^ARB1^+/+ retinoblastoma cell line (RB3823) over a MYCN^ARB1^-/- cell line (Y79). Such molecules may be leads for therapy of MYCN^ARB1^+/+ retinoblastoma, and will further elucidate the molecular differences between these two subtypes of retinoblastoma.

Methods: Compounds from the LOPAC1280 library were tested to judge their effect at 10 μM on cell proliferation in both Y79 and RB3823 cell lines using Alamar Blue after a 48 hour incubation in 384 well format. Compounds that reduced cell proliferation by at least 40% in one or both cell lines were tested twice more in both cell lines to confirm efficacy. Dose response testing was conducted using compound concentrations ranging from 100 pM to 1 mM in 384 well format.

Results: In initial screening, 95 compounds showed cytotoxicity to one or both cell lines. Secondary screening yielded 9 compounds that reduced RB3823 proliferation by at least 40% compared to untreated cells. Confirmatory screening identified 6 of those 9 compounds that reduced RB3823 proliferation by at least twice that of Y79. In dose-response assays of 5 of these compounds, one compound showed reproducibly higher efficacy in RB3823 than Y79. Dimethoxy-naphthoquinone (DMNQ) had a GI₅₀ in RB3823 at least 3 times lower than that for Y79. DMNQ is an oxidizing quinone that is thought to act by depleting cellular reduction potential leading to oxidation of proteins and DNA, causing cell death.

Conclusions: Given our findings, potential may exist for selective treatment options for aggressive, MYCN-driven retinoblastoma. DMNQ has not been used in humans, but a DMNQ derivative has previously shown efficacy in a mouse lung carcinoma xenograft. Further characterization of DMNQ’s mode of action in the retinoblastoma context is required, but the door is open to the family of quinones as potential treatment options for MYCN^ARB1^+/+ retinoblastoma.

Keywords: 703 retinoblastoma • 503 drug toxicity/drug effects