Epigenetic targeting of Waldenström macroglobulinemia cells with BET inhibitors synergizes with BCL2 or histone deacetylase inhibition

Abstract

Aim: Waldenström macroglobulinemia (WM) is a low-grade B-cell lymphoma characterized by overproduction of monoclonal IgM. To date, there are no therapies that provide a cure for WM patients, and therefore, it is important to explore new therapies. Little is known about the efficiency of epigenetic targeting in WM. Materials & methods: WM cells were treated with BET inhibitors (JQ1 and I-BET-762) and venetoclax, panobinostat or ibrutinib. Results: BET inhibition reduces growth of WM cells, with little effect on survival. This finding was enhanced by combination therapy, with panobinostat (LBH589) showing the highest synergy. Conclusion: Our studies identify BET inhibitors as effective therapy for WM, and these inhibitors can be enhanced in combination with BCL2 or histone deacetylase inhibition.

Keywords: BET inhibitors; Waldenström macroglobulinemia; epigenetics; panobinostat; venetoclax.

Figure 1.. BET bromodomain inhibitors reduce Waldenström macroglobulinemia cell growth.

(A) WM cells (0.25 × 10³) were treated with the indicated doses of iBET or DMSO control (0). (B) WM cells (0.25 × 10³) were treated with the indicated doses of JQ1 or DMSO control. Cells were cultured at 37°C for 72 h, followed by the determination of cell proliferation using an XTT assay. Each experiment was repeated at least five-times. Bars represent the mean ± SE of at least three biological replicates of each experiment. (C) WM cells (0.5 × 10⁶) were treated with 5 mM iBET, JQ1 or DMSO control for 72 h followed by cell-cycle analysis as described in the Materials & methods section. This experiment was repeated twice with similar results and shown is a representative experiment. ****p < 0.0001. DMSO: Dimethyl sulfoxide; iBET: I-BET-762; SM: Standard Error; WM: Waldenström macroglobulinemia.

Figure 2.. The tumor microenvironment does not protect against BET inhibitors.

(A) HS-5 stromal cells (0.1 × 10⁶) and WM cells (0.5 × 10⁶) were co-cultured on coverslips as described in the Materials & methods section for 2 days. Coverslips were carefully removed and used for imaging using SEM (top panel) or confocal microscopy (lower panel). (B) Bone marrow stromal cells (HS-5; 0.1 × 10³) were plated in 96-well plates and allowed to adhere overnight. Cells were fixed using 10 µg/ml of mitomycin C for 3 h, followed by washing with DPBS. WM cells (0.5 × 10³) were added to each well and treated with either 5 mM of iBET, JQ1 or DMSO control. Cells were incubated at 37°C for 72 h, and cell proliferation was determined by XTT assay, as described in Materials & methods section. (C) HS-5 cells (0.25 × 10³) were treated with 5 mM iBET, 5 mM JQ1 or DMSO control for 72 h, followed by the determination of cell proliferation using an XTT assay. These experiments were repeated at least three-times. Bars represent the mean ± SE of at least three biological replicates of each experiment. ***p < 0.001; ****p < 0.0001. DMSO: Dimethyl sulfoxide; DPBS: Dulbecco’s phosphate-buffered saline; iBET: I-BET-762; SC: Stromal cells; SE: Standard error; WM: Waldenström macroglobulinemia.

Figure 3.. BET inhibitors induce modest cell death.

WM cells (0.5 × 10⁶) were treated with 5 mM of iBET or JQ1 or DMSO control for 72 h. Cells were harvested and viability was determined by annexin-V/PI staining. (A) Percent annexin-V-positive cells were determined by flow cytometry. (B) Percent viable cells as determined by flow cytometry. Each experiment was repeated at least three-times. Bars represent the mean ± SE of all experiments. (C) MNCs (2 × 10⁶ cells) from BM biopsy specimens from WM patients were treated with the indicated doses of JQ1 or DMSO control as described in Materials & methods section. Cells were harvested and viability was determined by annexin-V/PI and CD19 staining. Gating was done on lymphocytes from total MNCs followed by gating on CD19⁺ cells. Annexin-V/PI was analyzed on CD19⁺ cells. (D) Annexin-V-positive cells were determined by flow cytometry in two WM patient samples (WM1 and WM2). (E) Percent viable cells were determined for primary WM patient samples. ****p < 0.0001. BM: Bone marrow; DMSO: Dimethyl sulfoxide; iBET: I-BET-762; MNC: Mononuclear cell; PI: Propidium iodide; SE: standard error; WM: Waldenström macroglobulinemia.

