Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Nov 1.
Published in final edited form as: Exp Hematol. 2015 Aug 6;43(11):951–962.e3. doi: 10.1016/j.exphem.2015.07.005

Targeting executioner procaspase-3 with the procaspase activating compound B-PAC-1 induces apoptosis in multiple myeloma cells

Shadia Zaman a, Rui Wang a, Varsha Gandhi a,b
PMCID: PMC4630139  NIHMSID: NIHMS713876  PMID: 26257207

Abstract

Multiple myeloma (MM) is a plasma cell neoplasm that has a low apoptotic index. We investigated a new class of small molecules that target the terminal apoptosis pathway, called procaspase activating compounds (PACs), in myeloma cells. PAC agents (PAC-1 and B-PAC-1) convert executioner procaspases (procaspase-3, -6 and -7) to active caspase-3, -6, and -7, which cleave target substrates to induce cellular apoptosis cascade. We hypothesized that targeting this terminal step will overcome survival and drug-resistance signals in myeloma cells and induce programmed cell death. Myeloma cells expressed executioner caspases. In concert, our studies demonstrated that B-PAC-1 is cytotoxic to chemotherapy resistant or sensitive myeloma cell lines (n=7) and primary patient cells (n=11). Exogenous zinc abrogated B-PAC-1-induced cell demise. B-PAC-1-treatment-induced apoptosis was similar in the presence or absence of growth-promoting cytokines such as interleukin-6 and hepatocyte growth factor. Presence or absence of anti-apoptotic proteins such as BCL-2, BCL-XL, or MCL-1 did not impact B-PAC-1-mediated programmed cell death. Collectively, our data demonstrate the proapoptotic effect of B-PAC-1 in MM and suggests that activating terminal executioner procaspase-3, -6 and -7 bypasses survival and drug-resistance signals in myeloma cells. This novel strategy has the potential to be an effective anti-myeloma therapy.

Keywords: procaspase activating compound, procaspase-3, multiple myeloma, B-PAC-1, executioner procaspase

Introduction

Multiple myeloma (MM) is currently the second most common hematological malignancy in the US, and accounts for approximately 10% of all hematologic cancers [1, 2]. In MM, the excess plasma cells in the bone marrow interfere with the production of normal blood cells and lead to devastating sequelae, including osteolytic bone lesions, renal disease, and immunodeficiency [3]. Currently, the most common treatment administered to myeloma patients include high dose chemotherapy of dexamethasone, bortezomib, and lenalidomide followed by autologous hematopoietic stem-cell transplantation [4]. However, these treatments are not curative and most patients eventually acquire resistance to these chemotherapeutic agents and relapse.

A main distinction of cancer is its resistance to natural apoptotic signals [5], enabling cancerous cells to survive and divide even in the presence of endogenous proapoptotic stimuli [6]. These signals result from either up or down regulation of key proteins in the apoptotic cascade that can occur in the intrinsic and extrinsic apoptotic pathways [5, 7, 8]. The intrinsic pathway is activated by cellular stress such as DNA damaging agents, leading to the release of cytochrome c from the mitochondria. Cytochrome c forms a complex with APAF1 and initiator caspase-9 called the apoptosome that converts the executioner procaspases-3, -6, and -7 to active caspase-3, -6 and -7. These active caspases target numerous substrates for cleavage, eventually leading to apoptosis. Similarly, the extrinsic pathway is activated upon stimulation of the tumor necrosis factor family death receptors, which convert procaspase-8 to caspase-8 and activates the executioner procaspases leading to apoptosis.

Myeloma cells have a low apoptotic index which decreases as the disease progresses from monoclonal gammopathy of undetermined significance (MGUS) to smoldering or indolent myeloma (SMM/IMM) to newly diagnosed MM [9]. This decrease in the apoptotic index is often due to overexpression of survival proteins including the BCL-2 family proteins, BCL-2, BCL-XL and MCL-1 [10–14], and the IAP family proteins, cIAP1, cIAP2 and XIAP [15–17]. The deregulation of these survival proteins prevents proapoptotic signals from being transmitted to activate executioner caspases and, thus, cause cancerous cells to proliferate uncontrollably [18].

The proteolytic conversion of procaspase-3 (-6 or -7) to caspase-3 (-6 or -7) results in the active ‘executioner’ caspase that subsequently catalyzes the hydrolysis of many protein substrates [18]. Active caspase-3 is a homodimer of heterodimers and is typically activated through the action of caspase-8 and caspase-9 [18]. To prevent premature activation of procaspase-3, the zymogen utilizes a triaspartic acid ‘safety catch.’

Procaspase-3 has been found to have elevated concentrations in cells from various cancerous tissues including certain neuroblastomas, lymphomas, leukemias, melanomas and liver cancers [18]. This allows for a therapeutic index to be achieved for caspase-3-directed agents. Due to the focal role of active caspase-3 in successfully inducing apoptosis, the use of a small molecule to directly activate procaspase-3 has been suggested as a tactical approach in targeted cancer therapy. Such an effort would bypass the damaged apoptotic cascade in cancer cells and directly convert procaspase-3 to caspase-3 [6].

Procaspase activating compound 1 (PAC-1) and its new generation analog (B-PAC-1) are a class of small molecules that stimulate the conversion of procaspase-3 to caspase-3 [6]. Mechanistically, PAC-1 induces activation of procaspase-3 in vitro via sequestration of inhibitory zinc ions. Evidences have shown that zinc binding is critical to the ability of PAC-1 to induce death in cancer cells [6]. PAC-1 induces the autoactivation of caspase-3 and caspase-3-mediated cleavage of anti-apoptotic proteins (such as BCL-2 and BCL-XL), which in turn may induce depolarization of the mitochondrial membrane and amplify the apoptotic effect. PAC-1 has entered Phase I trials and B-PAC-1 is being evaluated to move to clinic.

Procaspase-3 presents itself as a strategic therapeutic target capable of bypassing upstream mutational inactivation of proapoptotic proteins. Described herein are experiments testing the effectiveness and mechanism of action of B-PAC-1, a new investigational drug in multiple myeloma cells.

