Extrinsic interactions in the microenvironment in vivo activate an antiapoptotic multidrug-resistant phenotype in CLL

Abstract

The Bcl-2 inhibitor venetoclax has yielded exceptional clinical responses in chronic lymphocytic leukemia (CLL). However, de novo resistance can result in failure to achieve negative minimal residual disease and predicts poor treatment outcomes. Consequently, additional proapoptotic drugs, such as inhibitors of Mcl-1 and Bcl-xL, are in development. By profiling antiapoptotic proteins using flow cytometry, we find that leukemic B cells that recently emigrated from the lymph node (CD69+/CXCR4Low) in vivo are enriched for cell clusters simultaneously overexpressing multiple antiapoptotic proteins (Mcl-1High/Bcl-xLHigh/Bcl-2High) in both treated and treatment-naive CLL patients. These cells exhibited antiapoptotic resistance to multiple BH-domain antagonists, including inhibitors of Bcl-2, Mcl-1, and Bcl-xL, when tested as single agents in a flow cytometry-based functional assay. Antiapoptotic multidrug resistance declines ex vivo, consistent with resistance being generated in vivo by extrinsic microenvironmental interactions. Surviving "persister" cells in patients undergoing venetoclax treatment are enriched for CLL cells displaying the functional and molecular properties of microenvironmentally induced multidrug resistance. Overcoming this resistance required simultaneous inhibition of multiple antiapoptotic proteins, with potential for unwanted toxicities. Using a drug screen performed using patient peripheral blood mononuclear cells cultured in an ex vivo microenvironment model, we identify novel venetoclax drug combinations that induce selective cytotoxicity in multidrug-resistant CLL cells. Thus, we demonstrate that antiapoptotic multidrug-resistant CLL cells exist in patients de novo and show that these cells persist during proapoptotic treatment, such as venetoclax. We validate clinically actionable approaches to selectively deplete this reservoir in patients.

Graphical abstract

Figure 1.

Circulating leukemic B cells recently emigrated from the LN in patients with CLL are enriched for cells simultaneously overexpressing multiple antiapoptotic proteins. (A-B) Patient PBMCs (N = 20) were examined using FCM for expression of apoptotic proteins (Mcl-1, Bcl-xL, Bcl-2, Bim, Puma, Bak, and Bax) and markers of microenvironmental interactions (CD69 and CXCR4) in CLL cells (viability dye⁻/CD5⁺/CD19⁺). (A) Flow cytometry images present the gating strategy for identification of CLL cells, CD69/CXCR4-expressing CLL cells, and expression of apoptotic proteins in CD69⁻/CXCR4^High and CD69⁺/CXCR4^Low CLL cells. (B) The expression of antiapoptotic proteins Mcl-1, Bcl-xL, and Bcl-2 in CD69⁺/CXCR4^Low CLL cells as compared with CD69⁻/CXCR4^High CLL cells. Data are presented as fold difference in geometric mean fluorescence intensity (GMFLI). (C) FCS files were generated from pregated CD69⁺/CXCR4^Low- or CD69⁻/CXCR4^High-expressing CLL cells in every patient (N = 19) using FlowJo software, and cell clusters expressing different levels of antiapoptotic proteins (Mcl-1, Bcl-xL, and Bcl-2) were identified through unsupervised clustering analysis, as described in "Materials and methods." A heatmap was generated based on GMFLI values to show the expression of various antiapoptotic proteins in different clusters (left). The clusters were visualized using a bubble graph in which every bubble represents 1 cluster and the area of the bubble (size) is proportional to the mean percentage of cells in that cluster (middle). A table presenting the mean percentage of cells in each cluster identified in CD69⁺/CXCR4^Low- or CD69⁻/CXCR4^High-expressing CLL cells (right). The mean percentage of cells in a cluster was determined by calculating the average for that cluster across patients analyzed (N = 19). (D) PBMCs of patients with CLL (patients 25, 27, 52, 81, and 95), either fresh or after culturing ex vivo for 24 hours in RPMI containing 10% fetal calf serum without added agonists, were analyzed for expression of the antiapoptotic proteins Mcl-1, Bcl-xL, and Bcl-2 using FCM. Data are presented as fold difference in GMFLI in CD69⁺ or CD69⁻ CLL cells as compared with CD69⁻ CLL cells from fresh PBMCs (ie, processed without ex vivo culture). Statistical significance was determined by ANOVA with Sidak’s post-hoc test for multiple comparisons. *P < .05; **P < .01; ****P < .0001; ns, not significant. Data are presented as mean ± SD. Neg, negative; Pos, positive.

