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

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

  • Cell Death Differ. 2025 Aug;32(8):1382-1396. doi: 10.1038/s41418-025-01487-7.
Jia-Nan Gong 1 2 3,  Tirta M Djajawi 4 5 6 7,  Donia M Moujalled 4 5 8,  Giovanna Pomilio 4 8,  Tiffany Khong 8 9 10,  Li-Ping Zhang 11,  Pasquale L Fedele 4 5 12 13,  Michael S Low 4 5 12 13,  Mary Ann Anderson 4 5 14,  Christopher D Riffkin 4,  Christine A White 4 5 15,  Ping Lan 4 5 16,  Guillaume Lessene 4 5 17,  Marco J Herold 4 5 6 7,  Andreas Strasser 4 5,  Andrew Spencer 8 9 10,  George Grigoriadis 13,  Andrew H Wei 4 5 8 9 14,  Mark F van Delft 4 5,  Andrew W Roberts 18 19 20,  David C S Huang 21 22
Affiliations

Affiliations

  • 1 Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia. gongjianan@cnilas.org.
  • 2 Departments of Medical Biology, University of Melbourne, Melbourne, VIC, Australia. gongjianan@cnilas.org.
  • 3 NHC Key Laboratory of Human Disease Comparative Medicine, The Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, National Human Diseases Animal Model Resource Center, National Center of Technology Innovation for Animal Model, Beijing, China. gongjianan@cnilas.org.
  • 4 Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia.
  • 5 Departments of Medical Biology, University of Melbourne, Melbourne, VIC, Australia.
  • 6 Olivia Newton-John Cancer Research Institute, Heidelberg, VIC, Australia.
  • 7 School of Cancer Medicine, La Trobe University, Bundoora, VIC, Australia.
  • 8 Australian Centre for Blood Diseases, Alfred Health-Monash University, Melbourne, VIC, Australia.
  • 9 Department of Clinical Haematology, The Alfred Hospital, Melbourne, VIC, Australia.
  • 10 Malignant Haematology and Stem Cell Transplantation, The Alfred Hospital, Melbourne, VIC, Australia.
  • 11 NHC Key Laboratory of Human Disease Comparative Medicine, The Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, National Human Diseases Animal Model Resource Center, National Center of Technology Innovation for Animal Model, Beijing, China.
  • 12 Department of Haematology, Monash Health, Clayton, VIC, Australia.
  • 13 School of Clinical Sciences at Monash Health, Monash University, Clayton, VIC, Australia.
  • 14 Clinical Haematology, The Royal Melbourne Hospital and Peter MacCallum Cancer Centre, Melbourne, VIC, Australia.
  • 15 oNKo-Innate, Melbourne, VIC, Australia.
  • 16 Institute for Advanced and Applied Chemical Synthesis, Jinan University, Jinan, China.
  • 17 Department of Pharmacology and Therapeutics, University of Melbourne, Melbourne, VIC, Australia.
  • 18 Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia. roberts@wehi.edu.au.
  • 19 Departments of Medical Biology, University of Melbourne, Melbourne, VIC, Australia. roberts@wehi.edu.au.
  • 20 Clinical Haematology, The Royal Melbourne Hospital and Peter MacCallum Cancer Centre, Melbourne, VIC, Australia. roberts@wehi.edu.au.
  • 21 Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia. huang_d@wehi.edu.au.
  • 22 Departments of Medical Biology, University of Melbourne, Melbourne, VIC, Australia. huang_d@wehi.edu.au.
PMID: 40204951 DOI: 10.1038/s41418-025-01487-7
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.

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