Materials and methods

Cell cultures and reagents

All cell lines were maintained in a 37 °C humidified incubator with 5% CO2. Myeloma cell lines were grown in media as indicated in Table 1 [19–23]. HL-60/Neo, HL-60/BCL-2 and HL-60/BCL-XL cell lines were maintained in RPMI-1640 media supplemented with 10% fetal bovine serum and 1 mM sodium pyruvate. Mouse embryo fibroblasts that were wild type for MCL-1 (WT MCL-1) or deleted for MCL-1 (MCL-1Δ) were maintained in DMEM media with no glucose and was supplemented with 1× MEM non-essential amino acid (Gibco, Grand Island, NY), 1× penicillin/streptomycin, 0.2 mM β-mercaptoethanol (Sigma, St Louis, MO), 10% fetal bovine serum and 2mM L-glutamine. All cell lines were authenticated and tested for Mycoplasma contamination by the UT MD Anderson Cancer Center Characterized Cell Line Core. The procaspase-3 activating compounds (PAC-1, B-PAC-1 (previously known as L14R8) and PAC-1a) were a kind gift from Dr. Hergenrother (University of Illinois at Urbana-Champaign, IL).

Table 1.

Myeloma cell lines used in this study.

Cell Line Origin Medium + Supplements Characteristics
U266 ATCC RPMI-1640 + 10% FBS
MM.1S Dr. Rosen, Lurie Comprehensive Cancer Center, Chicago, IL RPMI-1640 + 10% FBS Glucocorticoid-and lenalidomide-sensitive
MM.1R Dr. Rosen, Lurie Comprehensive Cancer Center, Chicago, IL RPMI-1640 + 10% FBS Glucocorticoid-resistant
MM.1/R10R Dr. Orlowski, UT MD Anderson Cancer Center, Houston, TX RPMI-1640 + 10% FBS, 1% penicillin-streptomycin, 10 mM L-glutamine, 5 µg/ml gentamycin, 10 nM lenalidomide Lenalidomide-resistant
RPMI-8226 ATCC RPMI-1640 + 10% FBS, 1% penicillin-streptomycin, 10 mM L-glutamine
ANBL-6 Jelinek DF et al., Cancer Research 1993;53:5320–5327 RPMI-1640 + 10% FBS, 1% penicillin-streptomycin, 10 mM L-glutamine, 1 ng/ml IL-6 Bortezomib-senstitive
ANLB-6/V10R Dr. Orlowski, UT MD Anderson Cancer Center, Houston, TX RPMI-1640 + 10% FBS, 1% penicillin-streptomycin, 10 mM L-glutamine, 1 ng/ml IL-6, 10 nM bortezomib Bortezomib-resistant
KAS-6/1 Westendorf JJ et al., Leukemia 1996;10:866–876. RPMI-1640 + 10% FBS, 1% penicillin-streptomycin, 10 mM L-glutamine, 1 ng/ml IL-6 Bortezomib- and lenalidomide-sensitive
KAS-6/V10R Dr. Orlowski, UT MD Anderson Cancer Center, Houston, TX RPMI-1640 + 10% FBS, 1% penicillin-streptomycin, 10 mM L-glutamine, 1 ng/ml IL-6, 10 nM bortezomib Bortezomib-resistant
KAS-6/R10R Dr. Orlowski, UT MD Anderson Cancer Center, Houston, TX RPMI-1640 + 10% FBS, 1% penicillin-streptomycin, 10 mM L-glutamine, 1 ng/ml IL-6, 10 nM lenalidomide Lenalidomide-resistant
Open in a new tab

Cell apoptosis assays

Cells were harvested after treatment with PAC agents for 24 hours. Cells were centrifuged at 1500 rpm for 5 min, washed once with 1× phosphate buffered saline and incubated with annexin V-FITC (Pharmingen Biosciences, San Diego, CA) for 10 min. Cells were then treated with 0.5 µg/ml propidium iodide (PI) and analyzed on a BD FACSCalibur (BD Biosciences, San Jose, CA).

Co-culture of U266 cells with NKtert cells

U266 cells were co-cultured with NKtert stromal cells at a ratio of 20:1. NKtert cells were seeded in 12-well plates at a concentration of 2.5 × 104 cells/ml. After 14–16 hours, U266 cells were seeded on NKtert cells at a concentration of 5 × 105 cells/ml. Co-cultured cells were incubated for 2 hours before the treatments were added. After 24 hours, U266 cells, which are suspension cells, were removed for cell apoptosis analysis. NKtert cells were also analyzed for cell apoptosis after detaching them with Accutase Cell Detachment Solutions (Innovative Cell Technologies, San Diego, CA).

Immunoblot analysis

Whole-cell lysates were prepared using 1× RIPA buffer (Millipore, Billerica, MA), supplemented with protease and phosphatase inhibitors (Roche Diagnostics, Indianapolis, IN). Cells were sonicated in RIPA buffer for 2 × 3 min and centrifuged at 4°C for 10 min at 14,000 rpm. The supernatant was used for further analysis. Equal concentration of lysates was separated on 4–12% Criterion XT Bis-Tris precast gels (Bio-Rad, Hercules, CA).

Antibodies used in immunoblot analysis were caspase-3, -6 and -7 (Cell Signaling Technologies, Danvers, MA), cIAP2 (Epitomics, Burlingame, CA), MCL-1 (Santa Cruz Biotechnology, Dallas, TX), cIAP1 and GAPDH (Abcam, Cambridge, MA), cleaved PARP and XIAP (BD Biosciences, San Jose, CA). Immunoblots were scanned on an Odyssey imaging system (LI-COR Biosciences, Lincoln, NE) and quantitated using Image Studio Lite Ver 3.1 (LI-COR Biosciences, Lincoln, NE). Caspase positive and negative control cell lysates were obtained from Cell Signaling Technologies.

Cytotoxicity analysis of primary bone marrow CD138+ cells

Bone marrow samples were obtained from patients with myeloma through a protocol approved by the MD Anderson Cancer Center Institutional Review Board (IRB) and in accordance with the Declaration of Helsinki. CD138+ cells were isolated from bone marrow by the institutional myeloma core facility [22]. For some bone marrow samples, cells were received unsorted. For these samples, after drug treatment cells were centrifuged and were labeled with monoclonal CD138 antibody conjugated to APC (Miltenyi Biotec, Auburn, CA). Both CD138+ sorted and unsorted cells were stained with annexin V-FITC and PI and analyzed as described above.

Statistical analysis

Statistical analysis was done using GraphPad Prism (GraphPad software version 6, San Diego, CA). The analyses used are indicated in the figures.

