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Blood, 15 February 2007, Vol. 109, No. 4, pp. 1669-1677. Prepublished online as a Blood First Edition Paper on October 5, 2006; DOI 10.1182/blood-2006-08-042747.
NEOPLASIA Targeting PKC in multiple myeloma: in vitro and in vivo effects of the novel, orally available small-molecule inhibitor enzastaurin (LY317615.HCl)1 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA; 2 Department of Internal Medicine V, University of Heidelberg, Germany; and 3 Eli Lilly and Company, Indianapolis, IN
In multiple myeloma (MM) protein kinase C (PKC) signaling pathways have been implicated in cell proliferation, survival, and migration. Here we investigated the novel, orally available PKC-inhibitor enzastaurin for its anti-MM activity. Enzastaurin specifically inhibits phorbol ester–induced activation of PKC isoforms, as well as phosphorylation of downstream signaling molecules MARCKS and PKCµ. Importantly, it also inhibits PKC activation triggered by growth factors and cytokines secreted by bone marrow stromal cells (BMSCs), costimulation with fibronectin, vascular endothelial growth factor (VEGF), or interleukin-6 (IL-6), as well as MM patient serum. Consequently, enzastaurin inhibits proliferation, survival, and migration of MM cell lines and MM cells isolated from multidrug-resistant patients and overcomes MM-cell growth triggered by binding to BMSCs and endothelial cells. Importantly, strong synergistic cytotoxicity is observed when enzastaurin is combined with bortezomib and moderate synergistic or additive effects when combined with melphalan or lenalidomide. Finally, tumor growth, survival, and angiogenesis are abrogated by enzastaurin in an in vivo xenograft model of human MM. Our results therefore demonstrate in vitro and in vivo efficacy of the orally available PKC inhibitor enzastaurin in MM and strongly support its clinical evaluation, alone or in combination therapies, to improve outcome in patients with MM.
Characterized by clonal proliferation of immunoglobulin-secreting plasma cells within the bone marrow (BM), multiple myeloma (MM) is the most common hematologic malignancy in patients older than 65 years. Although conventional and novel therapies such as thalidomide, bortezomib, and lenalidomide have prolonged progression-free and overall survival, MM remains incurable. The delineation of signaling pathways, which mediate MM-cell growth, survival, and migration within the BM microenvironment, can both enhance our understanding of disease pathogenesis and identify molecular targets for novel MM therapies to improve patient outcome.
The PKC family of serine/threonine kinases is comprised of at least 12 isoforms, which are classified into 3 structurally and functionally distinct subgroups: conventional isoforms (cPKC including PKC
Given its proposed key role in tumorigenesis, PKC is a promising new target in cancer therapy. The macrocyclic bisindolylmaleimide enzastaurin (LY317615.HCl) is a novel orally available PKC inhibitor. Enzastaurin competes with ATP for the nucleotide triphosphate-binding site of PKC, thereby blocking its activation. Besides its major target PKCß, enzastaurin also potently inhibits other PKC isoforms including PKC
In MM, PKC isoform expression has been reported in several MM cell lines.29–33 Functionally, PKCs are: (1) involved in MM-cell apoptosis30,34; (2) required for VEGF- and Wnt-induced MM-cell migration33,35; and (3) associated with the control of IL-6 receptor-
Materials
Enzastaurin (LY317615.HCl) was provided by Eli Lilly (Lilly Research Labs, Eli Lilly and Company, Indianapolis, IN). Other reagents were obtained as follows: VEGF165 from R&D Systems (Minneapolis, MN); TPA (12-O-tetradecanoyl-phorbol-13-acetate); pPKC(Thr638/641), pPKC(Ser660), pPKC Cells and cell culture All human MM cell lines and primary patient MM cells were cultured as previously described.32 Human umbilical vein endothelial cells (HUVECs) from a pool of 5 healthy donors (kindly provided by Drs A. Cardoso and M. Tavares, Dana-Farber Cancer Institute [DFCI]) were maintained in EGM-2MV media (Clonetics BioWhittaker, Walkersville, MD) containing 2% FBS. Isolation of patient tumor cells