The second most commonly diagnosed hematologic malignancy, multiple myeloma, affects predominantly older patients (>60s) and is characterized by paraprotein in the serum or urine. Clinical manifestations include anemia, hypercalcaemia, progressive renal impairment, and osteolytic bone destruction. Despite promising new therapies, multiple myeloma eventually relapses in almost all patients. HSP are ubiquitous and highly conserved in prokaryotes and eukaryote organisms. Exposure to a broad range of stimuli results in increased HSP protein expression. These chaperone proteins are involved in protein transportation, prevent protein aggregation, and ensure correct folding of nascent and stress-accumulated misfolded proteins. In cancer, HSP expression is dysregulated, resulting in elevated expression, which promotes cancer by preventing programmed cell death and supporting autonomous cells growth, ultimately leading to resistance to heat, chemotherapy, and other stresses. Client proteins of HSP90 such as AKT, p53, MEK, STAT3, and Bcr-Abl are vital in tumor progression, including multiple myeloma, and their maturation and stability is dependent on HSP90. Therefore, inhibition of HSP90 via a HSP90 inhibitor (such as NVP-HSP990) should interrupt multiple signaling pathways essential for oncogenesis and growth in multiple myeloma. Our study showed that NVP-HSP990 triggered apoptosis in a panel of human multiple myeloma cells, induced cell-cycle arrest, PARP cleavage, downregulation of client proteins, the inability to reactivate phospho-STAT3 following exogenous IL-6 stimulation, and it synergized with azacytidine and bortezomib in cell lines and primary multiple myeloma samples. The mechanism of HSP90 inhibition in multiple myeloma warrants further evaluation. Mol Cancer Ther; 10(10); 1909–17. ©2011 AACR.

Multiple myeloma is an incurable clonal B-cell malignancy that comprises 1% of all cancers and approximately 10% of all hematologic malignancies. While an increasing range of active therapeutic approaches are available for the treatment of multiple myeloma, the inevitability of relapse underscores the present incurability of the disease. A key feature of multiple myeloma is the marked heterogeneity as recently defined by karyotypic and gene expression profiling data. This heterogeneity not only contributes to the significant variations in survival seen with presently available treatments but also highlights the challenge in identifying broadly effective newer therapeutic strategies. Finally, multiple myeloma has a complex relationship with the bone marrow microenvironment (BMME) that provides, via soluble and contact-mediated interactions, a niche that promotes the acquisition of greater levels of drug resistance during the course of the disease (1).

HSP were first discovered in 1962 (2) in Drosophila flies and then later in Escherichia Coli (3), yeast (4), plants (5), and mammalian cells (6). HSP have been shown to play a key role in a variety of cellular functions including cell cycling (7), cell growth (8), DNA transcription (9), and apoptosis (10). Furthermore, HSP are vital in mediating the maturation and stability of various proteins (11), inhibiting programmed cell death (12), and conferring resistance to hyperthermia (13). HSP expression is induced by heat and other stressors that include radiation and cytotoxic chemotherapy exposure. Mammalian HSPs have been classified into 5 major families HSP100, HSP90, HSP70, HSP60, and the small HSP based upon their molecular weight, amino acid sequence homology, and function.

HSP90 is the most abundant cytosolic HSP and regulates the fate of a variety of client proteins crucial for multiple cell signaling processes in eukaryotic cells. These include AKT (PI3K/AKT pathway), IL-6R (JAK/STAT pathway), FAK (integrin pathway), Bcr-Abl (RAS/ERK pathway), Cdk 4, 6, 9 (cell cycling), IκB kinases (NFκB pathway), and Apaf-1 (apoptosis). Enhanced or diminished expression of HSP90 promotes cell survival (14) or growth inhibition (15), respectively. Moreover, the expression of HSP in normal cells is lower than in cancer cells (16), suggesting an increased reliance in cancer cells on HSP for the maintenance of cellular viability, hence targeting HSP may represent a therapeutic strategy that could potentially interfere with multiple oncogenic pathways simultaneously but with a relevant therapeutic window.

