Immunoproteasome Activity in Chronic Lymphocytic Leukemia as a Target of the Immunoproteasome-Selective Inhibitors

Abstract

Targeting proteasome with proteasome inhibitors (PIs) is an approved treatment strategy in multiple myeloma that has also been explored pre-clinically and clinically in other hematological malignancies. The approved PIs target both the constitutive and the immunoproteasome, the latter being present predominantly in cells of lymphoid origin. Therapeutic targeting of the immunoproteasome in cells with sole immunoproteasome activity may be selectively cytotoxic in malignant cells, while sparing the non-lymphoid tissues from the on-target PIs toxicity. Using activity-based probes to assess the proteasome activity profile and correlating it with the cytotoxicity assays, we identified B-cell chronic lymphocytic leukemia (B-CLL) to express predominantly immunoproteasome activity, which is associated with high sensitivity to approved proteasome inhibitors and, more importantly, to the immunoproteasome selective inhibitors LU005i and LU035i, targeting all immunoproteasome active subunits or only the immunoproteasome β5i, respectively. At the same time, LU102, a proteasome β2 inhibitor, sensitized B-CLL or immunoproteasome inhibitor-inherently resistant primary cells of acute myeloid leukemia, B-cell acute lymphoblastic leukemia, multiple myeloma and plasma cell leukemia to low doses of LU035i. The immunoproteasome thus represents a novel therapeutic target, which warrants further testing with clinical stage immunoproteasome inhibitors in monotherapy or in combinations.

Keywords: LU005i; LU035i; activity-based probes; acute myeloid leukemia; chronic lymphocytic leukemia; immunoproteasome; multiple myeloma; plasma cell leukemia; proteasome activity; proteasome inhibitors.

Figure 1

Profile of the activity of the immunoproteasome subunits over constitutive proteasome subunits determined by ABP labelling in different hematological malignancies. (A) Comparison between the ratio of activity of proteasome β5i versus β5c, data represent mean ± SD. (B) Comparison between the ratio of activity of proteasome β1i versus β1c, data represent mean ± SD. (C) Comparison between the ratio of activity of proteasome β2i versus β2c, data represent mean ± SD. In all analyses, statistical significance was obtained with ANOVA and Tukey’s multiple comparison test, where * represents p < 0.05, *** represents p < 0.001 and **** represents p < 0.0001. AML = acute myeloid leukemia, B-ALL = B-cell acute lymphoblastic leukemia, B-CLL = B-cell chronic lymphocytic leukemia, MM = multiple myeloma, PCL = plasma-cell leukemia, PBMC = peripheral blood mononuclear cells.

Figure 2

Profile of the IC₅₀ values of the approved proteasome inhibitors in different hematological malignancies. (A) Comparison of IC₅₀ values of bortezomib determined 48 h after the continuous treatment in various hematological malignancies, data represent mean ± SD. (B) Comparison of IC₅₀ values of carfilzomib determined 48 h after the continuous treatment in various hematological malignancies, data represent mean ± SD. In all analyses, statistical significance was obtained with ANOVA and Tukey’s multiple comparison test, where * represents p < 0.05 and **** represents p < 0.0001. AML = acute myeloid leukemia, B-ALL = B-cell acute lymphoblastic leukemia, B-CLL = B-cell chronic lymphocytic leukemia, MM = multiple myeloma, PCL = plasma-cell leukemia, PBMC = peripheral blood mononuclear cells, BTZ = bortezomib; CFZ = carfilzomib.

