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. 2019 May 13;35(5):752-766.e9.
doi: 10.1016/j.ccell.2019.04.005.

BCL2 Amplicon Loss and Transcriptional Remodeling Drives ABT-199 Resistance in B Cell Lymphoma Models

Xiaohong Zhao  1 , Yuan Ren  1 , Matthew Lawlor  2 , Bijal D Shah  3 , Paul M C Park  2 , Tint Lwin  1 , Xuefeng Wang  4 , Kenian Liu  5 , Michelle Wang  1 , Jing Gao  1 , Tao Li  6 , Mousheng Xu  2 , Ariosto S Silva  7 , Kaplan Lee  8 , Tinghu Zhang  2 , John M Koomen  9 , Huijuan Jiang  10 , Praneeth R Sudalagunta  7 , Mark B Meads  1 , Fengdong Cheng  11 , Chengfeng Bi  12 , Kai Fu  12 , Huitao Fan  13 , William S Dalton  3 , Lynn C Moscinski  5 , Kenneth H Shain  3 , Eduardo M Sotomayor  11 , Gang Greg Wang  13 , Nathanael S Gray  14 , John L Cleveland  15 , Jun Qi  16 , Jianguo Tao  17
Affiliations

BCL2 Amplicon Loss and Transcriptional Remodeling Drives ABT-199 Resistance in B Cell Lymphoma Models

Xiaohong Zhao et al. Cancer Cell. .

Abstract

Drug-tolerant "persister" tumor cells underlie emergence of drug-resistant clones and contribute to relapse and disease progression. Here we report that resistance to the BCL-2 targeting drug ABT-199 in models of mantle cell lymphoma and double-hit lymphoma evolves from outgrowth of persister clones displaying loss of 18q21 amplicons that harbor BCL2. Further, persister status is generated via adaptive super-enhancer remodeling that reprograms transcription and offers opportunities for overcoming ABT-199 resistance. Notably, pharmacoproteomic and pharmacogenomic screens revealed that persisters are vulnerable to inhibition of the transcriptional machinery and especially to inhibition of cyclin-dependent kinase 7 (CDK7), which is essential for the transcriptional reprogramming that drives and sustains ABT-199 resistance. Thus, transcription-targeting agents offer new approaches to disable drug resistance in B-cell lymphomas.

Keywords: ABT-199; BCL2; CDK7; THZ1; double-hit lymphoma; drug persister; drug resistance; mantle cell lymphoma; super-enhancer remodeling; transcriptome reprogramming.