Clinical trials using HSP 90 inhibitors such as KOS-953 (tanespimycin), IPI-504 (retaspimycin), and IPI-493 (all are geldanamycin analogues) and other synthetic compounds such as MPC-3100, AV-142, SNX-5422, BIIB0214, NVP-922, STA-9090, KW-2478, and AT-13387 are currently under clinical evaluation. HSP90 inhibitors can be used as a single agent or in combination with other forms of treatment such as chemotherapy and radiotherapy (17).

In this study, we aimed to determine the effect of HSP990, a novel orally bioavailable, HSP90 inhibitor on multiple myeloma and to verify the molecular pathways responsible for affecting antiproliferative and proapoptotic effects in human multiple myeloma cell lines (HMCL) and primary multiple myeloma tumor samples.

Cells and reagents

HMCL U266, NCI H929, and RPMI 8226 were obtained from the American Type Culture Collection (ATCC). There were no further authentications on the cell lines other than by the ATCC using short tandem repeat analysis. OPM-2 and LP 1 cell lines were from Deutshe SammLung von Mikroorgaanismen und Zellculturen. These cells were authenticated by the supplier using cytogenetics, DNA typing, immunophenotyping, and cell line speciation. Commercially available cell lines are purchased every 2 to 3 years. ANBL6, OCI-MY1, and XG-1 were kind gifts from Dr. Frits Van Rhee (Winthrop P. Rockefeller Cancer Institute) in 2008. Cell identities were not authenticated by the authors other than confirming the cell lines are plasma cells by CD138, CD38, and CD45 by flow cytometry. All cell lines were cultured in RPMI-1640 media supplemented with 10% heat-inactivated bovine calf serum, 2 mmol/L l-glutamine, and 100 U/mL penicillin/streptomycin in a humidified atmosphere (37°C; 5% CO2) except ANBL6 and XG-1, which had 2 ng/mL and 5 ng/mL of recombinant human IL6, respectively, added to the culture media. Cell lines are routinely screened for mycoplasma contamination by VenorGeM Mycoplasma Detection Kit, a PCR-based method. The HSP90 inhibitor (NVP-HSP990) was kindly provided by Novartis and the molecular structure is as shown in Fig. 1A with molecular weight of 379.4 g/mole. The compound was resuspended in 100% DMSO solution to make a final stock solution of 10 mmol/L and was stored at −80°C. Azacytidine was kindly provided by Pharmion Pty Ltd. and was resuspended in 0.9% NaCl solution to make a final stock solution of 10 mmol/L, and bortezomib was purchased from Janssen-Cilag and dissolved in 0.9% saline.

Figure 1.

NVP-HSP990 affects proliferation and cell cycle of HMCL. A, the molecular structure of HSP990; (R)-2-amino-7-((R)-4-fluoro-2-(6-methoxypyridin-2-yl)phenyl)-4-methyl-7,8-dihydropyrido[4,3-d]pyrimidin-5(6H)-one, molecular weight of 374.9 grams per mole. B, a panel of HMCL was treated with various concentrations of NVP-HSP990 and proliferation was measured by MTS assay. Graph depicts proliferation at 72 hours. C, OCI-MY1 was treated with NVP-HSP990 at 100 nmol/L for 24, 48, and 72 hours. Cells were stained with Annexin V–FITC and PI followed by FACS analysis to measure cell death. D, cell-cycle posttreatment with NVP-HSP990 was also evaluated in OCI-MY1 at the same time points.

Figure 1.

NVP-HSP990 affects proliferation and cell cycle of HMCL. A, the molecular structure of HSP990; (R)-2-amino-7-((R)-4-fluoro-2-(6-methoxypyridin-2-yl)phenyl)-4-methyl-7,8-dihydropyrido[4,3-d]pyrimidin-5(6H)-one, molecular weight of 374.9 grams per mole. B, a panel of HMCL was treated with various concentrations of NVP-HSP990 and proliferation was measured by MTS assay. Graph depicts proliferation at 72 hours. C, OCI-MY1 was treated with NVP-HSP990 at 100 nmol/L for 24, 48, and 72 hours. Cells were stained with Annexin V–FITC and PI followed by FACS analysis to measure cell death. D, cell-cycle posttreatment with NVP-HSP990 was also evaluated in OCI-MY1 at the same time points.

