Functional genomics identifies new synergistic therapies for retinoblastoma

Arthur Aubry,Mohammad Ahmad,Jason Moffat,Rod Bremner,Madhavan Jagadeesan,Suying Lu,Kevin R Brown,Katherine Huang,Vikas Khetan,Joel D Pearson,Jeffrey L Wrana,Izhar Livne-Bar,Troy Ketela,Tao Yu

doi:10.1038/s41388-020-1372-7

Abstract

Local intravitreal or intra-arterial chemotherapy has improved therapeutic success for the pediatric cancer retinoblastoma (RB), but toxicity remains a major caveat. RB initiates primarily with RB1 loss or, rarely, MYCN amplification, but the critical downstream networks are incompletely understood. We set out to uncover perturbed molecular hubs, identify synergistic drug combinations to target these vulnerabilities, and expose and overcome drug resistance. We applied dynamic transcriptomic analysis to identify network hubs perturbed in RB versus normal fetal retina, and performed in vivo RNAi screens in RB1null and RB1wt;MYCNamp orthotopic xenografts to pinpoint essential hubs. We employed in vitro and in vivo studies to validate hits, define mechanism, develop new therapeutic modalities, and understand drug resistance. We identified BRCA1 and RAD51 as essential for RB cell survival. Their oncogenic activity was independent of BRCA1 functions in centrosome, heterochromatin, or ROS regulation, and instead linked to DNA repair. RAD51 depletion or inhibition with the small molecule inhibitor, B02, killed RB cells in a Chk1/Chk2/p53-dependent manner. B02 further synergized with clinically relevant topotecan (TPT) to engage this pathway, activating p53–BAX mediated killing of RB but not human retinal progenitor cells. Paradoxically, a B02/TPT-resistant tumor exhibited more DNA damage than sensitive RB cells. Resistance reflected dominance of the p53–p21 axis, which mediated cell cycle arrest instead of death. Deleting p21 or applying the BCL2/BCL2L1 inhibitor Navitoclax re-engaged the p53–BAX axis, and synergized with B02, TPT or both to override resistance. These data expose new synergistic therapies to trigger p53-induced killing in diverse RB subtypes.

Highlights

Retinoblastoma (RB) is an aggressive cancer of the infant retina initiated by homozygous RB1 tumor suppressor gene inactivation or, rarely, by MYCN amplification [1,2,3]
Network analysis prioritized 61 candidates for an in vivo shRNA screen in orthotopic xenografts of two RB1null lines, which were compared with 86 controls
Validation screens were run with 21 primary hits and 34 controls in three RB1null lines and an RB1wt;MYCNamp line, yielding 15 genes that were positive in both screens

Summary

Introduction

Retinoblastoma (RB) is an aggressive cancer of the infant retina initiated by homozygous RB1 tumor suppressor gene inactivation or, rarely, by MYCN amplification [1,2,3]. Blocking activation of the SCFSKP2 complex with the neddylation inhibitor MLN4924 (Pevonedistat) shows promise as a new RB therapy [17] Such studies illustrate the value in dissecting networks that drive RB cell growth and survival to identify novel therapeutic strategies. To identify high value candidates for in vivo screens, we employed Dynamic Network Modularity (DyNeMo) This tool combines transcriptomic and protein network information to define whether the stoichiometry of co-expressed hubs and partners is altered in cancer vs normal cells. DyNeMo pinpointed disrupted hubs influencing outcome in breast cancer [21] Applying this approach to RB transcriptome data, we identify candidates, establish hits through in vivo RNAi screens in RB1null and MYCNamp tumors, and exploit those insights to develop several drug combinations that synergistically kill RB. We identify a resistance mechanism and a strategy to resensitize affected RB cells

Results

Discussion

Materials and methods

Compliance with ethical standards