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Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.. Phenotypes of ABT-199 (Venetoclax)-Resistant Mantle Cell Lymphoma
(A) Top, experimental design for isolation of DTP cells and drug-tolerant expanded persisters (DTEP) of HBL-2 mantle cell lymphoma (MCL) cells. A total of 1,000 cells/well plated on 384-well collagen-coated plates were continuously treated with 20 nM ABT-199 (50–100× half maximal inhibitory concentration [IC50], IC50 was measured at 72 h after treatment). After 10–14 days, nearly all wells lacked viable cells as detected by live cell imaging, except for 12 wells, where drug-tolerant persisters (DTP) cells remained (bottom). Spatially separated colonies from these wells were then transferred to individual wells of a 96-well plate. Over the course of 3 months the selected colonies were expanded in medium containing 20 nM ABT-199 and three colonies survived and expanded to obtain the DTEP that are coined J4, L20, and I10. (B) Dose-response curves to ABT-199, for parental HBL-2 cells and DTEP clones (L20, J4, I10) showing drug response and resistance to ABT-199 treatment (72 h, with technical replicates, n = 4) at each indicated dose. (C) Drug-response curves of parental cells, DTEP cells, and DTEP cells after removing ABT-199 for a period of 6 weeks (“drug holiday” [dh]) to ABT-199 (100 nM) (technical replicates, n = 4). (D) BH3 profiling of parental HBL-2 and DTEP cells, as measured by mitochondrial membrane depolarization. BAD is indicative of BCL-2 or BCL-XL dependency, HRK is specifically indicative of BCL-XL dependency, BAD and HRK (BAD-HRK) are indicative of BCL-2 dependency, and MS1 is indicative of an MCL-1 dependency. (E) Levels of the indicated BCL-2 family proteins in parental HBL-2 cells versus ABT-199-resistant DTEP cells was determined by immunoblot analyses. Blots are representative of three independent experiments, which were quantified and the means are shown. (B–E) Data are shown as mean ± SD. See also Figure S1.
Figure 2.
Figure 2.. 18q21 Amplicon Loss and Super-Enhancer Remodeling Drive ABT-199 Resistance in Mantle Cell Lymphoma
(A) Unsupervised clustering of RNA-seq data from parental and DTEP cells in triplicate. Key genes involved in the evolution of DTEP are indicated. (B) Copy number variant (CNV) analysis of DTEP and parental cells. Copy-number loss (deletion), red; copy-number gain (amplification), blue. (C) Fluorescence in situ hybridization (FISH) analysis using a BCL2 probe in DTEP and parental cells. Cell nuclei are counterstained with DAPI in blue, the 5′ region of BCL2 gene was targeted with a red colored probe and the 3′ region of the BCL2 gene with a green colored probe. One isolated fusion signal represents one normal BCL2 gene, while five BCL2 fusion signals in cluster indicate BCL2 gene amplification (oval circle). (D) Viability of BCL-2, NOXA, or TCF4 knockdown (KD) versus control (GFP-KD) parental HBL-2 cells following treatment with ABT-199 (left, 3.0 μM; right, 0.04 μM) was assessed by live cell imaging. (E) Image-based cell-viability assays of DTEP control cells and cells overexpressing TCF4 and BCL2 in response to ABT-199 (upper left, 0.11 μM; upper right, 0.04 μM; middle/lower left, 1.1 μM; middle/lower right, 0.12 μM). (F) Enhancers ranked by H3K27ac signals. Super-enhancer (SE)-associated genes were identified as H3K27ac signal densities that surpass the inflection point by ChIP-seq and are indicated in red font; typical enhancer (TE)-associated genes are noted in black font. (G) Gene set enrichment analysis (GSEA). Top panels, gained (activated) SE-associated genes in DTEP cells. Bottom panels, SE-associated genes that are present in parental HBL-2 cells but that are suppressed (inactivated) in DTEP cells. Genes were ranked according to their expression fold change between DTEP and parental HBL-2 cells. NES, normalized enrichment score; FDR, false discovery rate. (D and E) Experiments were performed in triplicate and results are representative of three independent experiments. See also Figures S2 and S3 and Tables S2, S3, and S4.
Figure 3.
Figure 3.. ABT-199-Resistant DTEP Cells Are Highly Sensitive to CDK7 Inhibition
(A) Drug sensitivity shown as a heatmap of relative area under the curve differences between DTEP and parental cells for each indicated drug. (B) Top drug sensitivity hits for parental HBL-2 cells (top) and DTEP cells (bottom) are listed. (C) Image-based cell-viability assay of parental and DTEP cells treated with DMSO or THZ1 (100 nM) for the indicated time points. (D) Clonogenic growth assay of parental and DTEP cells treated with DMSO or THZ1 (50 nM) for the indicated time points. (E) Western blots of parental and DTEP cells treated with the indicated doses of THZ1 at different time points. CTD, C-terminal repeat domain of RNAPII; cPARP, cleaved PARP. (A–E) Data are shown as mean ± SD and are representative of three independent experiments. See also Figure S4.
Figure 4.
Figure 4.. CDK7 Inhibition Suppresses SE-Driven Transcriptional Reprogramming and Overcomes and Blocks the Evolution of ABT-199 Drug Resistance
(A) Quartile boxplots of Log2 fold changes of global gene expression following THZ1 treatment (50 nM, 250 nM, 6 h) in parental HBL-2 cells and DTEP cells. Top, middle, and bottom lines of the boxplot represent third quantile, median and first quantile of all values, respectively. The whiskers, the two lines outside the box, represent the highest and lowest observations excluding the possible outliers (dots above or below the whisker lines are outliers). (B) Global RNAPII ChIP-seq profiles showing the RNAPII-seq signal (y axis, RRPM/BP) across all RNAPII domains (>7 kb, x axis) in parental HBL-2 cells and DTEP cells (I10, J4) ± THZ1 treatment (50 nM, 6 h). The x axis represents size-scaled RNAPII domains, with or without flanking regions as indicated; TSS, transcription start site; PRPM/BP, Rx-normalized reads per million per base pair; BP, base pair. (C) Quartile boxplots of Log2 fold changes in the expression of genes that are regulated by gained SEs (with increased RNAPII binding) following 6 h THZ1 treatment (left, 50 nM; right, 250 nM) in parental and DTEP cells. Top, middle, and bottom lines of the boxplot represent third quantile, median and first quantile of all values, respectively. The whiskers, the two lines outside the box, represent the highest and lowest observations excluding the possible outliers (dots above or below the whisker lines are outliers). (D) GSEA of preferentially enriched for genes in DTEP cells sensitive to THZ1 treatment (50 nM) with NES at −2.06, −2.25, and −2.54, respectively. (E) Venn diagram of shared SE-associated/THZ1-sensitive genes in three DTEP cells. (F) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to identify the number of shared SE-associated/THZ1-sensitive genes among three DTEP cells. (G) Principal-component analysis (PCA) of gene expression for parental and DTEP cells with and without THZ1 treatment. See also Figures S5 and S6.
Figure 5.
Figure 5.. CDK7 and Super-Enhancer-Driven Genes Contribute to ABT-199 Resistance of DTEP Cells
(A) Image-based cell-viability assays (upper panel) and western blots (lower panel) of control (GFP) or CDK7 knockdown derivatives of parental (left), I10 (middle), and L20 (right) cells treated with THZ1 (220 nM). (B) Efficiency of CRISPR/Cas9-directed knockdown of BCL2A1, MCL-1, IKZF1, and IRF-5, and deletion of FOXC1, in DTEP cells, as determined by western blot. (C–F) Viability of I10 (left), J4 (middle), and L20 (right) DTEP cells in paired control (GFP) and BCL2A1 (C), MCL-1 (D), IRF5 (E) knockdown, and FOXC1 (F) knock out cells ± ABT-199 treatment (C), 10 μM and (D–F), 3.3 μM. (G) Image-based cell-viability assays of J4 in paired control (GFP) and IKZF1 knockdown cells ± ABT-199 treatment (3.3 μM). (A–G) Data shown are representative of at least three independent experiments.
Figure 6.
Figure 6.. CDK7 Inhibition Synergizes with ABT-199, Overrides ABT-199 Resistance and Prevents Emergence of ABT-199 Resistance
(A and B) Image-based cell-viability assays at different time points (upper) and doses (lower, at 48 h) of MCL cell lines (A) and primary MCL patient specimens (B). Combination index (CIs) for drug combinations were obtained with CalcuSyn software using percent inhibition (fraction affected [Fa]) resulting from combined action of the two drugs versus effects of either drug alone. CI values
Figure 7.
Figure 7.. BCL2 Amplicon Loss and CDK7 Vulnerability Are Hallmarks of ABT-199-Resistant DHL Cells and Primary DHL Specimens
(A) Image-based cell-viability assays of parental DHL VAL and VAL-DTEP cells treated with the indicated doses of ABT-199 (upper) or with 110 nM THZ1 (lower). Data shown are representative of three independent experiments. (B) Left, image-based cell viability of primary double-hit lymphoma (DHL) patient samples cultured on lymphoma stromal cells following treatment with ABT-199 (20 nM), THZ1 (50 nM), or ABT-199 + THZ1 (20 and 50 nM, respectively). Right, synergistic effects for drug combinations were obtained through combination indices (CIs) with CalcuSyn software using percent inhibition (fraction affected [Fa]) resulting from combined action of the two drugs versus effects of either drug alone. CI values BCL2 probe in parental DHL VAL cells (VAL, left), and ABT-199-resistant VAL-DTEP cells (right). Cell nuclei are counterstained with DAPI in blue, the 5′ region of the BCL2 gene was targeted with a red colored probe and the 3′ region of BCL2 gene with a green colored probe. (D) GSEA of DTEP-associated and THZ1-sensitive genes identified in DTEP MCL cell lines with THZ1-induced altered genes in primary ABT-199-resistant MCL patient samples. Genes were ranked according to their expression fold change between control and THZ1 treatment (50 or 250 nM, 6 h). NES, normalized enrichment score; FDR, false discovery rate. (E) Western blot showing THZ1-induced degradation of the large subunit of RNAPII in parental HBL-2 and DTEP cells, and that pre-treatment with proteasome inhibitor MG132 impaired THZ1-induced degradation of RNAPII in all DTEP cells. Data shown are representative of three independent experiments. See also Figures S7 and S8.
Figure 8.
Figure 8.. Model for the Evolution of ABT-199 Persistence/Resistance and CDK7 Vulnerability
Model for the evolution of ABT-199 persistence/resistance in 18q21-amplified mantle cell lymphoma and double-hit lymphoma models, which occurs through the selection for rare clones having loss or reductions in copy number of 18q21 amplicons that harbor BCL2, and via adaptive super-enhancer (SE)-driven transcriptional reprogramming. The latter mechanism confers vulnerability to transcriptional inhibition by targeting CDK7.
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References