After patients provided informed consent in accordance with the Declaration of Helsinki and approval by the Institutional Review Board of the DFCI, MM patient cells (96% CD38+CD45RA–) were obtained as previously described.32 Cell fractionation After washing 3 times with phosphate-buffered saline, MM.1S cells were transferred into 400 µL hypotonic lysis buffer followed by cell fractionation as previously described.32 Cell lysis, immunoprecipitation, and Western blotting Cell lysis, immunoprecipitation, and Western blot analysis were performed as described previously.32 Measurement of PKC activity PKC activity was measured with a PKC assay kit (Upstate Biotechnology, Lake Placid, NY) according to the manufacturer's instructions and as described previously.32 DNA synthesis and cell-proliferation assay Cell growth was assessed by measuring [3H]-thymidine uptake, as described in prior studies.32 Cell adhesion assays Adhesion assays were performed using the Vybrant cell-adhesion assay kit (Molecular Probes, Eugene, OR), as previously described.32 All experiments were done in triplicate. Annexin/PI stain, SYTOX green nucleic acid stain, MTT assay Enzastaurin in solution has an intense orange color. Besides method-related adequate baseline normalization, we therefore used a variety of apoptosis/cytotoxicity assays to confirm our results under different conditions. MM.1S cells were treated with either vehicle or enzastaurin for 24 hours and 48 hours, washed with PBS, and evaluated for annexin V (BD PharMingen) or SYTOX green nucleic acid staining (Invitrogen/Molecular Probes) using a fluorescence-activated cell sorter (FACS). MTT colorimetric survival assay The inhibitory effect of enzastaurin on MM cell lines was assessed using MTT assay, as described previously.38 Cell survival was estimated as a percentage of the value of untreated controls. Transwell migration assay Cell migration was assayed using a modified Boyden chamber assay, as described previously.32 In vitro angiogenesis assay The antiangiogenic potential of enzastaurin was studied using an in vitro angiogenesis assay kit (Chemicon, Temecula, CA), as per manufacturer's instructions. Tube formation was assessed using an inverted light/fluorescence microscope at x4 to x10 magnification. Photographs are representative of each group and 3 independent experiments. Isobologram analysis For combination studies, data from 3H[dT] uptake assays were converted into values representing the fraction of growth affected (FA) in drug-treated versus untreated cells and analyzed using CalcuSyn software program (Biosoft, Ferguson, MO) based on the Chou-Talalay method. A combination index (CI) smaller than 1 indicates synergism, whereas 0.9 to 1.1 indicates additive effects. MM xenograft mouse model
To determine the in vivo anti-MM activity of enzastaurin, beige-nude Xid mice were inoculated subcutaneously in the right flank with 3 x 107 MM.1S MM cells in 100 µL RPMI 1640 medium, together with 100 µL Matrigel (Becton Dickinson Biosciences, Bedford, MA). When tumor was measurable, mice were assigned to the enzastaurin treatment group or a control group. Enzastaurin was dissolved in 100% ethanol, diluted 1:10 in D5W (Sigma-Aldrich, St Louis, MO), and given twice daily by oral gavage for indicated periods. The control group received the carrier alone at the same schedule and route of administration. Tumor burden was measured every alternate day using a caliper (calculated volume = 4 Immunohistochemistry Sections (4 µm) of formalin-fixed tissue were used for TUNEL staining and staining with CD31 antibody (BD PharMingen, San Diego, CA) in a humid chamber at room temperature, as in prior studies.39 Leica N Plan 5x/0.12 PH0, Leica N Plan 10x/0.25 PH1, and Leica HCX PL Fluotar 40x/0.60 corr PH2 XT objective lenses were used. Statistical analysis Statistical significance of differences observed in enzastaurin-treated versus control-cell cultures was determined using an unpaired Student t test. The minimal level of significance was P below .05. Overall survival in animal studies was measured using the Kaplan-Meier method, and results are presented as the median overall survival, with 95% confidence intervals (Cls).