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Proliferation assay

MTS assays (CellTiter 96 aqueous one solution cell proliferation assay, Promega) were used to quantify the percentage of metabolically active NVP-HSP990–treated versus untreated cells. Briefly, 20,000 cells per well were plated onto a 96-well plate and NVP-HSP990 was added at a range of concentrations (100 pmol/L–10 μmol/L) at 0 hour. Then at 24 and 72 hours, 20 μL of MTS reagent was added and the cells were incubated for a further 4 hours at 37°C. The plates were then read at 490 nm using a microplate spectrometer (FLUOstar OPTIMA).

Annexin V/propidium iodide and cell-cycle analyses of HSP990-treated HMCLs

NCI H929 and OCI-MY1 were treated with 100 nmol/L NVP-HSP990 for 72 hours and the percentage of drug-specific apoptosis (adjusted against untreated controls) at 24, 48, and 72 hours was determined by flow cytometry using propidium iodide (PI) and Annexin V–fluorescein isothiocyanate (FITC) staining. Briefly, 2 × 106 NCI H929 and OCI-MY1 cells were washed with 0.01 mol/L PBS (0.0027 mol/L KCl and 0.137 mol/L NaCl, pH 7.4, at 25°C) and resuspended in 100 μL of binding buffer (10 mmol/L HEPES/NaOH, pH 7.4, 140 mmol/L NaCl, and 2.5 mmol/L CaCl2). An amount of 5 μL of Annexin V–FITC-conjugated antibody (Invitrogen) was added to the cells for 15 minutes in the dark before washing and resuspension in binding buffer with PI. The stained cells were analyzed on a BD FACSCalibur Flow Cytometer (Becton Dickinson). To determine the cellular DNA content of HSP990-treated cells, 106 cells each of NCI H929 and OCI-MY1 were cultured with or without 100 nmol/L HSP990 for 24, 48, or 72 hours. Cells were harvested and washed with ice-cold PBS at 8,000 g for 5 minutes and resuspended in ethanol 0.01 mol/L PBS (70/30 v/v). After 30 minutes, the cells were pelleted and resuspended in 100 μL lysis buffer (LPR; BD Biosciences) followed immediately by 0.5 μL RNase/PI. The samples were analyzed after 15 minutes on the BD FACSCalibur Flow Cytometer and the percentages of cells in G0–G1, S and G2 + M phases of the cell cycle were analyzed by EXPO 32 flow cytometry software (Beckman Coulter).

Protein blot analysis

Protein lysates were isolated using radioimmunoprecipitation assay (RIPA) buffer (50 mmol/L Tris-HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, and 1 mmol/L phenylmethylsulfonylfluoride), and the cytoplasmic and membrane [plasma, mitochondria, and endoplasmic reticulum (ER)/golgi] fractions were isolated using Subcellular Protein Fractionation Kit (Thermo Scientific) as per manufacturer's instructions. For proteins lysates using the RIPA buffer, 106 cells were pelleted (8,000 g for 5 minutes) and washed with ice-cold PBS. The pellet was then solubilized in ice-cold RIPA buffer and incubated on ice for 20 minutes. The resultant lysates were then isolated by centrifugation at 4°C for 10 minutes at 10,000 g (Eppendorf 5415R). Protein concentration was measured using a colorimetric assay (Bio-Rad Protein DC Assay) and supernatants were either collected and used immediately or stored at −20°C. Proteins were resolved by SDS-PAGE on 1.5 mm gels and electro-transferred onto polyvinylidene fluoride membrane (Pall) by Trans-Blot Semi-Dry Transfer Cell (Bio-Rad) using a transfer buffer containing 25 mmol/L Tris-HCl, 192 mmol/L glycine, and 20% (v/v) methanol. The membranes were blocked with 5% skim milk powder in TBST solution (TBS buffer and 0.05% Tween-20) and incubated with antibodies against PARP, STAT3, p-STAT3 (Tyr 705), IL-6Rα, p-MEK (Ser 217/221), HSP27, HSP70, MEK, AKT, p-p65 (Ser 536), p65, or α-tubulin overnight at 4°C followed by appropriate secondary antibodies linked to horse radish peroxidase. All primary antibodies were purchased from Cell Signalling Technologies and Santa Cruz Biotechnology Inc., and secondary antibodies were from Thermo Fisher Scientific. The blots were subjected to SuperSignal West Pico (Pierce Biotechnology) for 5 minutes then exposed on CL-Xposure film and developed using CP100 X-Ray Film Processor (Agfa).

