. 2022 Nov 17;140(20):2113-2126.

doi: 10.1182/blood.2021014304.

Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies

D Thomalla^{1

2}, L Beckmann^{1

2}, C Grimm^{3

4}, M Oliverio^{1

2}, L Meder^{1

5}, C D Herling^{1

2

6}, P Nieper¹, T Feldmann³, O Merkel^{1

2}, E Lorsy^{1

2}, A da Palma Guerreiro^{1

2}, J von Jan¹, I Kisis¹, E Wasserburger³, J Claasen^{1

2}, E Faitschuk-Meyer⁷, J Altmüller⁴, P Nürnberg^{4

8}, T-P Yang^{8

9}, M Lienhard¹⁰, R Herwig¹⁰, K-A Kreuzer^{1

2}, C P Pallasch^{1

2}, R Büttner¹¹, S C Schäfer^{11

12}, J Hartley¹³, H Abken¹³, M Peifer^{8

9}, H Kashkar^{2

8

14}, G Knittel¹⁵, B Eichhorst¹, R T Ullrich^{1

8}, M Herling^{1

2

6}, H C Reinhardt¹⁵, M Hallek^{1

2}, M R Schweiger^{3

4

8}, L P Frenzel^{1

2}

Affiliations

¹ Faculty of Medicine and Cologne University Hospital, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, University of Cologne, Cologne, Germany.
² Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany.
³ Institute for Translational Epigenetics, Medical Faculty, University of Cologne, Cologne, Germany.
⁴ Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany.
⁵ Mildred Scheel School of Oncology Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany.
⁶ Clinic of Hematology, Cellular Therapy and Hemostaseology, University of Leipzig, Leipzig, Germany.
⁷ Miltenyi Biotec B.V. & Co. KG, Bergisch-Gladbach, Germany.
⁸ Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.
⁹ Center of Integrated Oncology Cologne-Bonn, Medical Faculty, Department of Translational Genomics, University of Cologne, Cologne, Germany.
¹⁰ Department of Computational Molecular Biology, Max-Planck-Institute for Molecular Genetics, Berlin, Germany.
¹¹ Department of Pathology, University of Cologne, Cologne, Germany.
¹² Institut für Pathologie im Medizin Campus Bodensee, Friedrichshafen, Germany.
¹³ RCI, Regensburg Center for Interventional Immunology, University Hospital of Regensburg, Regensburg, Germany.
¹⁴ Institute for Molecular Immunologie, University of Cologne, Cologne, Germany.
¹⁵ Department of Hematology and Stem Cell Transplantation, University Hospital Essen, University Duisburg-Essen, German Cancer Consortium (DKTK Partner Site Essen), Essen, Germany.

PMID: 35704690
PMCID: PMC10653032
DOI: 10.1182/blood.2021014304

Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies

D Thomalla et al. Blood. 2022.

. 2022 Nov 17;140(20):2113-2126.

doi: 10.1182/blood.2021014304.

Authors

Affiliations

¹ Faculty of Medicine and Cologne University Hospital, Department I of Internal Medicine, Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf, University of Cologne, Cologne, Germany.
² Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany.
³ Institute for Translational Epigenetics, Medical Faculty, University of Cologne, Cologne, Germany.
⁴ Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany.
⁵ Mildred Scheel School of Oncology Cologne, Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany.
⁶ Clinic of Hematology, Cellular Therapy and Hemostaseology, University of Leipzig, Leipzig, Germany.
⁷ Miltenyi Biotec B.V. & Co. KG, Bergisch-Gladbach, Germany.
⁸ Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.
⁹ Center of Integrated Oncology Cologne-Bonn, Medical Faculty, Department of Translational Genomics, University of Cologne, Cologne, Germany.
¹⁰ Department of Computational Molecular Biology, Max-Planck-Institute for Molecular Genetics, Berlin, Germany.
¹¹ Department of Pathology, University of Cologne, Cologne, Germany.
¹² Institut für Pathologie im Medizin Campus Bodensee, Friedrichshafen, Germany.
¹³ RCI, Regensburg Center for Interventional Immunology, University Hospital of Regensburg, Regensburg, Germany.
¹⁴ Institute for Molecular Immunologie, University of Cologne, Cologne, Germany.
¹⁵ Department of Hematology and Stem Cell Transplantation, University Hospital Essen, University Duisburg-Essen, German Cancer Consortium (DKTK Partner Site Essen), Essen, Germany.