Results

B-PAC-1 induced apoptosis in a dose- and time-dependent manner

The procaspase-activating compounds (PAC), PAC-1 and B-PAC-1 (a potent analog of PAC-1), are small molecules that initiate apoptosis through targeting procaspase-3. PAC-1 is an ortho-hydroxy N-acyl hydrazine while B-PAC-1 is a second generation analog of PAC-1 that contains benzyloxy and di-t-butyl functional groups that increases its lipophilicity (Supplementary Figure 1). To examine if targeting procaspase-3 will be a viable therapeutic target in multiple myeloma, we first examined procaspase-3 protein expression in 8 multiple myeloma cell lines: RPMI-8226, ANBL-6, ANBL-6/V10R (bortezomib-resistant), KAS-6/1, KAS-6/R10R (lenalidomide-resistant), KAS-6/V10R (bortezomib-resistant), U266 and MM.1S (Fig. 1A). Procaspase-3 protein expression was readily detected at steady-state levels in all the tested cell lines, suggesting that PAC agents might be effective in inducing apoptosis in myeloma cells. In addition to procaspase-3, we also examined the protein expression of the other executioner procaspases, procaspase-6 and procaspase-7. As shown in Figs. 1B and 1C, both procaspases are readily detectable at steady-state levels in myeloma cell lines.

Figure 1. Multiple myeloma cell lines express procaspase-3, -6 and -7.

Figure 1

Open in a new tab

Immunoblot analysis of procaspase-3, -6 and -7 in multiple myeloma cell lines. GAPDH was used as internal loading control.

We examined the cytotoxicity of PAC-1 and B-PAC-1 in 2 myeloma cell lines, MM.1S and U266. A dose-response experiment with PAC-1, B-PAC-1 and PAC-1a (a structurally-related, inactive analog of PAC-1 that was used as a negative control) demonstrated that B-PAC-1 increased the percentage of cells stained with annexin V and propidium iodide (PI) in a dose-dependent manner compared to control cells treated with DMSO (Figs. 2A and 2B). Treatment of MM.1S cells with 3 and 10 µM B-PAC-1 increased annexin V+/PI+ cells by 70% and 93%, respectively (Fig. 2C). In U266 cells, 3 and 10 µM B-PAC-1 increased apoptotic cells by 39% and 84%, respectively (Fig. 2D). MM.1S cells displayed more sensitivity to B-PAC-1 (IC50 of 2.6 µM) than U266 cells (IC50 of 5µM). In contrast, neither PAC-1 nor PAC-1a induced apoptosis in these cell lines (Figs. 2 C–D). We, therefore, continued our investigations with B-PAC-1 in myeloma cells to further evaluate its efficacy.

Figure 2. Multiple myeloma cell lines are sensitive to B-PAC-1-induced apoptosis.

Figure 2

Figure 2

Open in a new tab

Multiple myeloma cell lines, MM.1S (A) and U266 (B), were treated with indicated concentration of B-PAC-1. Cells were stained for annexin V and propidium iodide (PI) and cell death was measured by flow cytometry. MM.1S (C) or U266 (D) cells were treated with indicated concentration of B-PAC-1, PAC 1 or PAC1a and cytotoxicity was measured as in A and B. Data are an average of three independent experiments and are displayed as Mean ± SEM. MM.1S (E) and U266 (F) cells were treated with 0, 3, and 10 µM B-PAC-1 for 3, 6, and 24 hours. Cells were stained as in (A and B) and % viable cells were measured by flow cytometry. Data are an average of three independent experiments and are displayed as Mean ± SEM.

To examine the kinetics of B-PAC-1 action in MM.1S and U266 cells, we conducted a time-course experiment and examined B-PAC-1 cytotoxicity after treatment with DMSO (control cells), 3, and 10 µM B-PAC-1 for 3, 6 and 24 hours (Figs. 2E and 2F). Both MM.1S and U266 cells demonstrated a time-dependent decrease in cell viability. Consistent with the dose-response experiments, MM.1S cells exhibited greater sensitivity than U266 cells to B-PAC-1. In MM.1S cells, cell viability decreased as early as 3 hours after treatment with 10 µM B-PAC-1. These data illustrate that B-PAC-1 induced apoptosis in myeloma cell lines in a dose- and time-dependent manner.

B-PAC-1-induced apoptosis is inhibited by zinc

Next, we examined the mechanism of action of B-PAC-1 in myeloma cell lines. Procaspase-3 is inhibited by labile zinc ions and becomes activated when cleaved by caspase-9 at specific aspartic acid residues. Studies have shown that PAC-1 activates procaspase-3 by chelating zinc ions, allowing it to auto-activate [6]. To examine if B-PAC-1 activated procaspase-3 in myeloma cells through chelating zinc ions, MM.1S cells were treated with 3 or 10 µM B-PAC-1 in the absence or presence of 100 µM zinc. As shown in Fig. 3A, 3 and 10 µM B-PAC-1 treatment reduced the viability of MM.1S cells; however, addition of excess exogenous zinc inhibited the function of B-PAC-1 and cell viability was restored to the levels observed in control cells. To examine if zinc-dependent inhibition of apoptosis was specific to B-PAC-1, we treated cells with staurosporine, a natural product isolated from the bacterium Streptomyces staurosporeus, that can induce apoptosis. Addition of exogenous zinc did not inhibit staurosporine-mediated cell apoptosis (Fig. 3A), suggesting that staurosporine does not depend on zinc for its cytotoxic activity.

Figure 3. B-PAC-1-induced apoptosis by cleaving procaspase-3, -6 and -7 and its action is inhibited by exogenous zinc.

Figure 3

Open in a new tab

(A) MM.1S cells were treated with 0, 3, and 10 µM B-PAC-1, with and without zinc, for 24 hours. Cells were also treated with 100 nM Staurosporin (a positive control for apoptosis), with and without zinc. Cells were stained for annexin V and PI and % viable cells were measured by flow cytometry. % viable cells represent the percentage of annexin V+ and PI+ cells. Data are presented as Mean ± SEM, n = 3. (B) Immunoblot analysis of MM.1S cells treated with 0, 5, and 10 µM B-PAC-1 with and without zinc for 24 hours. GAPDH was used as loading control. Caspase-3 positive and negative controls were used to identify the cleaved caspase-3 bands. (C) MM.1S cells were treated with 0, 3 or 10 µM B-PAC-1 for 24 hours, or treated with 100 µM ZnSO4, 10 µM B-PAC-1 or 10 µM B-PAC-1 + 100 µM ZnSO4 for 24 hours. Cell lysates were prepared and immunoblot analysis was done for procaspase-7 and caspase-7. Positive and negative controls were used to identify the cleaved bands. (D) MM.1S cells were treated with 0, 3 or 10 µM B-PAC-1 for 24 hours. Immunoblot analysis was done for procaspase-6 and caspase-6. Positive and negative controls were used to identify the cleaved bands. GAPDH was used as internal loading control in all the immunoblots.