Evaluation of IL-6Rα and p-STAT3 expression following treatment with NVP-HSP990

OCI-MY1 and U266 cells were cultured with and without both 100 nmol/L HSP990 and 10 ng/mL recombinant IL-6 (R&D Systems) for 24 hours to determine whether IL-6 could abrogate the effect of NVP-HSP990. Protein lysates (cells alone, cells with NVP-HSP990, cells with IL-6 or cells with NVP-HSP990 and IL-6) were isolated by RIPA buffer, the protein concentration was measured by DC Protein Assay (Bio-Rad) and 100 μg of protein was loaded per well. The levels of STAT3, p-STAT3, and IL-6Rα (OCI-MY1 only) were then determined with immunoblotting as previously described. Concurrently, cell death was also measured by PI and analyzed using EXPO 32 flow cytometry software.

Evaluation of synergistic anti-multiple myeloma effects when combining NVP-HSP990 with other anti-multiple myeloma agents

On the basis of our prior observation that azacytidine rapidly inhibits critical signaling pathways in multiple myeloma cells and the widespread use of bortezomib in a variety of combination treatment approaches for multiple myeloma, we studied NCI H929, OCI-MY1, and U266 to determine whether NVP-HSP990 induced synergistic anti-multiple myeloma effects when combined with either azacytidine or bortezomib. HMCLs were treated with NVP-HSP990 (100 nmol/L) and azacytidine (5 μmol/L) or bortezomib (5, 10, and 20 nmol/L) either simultaneously or scheduling the drugs 24 hours apart (NVP-HSP990 followed 24 hours later by either azacytidine or bortezomib and vice versa). Cell death was then measured by PI staining and EXPO 32 flow cytometry software and synergy was quantified by the Chou–Talalay equation using CalcuSyn software (18).

NVP-HSP990 treatment of primary myeloma tumor cells in autologous bone marrow cultures

Bone marrow mononuclear cells (MNC) from patients with relapsed/refractory multiple myeloma were isolated by Ficoll Paque Plus (Amersham Biosciences) after providing written informed consent as per an Institutional Review Board-approved protocol. Buffy layers containing the MNC were separated and the red blood cells were removed using red blood cell lysis buffer (10 mmol/L KHCO3, 150 mmol/L NH4Cl and 0.1 mmol/L EDTA, pH 8.0) for 5 minutes at 37°C followed by a wash with sterile PBS. Cells were then cultured overnight in RPMI 1640 media supplemented with 10% iron fortified bovine calf serum, 2 mmol/L l-glutamine, and 100 U/mL penicillin/streptomycin. The next day aliquots of 5 × 105 cells were treated with HSP990 (100, 500, and 1,000 nmol/L) for 48 hours. Following treatment the cells were stained with CD45-FITC (BD Biosciences) and CD38-PerCP (BD Biosciences) for 15 minutes at room temperature and washed with fluorescence-activated cell sorting (FACS) buffer (0.5% FCS/PBS). Cells were then fixed with 2% paraformaldehyde/PBS on ice for 20 minutes. After incubation, cells were washed and resuspended in permeabilization buffer (0.3% saponin, 1% FCS in PBS) in the presence of Apo 2.7-PE (BD Biosciences) for 20 minutes on ice. After a final wash in FACS buffer, the cells were resuspended in 300 μL of FACS buffer. Samples were acquired on a BD FACSCaliber Flow Cytometer and analyzed with EXPO 32 flow cytometry software. multiple myeloma cells were identified as CD45/CD38++ and apoptosis induction was detected via intracellular Apo 2.7 expression. For synergy experiments, NVP-HSP990 (500 nmol/L) was used either scheduled simultaneously or scheduled at 24-hour intervals as described above with either azacytidine (10 μmol/L; n = 2) or bortezomib (10 and 20 nmol/L; n = 1). Apoptosis induction was determined as described above.

Statistical methods

Mean and the SE of the mean from respective experiments are shown in the figures. Data were confirmed by at least 3 independent experiments except experiments using primary multiple myeloma samples. Determination of drug synergy was undertaken with CalcuSyn software.