Enzastaurin specifically inhibits TPA-triggered phosphorylation and activation of PKC isoforms in MM cells
PKC isoforms (PKC
The PKC inhibitor enzastaurin has already demonstrated marked preclinical and early clinical activity in a variety of tumors. In MM, PKCs are required for VEGF-induced MM-cell migration in our model cell line MM.1S as well as other MM cell lines and primary MM cells.35 The therapeutic potential of targeting PKC in MM has only recently been studied. Specifically, Rizvi et al showed that enzastaurin induced apoptosis in MM cell lines.40 In this study we further characterize the molecular mechanism whereby enzastaurin mediates in vitro and in vivo MM cytotoxicity. Typically, cPKCs and nPKCs change their conformation on membrane recruitment, thereby enabling both enzyme activation and subsequent substrate phosphorylation. We therefore first investigated whether enzastaurin abrogates TPA-induced PKC activation in MM.1S cells using both phospho-specific antibodies as well as immunoprecipitation kinase assays. Although enzastaurin did not inhibit TPA-induced recruitment of PKC isoforms to the plasma-cell membrane, it blocked TPA-induced homologous PKC activation loop Thr-514 and carboxy-terminal Ser-660 phosphorylation within the cytoplasm, Ser-660 phosphorylation at the plasma-cell membrane, as well as Thr-514 and Ser-660 phosphorylation within the nucleus. Less impressive differences in membrane-associated phosphorylation of Thr-514 may be due to enzastaurin-induced sustained PKC binding to the plasma-cell membrane, thereby blocking release of activated PKC into the cytoplasm (Figure 1A). In contrast to Thr-514 and Ser-660 phosphorylation, constitutive phosphorylation of regulatory PKC Thr-638 or Thr-641 (pPKC
Enzastaurin specifically inhibits TPA-triggered phosphorylation of signaling molecules downstream of PKC Having shown enzastaurin-mediated effects on TPA-induced PKC isoform activation, we next evaluated its effects on downstream signaling molecules. Our results demonstrate that enzastaurin inhibited TPA-induced phosphorylation of downstream PKCµ/PKD, MARCKS, GSK3ß, JNK1/2, ERK1/2, and c-Myc, but did not inhibit phosphorylation of PDK-1, a signaling molecule upstream of PKC, which triggers activation loop phosphorylation of cPKCs and nPKCs (Figure 2A).
Enzastaurin inhibits PKC signaling sequelae in MM cells triggered by BMSC-secreted growth factors and cytokines, costimulation with the extracellular matrix protein fibronectin, IL-6 or VEGF, as well as patient serum Growth factors and cytokines within the BM microenvironment (ie, IL-6 and VEGF) mediate MM-cell growth, survival, and drug resistance. We therefore next tested the impact of the BM microenvironment on MM PKC signaling. Our results show that PKCµ/PKD phosphorylation is triggered by conditioned medium from the BMSC line HS-5 (Figure 2B, SN), as well as by costimulation by both fibronectin/VEGF (Figure 2B, VEGF) and fibronectin/IL-6 (Figure 2, IL-6). Conversely, enzastaurin inhibited PKCµ/PKD activation triggered by these stimuli (Figure 2B). Furthermore, serum isolated from MM patient BM strongly triggered Thr-514 phosphorylation as well as activation of downstream signaling molecules including MARCKS, JNK, and c-Myc. Conversely, treatment with enzastaurin strongly inhibited serum-induced phosphorylation of catalytic Thr-514 PKC and downstream signaling molecules (Figure 2C). Taken together, these data confirm that enzastaurin inhibits PKC activation triggered by BMSC-secreted growth factors and cytokines, costimulation with the extracellular matrix protein fibronectin, VEGF or IL-6, as well as MM patient serum. Enzastaurin inhibits MM-cell growth We next examined the impact of PKC inhibition on MM-cell growth, survival, and migration. Enzastaurin induced marked dose-dependent growth inhibition in all MM cell lines investigated including MM.1S, MM.1R, RPMI 8226 (RPMI), RPMI-Dox40 (Dox40), NCI-H929, KMS-11, OPM-2, and U266 (Figure 3A). IC50 ranged from 0.6 to 1.6 µM, except for RPMI8226 (with an IC50 of 4 µM), which has markedly lower overall PKC expression than the other cell lines (Table S1). Importantly, enzastaurin also induced growth inhibition of (CD138+) MM cells isolated from 3 patients with multidrug-resistant progressive disease (Figure 3B).