To analyze B-PAC-1-mediated apoptosis at the molecular level, we conducted immunoblot analysis after treatment of MM.1S cells with DMSO (control cells), 5 or 10 µM B-PAC-1 for 24 hours (Fig. 3B and Supplementary Fig. 2). Compared to DMSO (vehicle) treatment, cells treated with 5 and 10 µM B-PAC-1 resulted in decrease in the levels of procaspase-3 and increase in the levels of cleaved caspase-3. We used Image Studio Lite Ver 3.1 to semi-quantitatively analyze the immunoblots and normalized the signal to GAPDH (Supplementary Figure 2). The decrease in procaspase-3 levels was associated with an increase in the levels of cleaved PARP, a target of caspase-3, thus confirming the cleaving of procaspase 3 and activation of caspase-3. As in the previous experiment, the addition of 100 µM exogenous zinc inhibited procaspase-3 cleavage. In contrast, staurosporine treatment increased procaspase-3 cleavage but was not affected by the addition of zinc.

We also examined the effects of B-PAC-1 treatment on the levels of survival proteins, including the IAP proteins (cIAP1, cIAP2 and XIAP) and the BCL-2 family protein MCL-1 (Fig. 3B and Supplementary Fig. 1). B-PAC-1 treatment led to a decrease in the levels of these anti-apoptotic proteins that was reversed by the addition of exogenous zinc, restoring the levels of these proteins to those of control cells.

Since previous studies with PAC-1 demonstrated that it targeted procaspase-7 [6], we examined if B-PAC-1 cleaved the other executioner procaspases, procaspase-7 and procaspase-6, in MM.1S myeloma cells. To examine the effect of B-PAC-1 on procaspase-7, MM.1S cells were treated with DMSO (control cells), 3 or 10 µM B-PAC-1 for 24 hours. As shown in Fig. 3C (left panel), compared to control-treated cells, 10 µM B-PAC-1 treatment reduced the levels of procaspase-7 and increased the levels of active caspase-7. To examine if zinc ions inhibited B-PAC-1-mediated cleavage of procaspase-7, MM.1S cells were treated with zinc (control cells), 10 µM B-PAC-1 or 10 µM B-PAC-1 in the presence of zinc ions. As shown in Fig. 3C (middle panel), B-PAC-1-mediated cleavage of procaspase-7 was inhibited by the addition of exogenous zinc. Jurkat cells treated with or without staurosporine were run in parallel as positive and negative controls, respectively.

Next, we examined if B-PAC-1 was able to cleave procaspase-6 into active caspase-6. MM.1S cells were treated with DMSO (control cells), 3 or 10 µM B-PAC-1 for 24 hours. As Fig. 3D shows, compared to control cells, 10 µM B-PAC-1 decreased the levels of procaspase-6. GAPDH was used as loading control to indicate the difference in expression was not due to differential loading. However, we were unable to detect the active caspase-6 band on the immunoblot. The absence of cleaved caspase-6 could be due to either low levels in myeloma cells or its further degradation. We have observed similar results in the prior published report [24]. Because we were unable to detect cleaved caspase-6 in myeloma cell lines, we used procaspase-6 expression to determine the activity of B-PAC-1 on caspase-6. In summary, these data indicate that in addition to targeting procaspase-3, B-PAC-1 also targeted procaspase-7 and -6 and converted procaspase-7 to active caspase-7, while reducing the levels of procaspase-6.

B-PAC-1 overcame microenvironment-mediated drug resistance

The bone marrow microenvironment supports the growth of myeloma cells and is a source of resistance to anti-myeloma therapeutics due to the release of growth-promoting cytokines from cells in the bone marrow milieu. To examine if B-PAC-1 can overcome microenvironment-related drug resistance, we investigated the effects of B-PAC-1 in the presence of high levels of the exogenous growth factors interleukin (IL)-6 and hepatocyte growth factor (HGF). MM.1S and U266 cells were treated with 5 µM B-PAC-1 in the presence of 10 or 50 ng/ml IL-6 (Figs. 4 A–B). Treatment with 5 µM B-PAC-1 reduced cell viability by >80% in MM.1S cells and >44% in U266 cells. Importantly, B-PAC-1 was able to induce similar levels of apoptosis in the absence and presence of IL-6. Similarly, B-PAC-1 was equally effective in inducing apoptosis in cells grown in the absence or presence of 50 ng/ml HGF (Figs. 4 A–B).

Figure 4. B-PAC-1 induces apoptosis in the presence of IL-6 and HGF.

Figure 4

Open in a new tab

MM.1S (A) and U266 (B) cells were treated as indicated for 24 hours. Cells were stained for annexin V and PI and % viable cells were measured by flow cytometry. Viable cells represent the percentage of annexin V+ and PI+ cells. Data are presented as Mean ± SEM, n = 3. **** p < 0.0001 by 1-way ANOVA when compared to DMSO-treated cells.

In addition to examining B-PAC-1 cytotoxicity in the presence of exogenous growth factors, we also examined its cytotoxicity when myeloma cells were co-cultured with NKtert cells, a human bone marrow stromal cell line. As shown in Supplementary Figure 3, B-PAC-1 was effective in reducing the viability of U266 cells when cultured alone and also, when co-cultured with NKtert cells, indicating that it is able to overcome the protective bone marrow microenvironment of NKtert cells. However, B-PAC-1 was also toxic to NKtert cells (data not shown).

B-PAC-1 was cytotoxic to drug-resistant myeloma cell lines

Next, we investigated if B-PAC-1 is effective in inducing apoptosis in cells that are resistant to current multiple myeloma therapeutics. FDA-approved drugs for multiple myeloma include dexamethasone, lenalidomide and bortezomib; therefore, we tested cell lines that are sensitive to these agents (MM.1S, KAS-6/1) and cells that are resistant to lenalidomide (MM1/R10R, KAS-6/R10R), dexamethasone (MM.1R) or bortezomib (KAS-6/V10R) for sensitivity to B-PAC-1. A dose-response experiment with B-PAC-1 demonstrated that apoptosis was induced in all of these cell lines (Fig. 5). The similarity in the response to B-PAC-1 was high between MM.1S and MM.1/R10R (Pearson correlation = 0.9806, p = 0.0032), and between MM.1S and MM.1R (Pearson correlation = 0.9778, p = 0.004). Similarly, the KAS-6/1 showed a similar response to B-PAC-1 as KAS-6/R10R (Pearson correlation = 0.9978, p = 0.0001) and as KAS-6/V10R (Pearson correlation = 0.9814, p = 0.003).

