Overcoming Gemcitabine Resistance in Pancreatic Cancer Using the BCL-XL-Specific Degrader DT2216

Abstract

Pancreatic cancer is the third most common cause of cancer-related deaths in the United States. Although gemcitabine is the standard of care for most patients with pancreatic cancer, its efficacy is limited by the development of resistance. This resistance may be attributable to the evasion of apoptosis caused by the overexpression of BCL-2 family antiapoptotic proteins. In this study, we investigated the role of BCL-X_L in gemcitabine resistance to identify a combination therapy to more effectively treat pancreatic cancer. We used CRISPR-Cas9 screening to identify the key genes involved in gemcitabine resistance in pancreatic cancer. Pancreatic cancer cell dependencies on different BCL-2 family proteins and the efficacy of the combination of gemcitabine and DT2216 (a BCL-X_L proteolysis targeting chimera or PROTAC) were determined by MTS, Annexin-V/PI, colony formation, and 3D tumor spheroid assays. The therapeutic efficacy of the combination was investigated in several patient-derived xenograft (PDX) mouse models of pancreatic cancer. We identified BCL-X_L as a key mediator of gemcitabine resistance. The combination of gemcitabine and DT2216 synergistically induced cell death in multiple pancreatic cancer cell lines in vitro In vivo, the combination significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice compared with the individual agents in pancreatic cancer PDX models. Their synergistic antitumor activity is attributable to DT2216-induced degradation of BCL-X_L and concomitant suppression of MCL-1 by gemcitabine. Our results suggest that DT2216-mediated BCL-X_L degradation augments the antitumor activity of gemcitabine and their combination could be more effective for pancreatic cancer treatment.

Figure 1.

BCL2L1 (BCL-X_L) provides resistance to gemcitabine treatment in pancreatic cancer. A, Representation of the Therapeutic Genome (RxG) CRISPR library screening to identify genes important for resistance to gemcitabine (GEM), 5FU, and niraparib (NIR) in the AsPC-1 pancreatic cancer cell line. B–D, Log₂ volcano plots showing the results of the screening for gemcitabine (B), 5FU (C), or niraparib (D). Several biologically interesting hits (including BCL2L1) identified from the screening are highlighted in red. Each gene targeted by the library was ranked based on the Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) positive selection score. FDR, False discovery rate; FC, Fold change.

Figure 2.

The combination of DT2216 and gemcitabine synergistically kills pancreatic cancer cells in vitro. A, Percentage of total apoptotic cells (the sum of early [Annexin V⁺] and late [PI⁺/Annexin V⁺] apoptotic cells) after the cells were treated with vehicle (Veh) or with the indicated concentrations of DT2216 (DT) or gemcitabine (GEM) alone or in combination for 72 hours. Data are presented as the mean ± SD of four independent experiments for each cell line and condition. a, b and c,P < 0.05 versus. Veh, DT and GEM, respectively. B, Percentage of total apoptotic G-68 primary pancreatic cancer cells after they were treated with vehicle (Veh) or the indicated concentrations of DT or gemcitabine alone or in combinations for 72 hours. Data are presented as mean ± SD (n = 3 independent experiments). *,P < 0.05; ****,P < 0.0001. Representative flow cytometric quadruple graphs for A and B are shown in Supplementary Fig. S2A and S2B, respectively. C, Percentage viability of G-68 cells after they were treated with increasing concentrations of DT, gemcitabine, or their combination (GEM+DT; 1:1 ratio) for 72 hours. The table shows the IC₅₀ values of DT, gemcitabine, or their combination (GEM+DT). IC₅₀ values are shown for a representative experiment out of three independent experiments. The CI value (< 1) indicates a synergy between DT and gemcitabine. D, G-68 cells were treated with the indicated concentrations of DT or gemcitabine alone or in combination for 72 hours followed by incubation in drug-free medium for another two weeks. Crystal violet staining was performed to visualize the colonies. Data are representative images from two independent experiments. E, G-68 cells were seeded in a 48-well plate and allowed 48 hours for spheroid formation before DT and/or gemcitabine treatment. Micrographs (magnification at 50×) of spheroids in the corresponding wells after they were treated with the indicated concentrations of DT or gemcitabine alone or in combination for 10 days are shown. F, Microphotographs (magnification 50×) of spheroids from the marked areas in E. G, Quantification of the number of spheroids from E as a percentage of Veh. a, b and c,P < 0.05 versus Veh, DT, and GEM, respectively. Data presented in E–G are representative of three independent experiments. Statistical significance in A, B, and G was determined by two-way ANOVA with Tukey post hoc test.

