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[Cancer Research 63, 8428-8436, December 1, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Antitumor Activity of Lysophosphatidic Acid Acyltransferase-ß Inhibitors, a Novel Class of Agents, in Multiple Myeloma

Teru Hideshima1, Dharminder Chauhan1, Toshiaki Hayashi1, Klaus Podar1, Masaharu Akiyama1, Constantine Mitsiades1, Nicholas MItsiades1, Baoqing Gong2, Lynn Bonham2, Peter de Vries2, Nikhil Munshi1, Paul G. Richardson1, Jack W. Singer2 and Kenneth C. Anderson1

1 Jerome Lipper Multiple Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts, and
2 Cell Therapeutics Inc., Seattle, Washington


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we examined the effects of isoform-specific functional inhibitors of lysophosphatidic acid acyltransferase (LPAAT), which converts lysophosphatidic acid to phosphatidic acid, on multiple myeloma (MM) cell growth and survival. The LPAAT-ß inhibitors CT-32176, CT-32458, and CT-32615 induced >95% growth inhibition (P < 0.01) in MM.1S, U266, and RPMI8226 MM cell lines, as well as MM cells from patients (IC50, 50–200 nM). We further characterized this LPAAT-ß inhibitory effect using CT-32615, the most potent inhibitor of MM cell growth. CT-32615 triggered apoptosis in MM cells via caspase-8, caspase-3, caspase-7, and poly (ADP-ribose) polymerase cleavage. Neither interleukin 6 nor insulin-like growth factor I inhibited CT-32615-induced apoptosis. Dexamethasone and immunomodulatory derivatives of thalidomide (IMiDs), but not proteasome inhibitor PS-341, augmented MM cell apoptosis triggered by LPAAT-ß inhibitors. CT-32615-induced apoptosis was associated with phosphorylation of p53 and c-Jun NH2-terminal kinase (JNK); conversely, JNK inhibitor SP600125 and dominant-negative JNK inhibited CT-32615-induced apoptosis. Importantly, CT-32615 inhibited tumor necrosis factor-{alpha}-triggered nuclear factor-{kappa}B activation but did not affect either tumor necrosis factor-{alpha}-induced p38 mitogen-activated protein kinase phosphorylation or interleukin 6-triggered signal transducers and activators of transcription 3 phosphorylation. Finally, although binding of MM cells to bone marrow stromal cells augments MM cell growth and protects against dexamethasone-induced apoptosis, CT-32615 induced apoptosis even of adherent MM cells. Our data therefore demonstrate for the first time that inhibiting LPAAT-ß induces cytotoxicity in MM cells in the bone marrow milieu, providing the framework for clinical trials of these novel agents in MM.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite the use of conventional therapies with alkylating agents, anthracyclines, and corticosteroids (1 , 2) , as well as high-dose therapy and stem cell transplantation (3, 4, 5) , MM3 remains incurable because of intrinsic and acquired drug resistance (6, 7, 8, 9, 10) . Furthermore, the BM microenvironment also confers drug resistance in MM cells via at least two different mechanisms: adhesion of MM cells to fibronectin confers cell adhesion-mediated drug resistance, associated with induction of p27Kip1 and G1 growth arrest (11 , 12) ; and cytokines (i.e., IL-6 and IGF-I) in the BM milieu induce Janus kinase 2/STAT3 and/or PI3-K/Akt signaling, which mediates resistance to conventional and novel therapies (13, 14, 15, 16) . Biologically based treatments targeting not only the MM cell but also MM cell-host interactions and the BM microenvironment can overcome drug resistance in both preclinical and early clinical studies (17, 18, 19) .

LPA and PA are phospholipids involved in signal transduction and in lipid biosynthesis. LPAAT, also known as 1-acyl sn-glycerol-3-phosphate acyltransferase, is encoded by a gene located in the class III region of the human MHC (20) . It catalyzes the conversion of LPA to PA, which mediates cytokine expression and inflammatory injury after hemorrhage (21) . Recent studies in EBV-transformed B-lineage line IM-9 cells have also shown that inhibition of LPAAT-ß activity disrupts Ras/Raf/MAPK and PI3-K/mTOR signaling, followed by apoptosis (22) . To date, however, the biological effect of LPAAT inhibition on MM cells has not been characterized.