Figure 2.

Circulating CLL cells that recently emigrated from the LN in vivo show upregulation of classical/alternative NF-κB signaling. NF-κB nuclear localization was examined in FFWB samples of patients with CLL using imaging FCM (ImageStream). (A) Representative cell images displaying staining for CD5, CD19, CD69, CXCR4, RelA/RelB/p100,52, 7AAD, 7AAD/RelA, 7AAD/RelB, or 7AAD/p100,52 (merge) in a CLL sample. The images were captured at ×60 magnification. (B) Fold difference in nuclear localization of RelA, RelB, and p100,52 in CD69⁺/CXCR4^Low CLL (CD5⁺/CD19⁺) cells as compared with CD69⁻/CXCR4^High CLL cells in multiple patient samples. Statistical significance was determined by Student t test. *P < .05; **P < .01; ***P < .001. Data are presented as mean ± SD.

Figure 3.

Circulating CLL cells with CD69⁺ activation phenotype that overexpress multiple antiapoptotic proteins in vivo display antiapoptotic multidrug resistance. (A-B) Freshly frozen PBMCs from various patients with CLL were screened using the ATA by incubating with inhibitor of Bcl-2 (venetoclax 12.5, 25, 50, or 100 nM), Mcl-1 (S63845 0.61, 0.91, 1.35, or 2.05 µM), or Bcl-xL (A1155463 4, 8, 16, or 32 µM) for 3 hours without added agonists. (A) Representative FCM images of cleaved caspase-9 staining in CD69⁺ or CD69⁻ CLL (viability dye⁻/CD5⁺/CD19⁺) cells in a patient PBMC (patient 07) incubated with dimethyl sulfoxide (DMSO) or venetoclax. (B) Percentage of CD69⁺ or CD69⁻ CLL cells positive for cleaved caspase-9 from multiple patient samples exposed to various proapoptotic agents in ATA. (C) To demonstrate ex vivo lability of microenvironmentally induced drug resistance, PBMCs of patients with CLL (patients 8, 25, 27, 41, and 52), either fresh or after culturing ex vivo in RPMI containing 10% fetal calf serum for 24 hours without added agonists, were processed in ATA by incubating with venetoclax (200 nM) or A1155463 (64 µM) for 1 hour (the patient 41 sample was incubated with drugs for 2 hours) or S63845 (6.9 µM) for 3 hours. Since patient 41 was not sensitive to Mcl-1 inhibitor, it was excluded from the S63845 panel. Data are presented as percentage CD69⁺ or CD69⁻ CLL (viability dye⁻/CD5⁺/CD19⁺) cells positive for cleaved caspase-9. The data in Figure 3B-C are presented after subtracting spontaneous apoptosis values from DMSO treatment controls. Statistical significance was determined by ANOVA with Sidak’s post-hoc test for multiple comparisons. *P < .05; **P < .01; ***P < .001; ****P < .0001. Data are presented as mean ± SD. SSC-A, side scatter area.

Figure 4.