Figure 5. B-PAC-1 induces apoptosis in drug resistant cell lines.

Figure 5

Open in a new tab

MM.1S (A), KAS-6/1 (B), lenalidomide-resistant MM1/R10R (C), lenalidomide-resistant KAS-6/R10R (D), dexamethasone-resistant MM.1R (E), and bortezomib-resistant KAS-6/V10R (F) cells were treated with 0, 1, 3, 10 and 20 µM B-PAC-1 for 24 hours and % viable cells was assessed after flow cytometry analysis of annexin V+/PI+ cells. Data are Mean ± SD of 3 biological replicates.

B-PAC-1 was cytotoxic in the presence of BCL-2 or BCL-XL overexpression

As described above, B-PAC-1 induces apoptosis by targeting procaspase-3 and chelating inhibitory zinc ions from procaspase-3 allowing it to auto-activate. This mode of B-PAC-1 action would suggest that inactivating gene mutations or overexpression of proteins upstream in the apoptosis pathway would not affect B-PAC-1-mediated apoptosis. To test this hypothesis, we use HL-60 cell lines that overexpressed BCL-2 (HL-60/BCL-2) or BCL-XL (HL-60/BCL-XL) and compared the sensitivities of these cell lines to vector control HL-60 cell line (HL-60/Neo) (Fig. 6A). As shown in Fig. 6B, increasing doses of B-PAC-1 decreased HL-60/Neo, HL-60/BCL-2 and HL-60/BCL-XL cell numbers in a dose-dependent manner. Furthermore, decreases in cell number was associated with decreases in cell viability in these 3 cell lines, as determined by measurement of annexin V+/PI+ cells (Fig. 6C). These data indicated that B-PAC-1 can bypass mutational inactivation of apoptosis that occurs due to overexpression of cell survival proteins, and induce apoptosis.

Figure 6. B-PAC-1 induces apoptosis in HL-60 cell lines overexpressing BCL-2 or BCL-XL and in mouse embryonic fibroblasts with wildtype or MCL-1Δ.

Figure 6

Open in a new tab

(A) Immunoblot analysis of HL-6/Neo, HL-60/BCL-2 and HL-60/BCL-XL cell lines. Immunoblots were probed for BCL-2 and BCL-XL. GAPDH was used as loading control. (B) HL-6/Neo, HL-60/BCL-2 and HL-60/BCL-XL cell lines were treated with 0, 1, 3, 10, and 20 µM B-PAC-1 for 24 hours. Cell numbers were measured and are displayed as % of control. Data are represented as Mean ± SEM, n = 3. (C) HL-6/Neo, HL-60/BCL-2 and HL-60/BCL-XL cell lines were treated as in (B). Viable cells were determined after flow cytometry analysis of annexin V+/PI+ cells. Data are displayed as % of control, Mean ± SEM, n = 3. (D) MEF wild type (WT) for MCL-1 or lacking MCL-1 (MCL-1Δ) were treated with 0, 1, 3, 10, and 20 µM B-PAC-1 for 24 hours. Cell numbers were measured and are displayed as % of control. Data are represented as Mean ± SEM, n = 3. (E) MEF wild type (WT) for MCL-1 or lacking MCL-1 (MCL-1Δ) were treated as in (D). Viable cells were determined after flow cytometry analysis of annexin V+/PI+ cells. Data are displayed as % of control, Mean ± SEM, n = 3.

B-PAC-1 was cytotoxic to mouse embryonic fibroblasts that were wild type or deleted for MCL-1

To examine if B-PAC-1 action was independent of MCL-1, we used mouse embryonic fibroblasts that were either wild type for MCL-1 (WT MCL-1) or deleted for MCL-1 (MCL-1Δ) [25]. Treatment of WT MCL-1 and MCL-1 Δ cells with B-PAC-1 demonstrated that it reduced cell numbers in a dose-dependent manner (Fig. 6D). The decrease in cell numbers was associated with decrease in the percentage of viable cells (cells staining negative for annexin V and propidium iodide) indicating that B-PAC-1 induced apoptosis in these cell lines (Fig. 6E).

B-PAC-1 was cytotoxic to primary myeloma cells

Finally, we examined the cytotoxicity of B-PAC-1 in primary CD138+ myeloma cells isolated from bone marrow aspirates of myeloma patients. CD138+ cells were treated with DMSO (control cells) or 20 µM B-PAC-1 for 72 hours. Cell apoptosis was measured by staining for annexin+/PI+ cells. Higher cell death was induced by 20 µM B-PAC-1 compared to control -treated cells (Fig. 7). These data suggest that B-PAC-1 is cytotoxic in patient myeloma cells.

Figure 7. B-PAC-1 induces apoptosis in CD138+ primary myeloma cells.

Figure 7

Open in a new tab

Mononuclear bone marrow cells were obtained and CD138+ cells were isolated as described in Methods. Cells were treated with 0 or 20 µM B-PAC-1 for 72 hours. Cell viability was assessed by flow cytometry analysis of annexin V+/PI+ cells. Data are displayed as ratio of control. n=11, p < 0.05

Discussion

In this study, we investigated the induction of apoptosis in multiple myeloma cells using a novel small molecule procaspase-activating compound called B-PAC-1. Our data showed that B-PAC-1 induced apoptosis in MM.1S and U266 cells in a dose- and time-dependent manner. Cell death was minimal in normal B cells and peripheral blood mononuclear cells [26].These data indicated that PAC agents such as B-PAC1 may have therapeutic efficacy in myeloma cells.

Executioner procaspases (3, 6, and 7) are cysteine-aspartic acid proteases that once activated, catalyze the terminal step in the apoptosis pathway. These proteases exist as dimers of inactive proenzymes and have to be cleaved at specific aspartic acid residues to be activated. Active caspase-3 (-6, or -7) cleaves numerous substrates that eventually lead to cell death.

Procaspase-3 is a proenzyme that is inhibited by zinc ions [6, 27]. It was previously shown that the PAC agent PAC-1 activated procaspase-3 through chelating inhibitory zinc ions [6]. This allowed procaspase-3 to auto-activate to caspase-3. Our studies show that B-PAC-1 also functions as a zinc chelator in multiple myeloma cells. 100 µM zinc was able to inhibit B-PAC-1-mediated cell death in MM.1S cells. Furthermore, immunoblot analysis indicated that this was due to the inhibition of the formation of active caspase-3 as zinc addition to B-PAC-1-treated cells reduced the levels of active caspase-3 and increased the levels of procaspase-3.