In this report, we examine the effects of isoform-specific functional inhibitors of LPAAT-ß on human MM cells. We demonstrate that the LPAAT inhibitor CT-32228, as well as its analogues CTI-32176, CT-32458, and CT-32615, induce potent cytotoxicity against MM cell lines and freshly isolated MM cells from patients (IC50, 50–100 nM). MM cell apoptosis triggered by CT-32615 is mediated via caspase-8, caspase-7, and PARP cleavage, an uncommon apoptotic pathway in MM (16) . As with proteasome inhibitor PS-341 (23) and immunomodulatory derivatives of thalidomide (IMiDs; Ref. 24 ), CT-32615 augments Dex-induced MM cell growth inhibition; it also enhances IMiD1 (CC-4047) and IMiD3 (CC-5013; Revimid) but not PS-341-induced MM cell cytotoxicity. IL-6 (13 , 14 , 25, 26, 27, 28, 29, 30, 31, 32) and IGF-I (15 , 33, 34, 35) induce MM cell growth and protection against Dex-induced apoptosis; however, neither exogenous IL-6 nor IGF-I overcomes CT-32615-induced cytotoxicity. Finally, adherence of MM cells to BMSCs augments MM cell growth and protects against Dex-induced tumor cell apoptosis (16 , 23 , 36, 37, 38, 39) , but CT-32615 induces apoptosis even of MM cells adherent to BMSCs. Our data therefore demonstrate for the first time that inhibiting LPAAT induces cytotoxicity in MM cells in the BM milieu, providing the framework for clinical trials of these novel agents to improve the outcome of patients with MM.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
MM-derived Cell Lines and MM Cells from Patients.
Dex-sensitive (MM.1S) and resistant (MM.1R) human MM cell lines were kindly provided by Dr. Steven Rosen (Northwestern University, Chicago, IL). RPMI8226 and U266 human MM cells were obtained from American Type Culture Collection (Rockville, MD). Doxorubicin-resistant (RPMI-Dox40) and melphalan-resistant (RPMI-LR5) cells were kindly provided by Dr. William Dalton (H. Lee Moffitt Cancer Center, Tampa, FL). All MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (Sigma Chemical Co., St. Louis, MO), 2 µM L-glutamine, 100 units/ml of penicillin, and 100 µg/ml of streptomycin (Life Technologies, Inc., Grand Island, NY). MM cells were purified from BM aspirates from eight patients with MM who had relapsed and were refractory to conventional therapies using the RosetteSep separation system (StemCell Technologies, Vancouver, British Columbia, Canada). The purity of MM cells was confirmed by flow cytometry using phycoerythrin-conjugated anti-CD138 Ab (BD PharMingen, San Diego, CA).

BMSC Cultures.
BM specimens were obtained from patients with MM. Mononuclear cells separated by Ficoll-Hipaque density sedimentation were used to establish long-term BM cultures, as described previously (38 , 40) . When an adherent cell monolayer had developed, cells were harvested in HBSS containing 0.25% trypsin and 0.02% EDTA, washed, and collected by centrifugation.

LPAAT-ß Inhibitors and Reagents.
The LPAAT-ß inhibitors used were CT-32228 [N-(4-bromophenyl)-6-(5-chloro-2-methylphenyl)-[1,3,5] triazine-2,4-diamine] and its analogues (CT-32169, CT-32176, CT-32185, CT-32458, and CT-32615; Cell Therapeutics, Inc., Seattle, WA). Each of these compounds is an isoform-specific, noncompetitive inhibitor of LPAAT-ß with an IC50 against a semipurified enzyme preparation of <200 nM.

Pan-caspase inhibitor Z-VAD-FMK and caspase-8 inhibitor Z-IETD-FMK, as well as granzyme inhibitor I (Calbiochem, San Diego, CA), were dissolved in DMSO and stored at -20°C and used at 25 µM and 300 nM, respectively. For inhibition of caspase cleavage, cells were incubated with caspase inhibitors for 1 h before incubation with CT-32615 (50–100 nM; 24-h culture). JNK inhibitor SP600125 (Refs. 41 , 42 ; Calbiochem, San Diego, CA) was also stored at -20°C and used at 5–20 µM.

Measurement of LPAAT Enzymatic Activity from Cell Lines.
For measurement of LPAAT activity in cell lines, cell pellets were homogenized in 200 µl of ice-cold homogenization buffer [20 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM benzamidine, and 1 mg/ml each soybean trypsin inhibitor, pepstatin A, and leupeptin] with six strokes of a Duall homogenizer. The samples were centrifuged at 1500 x g for 2 min at 4°C, and the supernatant was saved for protein quantification with the bicinchoninic assay (Pierce) and for the LPAAT assay. LPAAT activity was measured by formation of PA during a 7-min incubation period at 37°C in 12 x 75-mm borosilicate test tubes containing 75 µl of 50 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM EDTA, 1 mg/ml of fatty acid-free BSA, 0.25 mM 14C-labeled 18:1-CoA (about 40,000 cpm/assay), 0.25 mM sn-1–18:1 lysoPA, 0.001–0.05 mg/ml of cell homogenate, and 8% DMSO or DMSO containing LPAAT inhibitor. Assays were quenched by the addition of 1.3 ml of chloroform:methanol:HCl (48:51:0.7). Carrier lipids (10 µg) were added, and the phases were separated with the addition of 0.35 ml of water. The upper phase was discarded, and the lower phase was dried under nitrogen. 14C-labeled PA was separated from 14C-labeled 18:1-CoA by TLC on Analtech silica gel 60 HP-TLC plates in chloroform:methanol:acetic acid:water (85:12.5:12.5:3) and was quantitated by exposing the TLC plates to a phosphor screen (Molecular Dynamics). LPAAT activity was linear with time and protein.

Growth Inhibition Assay.
The inhibitory effect of LPAAT inhibitor on MM cell lines, MM cells from patients, PBMCs, and BMSC growth was assessed by measuring MTT (Chemicon International Inc., Temecula, CA) dye absorbance, as described previously (24) .

