Targeting Myc in KSHV-associated primary effusion lymphoma with BET bromodomain inhibitors

Abstract

Primary effusion lymphoma (PEL) is an aggressive form of non-Hodgkin's B-cell lymphoma associated with infection by Kaposi's sarcoma-associated herpes virus (KSHV). (+)-JQ1 and I-BET151 are two recently described novel small-molecule inhibitors of BET bromodomain chromatin-associated proteins that have shown impressive preclinical activity in cancers in which MYC is overexpressed at the transcriptional level due to chromosomal translocations that bring the MYC gene under the control of a super-enhancer. PEL cells, in contrast, lack structural alterations in the MYC gene, but have deregulated Myc protein due to the activity of KSHV-encoded latent proteins. We report that PEL cell lines are highly sensitive to bromodomain and extra-terminal (BET) bromodomain inhibitors-induced growth inhibition and undergo G0/G1 cell-cycle arrest, apoptosis and cellular senescence, but without the induction of lytic reactivation, upon treatment with these drugs. Treatment of PEL cell lines with BET inhibitors suppressed the expression of MYC and resulted in a genome-wide perturbation of MYC-dependent genes. Silencing of BRD4 and MYC expression blocked cell proliferation and cell-cycle progression, while ectopic expression of MYC from a retroviral promoter rescued cells from (+)-JQ1-induced growth arrest. In a xenograft model of PEL, (+)-JQ1 significantly reduced tumor growth and improved survival. Taken collectively, our results demonstrate that the utility of BET inhibitors may not be limited to cancers in which genomic alterations result in extremely high expression of MYC and they may have equal or perhaps greater activity against cancers in which the MYC genomic locus is structurally intact and c-Myc protein is deregulated at the post-translational level and is only modestly overexpressed.

Conflict of interest statement

Conflict of interest: The authors declare no conflict of interest.

Figures

Figure 1. BRD4 inhibitors reduce cell viability in PEL cells lines in a dose-dependent manner

(a) PEL cell lines (BC1, BC3, BCBL1 and JSC1) were treated with indicated doses of (-)-JQ1 or (+)-JQ1 for 5 days and cell viability measured using an MTS assay (refer to Materials and methods). Namalwa, a Burkitt's lymphoma cell line, was used as a control. (b) PEL cell lines (solid lines) and non-PEL cells lines (dotted lines) were treated in triplicate with the indicated concentrations of I-BET151 and cell viability measured after 5 days using the MTS assay. (c-d) Patient-derived UMP-EL-1 and UM-PEL-3 cells were treated with indicated doses of (-)-JQ1, (+)-JQ1 (c) or I-BET151 (d) for 5 days and cell viability measured using an MTS assay. The values shown are mean ± SD of a representative of two independent experiments performed in triplicate.

Figure 2. BET bromodomain inhibitors induce cell cycle arrest in PEL cells

(a, b) Cell-cycle analysis of (-/+)-JQ1-(500 nM for 48 hours) and I-BET151 (500 nM for 48 hours)-treated PEL cell lines showing significant G0/G1 arrest. A non-PEL cell line, Namalwa, was used as a control. Cells were stained with propidium iodide (PI) and analyzed by flow cytometry. Results are representative of two independent experiments.

Figure 3. Induction of apoptosis and cellular senescence by (+)-JQ1 in PEL cells

(a) PEL cells were exposed to 500 nM (+)-JQ1 for 48 hours, stained with Annexin-V-FITC/PI and analyzed for apoptosis by flow cytometry. (b) PEL cells were treated with 500 nM (+)-JQ1 for 72 hours and its effect on cellular senescence detected by β-galactosidase staining. Results are representative of two independent experiments. (c) Western blot analysis showing induction of p27 upon 48 hours of treatment with (+)-JQ1. GAPDH serves as a loading control.

Figure 4. (+)-JQ1 treatment affects expression of MYC-dependent genes

(a) Heat map representation of 1,443 genes that are up- or down-regulated ≥1.5 fold in two of the three PEL cell lines following 8 hour treatment with 500 nM (+)-JQ1. (b) Gene Set Enrichment Analysis showing enrichment of gene sets containing target genes of MYC among genes affected by (+)-JQ1 treatment in PEL cells; NES=normalized enrichment score, q= false discovery rate.