Enzastaurin inhibits MM-cell growth triggered by BMSCs In addition to the autocrine/paracrine effects mediated by growth factors and cytokines within the MM BM microenvironment, direct MM-BMSC contact also triggers tumor-cell growth. Importantly, enzastaurin inhibited both MM.1S adhesion to BMSCs (Figure 3C) and cell growth (Figure 3D) in a BMSC–MM-cell coculture system. Moreover, enzastaurin similarly inhibited growth of CD138+ MM cells isolated from a multidrug-resistant patient with clinically progressive disease (Figure 3E). Taken together, these data demonstrate that enzastaurin inhibits MM-cell growth in MM cells alone and when cocultured with BMSCs. Enzastaurin induces MM-cell apoptosis We next sought to evaluate whether enzastaurin induces MM-cell apoptosis. Due to its self-fluorescence within the propidium iodide spectrum, we stained cells separately with annexin V-FITC and SYTOX green nucleic acid stain. Our data demonstrate that enzastaurin induces 55% apoptosis in MM.1S cells after 48 hours (data not shown). Moreover, MTT assays show dose-dependent enzastaurin-induced cytotoxicity in MM cell lines, but not in PBMCs isolated from 3 healthy donors (Figure 4A). Consistent with these data, protein profiling in enzastaurin-treated MM cells shows decreased phosphorylation of Akt, GSK3ß, and cMyc as well as down-regulation of Mcl-1, but not Bcl-2 and Bcl-XL, protein expression. In addition, our data show cleavage of caspase-8, caspase-3, and PARP, but not of caspase-9 (Figure 4B). Taken together, these data confirm that enzastaurin triggers MM-cell apoptosis.
Enzastaurin inhibits MM-cell migration Increased microvessel density and tumor-cell expansion within the BM microenvironment, as well as tumor-cell egress into the peripheral blood, are indicators of MM progression. PKC activity is associated with VEGF- as well as IGF-1–induced endothelial and MM-cell migration.35 Our data show that enzastaurin markedly down-regulates MM-cell adhesion to the extracellular matrix protein fibronectin (Figure 5A). Moreover, enzastaurin abrogates VEGF-triggered MM-cell migration on fibronectin as well as IGF-1–induced MM-cell migration (Figure 5B).
Enzastaurin has synergistic cytotoxicity with bortezomib, lenalidomide, and melphalan Combined therapies can often enhance growth inhibition and cytotoxicity, overcome drug resistance, and avoid side effects. We therefore next investigated whether combinations of enzastaurin with novel (bortezomib and lenalidomide), or conventional (melphalan) therapies enhanced growth inhibition and cytotoxicity. Specifically, low-dose enzastaurin (0.5 and 1 nM) was combined with melphalan (0.5 and 2 µM), lenalidomide (0.5 and 2 µM), and bortezomib (0.5 and 2 nM), at concentrations below their in vitro IC50 values (8 µM, 5 µM, and 5 nM, respectively). Equivalent plasma concentrations are easily achievable in MM patients.41–43 Isobologram analysis of thymidine uptake demonstrated marked synergistic growth inhibition of low-dose enzastaurin and bortezomib (combination index > 0.5) and moderate synergistic (combination index, 0.7-0.9) or additive (combination index, 0.9-1.1) effects in combination with lenalidomide or melphalan, respectively (Table 1). Moreover, inhibition of survival achieved with low-dose single agent bortezomib, lenalidomide, and melphalan was markedly enhanced when given in combination with low-dose enzastaurin (Figure 6). Taken together, these results provide the framework for combination clinical trials to increase therapeutic efficacy and reduce toxicity.
