ALT neuroblastoma chemoresistance due to telomere dysfunction–induced ATM activation is reversible with ATM inhibitor AZD0156
Authors: Venkatesh Pilla Reddy
Trevor A Burrow
Joanne Wilson
Wan Hsi Chen
Cody Eslinger
Elaine Cadogan
Aaron Smith
Stephen T Durant
Balakrishna Koneru
Ashly Hindle
Monish Ram Makena
Thinh H Nguyen
Ahsan Farooqi
C Patrick Reynolds
Journal: Science Translational Medicine (2021)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: telomerase; alternative lengthening of telomeres (ALT); neuroblastoma; temozolomide; irinotecan
Affiliations: Cancer Center, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA., Early Oncology, R&amp;D AstraZeneca, Cambridge CB10 1XL, UK., Clinical Pharmacology and Quantitative Pharmacology, Biopharmaceuticals R&amp;D, AstraZeneca, Cambridge SG8 6EE, UK.

Abstract:  Cancers overcome replicative immortality by activating either telomerase or an alternative lengthening of telomeres (ALT) mechanism. ALT occurs in ~25% of high-risk neuroblastomas, and progression in patients with ALT neuroblastoma during or after front-line therapy is frequent and often fatal. Temozolomide + irinotecan is commonly used as salvage therapy for neuroblastoma. Patient-derived cell lines and xenografts established from patients with relapsed ALT neuroblastoma demonstrated de novo resistance to temozolomide + irinotecan [SN-38 in vitro, P &lt; 0.05; in vivo mouse event-free survival (EFS), P &lt; 0.0001] vs. telomerase-positive neuroblastomas. We observed that ALT neuroblastoma cells manifested constitutive ataxia-telangiectasia mutated (ATM) activation due to spontaneous telomere dysfunction which was not observed in telomerase-positive neuroblastoma cells. We demonstrated that induction of telomere dysfunction resulted in ATM activation that, in turn, conferred resistance to temozolomide + SN-38 (4.2-fold change in IC50, P &lt; 0.001). ATM knockdown (shRNA) or inhibition using a clinical-stage small-molecule inhibitor (AZD0156) reversed resistance to temozolomide + irinotecan in ALT neuroblastoma cell lines in vitro (P &lt; 0.001) and in four ALT xenografts in vivo (EFS, P &lt; 0.0001). AZD0156 showed modest to no enhancement of temozolomide + irinotecan activity in telomerase-positive neuroblastoma cell lines and xenografts. Ataxia telangiectasia and Rad3 related (ATR) inhibition using AZD6738 did not enhance temozolomide + SN-38 activity in ALT neuroblastoma cells. Thus, ALT neuroblastoma chemotherapy resistance occurs via ATM activation and is reversible with ATM inhibitor AZD0156. Combining AZD0156 with temozolomide + irinotecan warrants clinical testing for neuroblastoma. 

Retracted: No

PubPeer comments:
 My concern is related with figure 1A. In figure 1A, the authors report the survival fraction of 4 ALT neuroblastoma (NB) cell lines, and of 100 non-ALT NB cell lines, after the treatment at a single concentration of SN-38, topotecan, etoposide, 4-hydroperoxy cyclophosphamide (4-HC), L-PAM (melphalan), and carbo (carboplatin). The problem with figure 1A, is that the authors donu2019t provide the name of the cell lines used, to link to a corresponding survival fraction value. Therefore, no cell line identification is provided, nor has been provided in any supplementary figures S1 to S17, or data files S1 to S6. It is in this context that I have a doubt. Former members of the lab, which are not coauthors of this article, generated in and before 2014, an Excel table with IC90 values of 97 NB cell lines treated with SN-38, topotecan, 4-HC and L-PAM. Importantly, the version of the table is January 20th, 2015. To obtain an IC90 value you have to treat cells in a range of concentrations for a given drug. The Excel table provides IC90 values found experimentally, and their logical range of concentrations match perfectly with the fixed concentrations for each drug described in this article, of my concern. Importantly, the time of cell-drug interaction is a standard time used in that lab for everybody doing cytotoxicity assays, that is 96 hours. This time period was commonly used by the Pediatric Preclinical Testing Program (PPTP), as may be verified in PMID: 20922763. Importantly, as disclosed to me, any of the signing coauthors worked with L-PAM, carboplatin, or etoposide using 97 neuroblastoma cell lines. For instance, M.R.M. arrived January 2010 and finished in 2017, published 3 articles as a first author PMID: 28119491, and 33079733 using as cellular model T-cell lymphoid malignancies, and PMID: 30251395 studying DFMO in NB cells. A.F. arrived September 2010 and finished in 2015, publishing as first author one article PMID: 24792489 using NB cell lines studying alternative lengthening telomeres. A.H. arrived in 2013 and finished in 2019, publishing as first author an article, PMID: 33323683, where is tested O6-methylguanine in combination with temozolomide, and irinotecan against NB cell lines. T.H.N. arrived in 2014, and probably finished in 2019. He published as a first author an article PMID: 31484706, testing fenretinide and venetoclax against NB cell lines. W.S.C. arrived 2015, left in 2019, and performed u201cin vivou201d work. Finally, B.K. arrived in 2013, and continues nowadays performing molecular biology work. Certainly, it is true that other former members of the lab, not coauthors of this article, performed cytotoxicity assays treating dozens of NB cell lines with these drugs, generating the Excel table mentioned (97 NB cell lines). It is possible to provide names and their areas of expertise. My concern is about accountability. With the information provided, there are grounds to believe that the experimental data used to create figure 1A were generated by former lab members, which havenu2019t signed as coauthors, or even cited in acknowledgements. On the other hand, data may have been generated truly by the signing coauthors. To clarify this situation, and remove any doubt, the authors (especially the corresponding author), should provide experimental evidence of the u201cde novou201d drug testing, and link the name of the neuroblastoma cell line tested with the survival fraction for each drug. ,