Cell Cycle Analysis.
MM.1S and RPMI8226 cells were cultured for 24 and 48 h in the presence of CT-32615 (12.5–100 nM) or control medium. Cell cycle analysis was performed as in prior studies described previously (23 , 24) .

Immunoblotting.
MM cells were cultured with CT-32615 in the presence or absence of caspase inhibitors and then harvested, washed, and lysed, as in previous studies (38 , 43) . Cell lysates were subjected to SDS-PAGE, transferred to polyvinylidene difluoride membrane (Bio-Rad Laboratories, Hercules, CA), and immunoblotted with anti-caspase-3 (BD PharMingen, San Diego, CA), anti-caspase-6, anti-caspase-7, anti-caspase-8, anti-caspase-9, PARP, phospho (ser15)-p53, p53, phospho-JNK, phospho-p38 MAPK, and p38 MAPK Abs (Cell Signaling, Beverly, MA), as well as with JNK1, I{kappa}B{alpha}, p21, phospho-STAT3, and STAT3 (Santa Cruz Biotechnology, Santa Cruz, CA) Abs, and with anti-{alpha}-tubulin Ab (Sigma).

Transient Transfection.
Transient transfection of dominant-negative JNK construct was performed, as in our prior studies (44 , 45) . Briefly, MM.1S cells were transiently transfected with vector alone or DN-JNK and cotransfected with vector containing GFP using Cell Line NucleofectoTM Kit V, according to the manufacturer’s (Amaxa Biosystems, Koln, Germany) instructions. Following transfections, GFP-positive cells were sorted by flow cytometry and subjected to MTT assay.

EMSA.
MM.1S cells were pretreated with CT-32615 for 24 h before stimulation with TNF-{alpha} (10 ng/ml; R & D Systems, Minneapolis, MN) for 20 min. Nuclear extract was obtained using Nuclear Extract Kit (Active Motif, Carlsbad, CA). EMSA was carried out as in our previous study (23) .

Effect of LPAAT-ß Inhibitor on Paracrine MM Cell Growth in the BM.
To evaluate the effect of LPAAT-ß inhibitors on growth of MM cells adherent to BMSCs, MM.1S and U266 cells were cultured in BMSC-coated 96-well plates for 48 h, in the presence or absence of CT-32615. DNA synthesis was measured as described previously (24) . MM cells (3 x 104 cells/well) were also incubated in these 96-well culture plates (Costar, Cambridge, MA) in the presence of medium and/or Dex for 48 h at 37°C. DNA synthesis was measured by [3H]thymidine (Perkin Elmer, Boston, MA) uptake. Cells were pulsed with [3H]thymidine (0.5 µCi/well) during the last 8 h of 48-h cultures. All experiments were performed in quadruplicate.

Statistical Analysis.
Statistical significance of differences observed in drug-treated versus control cultures was determined using the Wilcoxon signed-ranks test. The minimal level of significance was P < 0.05.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
LPAAT-ß Inhibitors Directly Induce Growth Inhibition of MM Cell Lines and MM Cells from Patients.
We first determined the effect of LPAAT-ß inhibitors on growth of MM cell lines (MM.1S, U266, and RPMI8226), freshly isolated MM cells from patients, as well as PBMCs cultured for 48 h in either the presence or absence of LPAAT-ß inhibitors, using MTT assay. The LPAAT-ß inhibitors used in these studies are CT-32228 and its analogues (CT-32169, CT-32176, CT-32185, CT-32458, and CT-32615). Among these inhibitors, CT-32176, CT-32458, and CT-32615 demonstrated significant potent cytotoxicity (>95% inhibition; P < 0.001), with IC50 of 50–100 nM against MM.1S (Fig. 1B)Citation , U266 (Fig. 1C)Citation , and RPMI8226 (Fig. 1D)Citation MM cell lines. Importantly, these inhibitors were also effective in (doxorubicin-resistant) RPMI-Dox40 cells (Fig. 1E)Citation as well as (Dex-resistant) MM.1R and (melphalan-resistant) RPMI-LR5 cells (data not shown). These results demonstrate that LPAAT-ß inhibitors induced cytotoxicity even in cell lines resistant to conventional chemotherapy. As was true for MM cell lines, the growth of MM cells from eight patients was also inhibited by LPAAT-ß inhibitors in a dose-dependent fashion, with IC50 of LPAAT-ß inhibitors at 50–200 nM (Fig. 1F)Citation . We further characterized this LPAAT-ß inhibitory effect in MM cells using CT-32615, the most potent inhibitor of LPAAT-ß. Importantly, CT-32615 (500 nM) triggered only 5–10% cytotoxicity of PBMCs from three normal volunteers (Fig. 1G)Citation . These data demonstrate that LPAAT-ß inhibitors specifically induce cytotoxicity in MM cells but not in PBMCs.