Enzastaurin blocks angiogenesis and MM-cell growth triggered by endothelial-cell adhesion PKC activation induces endothelial-cell proliferation and migration, thereby inducing endothelial tube formation.44 Importantly, enzastaurin induced marked antiangiogenic activity in the rat corneal micropocket assay and decreased microvessel density in human tumor xenografts including human breast cancer, ovarian cancer, small-cell lung cancer, colon cancer, hepatocellular cancer, gastric cancer, and glioblastoma multiforme.45 Consistent with these data, our results showed marked dose-dependent in vitro down-regulation of endothelial-cell growth (Figure 7A) and tubule formation (Figure 7B) triggered by enzastaurin. Moreover, enzastaurin, similar to the PKC inhibitor bisindolylmaleimide (BIM) I, abrogated TPA-induced Thr-514 and Ser-660 phosphorylation (Figure 7C, upper 2 panels), as well as subsequent activation of downstream signaling molecules including MARCKS, PKCµ/PKD, GSK3ß, JNK1/2, ERK1/2, c-Myc, and S6 (Figure 7C). Similar to BMSCs, MM-cell growth is stimulated when bound to endothelial cells. Importantly, our results demonstrate marked enzastaurin-induced inhibition of cell growth in both the endothelial cell-MM.1S, as well as the endothelial cell-patient MM cell, coculture systems (Figure 7D).
Enzastaurin markedly decreases tumor growth in a xenograft mouse model of human MM Having demonstrated effects of enzastaurin on both MM cells and endothelial cells in vitro, we next sought to assess the in vivo efficacy of enzastaurin using a mouse model of human MM. Immune-deficient beige-nude-xid (BNX) mice were given subcutaneous inoculations into the flank with 3 x 107 MM.1S cells. When the tumors reached a palpable size, mice were treated with 30 mg/kg enzastaurin administered twice daily by oral gavage over an 8-week period. Tumor growth in treated mice was significantly delayed compared to the control group (Figure 8A). However, tumors rapidly regrew after cessation of treatment. Using Kaplan-Meier and log-rank analysis, the mean overall survival (OS) was 23 days (95% CI, 13-52 days) in the control cohort versus 77 days (95% CI, 50 to > 200 days) in the treatment group (Figure 8B). Statistically significant prolongation in mean OS was observed in treated animals compared with control mice (P = .002). Importantly, treatment with either the vehicle alone or enzastaurin did not affect body weight (Figure 8C). Large areas of cells with condensed nuclei were seen in hematoxylin and eosin stains of enzastaurin-treated tumors, consistent with tumor-cell apoptosis or necrosis (Figure 8D). Angiogenesis was markedly reduced within tumors of enzastaurin-treated versus nontreated mice, as evidenced by CD31 staining (Figure 8E). Finally, TUNEL assays on tumor sections from treated versus control mice showed significantly increased apoptosis (Figure 8F). Taken together, these results demonstrate that enzastaurin triggers in vivo inhibition of tumor growth, decreased angiogenesis, and increased MM-cell apoptosis, associated with prolongation of host survival.
PKC serves as the major cellular receptor for the powerful tumor-promoting phorbol esters (eg, TPA).8–11 Moreover, members of the PKC family have been implicated in pathogenesis of both solid as well as hematologic malignancies. PKC therefore represents an attractive and promising therapeutic target. However, due to the many isoforms and their cell-specific functions, thorough preclinical and clinical evaluation is needed assess the therapeutic potential of PKC inhibitors in various cancers. Specifically, although implicated in MM pathogenesis, the therapeutic value of targeting PKC signaling sequelae in MM is to date unknown.