NVP-HSP990 inhibits HMCL proliferation and induces G1 cell-cycle arrest

HSP990 inhibited HMCL proliferation in a time and dose-dependent fashion. Figure 1B shows that all 8 genetically heterogenous HMCL had median IC50s at 72 hours of more than 100 nmol/L NVP-HSP990. Furthermore, there were no obvious differences in sensitivity to the inhibitor between the IL6-dependent (U266, OCI-MY1, ANBL6, and XG-1) and -independent cell lines (LP-1, NCI H929, OPM2, and RPMI 8226). An accumulation of Annexin V–positive cells was shown following HSP990 treatment (Fig. 1C), confirming early stage apoptosis induction while subsequent PI positivity confirmed NVP-HSP990-induced cell death. Figure 1D shows representative results of a cell-cycle analysis at 24, 48, and 72 hours following treatment with 100 nmol/L NVP-HSP990 with both accumulation of cells at Go–G1 consistent with cell-cycle arrest and an increasing Go population over time consistent with apoptosis.

Apoptosis induction and downregulation of client proteins by NVP-HSP990

Both OCI-MY1 and NCI H929 showed PARP cleavage (Fig. 2A) at 24 hours following NVP-HSP990 treatment confirming apoptosis induction. Apoptosis was also evident in NVP-HSP990 treated CD45/CD38++ primary multiple myeloma cells as shown by Apo 2.7 staining. Aspirates from 5 relapsed/refractory multiple myeloma patients were treated with NVP-HSP990 (100 nmol/L to 1,000 nmol/L) for 48 hours with apoptosis induction occurring in a time and dose-dependent fashion (Fig. 2B). We then proceeded to characterize the effect of NVP-HSP990 on HSP90 client proteins with immunoblot analyses showing suppression of IL-6Rα, AKT, and MEK expression following exposure to 100 nmol/L NVP-HSP990 (Fig. 2C).

Figure 2.

NVP-HSP990 induced apoptosis in HMCL, primary sample and down regulation of client proteins. A, protein lysates from HSP990 treated OCI-MY1 were collected from various time points (2–24 hours) and blotted with PARP and α-tubulin (loading control) antibodies. B, two consented primary samples were treated with 3 doses of NVP-HSP990 and apoptosis of CD38+veCD45−ve cells were measured by Apo2.7. UT, untreated. C, client proteins of HSP90 (IL-6Rα, MEK and AKT) were downregulated by NVP-HSP990 in OCI-MY1.

Figure 2.

NVP-HSP990 induced apoptosis in HMCL, primary sample and down regulation of client proteins. A, protein lysates from HSP990 treated OCI-MY1 were collected from various time points (2–24 hours) and blotted with PARP and α-tubulin (loading control) antibodies. B, two consented primary samples were treated with 3 doses of NVP-HSP990 and apoptosis of CD38+veCD45−ve cells were measured by Apo2.7. UT, untreated. C, client proteins of HSP90 (IL-6Rα, MEK and AKT) were downregulated by NVP-HSP990 in OCI-MY1.

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p-MEK translocation and downregulation of pp65 post-NVP-HSP990

Subsequently, we evaluated the impact of NVP-HSP990 on MEK phosphorylation with sequential analysis of p-MEK showing obvious downregulation before any decrease in total MEK protein expression in the cytoplasmic fractions. p-MEK in the NVP-HSP990 treated cells were found to have translocated to the plasma, mitochondrial, and ER/golgi fractions from the cytoplasmic fractions (Fig. 3A). Voltage-dependent anion channel (VDAC) was used as the mitochondrial loading control. Similarly, examination of NF-κβ signaling showed that pp65 was downregulated in a sustained fashion in OCI-MY1 from 4 hours following NVP-HSP990 exposure with reduced expression of total p65 only evident at 24 hours (Fig. 3B).

Figure 3.

Translocation of p-MEK and downregulation of pp65 post-NVP-HSP990. A, the levels of MEK and p-MEK protein in the cytoplasmic fraction decreased with time post HSP990. α-Tubulin was used as a loading control. p-MEK protein expression in the plasma, mitochondrial, and ER/golgi membrane fraction increased with time while MEK gradually reduced. VDAC was used as a loading control. B, p65 and p-p65, which are downstream of HSP90 client protein Iκβ, were substantially reduced by NVP-HSP990 in OCI MY1.

Figure 3.

