Re-appraising assays on permeabilized blood cancer cells testing venetoclax or other BH3 mimetic agents selectively targeting pro-survival BCL2 proteins

BH3 mimetic drugs that selectively target the pro-survival BCL2 proteins are highly promising for cancer treatment, most notably for treating blood cancers. Venetoclax, which inhibits BCL2, is now approved for treating chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). Preferably, robust and validated assays would identify patients most likely to benefit from therapy with venetoclax itself or with inhibitors of other pro-survival proteins. A sophisticated method that has been developed is the BH3 profiling assay. In this assay, permeabilized, instead of intact, cells are treated for a few hours with inhibitors of the pro-survival BCL2 proteins, and the resultant mitochondrial depolarization measured. Sensitivity to a specific inhibitor (e.g., venetoclax or other BH3 mimetics) is then used to infer the reliance of a tumor (e.g., CLL) on one or more pro-survival BCL2 proteins. However, we found that this methodology cannot reliably identify such dependencies. In part, this is because almost all cells express multiple pro-survival BCL2 proteins that restrain BAX and BAK which must be inhibited before mitochondrial depolarization and apoptosis can proceed. Using genetic and pharmacological tools across multiple cell line models of blood cancer, we demonstrated that selective BCL2 inhibitors have important flow-on effects that includes the redistribution of BH3-only proteins to ancillary pro-survival proteins not directly engaged by the inhibitor. These secondary effects, critical to the biological action of selective inhibitors, were not accurately recapitulated in permeabilized cells, probably due to the limited time frame possible in such assays or the altered biophysical conditions when cells are permeabilized. While we could consistently define the sensitivity of a tumor cell to a particular BH3 mimetic drugs using intact cells, this was not reliable with permeabilized cells. These studies emphasize the need to carefully evaluate assays on permeabilized cells undertaken with inhibitors of the pro-survival BCL2 proteins.

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Publication Details

Journal
Cell Death and Differentiation
Published
2025-04-09
DOI
https://doi.org/10.1038/s41418-025-01487-7
Citations
1
Primary Topic
Chronic Lymphocytic Leukemia Research
Type
article
Field-Weighted Citation Impact
1.63

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article

Re-appraising assays on permeabilized blood cancer cells testing venetoclax or other BH3 mimetic agents selectively targeting pro-survival BCL2 proteins

Michael Löw, Andrew Spencer, Andrew H. Wei, Tirta M. Djajawi et al.
1 citations
Cell Death and Differentiation
Chronic Lymphocytic Leukemia Research
1.63
article

Re-appraising assays on permeabilized blood cancer cells testing venetoclax or other BH3 mimetic agents selectively targeting pro-survival BCL2 proteins

Michael Löw, Andrew Spencer, Andrew H. Wei, Tirta M. Djajawi, Donia M. Moujalled, Jianan Gong, Mark F. van Delft, Tiffany Khong, Andrew W. Roberts, David C.S. Huang, Andreas Strasser, Pasquale L. Fedele, Ping Lan, Marco J. Herold, Guillaume Lessène, Giovanna Pomilio, Mary Ann Anderson, Liping Zhang, Christine White, Christopher D. Riffkin, George Grigoriadis
article en
1 citations

Abstract

BH3 mimetic drugs that selectively target the pro-survival BCL2 proteins are highly promising for cancer treatment, most notably for treating blood cancers. Venetoclax, which inhibits BCL2, is now approved for treating chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML). Preferably, robust and validated assays would identify patients most likely to benefit from therapy with venetoclax itself or with inhibitors of other pro-survival proteins. A sophisticated method that has been developed is the BH3 profiling assay. In this assay, permeabilized, instead of intact, cells are treated for a few hours with inhibitors of the pro-survival BCL2 proteins, and the resultant mitochondrial depolarization measured. Sensitivity to a specific inhibitor (e.g., venetoclax or other BH3 mimetics) is then used to infer the reliance of a tumor (e.g., CLL) on one or more pro-survival BCL2 proteins. However, we found that this methodology cannot reliably identify such dependencies. In part, this is because almost all cells express multiple pro-survival BCL2 proteins that restrain BAX and BAK which must be inhibited before mitochondrial depolarization and apoptosis can proceed. Using genetic and pharmacological tools across multiple cell line models of blood cancer, we demonstrated that selective BCL2 inhibitors have important flow-on effects that includes the redistribution of BH3-only proteins to ancillary pro-survival proteins not directly engaged by the inhibitor. These secondary effects, critical to the biological action of selective inhibitors, were not accurately recapitulated in permeabilized cells, probably due to the limited time frame possible in such assays or the altered biophysical conditions when cells are permeabilized. While we could consistently define the sensitivity of a tumor cell to a particular BH3 mimetic drugs using intact cells, this was not reliable with permeabilized cells. These studies emphasize the need to carefully evaluate assays on permeabilized cells undertaken with inhibitors of the pro-survival BCL2 proteins.

Cell Death and DifferentiationVol. 32(8)
The Royal Melbourne Hospital (AU), The University of Melbourne (AU), Walter and Eliza Hall Institute of Medical Research (AU), La Trobe University (AU), Chinese Academy of Medical Sciences & Peking Union Medical College (CN), Peter MacCallum Cancer Centre (AU), The Alfred Hospital (AU), Melbourne Bioinformatics (AU), University of Jinan (CN), Monash Alfred Psychiatry Research centre (AU), Monash Health (AU), Institute of Laboratory Animal Science (CN), Olivia Newton-John Cancer Wellness & Research Centre (AU), Alfred Health (AU), Monash University (AU), RMIT University (AU)
Leukemia and Lymphoma Society, Australian Government, Australian Cancer Research Foundation, National Natural Science Foundation of China, Chinese Academy of Medical Sciences, Natural Science Foundation for Young Scientists of Shanxi Province, Medical Research Council, National Health and Medical Research Council, Beijing Nova Program, Young Scientists Fund
Good health and well-being
Openalex Percentile: Top 19%
Chronic Lymphocytic Leukemia Research
1.63
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