    1. (ANSI) (2012). Authentication of Human Cell Lines: Standardization of STR Profiling (ATCC Standards Development Organization (ATCC SDO)).
    1. Anderson MA, Deng J, Seymour JF, Tam C, Kim SY, Fein J, Yu L, Brown JR, Westerman D, Si EG, et al. (2016). The BCL2 selective inhibitor venetoclax induces rapid onset apoptosis of CLL cells in patients via a TP53-independent mechanism. Blood 127, 3215–3224. - PMC - PubMed
    1. Bea S, Salaverria I, Armengol L, Pinyol M, Fernandez V, Hartmann EM, Jares P, Amador V, Hernandez L, Navarro A, et al. (2009). Uniparental disomies, homozygous deletions, amplifications, and target genes in mantle cell lymphoma revealed by integrative high-resolution whole-genome profiling. Blood 113, 3059–3069. - PMC - PubMed
    1. Bradner JE, Hnisz D, and Young RA (2017). Transcriptional addiction in cancer. Cell 168, 629–643. - PMC - PubMed
    1. Brown JD, Lin CY, Duan Q, Griffin G, Federation A, Paranal RM, Bair S, Newton G, Lichtman A, Kung A, et al. (2014). NF-kappaB directs dynamic super enhancer formation in inflammation and atherogenesis. Mol. Cell 56, 219–231. - PMC - PubMed

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