Here we investigated the cytotoxicity of the orally available, small-molecule PKC inhibitor enzastaurin against MM. Promising preclinical and early clinical efficacy data have already led to a phase 3 clinical trial in DLBCL and early-phase clinical trials are ongoing in solid as well as hematologic malignancies. Due to our expression profiling studies in MM cells, we focused our present studies on PKC
Functionally, enzastaurin strongly inhibits growth and survival of a broad range of MM cell lines and patient MM cells, including those with the prognostic adverse t(4;14) translocation, including NCI-H929 (with wild-type FGFR3 overexpression), OPM-2 (with FGFR3 mutation K650E), and KMS-11 (with FGFR3 mutation Y373C). In contrast, PKC412 (N-benzoyl-staurosporine; Novartis Pharma, Basel, Switzerland), initially also developed as a PKC inhibitor, showed marked efficacy only in MM cells overexpressing wild-type FGFR3 or FGFR3 mutants.47 Importantly, marked up-regulation of PKCß was reported in MM patients with the t(4;14)(p16;q32) translocation.36,37 Ongoing studies are investigating whether PKCß up-regulation within this patient group is directly linked to overexpression of wild-type FGFR3 and MMSET and subsequent FGFR3-activating mutations and whether these effects can be modulated by enzastaurin. Molecular mechanisms by which PKC isoforms regulate cell growth and survival are still elusive. Studies to delineate downstream mechanisms by which the PKC isoforms Besides MM proliferation and survival, enzastaurin blocks VEGF- and IGF-1–triggered MM-cell migration, angiogenesis, as well as MM-cell growth triggered by MM-endothelial-cell binding. Recent advances in MM therapy have demonstrated advantages of combination therapy. Moreover, to increase antitumor effects, recent reports strongly suggest the use of PKC inhibitors together with chemotherapeutics or targeted therapies. Indeed, our in vitro studies in MM show synergistic effects for enzastaurin with the proteasome inhibitor bortezomib and moderate synergistic or additive effects when combined with melphalan or lenalidomide. Finally, oral dosing of enzastaurin in a MM xenograft mouse model markedly decreases tumor burden directly, via inhibition of MM-cell proliferation and induction of apoptosis, and indirectly, via inhibition of tumor angiogenesis. In summary, these in vitro and in vivo data support the pivotal role for members of the PKC family in MM pathogenesis and provide the framework for protocol evaluation of the novel, orally available PKC inhibitor enzastaurin, either alone or in combination with bortezomib, lenalidomide, or melphalan, to enhance therapeutic efficacy, reduce adverse side effects, and improve outcome in patients with MM.
Contribution: K.P. designed, performed, and analyzed research and wrote the manuscript; M.S.R. designed, performed and analyzed research; J.Z., D.M., and I.B. performed research; and Y.T.T., B.K.L., N.M., T.H., D.C., and K.C.A. analyzed data. Conflict-of-interest disclosure: B.K.L. is an employee of Eli Lilly and Company. K.P. and M.S.R. contributed equally to the study. Correspondence: Klaus Podar and Kenneth C. Anderson, Dana-Farber Cancer Institute, Department of Medical Oncology, Jerome Lipper Multiple Myeloma Center, 44 Binney St, Boston, MA 02115; e-mail: kenneth_anderson{at}dfci.harvard.edu and klaus_podar{at}dfci.harvard.edu.
This work was supported by a MMRF Senior Research grant award (K.P.); a grant from the Fritz-Thyssen Foundation (M.S. Raab); National Institutes of Health grants RO CA50947, PO-1 CA78378, and P50 CA100707; and the Doris Duke Distinguished Clinical Research Scientist Award (K.C.A.). We thank F. Abtahi for technical assistance and Drs A. Cardoso and M. Tavares for providing HUVECs. The authors further acknowledge the contribution of Dr P.G. Richardson and Dr R. Schlossman, as well as the patients, nursing staff, and clinical research coordinators of the Jerome Lipper Multiple Myeloma Center/Dana-Farber Cancer Institute for their help in providing primary tumor specimens for this study.
Submitted August 24, 2006; accepted September 25, 2006.
Prepublished online as Blood First Edition Paper, October 5, 2006
DOI: 10.1182/blood-2006-08-042747
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.
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Top
Abstract
Introduction
, PKCßI, PKCßII, PKC
); novel isoforms (nPKC including PKC
, PKC
PKC
, PKC
); and atypical isoforms (aPKC including, PKC
, PKC
/
). Based on homology within the catalytic domain, PKCµ/PKD and PKC
were recently added to the PKC superfamily. PKC isoforms show tissue- and cell-type–specific expression and function
/3 x (width/2)2 x (length/2)). Animals were killed when their tumor reached 2 cm or when the mice became moribund. Survival was evaluated from the first day of treatment until death. All animal studies were approved by the Dana-Farber Animal Care and Use Committee. 