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  		Fig. 1. LPAAT-ß inhibitors induce growth inhibition of MM cell lines and MM tumor cells from patients. A, structure of LPAAT-ß inhibitors. MM.1S (B), U266 (C), and RPMI8226 (D), and RPMI-Dox40 (E) MM cell lines, as well as MM tumor cells from patients (n = 8; F) were cultured in the presence of CT-32176 ({blacksquare}), CT-32458 ({blacktriangleup}), and CT-32615 ({bullet}; 0.8–500 nM) for 48 h. CT-32212 ({diamondsuit}) served as a negative control. G, PBMCs (n = 3) were cultured in the presence of CT-32615 (0.8–500 nM) for 48 h. Cell growth was assessed by MTT assay, and data represent means of quadruplicate cultures; bars, SD. H, LPAAT activity in RPMI 8226 and RPMI-Dox-40 cells was examined in the presence of CT-32615 (ß inhibitor, ), CT-11494 ({alpha} inhibitor, ), CT-32212 (non-inhibitor, {blacksquare}), or DMSO (vehicle control, {square}).

 
To confirm the effect of these inhibitors on in vitro LPAAT-ß activity in MM cells, LPAAT activity assays were performed using RPMI8226 and RPMI-Dox40 cells, which have high constitutive LPAAT-ß activity. CT-32615 significantly inhibited LPAAT-ß activity (65–70% inhibition), whereas either LPAAT-{alpha} inhibitor or control demonstrated only minimal effects (15–20% inhibition).

LPAAT-ß Inhibitor Augments Dex- and IMiD-induced Cytotoxicity in MM.
To determine whether LPAAT-ß inhibitor enhances cytotoxicity of conventional therapies or novel agents, we next examined the effect of either Dex or immunomodulatory drugs (IMiDs; Refs. 19 , 24 , 46 ) and CT-32615 on proliferation of MM.1S cells. As can be seen in Fig. 2Citation A, MTT assay at 48 h revealed that CT-32615 alone (25 and 50 nM) and Dex alone (0.04 and 0.2 µM) significantly inhibited MM.1S cell growth in a dose-dependent fashion; moreover, their growth inhibitory effects were additive. CT-32615 also augmented the cytotoxic effect of IMiD1 (Fig. 2B)Citation and IMiD3 (Fig. 2C)Citation . Interestingly, there was no additive or synergistic effect of CT-32615 with thalidomide or proteasome inhibitor PS-341 (Refs. 23 , 43 , 47 ; data not shown).



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  		Fig. 2. CT-32615 enhances the growth-inhibitory effect of Dex, IMiD1, and IMiD3. MM.1S cells were cultured with CT-32615 (25 and 50 nM) in the absence ({square}) or presence of 40 nM () and 200 nM ({blacksquare}) of Dex (A); 0.2 µM () and 1 µM ({blacksquare}) of IMID1 (B): or 0.2 µM () and 1 µM ({blacksquare}) of IMiD3 (C) for 48 h. Cell growth was assessed by MTT assay, and data represent means of quadruplicate cultures; bars, SD.

 
LPAAT-ß Inhibitor Overcomes the Antiapoptotic Effect of IL-6 or IGF-I.
Because we and others have shown that both IL-6 (13 , 14 , 25, 26, 27, 28 , 31 , 32 , 44 , 48) and IGF-I (15 , 33, 34, 35) mediate growth and antiapoptosis in MM cells, we next examined whether LPAAT-ß inhibitor could overcome these effects of exogenous IL-6 or IGF-I. Although IL-6 (50 ng/ml) and IGF-I (50 ng/ml) triggered a 1.2–1.3-fold increase in MM.1S cell growth in cultures relative to medium alone, CT-32615 inhibited this response in a dose-dependent fashion (Fig. 3)Citation , indicating that neither IL-6 nor IGF-I overcomes the inhibitory effect of CT-32615 on MM cell growth.



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  		Fig. 3. Neither IL-6 nor IGF-I protects against CT-32615-induced apoptosis. MM.1S cells were cultured with DMSO control medium ({square}) and with 25 µM () or 50 nM ({blacksquare}) CT-32615, in the presence or absence of IL-6 or IGF-I (10 and 50 ng/ml). Cell growth was assessed by MTT assay, and data represent means of quadruplicate cultures; bars, SD.

 
LPAAT-ß Inhibitor Increases G2-M Phase, Followed by Apoptosis.
To further analyze the mechanism of CT-32615-induced cytotoxicity in MM cells, we examined the cell cycle profile of MM.1S cells cultured with medium or CT-32615 (25–100 nM) for 24 and 48 h. After incubation, cells were harvested and stained with propidium iodide. As shown in Fig. 4Citation A, culture with CT-32615 for 24 h induced a progressive increase in G2-M phase in MM.1S cells in a dose-dependent manner. Importantly, culture with CT-32615 significantly increased sub-G0-G1 phase cells in a dose-dependent fashion by 48 h (Fig. 4B)Citation . Similar results were observed for U266 and RPMI8226 cells (data not shown). As in Fig. 4Citation A, p21Cip1 expression was up-regulated by CT-32615 in a time-dependent fashion (Fig. 4C)Citation . No significant change of p27 protein expression was observed (data not shown).



