Research ArticleHematologyOncology Open Access | 10.1172/JCI170169

Stephen Jun Fei Chong,1 Fen Zhu,1 Olga Dashevsky,1 Rin Mizuno,1 Jolin X.H. Lai,2 Liam Hackett,1 Christine E. Ryan,1 Mary C. Collins,1 J. Bryan Iorgulescu,1,3 Romain Guièze,1 Johany Penailillo,1,3 Ruben Carrasco,1,3 Yeonjoo C. Hwang,4 Denise P. Muñoz,4 Mehdi Bouhaddou,5 Yaw Chyn Lim,6 Catherine J. Wu,1 John N. Allan,7 Richard R. Furman,7 Boon Cher Goh,6 Shazib Pervaiz,2 Jean-Philippe Coppé,4 Constantine S. Mitsiades,1 and Matthew S. Davids1

1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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1Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA.

2Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.

3Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA.

4Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California, USA.

5Department of Microbiology, Immunology and Molecular Genetics, UCLA, Los Angeles, California, USA.

6Cancer Science Institute, National University of Singapore, Singapore.

7Division of Hematology and Medical Oncology, Weill Cornell Medicine, New York, New York, USA.

Address correspondence to: Matthew S. Davids, Department of Medical Oncology, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston, Massachusetts 02215, USA. Phone: 617.632.6331; Email: matthew_davids@dfci.harvard.edu.

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Published September 26, 2023 - More info

Published in Volume 133, Issue 22 on November 15, 2023
J Clin Invest. 2023;133(22):e170169. https://doi.org/10.1172/JCI170169.
© 2023 Chong et al. This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Published September 26, 2023 - Version history
Received: March 3, 2023; Accepted: September 19, 2023 View PDF Abstract

The B cell leukemia/lymphoma 2 (BCL-2) inhibitor venetoclax is effective in chronic lymphocytic leukemia (CLL); however, resistance may develop over time. Other lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax. Although genomic resistance mechanisms such as BCL2 mutations have been described, this probably only explains a subset of resistant cases. Using 2 complementary functional precision medicine techniques — BH3 profiling and high-throughput kinase activity mapping — we found that hyperphosphorylation of BCL-2 family proteins, including antiapoptotic myeloid leukemia 1 (MCL-1) and BCL-2 and proapoptotic BCL-2 agonist of cell death (BAD) and BCL-2 associated X, apoptosis regulator (BAX), underlies functional mechanisms of both intrinsic and acquired resistance to venetoclax in CLL and DLBCL. Additionally, we provide evidence that antiapoptotic BCL-2 family protein phosphorylation altered the apoptotic protein interactome, thereby changing the profile of functional dependence on these prosurvival proteins. Targeting BCL-2 family protein phosphorylation with phosphatase-activating drugs rewired these dependencies, thus restoring sensitivity to venetoclax in a panel of venetoclax-resistant lymphoid cell lines, a resistant mouse model, and in paired patient samples before venetoclax treatment and at the time of progression.

Graphical Abstract Introduction

The incorporation of the B cell leukemia/lymphoma 2 (BCL-2) inhibitor venetoclax into the treatment paradigm for chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML) has revolutionized the treatment of these diseases (1, 2). However, this therapy is not thought to be curative, and with longer-term follow-up, resistance has been increasingly recognized as a major challenge. Furthermore, common lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax (3). Thus, there is an urgent need to identify potential resistance mechanisms and alternative therapeutic approaches to overcome them. While acquired venetoclax resistance is explained in part by genetic mechanisms, such as the BCL2 p.G101V that reduces binding of venetoclax to BCL-2, these mutations are only observed in about half of the patients with CLL on long-term venetoclax therapy, and when these mutations do occur, they are often found at a low variant allele frequency (VAF) (4–6). Thus, the venetoclax resistance mechanism(s) in 2 of the most common lymphoid malignancies (CLL and DLBCL) remain incompletely understood, particularly with regard to functional (i.e., nongenetic) resistance mechanisms.

Prior studies have suggested that posttranslational modification(s) of the BCL-2 family proteins could alter their functions in governing the intrinsic apoptotic pathway. In particular, phosphorylation of antiapoptotic BCL-2 at serine 70 (S70pBCL-2) has been shown to promote cancer cell survival in response to proapoptotic stimuli and to enhance its sequestration of the proapoptotic effector BAX (7–12). Phosphorylation of the antiapoptotic protein myeloid leukemia 1 (MCL-1) at threonine 163 (T163pMCL-1) has been shown to increase MCL-1 protein stability, thereby increasing the sequestration of proapoptotic proteins and inducing chemoresistance in lymphoid malignancies (13–16). In contrast, phosphorylation of BCL-2 agonist of cell death (BAD) at serine 112 (S112pBAD) or BCL-2 associated X, apoptosis regulator (BAX) at serine 184 (S184pBAX) inhibits its proapoptotic function (17–20). Given their relevance in promoting survival and chemoresistance, we hypothesized that increased S70pBCL-2, T163pMCL-1, S112pBAD, and/or S184pBAX may be present in resistant tumor cells from patients with lymphoid malignancies and, if so, may be an important factor in driving venetoclax resistance.