Surviving persister cells in venetoclax-treated patients with CLL are enriched for leukemic B cells displaying functional and molecular properties of multidrug resistance. (A) Schema of the studied patients with CLL (supplemental Table 4) with timing of sample collections taken before and during venetoclax treatment. (B-C) PBMCs of patients with CLL isolated prior to or during treatment with venetoclax were analyzed for multidrug resistance in ATA by incubating ex vivo with inhibitors of Bcl-2 (venetoclax; 25, 50, 100, or 200 nM), Bcl-xL (A1155463; 8, 16, 32, or 64 µM), or Mcl-1 (S63845; 2.05, 3.07, 4.6, or 6.9 µM) for 3 hours without agonists. (B) Representative flow images showing caspase-9 cleavage in CLL (viability dye⁻/CD5⁺/CD19⁺) cells following ex vivo incubation with venetoclax (200 nM), A1155463 (64 μM), or S63845 (6.9 μM) of a patient PBMC (patient 71) taken prior to or at 3 and 27 days of treatment with venetoclax. (C) Percentage CLL cells positive for cleaved caspase-9 following ex vivo incubation with venetoclax, A1155463, or S63845 of multiple PBMCs of patients with CLL (N = 5) taken prior to or during treatment with venetoclax, as shown in panel A. Data are presented after subtracting spontaneous apoptosis values from DMSO treatment controls. (D) The expression of antiapoptotic proteins in PBMCs of patients with CLL (N = 5) taken prior to or during treatment with venetoclax was analyzed by FCM. FCS files were generated from pregated CLL (viability dye⁻/CD5⁺/CD19⁺) cells, and cell clusters expressing different levels of antiapoptotic proteins in before and during therapy samples were identified using an unsupervised clustering analysis as described in Figure 1C. A heatmap was generated based on GMFLI values to show expression of various antiapoptotic proteins in different clusters (left). Clusters were visualized using a bubble graph in which every bubble represents 1 cluster and the area of the bubble (size) is proportional to the mean percentage of cells in that cluster (middle). A table showing mean percentage cells in every cluster in samples taken before and during venetoclax treatment (right). The mean percentage of cells in a cluster was determined by calculating the average for that cluster across patients analyzed (N = 5). Statistical significance was determined by ANOVA with Sidak’s post-hoc test for multiple comparisons. *P < .05; **P < .01; ***P < .001; ****P < .0001. Data are presented as mean ± SD. Pt, patient; VEN, venetoclax.

Figure 5.

Drug screen performed using patient PBMCs cultured in an ex vivo microenvironment model identifies novel venetoclax combinations that exhibit selective cytotoxicity in multidrug-resistant CLL cells. (A) Diagram presenting the experimental approach for combination drug screening in microenvironmentally induced multidrug-resistant CLL cells ex vivo. PBMCs of patients with CLL were preincubated with the combination of CpG-ODN, sCD40L, and IL-10 (agonist mix) for 12 hours. Then, samples were treated with venetoclax (25 nM) in combination with increasing concentrations of the IKKα/β inhibitor BMS345541 (2, 4, 6, or 8 µM), the MALT1 inhibitor MI-2 (0.18, 0.37, 0.75, or 1.5 µM), the proteasome inhibitor bortezomib (1, 2, 4, or 8 nM), or the Nedd8 activating enzyme inhibitor pevonedistat/MLN4924 (0.25, 0.5, 0.75, or 1 µM), as well as a second dose of agonist mix for 24 hours. (B) The cleaved PARP in CD5⁺/CD19⁺ (CLL) cells was analyzed by FCM. (C) Bar graphs presenting Bliss predicted and actual (ie, observed) cytotoxicity for the combination of venetoclax (25 nM) with BMS345541 (8 µM), MI-2 (1.5 µM), bortezomib (8 nM), or pevonedistat/MLN4924 (1 µM) in CLL cells. The Bliss predicted cytotoxicity was determined as described earlier.^, Synergistic benefit is expected when the actual cytotoxicity is more than Bliss-predicted cytotoxicity. (D) The cleaved PARP in CD3⁺/CD5⁺ cells (normal T lymphocytes) was analyzed by FCM. The data in panels B and D are presented after subtracting spontaneous apoptosis values from DMSO treatment controls. Statistical significance was determined by ANOVA with Sidak’s post-hoc test for multiple comparisons. *P < .05; **P < .01; ***P < .001; ****P < .0001. Data are presented as mean ± SD.