A synthetic peptide targeting the BH4 domain of Bcl-2 induces apoptosis in multiple myeloma and follicular lymphoma cells alone or in combination with agents targeting the BH3-binding pocket of Bcl-2

Abstract

Bcl-2 inhibits apoptosis by two distinct mechanisms but only one is targeted to treat Bcl-2-positive malignancies. In this mechanism, the BH1-3 domains of Bcl-2 form a hydrophobic pocket, binding and inhibiting pro-apoptotic proteins, including Bim. In the other mechanism, the BH4 domain mediates interaction of Bcl-2 with inositol 1,4, 5-trisphosphate receptors (IP3Rs), inhibiting pro-apoptotic Ca2+ signals. The current anti-Bcl-2 agents, ABT-263 (Navitoclax) and ABT-199 (Venetoclax), induce apoptosis by displacing pro-apoptotic proteins from the hydrophobic pocket, but do not inhibit Bcl-2-IP3R interaction. Therefore, to target this interaction we developed BIRD-2 (Bcl-2 IP3 Receptor Disruptor-2), a decoy peptide that binds to the BH4 domain, blocking Bcl-2-IP3R interaction and thus inducing Ca2+-mediated apoptosis in chronic lymphocytic leukemia, multiple myeloma, and follicular lymphoma cells, including cells resistant to ABT-263, ABT-199, or the Bruton's tyrosine kinase inhibitor Ibrutinib. Moreover, combining BIRD-2 with ABT-263 or ABT-199 enhances apoptosis induction compared to single agent treatment. Overall, these findings provide strong rationale for developing novel therapeutic agents that mimic the action of BIRD-2 in targeting the BH4 domain of Bcl-2 and disrupting Bcl-2-IP3R interaction.

Keywords: ABT-199; Bcl-2; Bruton's tyrosine kinase; inositol 1,4,5-trisphosphate receptor; lymphoid malignancy.

Figure 1. Differential sensitivity of various malignant and non-malignant cell lines to BIRD-2-induced cell death

A. Primary human CLL cells, cell lines representing a variety of lymphoid malignancies, and non-lymphoid cell lines were treated with 10 μM BIRD-2 or BIRD-2ctrl for 24 hr and viability was assessed using an MTS assay. Findings represent mean ± SD from three independent experiments. B. and C. HMCLs were incubated with BIRD-2 at multiple dose levels for 24 hr and viability assessed using the MTS assay. BIRD-2ctrl did not induce cell death at the same dose levels (not shown). Symbols represent mean ± SD in at least two separate experiments performed in triplicate.

Figure 2. Apoptosis induction by BIRD-2

A. and B. NCI-H929 (A) and KMS-12-BM (B) cell viability by MTS assay 24 hr after treatment with 10 μM BIRD-2 vs BIRD-2ctrl (mean ± SD of at least 3 experiments). C. and D. Left: representative epifluorescence images of Hoechst 33342-stained NCI-H929 (C) and KMS-12-BM (D) cells treated with 10 or 20 μM BIRD-2 or BIRD-2ctrl for 24 hr. Bright staining of nuclei is due to nuclear condensation and is characteristic of apoptosis, while dim staining indicates live cells. Right: quantification of apoptotic Hoechst 33342-stained nuclei. Data expressed as mean ± SD of over 200 cells per treatment group. E. Immunoblotting displaying relative levels of PARP, cleaved PARP*, and cleaved Caspase-3* in NCI-H929 cells treated with either vehicle (Veh) water, 10 μM BIRD-2 or BIRD-2ctrl for the indicated times. Beta actin is loading control.

Figure 3. Bax activation by BIRD-2 and protection from BIRD-2-induced cell death by caspase inhibition

A. Representative immunocytochemistry images demonstrating the activation and oligomerization of Bax in NCI-H929 cells 4 hr post treatment with 10 μM BIRD-2, 10 μM BIRD-2ctrl, or 2.5 μM staurosporine. Cells were stained with the Bax 6A7 antibody, which specifically detects the active form of Bax, or with normal mouse IgG (control), and were imaged by brightfield (top row) or fluorescence (bottom row) microscopy. Arrows indicate active oligomerized Bax. Scale bar, 50 μm. B. Quantification of apoptotic Hoechst 33342-stained nuclei in NCI-H929 cells after 1 hr pretreatment with 200 μM Z-VAD-FMK or DMSO (vehicle) followed by 4 hr treatment with 10 μM BIRD-2. Data expressed as mean ± SEM of three quantifications of over 200 cells per treatment group. C. CellTiter-Glo viability assay in NCI-H929 cells pretreated for 1 hr with 200 μM Z-VAD-FMK or DMSO followed by 4 hr treatment with 10 μM BIRD-2. Data expressed as mean ± SEM of triplicate measurements.

Figure 4. BIRD-2 induces HMCL cell death in vivo

NCI-H929 cells (9 × 10⁶) were injected subcutaneously into the flank region of NCR nu/nu mice. When tumor volume reached an average of 60–80 mm³, mice were injected at the site of tumors with 20 μL saline or BIRD-2 at a dose of 10 mg per kg body weight. A. Growth of xenograft tumors in mice treated with either saline or BIRD-2. Arrows indicate time of treatment. Data points indicate mean ± SEM of three independent experiments with at least three mice per treatment group; *, p < 0.05; **, p < 0.01. B. Representative saline- and BIRD-2-treated mice imaged at the completion of an experiment. C. Weight of tumors excised from saline-treated or BIRD-2-treated mice. Data are expressed as mean ± SEM. D. Body weight of mice treated with either saline or BIRD-2. p > 0.05 for all data points.

