TDP1 and PARP1 Deficiency Are Cytotoxic to Rhabdomyosarcoma Cells

Hok Khim Fam,Cornelius F Boerkoel,Kunho Choi,Maureen J O'Sullivan,Gulisa Turashvili,Guobin Sun,Mason Bond,Miraj Chowdhury,Timothy J Triche,Nichola Osborne,Samuel Aparicio,Patrick C.W Wong,Sheetal A Mitra,Cheryl Walton,Catherine J Pallen

doi:10.1158/1541-7786.mcr-12-0575

Abstract

Rhabdomyosarcoma is the most common soft tissue sarcoma in children. Metastatic rhabdomyosarcoma in children has a 5-year event-free survival rate of <30%, and a recent clinical trial with irinotecan, a topoisomerase I inhibitor, failed to improve outcome. Therefore, it was surmised that failure of irinotecan may be the result of overexpression of the DNA repair enzyme tyrosyl-DNA phosphodiesterase (TDP1), which processes topoisomerase I-DNA complexes resulting from topoisomerase I inhibitor treatment. Using human tissue microarrays and gene expression arrays, a marked overexpression of TDP1 protein and mRNA in RMS tumors was observed. Critically, knockdown of TDP1 or inhibition of poly(ADP-ribose) polymerase-1 (PARP-1), an enzyme in the same complex as TDP1, sensitized rhabdomyosarcoma cell lines to analogues of irinotecan. Interestingly, BRCA1/2 mutations or altered expression was not detectable in rhabdomyosarcoma cells; however, TDP1 knockdown and PARP-1 inhibition alone were cytotoxic to a subset of rhabdomyosarcoma cells, suggesting that they harbor genetic lesions in DNA repair components that have synthetic lethal interactions with loss of TDP1 or PARP1 function. Furthermore, culturing embryonal rhabdomyosarcoma cells in serum/nutrient-restricted medium increased cellular cytotoxicity upon PARP-1 inhibition and was intrinsically cytotoxic to alveolar, though not embryonal rhabdomyosarcoma cells. The results of these studies suggest a compensatory role for TDP1 in rhabdomyosarcoma after topoisomerase-I based therapy and further demonstrate that TDP1 knockdown, PARP-1 inhibition, and dietary restriction have therapeutic validity. Selective targeting of TDP1 and/or PARP-1 in rhabdomyosarcoma induces cytotoxicity and sensitizes to DNA damaging agents.

Highlights

Conventional chemotherapeutic agents have targeted general cellular processes such as nucleotide synthesis, DNA replication, and cell division
Tdp1 is highly expressed in rhabdomyosarcoma tumors To determine whether Tdp1 is expressed in rhabdomyosarcoma and other pediatric solid tumors, we immunohistochemically screened a pediatric tumor microarray consisting of 24 embryonal rhabdomyosarcoma, 18 alveolar rhabdomyosarcoma, 12 ganglioneuroma, 23 neuroblastoma, 10 Ewing sarcoma, 9 medulloblastoma, and 24 Wilms tumor readable tissue cores
Investigating whether TDP1 knockdown and poly(ADP-ribose) polymerase-1 (PARP-1) inhibition are additive, we found that the combination of 70% to 90% knockdown of TDP1 and 10 mmol/L Rucaparib decreased A204, Birch, RH30, and CW9019 cell proliferation more than either treatment alone; this treatment had no effect on control myoblasts (Fig. 4A)

Summary

Introduction

Conventional chemotherapeutic agents have targeted general cellular processes such as nucleotide synthesis, DNA replication, and cell division. Authors' Affiliations: 1Child and Family Research Institute; and 2Department of Medical Genetics, University of British Columbia, Vancouver, Canada; 3Department of Pathology, Children's Hospital Los Angeles, Los Angeles, CA; 4Department of Pediatrics, University of British Columbia, Vancouver, Canada; 5Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada; †6National Children's Research Centre and Histology Laboratory, Our Lady's Children’s Hospital, Crumlin, Dublin; †7Trinity College Dublin, Ireland; 8Molecular Oncology and Breast Cancer Program, BC Cancer Research Centre, Vancouver, BC, Canada. Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/).

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