Figure 5. BET inhibitors block MYC transcription programs in PEL

(a) Quantitative RT-PCR analysis of MYC levels in PEL and non-PEL (Namalwa) cells treated with 250 nM of (-)- or (+)-JQ1 for 48 hours. Real-time PCR reactions were performed in triplicate and the data presented as fold change in target gene expression (Mean ± SD) after normalization with GAPDH as a housekeeping gene. The results shown are representative of two independent experiments. (b) PEL and non-PEL (Namalwa) cells treated with indicated doses of (+)-JQ1 for 48 hours were lysed and analyzed by Western blot for the expression of c-Myc. GAPDH was used as a loading control. (c) PEL cell lines (BC1, BC3 and BCBL1) and non-PEL (Namalwa) cells were treated with 250 nM of (-)- or (+)-JQ1 for 72 hours and nuclear extracts were isolated to study the effect of (+)-JQ1 on nuclear c-Myc protein level as measured by an ELISA-based DNA-binding assay. The results shown are representative of two independent experiments performed in triplicate. ** indicates p > 0.01 calculated using a two-tailed Student's t-test. (d) Western blot analysis of c-Myc upon 48 hours of indicated concentrations of I-BET151 treatment with Tubulin as a loading control. (e) Quantitative RT-PCR analysis of MYC downstream target genes – MYB and TERT, and TYRO3 in the indicated cell lines treated with 250 nM of (-)- or (+)-JQ1 for 48 hours. GAPDH was used as a housekeeping gene to normalize gene expression data. The results shown are a representative of two independent experiments performed in triplicate. ** indicates p > 0.01 calculated using a two-tailed Student's t-test.

Figure 6. Silencing of BRD4 and MYC blocks proliferation of PEL cells

(a) Western blot of whole cell lysates from BC1 cells stably expressing tetracycline-inducible shRNAs targeting BRD4 and MYC. Cells were grown in the absence and presence of doxycycline (500 ng/mL) for 11 days. GAPDH was used as a loading control. (b) BC1 cells stably transduced with control, BRD4 or MYC shRNAs were treated with doxycycline (500 ng/mL, 5 days) and cell survival was measured by MTS assay. The values shown are mean ± S.D of a representative of two independent experiments performed in triplicate. (c) BC1 cells stably transduced with control, BRD4 or MYC shRNAs were treated with doxycycline (500 ng/mL, 5 days) and stained with propidium iodide for cell-cycle analysis. The data represents the mean ± S.D of two independent experiments.

Figure 7. Reconstitution of MYC rescues (+)-JQ1-treated cells from growth-suppression

(a) BC1 cells infected with retroviral vectors expressing an empty vector, Myc or FLAG-tagged Myc T58A were analyzed for expression of the transduced proteins by Western blotting using antibodies against c-Myc and FLAG. GAPDH was used as a loading control. (b) BC1 cells stably expressing Myc or Myc T58A were treated with 250 nM of (+)-JQ1 for 48 hours and the effect of JQ1 on c-Myc expression was examined by Western blotting. GAPDH was used to show equal protein loading. (c) PEL cells expressing vector control or exogenous Myc or Myc T58A were treated with 500 nM of (+)-JQ1 for 5 days and the cell survival was measured by an MTS assay. The values shown are mean ± SD of three independent experiments performed in triplicate. (d) BC1 cells stably expressing vector control or exogenous Myc or Myc T58A were treated with 250 nM of (+)-JQ1 for 48 hours and stained with propidium-iodide for cell cycle analysis. The data represents one of three independent experiments.

Figure 8. Lack of induction of KSHV lytic replication by (+)-JQ1 in PEL cells

(a) Immunoblot analysis of RTA in PEL cells treated with (+)-JQ1 (48 hours, 500 nM). RTA expression in BCBL1-TREx-RTA cells upon treatment with doxycycline (48 hours, 100 ng/mL) serves as a positive control for lytic induction. GAPDH is used as a loading control. (b) (+)-JQ1-treatment (72 hours, 500 nM) of PEL cells does not induce production of infectious virions. Cell-free supernatants were collected from PEL cells treated with 500 nM (+)-JQ1 for 72 hours and used to infect 293PAN-Luc cells containing a luciferase reporter construct driven by PAN promoter. Supernatant from doxycycline-treated BCBL-Trex-RTA cells was used a positive control. Data represents one of two experiments performed in duplicate (mean +/- SD, n=2).

Figure 9. (+)-JQ1 impairs in vivo growth of PEL in a mouse xenograft model

(a) 5 week old female NOD/SCID mice were injected intra-peritoneally (IP) with 2 × 107 BC1-Luc cells. Upon establishment of tumors, mice were randomly segregated into vehicle control or (+)-JQ1 (50 mg/kg IP twice daily × 11 days) groups. Change in body weight of mice assigned to vehicle control or (+)-JQ1 (p <0.0001) are shown. (b) Tumor burden of vehicle control or (+)-JQ1-treated mice as measured by bioluminescence imaging (p<0.0001, day 11). (c) Representative whole body bioluminescence images of mice injected with BC1-Luc cells 1-, 5- and 11-days post vehicle control or (+)-JQ1 treatment. (d) Survival curves (Kaplan-Meier) of mice injected with PEL cells showing an increase in survival of (+)-JQ1 treated mice when compared with vehicle control (p = 0.008, log-rank test).