American Association for Cancer Research
Browse

Supplementary Figures from TDP1 and PARP1 Deficiency Are Cytotoxic to Rhabdomyosarcoma Cells

Download (381.13 kB)
journal contribution
posted on 2023-04-03, 16:04 authored by Hok Khim Fam, Cheryl Walton, Sheetal A. Mitra, Miraj Chowdhury, Nichola Osborne, Kunho Choi, Guobin Sun, Patrick C.W. Wong, Maureen J. O'Sullivan, Gulisa Turashvili, Samuel Aparicio, Timothy J. Triche, Mason Bond, Catherine J. Pallen, Cornelius F. Boerkoel

Supplementary Figures - PDF file 381K, S1. Specificity of the Tdp1 antiserum. S2. Tdp1 expression in pediatric solid tumors. S3. Generation of transient and constitutive TDP1 knockdown by treatment with siRNA and shRNA and assay for sensitivity of Tdp1-deficient MEFs to DNA breaks. S4. Detection of Tdp1 cleavage activity in cell lysates by kinetic determination of Tdp1 cleavage activity in nuclear lysates using a real-time fluorophorequencher coupled DNA substrate. S5. Effect of cellular growth rate vs. Tdp1 expression on sensitivity to CPT treatment. S6. Analysis of PARP-1 and PARP-2 expression levels in RMS and normal myoblasts. S7. PARP-1 inhibitor treatment sensitizes RMS cell lines to CPT. S8. RMS cells develop resistance to PARP-1 inhibitors posttreatment with TDP1 shRNA

History

ARTICLE 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.Implications: Selective targeting of TDP1 and/or PARP-1 in rhabdomyosarcoma induces cytotoxicity and sensitizes to DNA damaging agents. Mol Cancer Res; 11(10); 1179–92. ©2013 AACR.