The protein phosphatase 2A (PP2A) is a serine/threonine phosphatase that dephosphorylates its target proteins. Numerous studies have demonstrated a tumor suppressor role of PP2A, and PP2A inactivation contributes to disease progression in several hematologic malignancies (12, 21–26). Recent work has linked PP2A inactivation to increased S70pBCL-2 and suggested a strong association between S70pBCL-2 and poor prognosis in certain lymphomas (12). Similarly, the stability and function of MCL-1, BAD, and BAX are governed in part by PP2A (18, 19, 27–30). Thus, we hypothesized that drugs that activate PP2A could reverse venetoclax resistance via dephosphorylation of BCL-2, MCL-1, BAD, and BAX, thereby resensitizing tumor cells to BCL-2 inhibition.

Here, we report data comparing venetoclax-sensitive with venetoclax-resistant malignant lymphoid cells, including a panel of cell lines, a lymphoid mouse model, and primary samples from patients with CLL before venetoclax treatment and at the time of progression while on venetoclax. We found that hyperphosphorylated BCL-2 family proteins were a crucial mechanism of intrinsic and acquired venetoclax resistance in lymphoid malignancies and we provide evidence supporting future clinical investigation of a therapeutic strategy utilizing PP2A-activating drugs (PADs) to resensitize resistant cells to venetoclax.

Results

BCL-2 family protein phosphorylation contributes to intrinsic and acquired venetoclax resistance in lymphoid malignancies. To evaluate the potential contribution of BCL-2 family protein phosphorylation to venetoclax resistance, we examined lymphoid malignant cell lines that were intrinsically sensitive and resistant to venetoclax. We first identified 3 DLBCL cell lines — double-hit Su-DHL4, germinal center B cell TOLEDO, and activated B cell TMD8 — that were intrinsically resistant to venetoclax in relation to the intrinsically sensitive DLBCL cell line OCI-Ly1-S (Figure 1A). We also used 2 cell lines with acquired venetoclax resistance, generated from their parental OCI-Ly1-S and Su-DHL4 (OCI-Ly1-R, Su-DHL4-R) lines (Figure 1A).

Figure 1

Venetoclax-resistant malignant lymphoid cells display hyperphosphorylated BCL-2 family proteins. (A) Cell viability of intrinsically resistant Su-DHL4 (n = 3), TOLEDO (n = 4), and TMD8 (n = 4) cells or acquired-resistance OCI-Ly1-R (n = 3) and Su-DHL4-R (n = 3) cells in comparison with OCI-Ly1-S or Su-DHL4 cells following treatment with increasing concentrations of ABT199/venetoclax (Ven) at 48 hours, measured by CTG assay. (B) Western blots showing increased T163pMCL-1, S70pBCL-2, S112pBAD, and MCL-1 levels in intrinsically resistant and acquired-resistance malignant lymphoid cells. Bands were quantified by ImageJ software (NIH). Normalized expression values derived from T163pMCL-1/β-actin, S70pBCL-2/BCL-2, S112pBAD/BAD, and MCL-1/β-actin are displayed below the targets in this and subsequent specific figures. (C) Western blots showing increased T163pMCL-1 (n = 8), S70pBCL-2 (n = 9), S112pBAD (n = 6), and MCL-1 (n = 9) levels in primary CLL samples from patients on venetoclax with progressive disease (PD) compared with paired pre-venetoclax primary CLL patient samples (human, in vivo). Quantification is displayed in Supplemental Figure 1C. The sample number is different due to sample availability. PT, patient.

Next, we measured the phosphorylation status of the BCL-2 family members to evaluate for an association between their phosphorylation levels and resistance to venetoclax. We observed a selective increase in T163pMCL-1 (antiapoptotic activation) and S112pBAD (proapoptotic inhibition) in intrinsically resistant Su-DHL4, TOLEDO, and TMD8 cells, and a further increase in T163pMCL-1, S70pBCL-2 (antiapoptotic activation), and S112pBAD in the acquired-resistance Su-DHL4-R and OCI-Ly1-R lines. There was, however, no consistent increase in S184pBAX (proapoptotic inhibition) (Figure 1B). These findings suggest that the intrinsically higher T163pMCL-1 and S112pBAD levels in Su-DHL4, TOLEDO, and TMD8 cells may have contributed to intrinsic venetoclax resistance, whereas a further increase in T163pMCL-1, S70pBCL-2, and S112pBAD levels was required for acquired venetoclax resistance following continuous exposure to venetoclax. We also observed an increase in MCL-1 protein levels across all intrinsic and acquired-resistance lines (Figure 1B), supporting previous observations of MCL-1 stabilization by T163pMCL-1 (13–15). No obvious changes in protein expression of PP2A subunits (B56/PP2A catalytic subunits) were observed in these cell lines (Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI170169DS1).

We next utilized high-throughput kina