Translocation of p-MEK and downregulation of pp65 post-NVP-HSP990. A, the levels of MEK and p-MEK protein in the cytoplasmic fraction decreased with time post HSP990. α-Tubulin was used as a loading control. p-MEK protein expression in the plasma, mitochondrial, and ER/golgi membrane fraction increased with time while MEK gradually reduced. VDAC was used as a loading control. B, p65 and p-p65, which are downstream of HSP90 client protein Iκβ, were substantially reduced by NVP-HSP990 in OCI MY1.

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Modulation of molecular chaperones and p-STAT3 by NVP-HSP990

The known molecular chaperones of HSP90, HSP27, and HSP70 exhibited significant upregulation subsequent to NVP-HSP990 treatment consistent with prior observations with HSP90 inhibition (Fig. 4A). We then evaluated the JAK/STAT pathway, considered a key pathway regulating multiple myeloma cell survival and proliferation. Importantly, the HSP90 client protein STAT3 was markedly downregulated following NVP-HSP990 treatment coincident with abolition of p-STAT3 expression. Moreover, the addition of exogenous IL6 failed to restore either total STAT3 or p-STAT3 protein expression (Fig. 4B) nor overcome the ability of HSP990 to abrogate IL-6 induced IL-6Rα expression (Fig. 4C).

Figure 4.

Modulation of HSP90 molecular chaperones and p-STAT3. A, inhibition of HSP90 by NVP-HSP990 elevated HSP70 and HSP27 protein expression. B, exogenous IL-6 failed to restore STAT3 and p-STAT3 in OCI-MY1 and U266 when combined with HSP990. C, reestablishment of IL-6R was not observed when OCI-MY1 was treated with HSP990 in the presence of IL-6.

Figure 4.

Modulation of HSP90 molecular chaperones and p-STAT3. A, inhibition of HSP90 by NVP-HSP990 elevated HSP70 and HSP27 protein expression. B, exogenous IL-6 failed to restore STAT3 and p-STAT3 in OCI-MY1 and U266 when combined with HSP990. C, reestablishment of IL-6R was not observed when OCI-MY1 was treated with HSP990 in the presence of IL-6.

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NVP-HSP990 induces synergistic cell death of both HMCL and primary multiple myeloma cells when combined with azacytidine or bortezomib

Three HMCL, NCI H929, OCI-MY1, and U266 were treated with azacytidine either in combination with NVP-HSP990 for 48 hours or treated with azacytidine for 24 hours followed by NVP-HSP990 for a further 24 hours or the reverse schedule before measuring for drug-induced cell death with PI staining. Using CalcuSyn software, a combination index (CI) of less than 1 was defined as synergistic and in all 3 HMCL irrelevant of drug scheduling, all tested combinations were synergistic (Table 1). HMCL were also treated with bortezomib in combination with HSP990. Treatment was for 24 hours with both compounds added concurrently. The CI from these experiments was very low consistent with strong to very strong synergism (Table 1). Similarly, 3 multiple myeloma primary samples representing advanced disease were tested ex vivo with NVP-HSP990 in combination with either azacytidine or bortezomib. In patient 1, addition of azacytidine followed by NVP-HSP990 24 hours later displayed strong synergism while the reverse scheduling was additive. With patient 2, both schedules displayed moderate synergism (Table 2). The third primary sample was treated with NVP-HSP990 and 2 differing concentrations of bortezomib (10 and 20 nmol/L) and synergy was clearly observed in both instances (Table 2). Collectively these data suggest that NVP-HSP990 induced inhibition of HSP90 enhances the anti-multiple myeloma effect of both azacytidine and bortezomib against either HMCL or primary multiple myeloma cells.

Table 1.

