Targeting Autophagy Triggers Apoptosis and Complements the Action of Venetoclax in Chronic Lymphocytic Leukemia Cells

Abstract

Continuous treatment of patients with chronic lymphocytic leukemia (CLL) with venetoclax, an antagonist of the anti-apoptotic protein Bcl-2, can result in resistance, which highlights the need for novel targets to trigger cell death in CLL. Venetoclax also induces autophagy by perturbing the Bcl-2/Beclin-1 complex, so autophagy might represent a target in CLL. Diverse autophagy inhibitors were assessed for cytotoxic activities against patient-derived CLL cells. The AMPK inhibitor dorsomorphin, the ULK1/2 inhibitor MRT68921, and the autophagosome-lysosome fusion inhibitor chloroquine demonstrated concentration-dependent and time-dependent cytotoxicity against CLL cells, even in those from hard-to-treat patients who carried del(11q) and del(17p). Dorsomorphin and MRT68921 but not chloroquine triggered caspase-dependent cell death. According to the metabolic activities of CLL cells and PBMCs following treatments with 10 µM dorsomorphin (13% vs. 84%), 10 µM MRT68921 (7% vs. 78%), and 25 µM chloroquine (41% vs. 107%), these autophagy inhibitors are selective toward CLL cells. In these CLL cells, venetoclax induced autophagy, and addition of dorsomorphin, MRT68921, or chloroquine showed potent synergistic cytotoxicities. Additionally, MRT68921 alone induced G2 arrest, but when combined with venetoclax, it triggered caspase-dependent cytotoxicity. These data provide the rationale to target autophagy and for autophagy inhibitors as potential treatments for patients with CLL.

Keywords: AMPK/ULK1; autophagy; chronic lymphocytic leukemia; drug resistance; targeted therapy; venetoclax.

Figure 1

Targeting autophagy with distinct autophagy inhibitors induces concentration-dependent cytotoxicity in patient-derived CLL cells. (a) Overview of targets in the autophagic process and their pharmacological modulators; Determination of EC₅₀ values of autophagy inhibitors (b) mTOR activator MHY1485, (c) AMPK inhibitor dorsomorphin, (d) ULK1/2 inhibitor MRT68921, (e) PI3K class III inhibitor wortmannin, (f) autophagosome-lysosome fusion inhibitor chloroquine, and (g) late-stage autophagy inhibitor bafilomycin A1 in CLL cells derived from five patients after 48 h of treatment using the PrestoBlue (PB) assay.

Figure 2

The potency of autophagy inhibitors in cells of CLL patients with different genetic characteristics. The EC₅₀ values of autophagy inhibitors after 48 h with respect to (a) del(13q), trisomy 12 (+12), del(11q), del(17p), and (b) mutational status of IGHV. Data are means ± SEM. Each dot corresponds to the cells derived from a specific patient with CLL. One-way ANOVA with Dunnett’s multiple comparisons test and unpaired Student’s t-test were used, respectively. Not significant (ns) denotes p > 0.05.

Figure 3

Autophagy inhibitors dorsomorphin, MRT68921, and chloroquine trigger apoptotic cell death in primary CLL cells. CLL cells (1 × 10⁶ cells/mL) were treated with (a) dorsomorphin (n = 4), (b) MRT68921 (n = 3), and (c) chloroquine (n = 3) for 24 h without or with the pan-caspase inhibitor QVD-OPh. Afterward, cells were stained with propidium iodide (PI) and the cell viability was determined by flow cytometry. Data are means ± SEM of ≥3 independent experiments, each carried out in duplicate. Student’s t-test was used. Not significant (ns), ∗, and ∗∗ denote p > 0.05, p < 0.05, and p < 0.01, respectively. (d) Representative dot plot diagram for phosphatidylserine exposure as a measure of cell death. Patient-derived cells were treated with dorsomorphin, MRT68921, and chloroquine for 24 h, followed by ANV/SB staining.

Figure 4

Autophagy inhibitors dorsomorphin, MRT68921, and chloroquine treatments for 24 h disrupt mitochondrial membrane potential and decrease lysosomal integrity in patient-derived CLL cells. (a) Representative dot plot diagram of mitochondrial membrane potential and cell viability. (b) Autophagy inhibitors perturb lysosomal integrity and decrease cell viability of primary CLL cells. The proportions of LysoTracker+ cells (high lysosomal integrity, viable cells) and DAPI− cells (viable cells) were determined using imaging flow cytometry. Representative cell images are shown. Data are means ± SEM of 3 independent experiments. Not significant (ns) and ∗ denote p > 0.05 and p < 0.05, respectively.