Figure 4.. BET inhibitors reduce MYC but induce BCL-2 expression.

(A) WM cells (2 × 10⁶) were treated with 5 mM of either iBET or JQ1 or DMSO control for 24 h. Cells were harvested and RNA was extracted and used to determine MYC expression by qRT-PCR. This experiment was repeated at least three-times and data were presented as mean ± SE of all experiments. (B) WM cells (5 × 10⁶) were treated with either 5 mM of iBET, JQ1 or DMSO control for 24 h. Cells were harvested and lysed and lysates were used to determine protein levels by western blot. (C) WM cells (5 × 10⁶) were treated with either 5 mM of iBET, JQ1 or DMSO control for 24 h. Cells were lysed and lysates were used to determine BCL-2 protein levels by Western blot. Western blot experiments were repeated at least three-times with similar results. DMSO: Dimethyl sulfoxide; iBET: I-BET-762; SE: Standard error; WM: Waldenström macroglobulinemia.

Figure 5.. BET inhibitor reduces IgM levels.

(A) WM cells (2 × 10⁶) were treated with 5 mM of iBET or DMSO control for 24 h. Cells were harvested and RNA was extracted and used in qRT-PCR to determine IgM expression. (B) WM cells (2 × 10⁶) were treated with either 5 mM iBET or DMSO control for 72 h. Supernatant was used to quantify IgM secretion by ELISA. Each experiment was repeated at least three-times. Bars represent the mean ± SE of all experiments. **p < 0.01; ***p < 0.001; ****p < 0.0001. DMSO: Dimethyl sulfoxide; iBET: I-BET-762; qRT: quantitative reverse-transcription; SE: Standard error; WM: Waldenström macroglobulinemia.

Figure 6.. Combined targeting of BET proteins and BCL-2/histone deacetylase enhances cell death in Waldenström macroglobulinemia cells.

(A & B) WM cells (0.5 × 10⁶) were treated with either 500 nM of ABT-199, 5 mM of JQ1, a combination of venetoclax (ABT-199) and JQ1, or DMSO control for 72 h. Cells were harvested and stained with annexin-V/PI followed by data acquisition using flow cytometry. Annexin-V-FITC positive cells are shown in (A) and viable cells are shown in (B). (C & D) WM cells (0.5 × 10⁶) were treated with either 25 nM of panobinostat (LBH589), 5 mM of JQ1, a combination of LBH589 and JQ1, or DMSO control for 72 h. Cells were harvested and stained with annexin-V/PI followed by data acquisition using flow cytometry. Annexin-V-FITC positive cells are shown in (C) and viable cells are shown in (D). Each experiment was repeated at least three-times. Bars represent the mean ± SE of all experiments. ****p < 0.0001. DMSO: Dimethyl sulfoxide; PI: Propidium iodide; SE: Standard error; WM: Waldenström macroglobulinemia.

Figure 7.. Combined targeting of BET proteins and Bruton’s tyrosine kinase has a modest effect on cell viability in Waldenström macroglobulinemia cells.

(A) WM cells (0.5 × 10⁶) were treated with either 5 mM of ibrutinib, 5 mM of JQ1, combination of ibrutinib and JQ1 or DMSO control for 72 h, followed by determination of cell viability by annexin-V/PI double staining. (B) WM cells (0.25 × 10³) were treated with either 5 mM of ibrutinib, 5 mM of JQ1, combination of ibrutinib and JQ1 or DMSO control for 72 h, followed by the determination of cell proliferation using an XTT assay as described in Materials & methods section. Each experiment was repeated at least three-times. Bars represent the mean ± SE of all experimental. ****p < 0.0001. DMSO: Dimethyl sulfoxide; PI: Propidium iodide; SE: Standard error; WM: Waldenström macroglobulinemia.