B-PAC-1 chelates zinc ions specifically from procaspase-3 and other executioner procaspases, thereby activating apoptosis [26]. Consistent with this statement, in biochemical assay systems, B-PAC-1 did not alter the activity of other zinc-dependent enzyme such as carboxypeptidase A and HDAC [26]. Similarly, inflammatory procaspase (procaspase-1) was not affected by B-PAC-1 [26]. These studies suggest that B-PAC-1 may not have off-target effects on other zinc-dependent enzymes.

Our studies show that B-PAC-1 reduced the levels of the IAP proteins cIAP1, cIAP2 and XIAP. We also observed decreases in the levels of the BCL-2 family survival protein MCL-1 upon treatment with B-PAC-1. These proteins are involved in myeloma cell survival [10, 13, 15–17]. Therefore, these results further indicate that B-PAC-1 reduces the survival of myeloma cells.

The bone marrow microenvironment is a major source of drug resistance in myeloma therapeutics. Soluble factors released by the bone marrow stimulate the growth and survival of myeloma cells [28, 29]. IL-6 is one such factor that is known to induce proliferation of myeloma cells and resistance to drugs [30–32]. IL-6 and similar factors induce BCL-2 family-dependent [13, 33] and –independent [34] survival pathways in myeloma cells. We showed that B-PAC-1 is equally effective in inducing apoptosis in MM.1S and U266 cells, irrespective of the presence or absence of IL-6.

HGF is another growth factor that has been implicated in myeloma disease. Myeloma patients have high levels of serum HGF, when compared with healthy donors [35], which has been correlated with poor prognosis [36–38]. Studies in our lab have further revealed that HGF levels increase as myeloma disease progresses from MGUS to SMM to newly diagnosed MM to Relapsed/Refractory MM [39]. Our studies show that B-PAC-1 effectively induced apoptosis in the presence of excess exogenous HGF.

MM is currently treated with glucocorticoids such as dexamethasone, immunomodulatory drugs such as lenalidomide, and proteasome inhibitors such as bortezomib. However, many patients who are on these treatments eventually acquire resistance to these therapies leading to disease relapse. The MM.1R cell line acquires resistance to dexamethasone because it expresses low levels, including alternative splice variants, of the glucocorticoid receptor [23, 40]. Therefore, this cell line does not undergo cytolysis upon glucocorticoid treatment. The resistance phenotype of the KAS-6/R10R lenalidomide-resistant cell line has been attributed to activation of the Wnt/β-catenin pathway [22] and also, the HGF/MET pathway [41]. For acquired resistance to bortezomib, several factors have been implicated including upregulation of the anti-apoptotic protein MCL-1 and downregulation of the proapoptotic protein BIM [42, 43]. Our studies indicate that B-PAC-1 treatment is able to counter these resistant mechanisms and is equally effective in inducing apoptosis in myeloma cell lines that are either sensitive or resistant to these agents.

Since B-PAC-1 targets at the level of procaspase-3, we hypothesized that overexpression of survival proteins upstream in the apoptosis pathway would have no effect on B-PAC-1-mediated apoptosis. We showed that B-PAC-1 was equally effective in inducing apoptosis in cell lines irrespective of the level of BCL-2 or BCL-XL. These data demonstrate that targeting myeloma cells at the level of procaspase-3 is an effective anti-myeloma therapy for cells that are mutated or overexpress upstream survival proteins in the apoptosis pathway. Consistent with these results, lack of BAX and BAK did not impact B-PAC-1-mediated cytotoxicity [26], further demonstrating a mechanism of cell death that is oblivious to the BCL-2 family-defined process, an important attribute since several studies have documented resistance mechanisms in myeloma cell lines due to overexpression of BCL-2 family proteins [44–47]. Furthermore, compared to myeloma cell lines, malignant myeloma plasma cells show even higher levels of these factors [48]. Consequently, agents that neutralize BCL-2 family antiapoptotic proteins such as ABT- 737 [49] or obatoclax [48] were effective in inducing cell death in most primary patient samples. However, there was resistance to ABT-737 in samples with high MCL-1 and obatoclax was also cytotoxic to normal bone marrow cells. Bypassing these pathways using procaspase activating compounds appears to be a novel alternative approach.

We examined if B-PAC-1 can effectively induce apoptosis in primary CD138+ myeloma cells obtained from bone marrow aspirates of patients. Our data shows that myeloma cells are highly sensitive to B-PAC-1 and showed statistically significant increases in apoptotic cells compared to control cells. These studies therefore indicate that B-PAC-1 might have therapeutic efficacy in myeloma treatment.

In conclusion, we have tested a novel agent, B-PAC-1, that targets the terminal executioner procaspases, induces apoptosis in chemo-resistant and sensitive myeloma cell lines, and obviates upstream survival signal.

Supplementary Material

01

Highlights.

  1. The procaspase activating compound B-PAC-1 activates procaspase-3, -6 and -7.

  2. B-PAC-1 is cytotoxic to chemotherapy-naïve and –resistant myeloma cells.

  3. B-PAC-1 is cytotoxic in presence of hepatocyte growth factor and interleukin 6.

  4. The action of B-PAC-1 is inhibited by exogenous zinc in myeloma cells.

  5. Original PAC-1 is in phase I trial; B-PAC-1 (an analog) is evaluated for clinic.

Acknowledgements

The authors thank Dr. Paul Hergenrother (University of Illinois, Urbana-Champaign, IL), Dr. Ted Tarasow (Vanquish Oncology, Champaign, IL) and Dr. Mark Gilbert (Vanquish Oncology, Champaign, IL) for providing PAC agents to conduct this study. We thank the UT MD Anderson Myeloma Core (Ivory J. Ellis, Saaiqa Maredia, Rebecca A. Murray, Kavita S. Chauhan, Trang T. Nguyen, Adam Stein, and Catherine M. Claussen) for coordinating collection and processing of primary CD138 positive myeloma cells. This work was supported by a Myeloma SPORE (CA142509) Development Research Project and a Sponsored Research Agreement from Vanquish Oncology. We thank Suzanne E. Davis for editorial help in preparing this manuscript.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Author contributions: S.Z. designed experiments, conducted most experiments, and wrote manuscript; R.W. conducted experiments, and wrote manuscript; V.G. conceptualized the research, directed SZ, obtained funding, wrote and reviewed manuscript; and all authors approved the final version.