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  		Fig. 4. Effect of CT-32615 on cell cycle profile in MM.1S cells. A, MM.1S cells were cultured with DMSO control medium and with 50 or 100 nM CT-32615 for 24 h. B, MM.1S cells were cultured with DMSO control medium and with 25 or 50 nM CT-32615 for 48 h. Cells were harvested and subjected to propidium iodide staining for cell cycle profile analysis. C, MM.1S cells treated with 100 nM CT-32615 for 12, 24, and 36 h were subjected to Western blotting to assess p21 expression.

 
Apoptosis Triggered by CT-32615 Is Mediated via Caspase-3, Caspase-7, and PARP Cleavage.
Apoptosis triggered by CT-32615 was further characterized by examining cleavage of caspases, as in our previous studies (23 , 24 , 30 , 43) . MM.1S cells treated with control medium and with 50 or 100 nM CT-32615 for 24 h were subjected to Western blotting to assess cleavage of caspase-3, caspase-7, caspase-8, caspase-9, and PARP. Caspase-3 cleavage is a hallmark of apoptosis triggered by chemotherapeutic agents in MM (14 , 23 , 28 , 30 , 43 , 46 , 49) , but only low levels of caspase-3 cleavage were observed in MM.1S cells treated with CT-32615 (Fig. 5A)Citation . However, CT-32615 (50 nM) treatment triggered caspase-7 followed by PARP, cleavage. Furthermore, caspase-8, but not caspase-9, was cleaved by CT-326215 treatment (Fig. 5A)Citation . In a time course experiment of CT-32615 (100 nM) treatment of MM.1S and RPMI8226 cells, both caspase-7 and PARP were cleaved after 24 h with only low levels of caspase-3 cleavage (Fig. 5B)Citation , consistent with the dose-dependent effects on cell cycle profile (Fig. 4A)Citation .



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  		Fig. 5. CT-32615 induces caspase-8, caspase-7, and PARP cleavage. A, MM.1S cells were cultured with DMSO medium control and with 50 or 100 nM CT-32615 for 24 h. B, MM.1S and RPMI8226 cells were cultured for 12, 24, and 36 h in the presence of 100 nM CT-32615. Cells were then harvested, lysed, and subjected Western blotting for detection of cleavage of caspase-3, caspase-8, caspase-9, caspase-7, and PARP using specific Abs.

 
Caspase Inhibitors Block Caspase-7/PARP Cleavage and Cytotoxicity Induced by CT-32615.
To examine whether inhibition of caspase cleavage blocks CT-32615-induced apoptosis, we next examined caspase-7 and PARP cleavage in 12- and 24-h cultures of MM cells with CT-32615 (100 nM) in the presence or absence of Z-VAD-FMK (25 µM). Z-VAD-FMK completely abrogated caspase-7 cleavage and inhibited PARP cleavage triggered by CT-32615 treatment (12 and 24 h; Fig. 6ACitation ). Importantly, Z-VAD-FMK also significantly (P < 0.01) inhibited CT-32615-induced cytotoxicity in both MM.1S (Fig. 6B)Citation and RPMI8226 (Fig. 6C)Citation cells, assessed by MTT assay after 48 h treatment.



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  		Fig. 6. Caspase inhibitors block CT-326615-induced cleavage of caspase-7, PARP, and growth inhibition of MM cells. A, MM.1S cells were cultured with CT-32615 (100 nM) for 12 and 24 h in the presence or absence of Z-VAD-FMK (25 µM). Cells were then harvested, lysed, and subjected to Western blotting for detection of cleavage of caspase-7 and PARP. MM.1S (B) and RPMI8226 (C) MM cells were cultured with 50 and 100 nM CT-32615 in the presence ({blacksquare}) or absence ({square}) of Z-VAD-FMK (25 µM) for 48 h. In each case, cell growth was assessed by MTT assay, and data represent means of quadruplicate cultures; bars, SD. D, MM.1S cells were cultured with CT-32615 (100 nM) for 24 h in the presence or absence of Z-VAD-FMK (25 µM), Z-IETD-FMK (25 µM), or granzyme B inhibitor (300 nM). Cells were then harvested, lysed, and subjected to Western blotting for detection of cleavage of caspase-8, caspase-7, and PARP. *, P < 0.01.

 
Because caspase-7 (50) and PARP (51) are also cleaved by granzyme B, we further examined whether a granzyme B inhibitor could block CT-32615-induced caspase-7 or PARP cleavage. Granzyme B inhibitor did not block CT-32615-induced caspase-7 or PARP cleavage, although Z-VAD-FMK and Z-IETD-FMK completely abrogated caspase-7 and inhibited PARP cleavage (Fig. 6D)Citation . Protein expression of granzyme B was not changed after CT-32615 treatment (data not shown). These data suggest that apoptotic signaling induced by CT-32615 is initiated via caspase-8/caspase-7/PARP cleavage and does not involve granzyme B.