Figure 3.

Synergy between DT2216 and gemcitabine attributes to a combined depletion of BCL-X_L and MCL-1. A, Immunoblot analysis of BCL-X_L, BCL-2, and MCL-1 in G-68 cells after they were treated with the indicated concentrations of DT2216 (DT) for 16 hours. B, Immunoblot analysis of BCL-X_L, BCL-2, MCL-1, and NOXA in G-68 cells after they were treated with the indicated concentrations of gemcitabine (GEM) for 48 hours. C, Immunoblot analysis of BCL-X_L, BCL-2, MCL-1, NOXA, and apoptosis markers – cleaved (C) and full-length caspase-3 and PARP in G-68 cells after they were treated with the indicated concentrations of gemcitabine and/or DT for 48 hours. Immunoblots presented in A-C are representative of three independent experiments. D, G-68 cells were treated with gemcitabine (1 μmol/L) and/or DT (0.1 μmol/L) for 48 hours. Cell lysates were subjected to co-immunoprecipitation (Co-IP) using BCL-X_L or MCL-1 antibodies followed by immunoblotting to detect BCL-X_L, MCL-1, and BIM. E, The levels of BCL-X_L, MCL-1, and BIM in the inputs for D. β-actin was used as an equal loading control for all immunoblot analyses presented in A–E. F, Viability of G-68 cells after they were treated with increasing concentrations of DT or S63845 (S) individually or in combination (DT+S) for 72 hours. IC₅₀ values are shown in the table. The CI value indicates a synergy between DT and S. G, G-68 cells were treated with 0.1 μmol/L of DT or 1 μmol/L of gemcitabine alone or in combination (gemcitabine + DT) for 48 hours. The fold changes in the expressions of BCL2L1 (encodes BCL-X_L), BCL2, MCL1, and PMAIP1 (encodes NOXA) mRNA are shown. GAPDH was used as an internal control. Data are from a single experiment performed in triplicate (mean ± SD).

Figure 4.

DT2216 increased the antitumor efficacy of gemcitabine in a patient-derived G-68 pancreatic cancer cell xenograft model. A, Representation of the experimental design of the G-68 xenograft study. Tumor-bearing mice were administered Veh, DT2216 (DT), gemcitabine (GEM), or a combination of DT and gemcitabine at the indicated dosing regimen. B, Change in body weight during the course of treatment. C, Graph showing the tumor volume changes in each group after the start of treatment until the control animals were euthanized. Data are presented as mean ± SEM (n  =  7 mice in each group at the start of treatment). Statistical significance was determined by unpaired two-sided Student t test test. **, P < 0.01; ****, P < 0.0001. D, Kaplan–Meier survival analysis with medium survival time of mice in each group. Statistical significance was determined by Mantel–Cox test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. E, Representative H&E staining images of tumors in each treatment group at 200x magnification, scale bar = 50 μm.

Figure 5.

DT2216 increased the antitumor efficacy of gemcitabine in pancreatic cancer PDX models. A, Representation of the experimental design of the PDX studies. Tumor-bearing mice were administered Veh, DT2216 (DT), gemcitabine (GEM), or a combination of DT and gemcitabine at the indicated dosing regimen. B, Graph showing the tumor volume changes in G192-p4 PDXs in each group after the start of treatment. C, Representative H&E staining images of G192-p4 PDX tumors in each treatment group at 200 × magnification, scale bar = 50 μm. D and E, Graphs showing the tumor volume changes in G176-p4 (D) and LM12-p3 (E) PDXs in each group after the start of treatment. The data presented in (B), (D) and E are mean ± SEM (n  =  7 mice in each group at the start of treatment). Statistical significance was determined by unpaired two-sided Student t test. *, P < 0.05; **, P < 0.01; ns, not significant.