PMID: 35704690
PMCID: PMC10653032
DOI: 10.1182/blood.2021014304

Abstract

The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.

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Conflict of interest statement

Conflict-of-interest disclosure: L.P.F. received research funding from Abbvie, Hofmann-La Roche, and Gilead; obtained consulting and/or speaker’s honoraria and travel support from AbbVie. H.C.R. received consulting and lecture fees from Abbvie, AstraZeneca, Vertex, and Merck; received research funding from Gilead Pharmaceuticals; is a cofounder of CDL Therapeutics GmbH. M. Herling received honoraria and research funding unrelated to the data presented here by Abbvie, EDO-Mundipharma, Janpix, Janssen-Cilag, Jazz, Novartis, Roche Stemline Therapeutics, and Takeda; holds nonexclusive licenses to clone 1-21 of diagnostic TCL1A antibodies. The remaining authors declare no competing financial interest.

Figures

**Figure 1.**
**Downregulation of BAX and PUMA and upregulation of MCL-1 in vitro and in vivo.** (A) Top: IC₅₀ values for VEN in 10 sensitive (blue) vs VEN-resistant (red) B-cell lymphoma cell lines: WSU-NHL, OCI-LY-19, DOHH-2, DB, KARPAS-422, HBL-1, 697, P30-OH-KUBO, Nalm6, and OSU. Middle: densitometric analyses of immunoblots of BCL2 proteins (supplemental Figure 1A-B). Mean ± SD of at least 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001, compared with parental (VEN naïve) cells, Students t test. Lower: results from WES. Genomic alterations are annotated according to the color panel below the image. (B) Immunohistochemistry of BAX in 6 primary CLL samples. Scale bar, 100 μm. Pictures 1 through 5: lymph node sections posttherapy; picture 6: bone marrow post–VEN therapy. Relative *MCL1* (left panel) and *BBC3* (*PUMA*) (right panel) and *BAX* mRNA expression level for CLL patients 1 and 2 pre- (blue) and post (red)-VEN therapy determined by bulk 3′RNA-seq. (C) Results for *Bax, Bbc3*, and *Mcl1* guide RNAs from CRISPR/Cas9-Screen in murine lymphoma cell line after 28 days with/without VEN (10 nM). (D) Immunoblot for MCL1, BCL-xL, BCL2, PUMA, and BAX in 2 T-PLL patients before and after VEN resistance. (E) Immunoblot for MCL1, BAK1, PUMA, and BAX in 3 sensitive and S63845-resistant B-cell lymphoma cell lines, respectively. (F) Densitometric analyses of immunoblots against MCL1, BAK1, PUMA, and BAX normalized to β-actin. Data illustrated as mean ± SD of at least 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .001, compared with parental (blue) cells, Student t test. 3′RNA-seq, 3’RNA-sequencing; IC₅₀, median inhibition concentration.