Figure 5

Autophagy inhibitors dorsomorphin, MRT68921, and chloroquine are selectively cytotoxic to CLL cells. CLL cells from 10 patients and PMBCs of five healthy donors (1 × 10⁶ cells/mL) were treated with increasing concentrations of (a) dorsomorphin, (b) MRT68921, or (c) chloroquine for 24 h. Afterward, the metabolic activities of cells were determined using the PrestoBlue assay. Data are means ± SEM of ≥3 independent experiments, each carried out in duplicate. Two-way ANOVA with Sidak’s multiple comparisons test was used. ∗, ∗∗∗, and ∗∗∗∗ denote p < 0.05, p < 0.001, and p < 0.0001 respectively.

Figure 6

Venetoclax induces autophagy in CLL cells. (a) THP1-Difluo hLC3 cells (5 × 10⁵ cells/mL) following the treatments with venetoclax (VE; 1–25 µM), the autophagy inducer rapamycin (RAPA; 200 nM), and the autophagy inhibitor bafilomycin A1 (BafA1; 100 nM) for 24 h. The expression of GFP and RFP were determined by imaging flow cytometry. Data are means ± SEM of 5 independent experiments. Representative cell images for each sample are shown; (b) Immunoblot analysis of p62/SQSTM1 in MEC-1 cells (1 × 10⁶ cells/mL) treated with vehicle control (0.2% DMSO) and 1 µM MRT68921 for 24 h, 10 µM venetoclax for 8 h, or HBSS for 2 h. The cells were harvested and lysed, and the proteins were separated by SDS PAGE, blotted onto nitrocellulose membranes, and probed for the expression of the autophagy marker p62/SQSTM1, with the loading control of β-actin. Data are means ± SEM of 3 independent experiments. A representative immunoblot is also shown; (c) Representative cell images (left) and quantification (right) of TFEB translocation in MEC-1 cells. The cells (5 × 10⁵ cells/mL) were treated with the vehicle control (0.2% DMSO) or to 10 µM venetoclax, HBSS, 1 µM MRT68921 or 100 nM Torin-1, for 1 h. After, the cells were fixed and permeabilized, and stained with an anti-TFEB antibody and the nuclear stain DAPI. Nuclear translocation of TFEB was monitored using imaging flow cytometry. Data are means ± SEM of ≥3 independent experiments. One-way ANOVA with Dunnett’s multiple comparisons test was used. Not significant (ns), ∗, ∗∗, ∗∗∗, and ∗∗∗∗ denote p > 0.05, p < 0.05, p < 0.01, p < 0.001, and p < 0.0001 respectively.

Figure 7

Synergistic effects of autophagy inhibitors and venetoclax. Synergistic effects of dorsomorphin and venetoclax in (a) MEC-1 cells and (b) primary CLL cells derived from 10 patients; Synergistic effects of MRT68921 and venetoclax in (c) MEC-1 cells and (d) primary CLL cells derived from 10 patients; Synergistic effects of chloroquine and venetoclax in (e) MEC-1 cells and (f) primary CLL cells derived from 10 patients. Each square represents the mean relative metabolic activity of cells. For MEC-1 cells experiments were repeated ≥3, each carried out in duplicate. For CLL cells, each column represents responses of individual patient’s cells to different treatments, while each row represents the responses of CLL cells from 10 patients to a specific treatment. One-way ANOVA with Dunnett’s multiple comparisons test was used. *, *** and **** denote p < 0.05, p < 0.001, and p < 0.0001, respectively.

Figure 8

Mechanistic insight into the synergistic cytotoxicity of venetoclax and MRT68921 in CLL cells. (a) Cell cycle analysis of MEC-1 cells (3 × 10⁵ cells/mL) treated with 5 µM venetoclax, 1 µM MRT68921, and their combination for 24 h. A representative experiment is shown; (b) Caspases are essential for synergistic cytotoxicity of venetoclax and MRT68921. MEC-1 cel cellsls (3 × 10⁵/mL) were treated with a combination of 5 µM venetoclax and 1 µM MRT68921, 10 µM QVD-OPh, and their combination for 24 h. The cell viabilities were determined by 5 µM propidium iodide staining and analysis by flow cytometry. Data are means ± SEM of 3 independent experiments. Student’s t-test was used. ∗∗ denotes p < 0.01.