Figure 5. Reciprocal sensitivity of HMCLs to BIRD-2 and ABT-263, ABT-199, or Ibrutinib

A. Differential sensitivity of HMCLs to treatment with 10 μM BIRD-2 for 24 hr measured by CTG viability assay, consistent with preceding findings (Figure 1) using MTS assay. B. HMCLs were treated for 24 hr with a range of concentrations of ABT-263. Findings indicate that KMS-12-BM and RPMI-8226 cells are much more sensitive to ABT-263 than are NCI-H929 or JJN-3 cells. C. HMCLs were treated for 24 hr with a range of concentrations of ABT-199. Findings indicate that KMS-12-BM cells are much more sensitive to ABT-199 than are NCI-H929, JJN-3 and RPMI-8226 cells. In panels A–C, cell viability was measured by CellTiter-Glo Assay, performed in triplicate in at least three experiments and displayed as mean ± SD. D. HMCLs were incubated with Ibrutinib at multiple dose levels for 24 hr and viability was assessed by CellTiter-Glo assay. Symbols represent mean ± SD of triplicate measurements; a representative experiment is shown.

Figure 6. Bim levels and their regulation by BIRD-2 in HMCLs

A. Immunoblot showing Bim, Bcl-2, and Mcl-1 levels in HMCLs. The sensitivity of cell lines to BH3 mimetic agents and to BIRD-2 is summarized at bottom. B. Immunoblot showing relative levels of Bim (long form) and Bcl-2 in NCI-H929 cells treated with either Veh, 10 μM BIRD-2 or BIRD-2ctrl for the indicated times. C. Relative Bim and Bcl-2 mRNA levels in NCI-H929 cells treated with 10 μM BIRD-2 or BIRD-2ctrl for times shown. Beta-actin mRNA levels are used in data normalization. Data are presented as mean ± SD of triplicate samples from a representative of two independent experiments. D. Immunoblot showing levels of Bim (long form) in KMS-12-BM cells treated with either Veh or BIRD-2 for the indicated times. E. Representative Ca²⁺ traces by single cell digital imaging documenting Ca²⁺ elevation induced by BIRD-2 (left panel) and its inhibition by EGTA + BAPTA (right panel). F. Immunoblot showing Bim elevation in NCI-H929 cells induced by BIRD-2 treatment for 4 hr. EGTA (10 mM) and BAPTA-AM (1 μM) were added to cells 30 min before BIRD-2 addition to chelate Ca²⁺. Beta-actin is loading control in each of the immunoblots. G. and H. Hoechst 33342 staining for apoptotic nuclei (G) and CTG viability assay (H) of NCI-H929 cells pre-treated for 30 min with EGTA (10 mM) and BAPTA-AM (1 μM) to chelate Ca²⁺ followed by 4 hr treatment with 10 μM BIRD-2. Data represent mean ± SEM of triplicate measurements.

Figure 7. Combined activity of BIRD-2 and BH3 mimetic agents

A. and B. RPMI-8226 (A) or NCI-H929 (B) cells were treated with the indicated concentrations of ABT-263, BIRD-2, or ABT-263 and BIRD-2 in combination. Cell viability was measured after 24 hr by CellTiter-Glo assay. Results are mean ± SEM of triplicate measurements. C. and D. KMS-12-PE (C) or U266 (D) cells were treated with the indicated concentrations of ABT-199, BIRD-2, or ABT-199 and BIRD-2 in combination. Cell viability was measured after 24 hr by CellTiter-Glo assay. Results are mean ± SEM of triplicate measurements.

Figure 8. Combined activity of BIRD-2 and ABT-199 in follicular lymphoma cells

A. WSU-FSCCL follicular lymphoma cells were treated with the indicated concentrations of ABT-199, BIRD-2, or ABT-199 and BIRD-2 in combination. Cell viability was measured after 24 hr by CellTiter-Glo assay. Results are mean ± SEM of triplicate measurements. B. Representative epifluorescence images of Hoechst 33342-stained WSU-FSCCL cells treated with 0.1 μM ABT-199 and 10 μM BIRD-2 either alone or in combination for 20 hr. Bright staining of nuclei is due to nuclear condensation and is characteristic of apoptosis, while dim staining indicates live cells. C. Quantification of apoptotic Hoechst 33342-stained nuclei. Data expressed as mean ± SEM of over 200 cells per treatment group.

Figure 9. Targeting Bcl-2′s two anti-apoptotic mechanisms

Left: Bcl-2 binds IP₃Rs and prevents Ca²⁺ elevation capable of inducing apoptosis. BIRD-2 inhibits Bcl-2-IP₃R interaction, thereby inducing apoptosis. Right: Bcl-2 binds BH3-only proteins and prevents them from inducing apoptosis. BH3 mimetic agents, such as ABT-263 and ABT-199, release BH3-only proteins from Bcl-2, thereby inducing apoptosis.