**Figure 2.**
**Effect of VEN on the expression of *BBC3* in B-cell lymphoma cell lines and primary CLL cells.** (A) Schematic representation of MeDip-seq results. (B) Schematic drawing of *BBC3* promoter region. For the Dual-Glo Luciferase Assay (Figure 2E), a 917 bp big region containing the CpGs of interest was cloned in a CpG-free vector, followed by the luciferase reporter. (C-D) Methylation changes detected by pyrosequencing for the CpG of interest in cell lines (C) and primary CLL cells before and after VEN resistance (D). (E) Dual-Glo Luciferase Assay analysis of methylated (meth) and unmethylated (unmeth) versions of the promoter region of *BBC3*. Mean ± SD, N = 10. ∗∗∗∗P < .0001, compared with unmethylated reporter construct, Student t test. (F) Methylation of BBC3 promoter region in 5′AZA-treated (5 passages) VEN-resistant B-cell lymphoma cell lines, determined by pyrosequencing. (G) Immunoblot for PUMA in 3 VEN-sensitive and -resistant B-cell lymphoma lines treated with or without 5′AZA (0.1 μM) for 5 passages. (H) Viability assay of 3 VEN-treated cell lines (24 hours, 1 μM) after incubation with 5′AZA (5 passages). Mean ± SD of 3 independent experiments, viability determined by flow cytometry. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001, compared with untreated (-5-AZA) cells, Student t test. (I) Schematic analysis of OCR analysis. (J-K) Mitochondrial respiration and glycolysis in *PUMA*-KO KARPAS-422 (J) and OSU (K) cells upon injection of the Seahorse Mito Stress test drugs. Data are shown as floating bars (min. to max.) and are representative of 3 to 6 independent experiments. Paired 2-tailed Student t test: ∗P < .05; ∗∗P < .01. ECAR, extracellular acidification rate; KO, knockout; w/o, without.

**Figure 3.**
**MCL1 inhibition (S63845) cannot eliminate BAX-deficient VEN-resistant cells.** (A) Top: mean IC₅₀ for S63845 of 9 VEN-sensitive (blue) and VEN-resistant cell lines (red) determined by flow cytometry after 48 hours. N ≥3. Lower part: heat map with relative BAX and MCL1 protein level in VEN-resistant cell lines. (B) Validation of *BAX* KO in OSU cells by immunoblot. N = 3. (C) Sensitivity of OSU KO cells toward VEN determined by flow cytometry after VEN treatment for 48 hours. Mean ± SD of 4 experiments. (D) Allelic fraction of *TP53* (c.515T>A) and *BAX* (c.361del) before and after VEN treatment (24 hours, 5 nM) in a high-risk CLL patient. Mean plus SD, 3 technical replicates; P = .0061 (E) Viability of purified, malignant splenic B cells of *Eμ-TCL1*^tg/wt; Cd19Cre^+/wt; Bax^wt/wt (blue), *Eμ-TCL1*^tg/wt; Cd19Cre^+/wt; Bax^fl/wt (red) and *Eμ-TCL1*^tg; Cd19Cre^+/wt; Bax^fl/fl (purple) mice treated with VEN, S63845 (24 hours), or fludarabine (48 hours), determined by MTT assays. Mean ± SD of 2 experiments; 3 technical replicates each. (F) Immunophenotyping of splenocytes of a 50-week-old *Eμ-TCL1*^tg; Cd19Cre^+/wt; Bax^fl/fl mouse. Gating strategy of viable, single cells on FSC/SSC dot plot and FSC-area/FSC-height dot plot. Analysis for Cd45, Cd19, IgM, and IgD expression. (G) Spleen weight and length of *Eμ-TCL1*^tg/wt; Cd19Cre^+/wt; Bax^wt/wt (blue; n = 6), *Eμ-TCL1*^tg/wt; Cd19Cre^+/wt; Bax^fl/wt (red; n = 6), and *Eμ-TCL1*^tg; Cd19Cre^+/wt; Bax^fl/fl (purple; n = 5) mice. Mean ± SD. ∗P < .05, compared with *Bax* wild-type mice, Student t test. (H) Determination of the amount of Cd19⁺/B220^dim/neg-positive cells in the blood (left panel; 32-week-old animals; n = 5, n = 8, and n = 2, respectively) and splenocytes (right panel; 44 weeks old animals; n = 4, n = 5, and n = 4, respectively) of *Eμ-TCL1*^tg/wt; Cd19Cre^+/wt; Bax^wt/wt (blue), *Eμ-TCL1*^tg/wt; Cd19Cre^+/wt; Bax^fl/wt (red), and *Eμ-TCL1*^tg; Cd19Cre^+/wt; Bax^fl/fl (purple) mice. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001, compared with *Bax* wild-type mice, Student t test. (I) Kaplan-Meier curves of overall survival of *Eμ-TCL1*^tg/wt; Cd19Cre^+/wt; Bax^wt/wt (blue; 48 weeks; n = 14), *Eμ-TCL1*^tg/wt; Cd19Cre^+/wt; Bax^fl/wt (red; 56 weeks; n = 7), and *Eμ-TCL1*^tg; Cd19Cre^+/wt; Bax^fl/fl (purple; 59.5 weeks; n = 6) mice. Survival of *Eμ-TCL1*^tg/wt; Cd19Cre^+/wt; Bax^fl/wt (red), and *Eμ-TCL1*^tg; Cd19Cre^+/wt; Bax^fl/fl (purple) compared with the respective controls (log-rank test; P = .0914). FS, forward scatter integral; FSC, forward scatter; IgM, immunoglobulin M; INT, integral; KO, knockout; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid; ns, not significant; SSC, side scatter integral.