Figure 3

Cytotoxicity of the immunoproteasome-selective proteasome inhibitors in hematological malignancies. (A) Comparison of IC₅₀ values of LU005i determined 48 h after the continuous treatment in various hematological malignancies. Data represent geometric mean ± geometric SD, statistical significance was obtained with ANOVA and Tukey’s multiple comparison test, where * represents p < 0.05 and **** represents p < 0.0001. Line represents a 2.5 µM dose, to which the inhibitor retains its selectivity. (B) Correlation between the activities of constitutive vs. the immunoproteasome β5 subunits and the cytotoxicity of LU005i in 15 AML, 3 B-ALL, 17 CLL, 6 MM and 5 PCL samples. Correlation and statistical significance were obtained using Spearman’s rank correlation. (C) Comparison of IC₅₀ values of LU035i determined 48 h after the continuous treatment in various hematological malignancies, data represent geometric mean ± geometric SD. In samples, where the IC₅₀ value was not reached, it was arbitrarily given an IC₅₀ = 100 µM. Statistical significance was obtained with ANOVA and Tukey’s multiple comparison test, where ** represents p < 0.01 and **** represents p < 0.0001. Line represents a 5 µM dose, to which the inhibitor retains its selectivity. (D) Correlation between the activities of constitutive vs. the immunoproteasome β5 subunits and the cytotoxicity of LU035i in 17 CLL samples. Correlation and statistical significance were obtained using Spearman’s rank correlation. (E) Dose-response curves of AMO-1 and AMO-1 PSMB5 knock-out cells to LU035i determined 48 h after the treatment. Data represent mean ± SD of three independent experiments. (F–H) Dose-response curves of three B-CLL samples to LU035i alone or in combination with 1 µM LU025c determined 48 h after the treatment. Data represent mean ± SD of tetraplicate. AML = acute myeloid leukemia, B-ALL = B-cell acute lymphoblastic leukemia, B-CLL = B-cell chronic lymphocytic leukemia, MM = multiple myeloma, PCL = plasma-cell leukemia, PBMC = peripheral blood mononuclear cells; LU005i = proteasome β5i + β2i + β1i selective inhibitor; LU035i = proteasome β5i selective inhibitor; LU025c = proteasome β5c selective inhibitor; i = immunoproteasome, c = constitutive proteasome.

Figure 4

β2-selective inhibitor sensitizes hematological malignancies to immunoproteasome-selective inhibitors. (A) Comparison of IC₅₀ values of LU102 determined 48 h after the continuous treatment in various hematological malignancies, data represent geometric mean ± geometric SD. Statistical significance was obtained with ANOVA and Tukey’s multiple comparison test. (B) Comparison of IC₅₀ values of LU035i combined with fixed dose of LU102 (2.5 µM) determined 48 h after the continuous treatment in various hematological malignancies. Data represent geometric mean ± geometric SD. Statistical significance was obtained with ANOVA and Tukey’s multiple comparison test. (C) Paired comparison of IC₅₀ values of LU035i combined with fixed dose of LU102 (2.5 µM) determined 48 h after the continuous treatment in AML samples. Statistical significance was obtained with paired t-test, where *** represents p < 0.001. (D) Paired comparison of IC₅₀ values of LU035i combined with fixed dose of LU102 (2.5 µM) determined 48 h after the continuous treatment in B-ALL samples. Statistical significance was obtained with paired t-test, where * represents p < 0.05. (E) Paired comparison of IC₅₀ values of LU035i combined with fixed dose of LU102 (2.5 µM) determined 48 h after the continuous treatment in MM samples. Statistical significance was obtained with paired t-test, where ** represents p < 0.01. (F) Paired comparison of IC₅₀ values of LU035i combined with fixed dose of LU102 (2.5 µM) determined 48 h after the continuous treatment in MM samples. Statistical significance was obtained with paired t-test, where * represents p < 0.05. (G) Dose-response curves of AMO-1 wt and AMO-1 PSMB5 knock-out cells to LU102 determined 48 h after the treatment. Data represent mean ± SD. (H) Dose-response curves of AMO-1 cells to LU035i alone or in combinations with 2.5 µM LU102, determined 48 h after the treatment. Data represent mean ± SD. AML = acute myeloid leukemia, B-ALL = B-cell acute lymphoblastic leukemia, B-CLL = B-cell chronic lymphocytic leukemia, MM = multiple myeloma, PCL = plasma-cell leukemia, PBMC = peripheral blood mononuclear cells; LU035i = proteasome β5i selective inhibitor; LU102 = proteasome β2c and β2i selective inhibitor; i = immunoproteasome, c = constitutive proteasome.