CT-32615 Induces Phosphorylation of p53 and JNK.
Because we found that proteasome inhibitor PS-341 induces phosphorylation of p53 (43) , we similarly examined whether CT-32615 induces phosphorylation (serine 15) of p53. Interestingly, CT-32615 induced phosphorylation of p53 in a dose-dependent manner; however, in contrast to PS-341, protein expression of p53 was not altered (Fig. 7A)Citation . Importantly, phosphorylation of JNK was also triggered by CT-32615 treatment (Fig. 7A)Citation , suggesting that CT-32615 induced a stress response in MM cells. To examine the role of activation of JNK in CT-32615-induced cytotoxicity, we next cultured MM cells with 50 nM CT-32615, in the presence or absence of specific JNK inhibitor SP600125 (41 , 42) . SP600125 significantly (P < 0.01) inhibited CT-32615-induced phosphorylation of JNK and caspase-8 cleavage in a dose-dependent fashion (Fig. 7B)Citation , thereby inhibiting CT-32615 dose-dependent cytotoxicity in RPMI8226 (Fig. 7C)Citation and MM.1S (Fig. 7D)Citation cells. Moreover, transfection of DN-JNK into MM.1S cells conferred CT-32615 resistance (Fig. 7E)Citation . Taken together, these results indicate that JNK mediates CT-32615-induced apoptosis.



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  		Fig. 7. CT-32615 induces phosphorylation of p53 and JNK in MM.1S cells. A, MM.1S cells were cultured with CT-32615 (12.5–100 nM) for 24 h. B, MM.1S cells were cultured with CT-32615 (50 nM) and SP600125 (2.5–20 µM) for 24 h. Cells were then harvested, lysed, and subjected to Western blotting to assay for phosphorylation of p53 and JNK and caspase-8 cleavage. RPMI8228 (C) and MM.1S (D) cells were cultured with or without CT-32615 (50 µM) in the presence of DMSO medium control ({square}) and with 2.5 µM ({square}), 5 µM (), or 10 µM ({blacksquare}) SP600125. In each case, cell growth was assessed by MTT assay. E, CT-32615-induced apoptosis was also examined in MM.1S cells transfected with DN-JNK ({bullet}), GFP control ({blacktriangleup}), or in nontransfected cells ({blacksquare}). The data represent means of quadruplicate cultures; bars, SD. *, P < 0.01; **, P < 0.05.

 
CT-32615 Inhibits TNF-{alpha}-induced NF-{kappa}B Activation.
We have recently shown the importance of NF-{kappa}B activity in promoting growth, survival, and drug resistance in MM cells in the MM BM milieu (16 , 37 , 38) . Because other novel agents demonstrate an inhibitory effect on NF-{kappa}B activation in MM cells, we similarly examined whether CT-32615 could inhibit NF-{kappa}B activation in MM.1S cells using EMSA, as in our previous studies (23 , 37 , 38 , 46 , 49) . CTI-32615 pretreatment (50 and 100 nM for 12 h) significantly inhibited DNA-binding activity of p65/p50 NF-{kappa}B in both nonstimulated and TNF-{alpha}-stimulated MM.1S cells (Fig. 8A)Citation . To exclude a nonspecific effect, we stimulated MM.1S cells with TNF-{alpha} and IL-6 after 12-h CT-32615 pretreatment and probed for phosphorylation of p38 MAPK and STAT3, respectively. Importantly, neither phosphorylation of p38 MAPK triggered by TNF-{alpha} (Fig. 8B)Citation nor phosphorylation of STAT3 triggered by IL-6 (Fig. 8C)Citation was altered by CT-32615 pretreatment.



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  		Fig. 8. CT-32615 inhibits NF-{kappa}B activation in MM.1S cells. MM.1S cells were cultured with either DMSO medium control or CT-32615 (50 and 100 nM) for 12 h before stimulation with TNF-{alpha} or IL-6. A, after 20 min stimulation with TNF-{alpha} (10 ng/ml), nuclear protein from the cells was subjected to EMSA to assess DNA binding activity of NF-{kappa}B. After 5 min stimulation with TNF-{alpha} (10 ng/ml) or 10 min stimulation with IL-6 (50 ng/ml), whole-cell lysates were subjected to Western blotting and probed with anti-phospho-p38 MAPK (A) or phospho-STAT3 (C) Abs, respectively.

 
CT-32615 Inhibits Paracrine MM Cell Growth and Abrogates the Protective Effect of BMSCs against Dex-induced Apoptosis.
Because we found that the BM microenvironment confers cell growth and drug resistance in MM cells (16 , 37 , 38) , we next studied the effect of CT-32615 on paracrine MM cell growth in the BM milieu. We first examined the direct toxicity of CT-32615 on BMSCs from patients (n = 4) using MTT assay, as in our previous studies (23 , 38) , and observed no significant growth inhibition (5–10%) triggered by CT-32615 treatment (Fig. 9A)Citation . MM.1S and U266 cells were then cultured with or without BMSCs in the presence or absence of CT-32615. Tumor cell adherence to BMSCs triggered increased [3H]thymidine uptake of MM.1S cells (1.44-fold; P < 0.01; Fig. 9BCitation ) and U266 cells (1.42-fold; P < 0.01; Fig. 9CCitation ), and CT-32615 inhibited this up-regulation of growth in a dose-dependent fashion (P < 0.01). Because adherence of MM cells to BMSCs triggers increased secretion of IL-6 and IGF-I in culture supernatants (15 , 36 , 38 , 39) , these results are consistent with the observed inability of exogenous IL-6 or IGF-I to overcome the growth-inhibitory effects of CT-32615 (Fig. 3)Citation .