**Figure 4.**
**VEN resistance can by overcome by activation of the extrinsic apoptotic pathway.** (A) Heat map showing IC₅₀ values of sensitive and VEN-resistant Nalm6 cells for 45 compounds: red, IC₅₀ ≥10 μM; yellow, IC₅₀ ≥5 μM; and green, IC₅₀ ≤1 μM. Cumulative results from 3 experiments. (B) Close-up view of the 3 most potent drugs identified for Nalm6 and corresponding Nalm6-VEN–resistant cells. (C) Cell death assay of sensitive and VEN-resistant Nalm6 for TRAIL (48 hours) determined by flow cytometry. Mean ± SD. N = 3. (D) Results from Caspase-Glo 8 Luminescent Assay in sensitive and VEN-resistant Nalm6 cells after treatment with TRAIL (50 ng/mL, 4 hours). N = 1. (E) Viability of sensitive and VEN-resistant Nalm6 cells after incubation with caspase-8 inhibitor Z-IETD-FMK (25 μM, 6 hours) and/or TRAIL treatment (200 ng/mL, 4 hours) determined by flow cytometry. N = 2. (F) Immunoblots for (t)BID, caspase-3, caspase-8, and PARP isoforms in Z-IETD-FMK and/or TRAIL treated cells. N = 2. DMSO, dimethyl sulfoxide.

**Figure 5.**
**Immunotherapeutic approaches are able to overcome BAX-dependent and -independent VEN resistance.** (A-B) Viability assays of Nalm6, DOHH-2, 697, and OSU cells (Ctrl, *BAX*^−/− #7, *BAX*^−/− #12) incubated with activated PBMCs (tumor:effector ratio of 10:1) and blinatumomab (50 ng/mL, 24/48 hours). Percentage of annexin-V⁺, CD4, and CD8⁻ cells was determined by flow cytometry, normalized to PBMC cocultured, untreated cells. N = 4; P < .001; paired t test. (C) Viability of sensitive and VEN-resistant Nalm6 cells and OSU-CLL wild-type and *BAX*^−/− clones after treatment with anti-CD19 CAR T cells determined by an XTT assay. Representative experiment of N = 3 experiments shown. Susceptibility toward CAR T-cell killing was not statistically different between sensitive and resistant cells. ∗∗P < .01; ∗∗∗P < .001. Ctrl, control; w/o, without.

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Comment in

Mechanisms of resistance to venetoclax.
Condoluci A, Rossi D. Condoluci A, et al. Blood. 2022 Nov 17;140(20):2094-2096. doi: 10.1182/blood.2022017341. Blood. 2022. PMID: 36394906 No abstract available.

References

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Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies

Affiliations

Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies

Authors

Affiliations

Abstract

Conflict of interest statement

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Comment in

References

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Full Text Sources

Molecular Biology Databases