Conflicts of interest

VG received Sponsored Research Agreement from Vanquish Oncology. All other authors declare no financial competing interests.

Contributor Information

Shadia Zaman, Email: shadia11@hotmail.com.

Rui Wang, Email: ruiw1992@gmail.com.

Varsha Gandhi, Email: vgandhi@mdanderson.org.

References

  • 1.Kuehl WM, Bergsagel PL. Multiple myeloma: evolving genetic events and host interactions. Nat Rev Cancer. 2002;2(3):175–187. doi: 10.1038/nrc746. [DOI] [PubMed] [Google Scholar]
  • 2.Kyle RA, Rajkumar SV. Multiple myeloma. N Engl J Med. 2004;351(18):1860–1873. doi: 10.1056/NEJMra041875. [DOI] [PubMed] [Google Scholar]
  • 3.Raab MS, et al. Multiple myeloma. Lancet. 2009;374(9686):324–339. doi: 10.1016/S0140-6736(09)60221-X. [DOI] [PubMed] [Google Scholar]
  • 4.Kyle RA, Rajkumar SV. Multiple myeloma. Blood. 2008;111(6):2962–2972. doi: 10.1182/blood-2007-10-078022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100(1):57–70. doi: 10.1016/s0092-8674(00)81683-9. [DOI] [PubMed] [Google Scholar]
  • 6.Peterson QP, et al. PAC-1 activates procaspase-3 in vitro through relief of zinc-mediated inhibition. J Mol Biol. 2009;388(1):144–158. doi: 10.1016/j.jmb.2009.03.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Lowe SW, Cepero E, Evan G. Intrinsic tumour suppression. Nature. 2004;432(7015):307–315. doi: 10.1038/nature03098. [DOI] [PubMed] [Google Scholar]
  • 8.Zaman S, Wang R, Gandhi V. Targeting the apoptosis pathway in hematologic malignancies. Leuk Lymphoma. 2014;55(9):1980–1992. doi: 10.3109/10428194.2013.855307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Witzig TE. Measurement of apoptosis and proliferation of bone marrow plasma cells in patients with plasma cell proliferative disorders. Br J Haematol. 1999;104(1):131–137. doi: 10.1046/j.1365-2141.1999.01136.x. [DOI] [PubMed] [Google Scholar]
  • 10.Wuilleme-Toumi S, et al. Mcl-1 is overexpressed in multiple myeloma and associated with relapse and shorter survival. Leukemia. 2005;19(7):1248–1252. doi: 10.1038/sj.leu.2403784. [DOI] [PubMed] [Google Scholar]
  • 11.Bodet L, et al. ABT-737 is highly effective against molecular subgroups of multiple myeloma. Blood. 2011;118(14):3901–3910. doi: 10.1182/blood-2010-11-317438. [DOI] [PubMed] [Google Scholar]
  • 12.Gauthier ER, et al. Role of bcl-X(L) in the control of apoptosis in murine myeloma cells. Cancer Res. 1996;56(6):1451–1456. [PubMed] [Google Scholar]
  • 13.Jourdan M, et al. A major role for Mcl-1 antiapoptotic protein in the IL-6-induced survival of human myeloma cells. Oncogene. 2003;22(19):2950–2959. doi: 10.1038/sj.onc.1206423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Miguel-Garcia A, et al. bcl-2 expression in plasma cells from neoplastic gammopathies and reactive plasmacytosis: a comparative study. Haematologica. 1998;83(4):298–304. [PubMed] [Google Scholar]
  • 15.Landowski TH, et al. Cell adhesion-mediated drug resistance (CAM-DR) is associated with activation of NF-kappa B (RelB/p50) in myeloma cells. Oncogene. 2003;22(16):2417–2421. doi: 10.1038/sj.onc.1206315. [DOI] [PubMed] [Google Scholar]
  • 16.Mitsiades CS, et al. Activation of NF-kappaB and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: therapeutic implications. Oncogene. 2002;21(37):5673–5683. doi: 10.1038/sj.onc.1205664. [DOI] [PubMed] [Google Scholar]
  • 17.Abe S, et al. Rapid induction of IAP family proteins and Smac/DIABLO expression after proapoptotic stimulation with doxorubicin in RPMI 8226 multiple myeloma cells. Exp Mol Pathol. 2007;83(3):405–412. doi: 10.1016/j.yexmp.2007.04.001. [DOI] [PubMed] [Google Scholar]
  • 18.Putt KS, et al. Small-molecule activation of procaspase-3 to caspase-3 as a personalized anticancer strategy. Nat Chem Biol. 2006;2(10):543–550. doi: 10.1038/nchembio814. [DOI] [PubMed] [Google Scholar]
  • 19.Jelinek DF, et al. Coexistence of aneuploid subclones within a myeloma cell line that exhibits clonal immunoglobulin gene rearrangement: clinical implications. Cancer Res. 1993;53(21):5320–5327. [PubMed] [Google Scholar]
  • 20.Westendorf JJ, et al. Establishment and characterization of three myeloma cell lines that demonstrate variable cytokine responses and abilities to produce autocrine interleukin-6. Leukemia. 1996;10(5):866–876. [PubMed] [Google Scholar]
  • 21.Bjorklund CC, et al. Evidence of a role for CD44 and cell adhesion in mediating resistance to lenalidomide in multiple myeloma: therapeutic implications. Leukemia. 2013 doi: 10.1038/leu.2013.174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Bjorklund CC, et al. Evidence of a role for activation of Wnt/beta-catenin signaling in the resistance of plasma cells to lenalidomide. J Biol Chem. 2011;286(13):11009–11020. doi: 10.1074/jbc.M110.180208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Moalli PA, et al. A mechanism of resistance to glucocorticoids in multiple myeloma: transient expression of a truncated glucocorticoid receptor mRNA. Blood. 1992;79(1):213–222. [PubMed] [Google Scholar]
  • 24.Mitsiades N, et al. Intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human multiple myeloma cells. Blood. 2002;99(6):2162–2171. doi: 10.1182/blood.v99.6.2162. [DOI] [PubMed] [Google Scholar]
  • 25.Stewart DP, et al. Ubiquitin-independent degradation of antiapoptotic MCL-1. Mol Cell Biol. 2010;30(12):3099–3110. doi: 10.1128/MCB.01266-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Patel V, et al. Expression of executioner procaspases and their activation by a procaspase-activating compound in chronic lymphocytic leukemia cells. Blood. 2015;125(7):1126–1136. doi: 10.1182/blood-2014-01-546796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Perry DK, et al. Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis. J Biol Chem. 1997;272(30):18530–18533. doi: 10.1074/jbc.272.30.18530. [DOI] [PubMed] [Google Scholar]
