Registered report: A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations

Abstract

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a substantial number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of experiments from “A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations” by Sharma and colleagues, published in Cell in 2010 (Sharma et al., 2010). Sharma and colleagues demonstrated that prolonged exposure of cancer cells to TKIs give rise to small populations of “drug tolerant persisters” (DTPs) (Figure 1B-C) that were reversed during subsequent maintenance under drug-free conditions (Figures 1E, 2B and 2E). DTPs exhibited reduced histone acetylation and sensitivity to HDAC inhibitors (HDIs) (Figure 4A-B). Drug sensitivity was restored with co-treatment of either HDIs or an IGF-1R inhibitor, in combination with TKIs (Figure 5A-B). Inhibition of IGF-1R activation also led to decreased KDM5A expression and restoration of H3K4 methylation, suggesting a direct link between the IGF-1R signaling pathway and KDM5A function (Figure 7A, 7C, and 7I). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.DOI: http://dx.doi.org/10.7554/eLife.09462.001

KDM5A and KDM5B histone-demethylases contribute to HU-induced replication stress response and tolerance

“…KDM5A is involved in the emergence of the so-called DTPs in cancer ( Sharma et al, 2010 ; Vinogradova et al, 2016 ). KDM5A upregulation allows the emergence of cells tolerant to cisplatin, a DNA damaging agent ( Haven et al, 2016 ; Vinogradova et al, 2016 ). In contrast, we show that KDM5A levels do not increase in cells becoming resistant to the replication stress-inducer HU.…”

Experiments from unfinished Registered Reports in the Reproducibility Project: Cancer Biology

“…Importantly, conducting experiments under different conditions could help provide insight into what conditions are necessary to obtain the original results. Interestingly, this was a point discussed during review of the Registered Report ( Haven et al, 2016 ). To summarize, this replication attempt obtained preliminary outcomes although the experiments were not continued and thus these data do not address whether DTPs maintained viability through IGF-1 receptor signaling and an altered chromatin state dependent on the histone demethylase KDM5A.…”

“…The decision to stop any individual experiment was influenced partly by the mundane technical or unanticipated methodological challenges unique to that experiment, and partly by factors related to time and cost estimates across all the replications that were ongoing as part of the Reproducibility Project: Cancer Biology as a whole. This includes four Registered Reports ( Blum and LaBarge, 2014 ; Evans and Griner, 2015 ; Greenfield and Griner, 2014 ; Incardona et al, 2015 ) where experimental work was initiated but ultimately stopped without results, one Registered Report ( Bhargava et al, 2016b ) where the results were submitted as a Replication Study and rejected (which has been posted as a preprint [ Pelech et al, 2021 ]), two Registered Reports ( Haven et al, 2016 ; Raouf et al, 2015 ) where experimental work began with preliminary outcomes although experiments were not completed, and five Registered Reports ( Bhargava et al, 2016a ; Chroscinski et al, 2015 ; Richarson et al, 2016 ; Sharma et al, 2016a ; Sharma et al, 2016b ) where some experiments were completed. The present paper reports the preliminary outcomes and completed experiments for those seven partially completed attempts.…”

“…This is complicated by the challenges persister cell populations pose to therapy, drawing parallels to similar challenges in fighting microbial infections, with new bioinformatic approaches to decipher the complexities of heterogeneous cell populations, with a focus on drug-tolerant cells in cancers needed ( Vallette et al, 2019 ). As part of the Reproducibility Project: Cancer Biology, we published a Registered Report ( Haven et al, 2016 ) that described how we intended to replicate selected experiments from the paper ‘A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations’ ( Sharma et al, 2010 ).…”

“…We attempted to independently replicate experiments that tested the phenotype of a small subpopulation of ‘drug-tolerant persister’ cells (DTPs) that are generated when PC9 cells, a protein-tyrosine kinase inhibitor (TKI)-sensitive non-small cell lung cancer cell line, is exposed to high concentrations of TKIs. As described in Protocol 1 in the Registered Report ( Haven et al, 2016 ), we first attempted to determine the growth characteristics of DTPs. When attempting to determine the percentage of erlotinib-treated PC9 cells that became DTPs we used the same concentration (2 µM) and timing (9 days with media and drug replaced every 3 days) as the original study.…”