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  		Fig. 9. CT-32615 inhibits paracrine MM cell growth and abrogates the protective effect of BMSCs against Dex-induced apoptosis in MM.1S cells. A, BMSCs (n = 4) were cultured with CT-32615 (0.8–500 nM) for 24 h. Cell growth was assessed by MTT assay, and data represent means of quadruplicate cultures; bars, SD. MM.1S (B) and U266 (C) MM cells were cultured in BMSC-coated or noncoated plates for 48 h in the presence of DMSO control ({square}), 12.5 nM ({square}), 25 nM (), and 50 nM ({blacksquare}) CT-32615. D, MM.1S cells were cultured with Dex (0.2 µM) in BMSC-coated or noncoated plates for 48 h, in the presence or absence of DMSO control ({square}), 12.5 nM ({square}), 25 nM (), and 50 nM ({blacksquare}) CT-32615. DNA synthesis was assessed by [3H]thymidine uptake, and data represent means of quadruplicate cultures; bars, SD.

 
To define the functional sequelae of CT32615-related NF-{kappa}B blockade in MM.1S cells, we examined its inhibitory effect on growth of MM cells in the presence of Dex and with or without BMSCs. Dex significantly inhibited MM.1S cell growth in a dose-dependent fashion, and BMSCs completely abrogated Dex-induced cytotoxicity. However, this protective effect of BMSCs against Dex-induced growth inhibition was abrogated in the presence of CT-32615 (Fig. 9D)Citation . These data suggest that LPAAT-ß inhibitors retain their antitumor activity against MM cells in the BM milieu.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
MM is currently incurable with either conventional or high-dose chemotherapy, and novel biological-based treatment strategies targeting both the MM cells and the BM microenvironment can overcome drug resistance in preclinical and clinical studies (16) . In this report, we first demonstrate that LPAAT-ß inhibitors, a novel class of agents, induce apoptosis in MM cell lines as well as freshly isolated MM cells from patients. A recent study also demonstrates elevated expression and activity of LPAAT-ß in a spectrum of human tumors, and conversely, that inhibition of LPAAT-ß activity induces apoptosis (52) .

In our study, the IC50 of LPAAT-ß inhibitors in MM cell lines is 50–100 nM and 70–200 nM in MM cells from patients. Importantly, we observed only 5–10% growth inhibition of PBMCs at a concentration of 500 nM, suggesting potential selective activity of these inhibitors in vivo. As is true for thalidomide/IMiDs-treated MM cells (24) and PS-341-treated MM cells (23) , Dex enhances CT-32615-induced cytotoxicity in MM.1S cells, suggesting differential apoptotic signaling cascades. For example, Dex induces caspase-9 activation (14) via a cytochrome c-independent, second mitochondria-derived activator of caspases (Smac) dependent pathway (29) . Dex also up-regulates I{kappa}B{alpha} protein expression (38) , thereby augmenting the inhibitory effect of CT-32615 on NF- {kappa}B activation.

IL-6 triggers proliferation of MM cells via activation of the Ras/Raf/MAPK kinase/p42/44 MAPK signaling cascade (27) and survival of MM cells via Janus kinase 2/STAT3 activation with downstream induction of Bcl-xL (53) and Mcl-1 (54, 55, 56) . Specifically, Dex resistance conferred by IL-6 is mediated via the PI3-K/Akt signaling cascade (13 , 14) . We therefore next examined whether exogenous IL-6 inhibited LPAAT-ß-induced cytotoxicity in MM cells. Importantly, neither IL-6 nor IGF-I, which promotes survival of MM cells (15) , abrogated CT-32615-induced cytotoxicity, suggesting that LPAAT-ß inhibitors can induce cytotoxicity of MM cells in their BM milieu.

In our study, CT-32615 induces G2-M arrest in MM cells, associated with up-regulation of p21Cip1, followed by apoptosis. We have demonstrated previously that apoptosis triggered by conventional or novel agents is mediated via caspase-8 and/or caspase-9, followed by caspase-3 and PARP cleavage (29) (16 , 23 , 30 , 31 , 43 , 46 , 47 , 57 , 58) . Although PARP cleavage occurs as early as 12 h after CT-32615 treatment, only low levels of caspase-3 cleavage were recognized, without caspase-9 cleavage. Importantly, CT-32615 triggers activation of caspase-8 and caspase-7 in a time- and dose-dependent fashion, associated with PARP cleavage; conversely, Z-VAD-FMK inhibits both caspase-7 and PARP cleavage, as well as CT-32615-induced MM cell cytotoxicity. Because granzyme B also triggers caspase-7 (50) or PARP (51) cleavage, we also examined the effect of granzyme B inhibitor on CT-32615-induced caspase-7 and PARP cleavage. The caspase-8 inhibitor IETD-FMK, but not the granzyme B inhibitor, blocks CT-32615-induced caspase-8, caspase-7, and PARP cleavage, confirming that LPAAT-ß inhibitors trigger MM cell apoptosis via caspase-8/caspase-7/PARP cleavage.