  • 28.Perez LE, et al. Bone marrow stroma confers resistance to Apo2 ligand/TRAIL in multiple myeloma in part by regulating c-FLIP. J Immunol. 2008;180(3):1545–1555. doi: 10.4049/jimmunol.180.3.1545. [DOI] [PubMed] [Google Scholar]
  • 29.Nefedova Y, Landowski TH, Dalton WS. Bone marrow stromal-derived soluble factors and direct cell contact contribute to de novo drug resistance of myeloma cells by distinct mechanisms. Leukemia. 2003;17(6):1175–1182. doi: 10.1038/sj.leu.2402924. [DOI] [PubMed] [Google Scholar]
  • 30.Catlett-Falcone R, et al. Constitutive activation of Stat3 signaling confers resistance to apoptosis in human U266 myeloma cells. Immunity. 1999;10(1):105–115. doi: 10.1016/s1074-7613(00)80011-4. [DOI] [PubMed] [Google Scholar]
  • 31.Frassanito MA, et al. Autocrine interleukin-6 production and highly malignant multiple myeloma: relation with resistance to drug-induced apoptosis. Blood. 2001;97(2):483–489. doi: 10.1182/blood.v97.2.483. [DOI] [PubMed] [Google Scholar]
  • 32.Voorhees PM, et al. Inhibition of interleukin-6 signaling with CNTO 328 enhances the activity of bortezomib in preclinical models of multiple myeloma. Clin Cancer Res. 2007;13(21):6469–6478. doi: 10.1158/1078-0432.CCR-07-1293. [DOI] [PubMed] [Google Scholar]
  • 33.Jourdan M, et al. Regulation of Bcl-2-family proteins in myeloma cells by three myeloma survival factors: interleukin-6, interferon-alpha and insulin-like growth factor 1. Cell Death Differ. 2000;7(12):1244–1252. doi: 10.1038/sj.cdd.4400758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Brocke-Heidrich K, et al. Interleukin-6-dependent gene expression profiles in multiple myeloma INA-6 cells reveal a Bcl-2 family-independent survival pathway closely associated with Stat3 activation. Blood. 2004;103(1):242–251. doi: 10.1182/blood-2003-04-1048. [DOI] [PubMed] [Google Scholar]
  • 35.Borset M, et al. Hepatocyte growth factor and its receptor c-met in multiple myeloma. Blood. 1996;88(10):3998–4004. [PubMed] [Google Scholar]
  • 36.Seidel C, et al. Elevated serum concentrations of hepatocyte growth factor in patients with multiple myeloma. The Nordic Myeloma Study Group. Blood. 1998;91(3):806–812. [PubMed] [Google Scholar]
  • 37.Seidel C, et al. Hepatocyte growth factor in myeloma patients treated with high-dose chemotherapy. Br J Haematol. 2002;119(3):672–676. doi: 10.1046/j.1365-2141.2002.03898.x. [DOI] [PubMed] [Google Scholar]
  • 38.Turesson I, et al. Prognostic evaluation in multiple myeloma: an analysis of the impact of new prognostic factors. Br J Haematol. 1999;106(4):1005–1012. doi: 10.1046/j.1365-2141.1999.01651.x. [DOI] [PubMed] [Google Scholar]
  • 39.Phillip CJ, et al. Targeting MET kinase with the small-molecule inhibitor amuvatinib induces cytotoxicity in primary myeloma cells and cell lines. J Hematol Oncol. 2013;6:92. doi: 10.1186/1756-8722-6-92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Moalli PA, et al. Alternatively spliced glucocorticoid receptor messenger RNAs in glucocorticoid-resistant human multiple myeloma cells. Cancer Res. 1993;53(17):3877–3879. [PubMed] [Google Scholar]
  • 41.Zaman S, et al. Bortezomib and lenalidomide resistant myeloma cells overexpress the hepatocyte growth factor/MET signaling axis and respond to MET kinase inhibitors in American Association of Cancer Research 105th Annual Meeting. Cancer Research; San Diego, CA. 2014. [Google Scholar]
  • 42.Chen S, et al. A Bim-targeting strategy overcomes adaptive bortezomib resistance in myeloma through a novel link between autophagy and apoptosis. Blood. 2014;124(17):2687–2697. doi: 10.1182/blood-2014-03-564534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Pei XY, et al. Circumvention of Mcl-1-dependent drug resistance by simultaneous Chk1 and MEK1/2 inhibition in human multiple myeloma cells. PLoS One. 2014;9(3):e89064. doi: 10.1371/journal.pone.0089064. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Podar K, et al. A pivotal role for Mcl-1 in Bortezomib-induced apoptosis. Oncogene. 2008;27(6):721–731. doi: 10.1038/sj.onc.1210679. [DOI] [PubMed] [Google Scholar]
  • 45.Gomez-Bougie P, et al. Noxa up-regulation and Mcl-1 cleavage are associated to apoptosis induction by bortezomib in multiple myeloma. Cancer Res. 2007;67(11):5418–5424. doi: 10.1158/0008-5472.CAN-06-4322. [DOI] [PubMed] [Google Scholar]
  • 46.Hu J, et al. Activation of ATF4 mediates unwanted Mcl-1 accumulation by proteasome inhibition. Blood. 2012;119(3):826–837. doi: 10.1182/blood-2011-07-366492. [DOI] [PubMed] [Google Scholar]
  • 47.Nencioni A, et al. Evidence for a protective role of Mcl-1 in proteasome inhibitor-induced apoptosis. Blood. 2005;105(8):3255–3262. doi: 10.1182/blood-2004-10-3984. [DOI] [PubMed] [Google Scholar]
  • 48.Trudel S, et al. Preclinical studies of the pan-Bcl inhibitor obatoclax (GX015-070 in multiple myeloma. Blood. 2007;109(12):5430–5438. doi: 10.1182/blood-2006-10-047951. [DOI] [PubMed] [Google Scholar]
  • 49.Kline MP, et al. ABT-737, an inhibitor of Bcl-2 family proteins, is a potent inducer of apoptosis in multiple myeloma cells. Leukemia. 2007;21(7):1549–1560. doi: 10.1038/sj.leu.2404719. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS713876-supplement-01.pdf (36.2KB, pdf)

RESOURCES