Because we have shown recently that proteasome inhibitor PS-341 induces phosphorylation of p53 (Ser-15) and JNK during MM cell apoptosis (43) , we next examined whether CT-32615 also triggered phosphorylation of these molecules. Interestingly, CT-32615 induces phosphorylationSer15 of p53 and JNK, suggesting that DNA damage and a stress response is triggered by CT-32615. Because p21Cip1 is a downstream target of p53, phosphorylationSer15 of p53 may up-regulate p21Cip1 expression in CT-32615-treated MM.1S cells. JNK is a stress-response protein that mediates apoptosis triggered by unfolded proteins (59) ; conversely, IL-6 inhibits MM cell apoptosis by inhibiting the JNK/SAPK pathway (60) . Importantly, in our study, the JNK-specific inhibitor SP600125 blocks CT-32615-induced phosphorylation of JNK and caspase-8 cleavage. Furthermore, both SP600125 and DN-JNK inhibited cytotoxicity in MM cells, indicating that JNK mediates LPAAT-ß inhibitor-induced apoptosis.

We have demonstrated that Dex (38) , arsenic trioxide (49) , proteasome inhibitor PS-341 (23 , 37) , IMiDs (61) , and I{kappa}B kinase inhibitor PS-1145 (38) block NF-{kappa}B activation in MM cells and BMSCs, resulting in MM cell growth inhibition (38 , 61) ; decreased MM cell adherence to BMSCs because of down-regulation of adhesion molecules (i.e., ICAM-1 and VCAM-1; Ref. 37 ); inhibition of IL-6 secretion in BMSCs (23 , 37 , 38 , 62) ; and decreased telomerase activity (63) . In this study, we therefore examined the inhibitory effect of LPAAT-ß inhibitor on NF-{kappa}B activation in MM cells and demonstrated, using EMSA, that CT-32615 markedly down-regulated DNA binding activity of NF-{kappa}B in MM.1S cells before cell death. Importantly, neither TNF-{alpha}-triggered p38 MAPK nor IL-6-triggered STAT3 phosphorylation was affected by CT-32615. Recent studies have shown that PKC{zeta} is a downstream molecule of RelA and c-Rel members of the NF-{kappa}B family, because dominant-negative mutant PKC{zeta} inhibits RelA or c-Rel transactivating activity in primary endothelial cells (64) or COS cells (65) , respectively. Moreover, PKC{zeta} plays a critical role in regulating TNF-{alpha}-induced NF-{kappa}B activation and ICAM-1 transcription in endothelial cells (66) . Because PA is a physiological activator of PKC{zeta} (67) , LPAAT-ß inhibitors that block conversion of LPA to PA may inhibit NF-{kappa}B activation via down-regulation of PA in MM cells.

Given that neither IL-6 nor IGF protect against LPAAT inhibition-induced cytotoxicity and that LPAAT-ß inhibitor down-regulates NF-{kappa}B activation, we next examined whether LPAAT-ß inhibitor can overcome MM cell growth, survival, migration, and drug resistance conferred by the BM microenvironment (11 , 12 , 16 , 36, 37, 38 , 68, 69, 70) . We demonstrated that CT-32615 abrogated paracrine growth of MM.1S and U266 cells in the presence of BMSCs, without cytotoxicity on BMSCs. Furthermore, although binding of MM cells to BMSCs protects against Dex-induced apoptosis in MM.1S cells, CT-32615 induced apoptosis, even of adherent MM cells. Our results therefore demonstrate for the first time that inhibiting LPAAT activity induces cytotoxicity in MM cells in their BM milieu, providing the framework for clinical trials of these agents to improve the outcome of patients with MM.


   FOOTNOTES
 
Grant support: NIH Grant Specialized Programs of Research Excellence (SPORE) IP50 CA10070-01, PO-1 78378, and RO-1 CA 50947; the Doris Duke Distinguished Clinical Research Scientist Award (to K. C. A.); the Multiple Myeloma Research Foundation (to T. H.); and the Cure for Myeloma Research Fund (to K. C. A.).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Kenneth C. Anderson, Dana-Farber Cancer Institute, Mayer 557, 44 Binney Street, Boston, MA 02115. Phone: (617) 632-2144; Fax: (617) 632-2140; E-mail: kenneth_anderson{at}dfci.harvard.edu

3 The abbreviations used are: MM, multiple myeloma; BM, bone marrow; BMSC, BM stromal cell; IL, interleukin; IGF, insulin-like growth factor; STAT3, signal transducers and activators of transcription 3; PI3-K, phosphatidylinositol 3-kinase; LPA, lysophosphatidic acid; LPAAT, LPA acyltransferase; PA, phosphatidic acid; MAPK, mitogen-activated protein kinase; PARP, poly(ADP-ribose) polymerase; Dex, dexamethasone; Ab, antibody; Z-VAD-FMK, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; Z-IETD-FMK, benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone; PBMC, peripheral blood mononuclear cell; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; JNK, c-Jun NH2-terminal kinase; GFP, green fluorescent protein; EMSA, electrophoretic mobility shift analysis; TNF, tumor necrosis factor; NF-{kappa}B, nuclear factor-{kappa}B. Back

Received 5/ 5/03. Revised 9/17/03. Accepted 9/18/03.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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