NVP-HSP990 synergizes with azacytidine and bortezomib in HMCL

Cell lineTreatmentCI
NCI H929 HSP990 + AZA 0.530 
 HSP990 then AZA 0.531 
 AZA then HSP990 0.561 
OCI MY1 HSP990 + AZA 0.636 
 HSP990 then AZA 0.387 
 AZA then HSP990 0.885 
U266 HSP990 + AZA 0.390 
 HSP990 then AZA 0.394 
 AZA then HSP990 0.694 
NCI H929 HSP990 + Bort(A) 0.089 
 HSP990 + Bort(B) 0.141 
 HSP990 + Bort(C) 0.240 
OCI MY1 HSP990 + Bort(A) 0.360 
 HSP990 + Bort(B) 0.123 
 HSP990 + Bort(C) 0.200 
CI 
<0.1 +++++  
0.1–0.3 ++++  
0.3–0.7 +++  
0.7–0.85 ++  
0.85–0.9  
0.9–1.1 ±  
Cell lineTreatmentCI
NCI H929 HSP990 + AZA 0.530 
 HSP990 then AZA 0.531 
 AZA then HSP990 0.561 
OCI MY1 HSP990 + AZA 0.636 
 HSP990 then AZA 0.387 
 AZA then HSP990 0.885 
U266 HSP990 + AZA 0.390 
 HSP990 then AZA 0.394 
 AZA then HSP990 0.694 
NCI H929 HSP990 + Bort(A) 0.089 
 HSP990 + Bort(B) 0.141 
 HSP990 + Bort(C) 0.240 
OCI MY1 HSP990 + Bort(A) 0.360 
 HSP990 + Bort(B) 0.123 
 HSP990 + Bort(C) 0.200 
CI 
<0.1 +++++  
0.1–0.3 ++++  
0.3–0.7 +++  
0.7–0.85 ++  
0.85–0.9  
0.9–1.1 ±  

NOTE: Human myeloma cell lines NCI H929, OCI-MY1, and U266 were treated with NVP-HSP990 in combination with AZA and Bort and all combinations displayed low CI which indicated synergy. + indicates compounds added simultaneously while “then” indicates 24 hours for the first compound followed by second compound for a further 24 hours. Drug dosages used in the treatments were AZA = 5 μmol/L, NVP-HSP990 = 100 nmol/L, Bort(A) = 5 nmol/L, Bort(B) = 10 nmol/L, and Bort(C) = 20 nmol/L. Cell death was measured by PI staining.

Abbreviation: CI, combination index.

Table 2.

Azacytidine combines synergistically with NVP-HSP990 in primary myeloma cells

PatientTreatmentCI
HSP990 then AZA 1.06 
 AZA then HSP990 0.293 
HSP990 then AZA 0.814 
 AZA then HSP990 0.799 
HSP990 then AZA 0.855 
 AZA then HSP990 0.677 
PatientTreatmentCI
HSP990 then AZA 1.06 
 AZA then HSP990 0.293 
HSP990 then AZA 0.814 
 AZA then HSP990 0.799 
HSP990 then AZA 0.855 
 AZA then HSP990 0.677 

NOTE: Primary myeloma cells from consented patients were treated with NVP-HSP990 in combination with AZA. Samples were exposed to the first compound for 24 hours followed by the second compound for an additional 24 hours. Cells were stained with CD38, CD45, and Apo2.7. Apoptosis of multiple myeloma cells (CD38+veCD45-ve) were measured by Apo2.7. Drug dosages used in the treatments were AZA = 10 μmol/L and NVP-HSP990 = 500 nmol/L. CI < 1 indicates synergism.

Abbreviation: CI, combination index.

multiple myeloma is characterized by marked genetic heterogeneity and remains an incurable disease despite recent advances in treatment. Acquired accumulative genetic aberrations within the multiple myeloma cells and critical interactions with the BMME (19) aid in maintaining the survival and propagation of multiple myeloma cells. Thus a therapeutic strategy capable of simultaneously targeting multiple survival and proliferation pathways remains an attractive paradigm for preclinical evaluation. The wide-ranging cellular sequelae of HSP inhibition coupled with the demonstrable overexpression of HSP in a range of malignancies (20) suggests that targeted inhibition of HSP may satisfy this paradigm. HSP90 is known to mediate the maturation and stability of proteins crucial for cell survival and homeostasis. Client proteins such as IL-6Rα, STAT3, IGFR, MEK, FAK, IκB kinases α and β, BCR-ABL, EGFR, VEGFR, and FLT3 form complexes with HSP90 and co-chaperones to achieve active conformation and/or increased stability. These client proteins are then available to promote growth factor independence, resistance to drugs, proliferation, tissue invasion, metastasis and angiogenesis, all critical components for tumor progression, and survival (21).

HMCL treatment with NVP-HSP990 recapitulated the molecular sequelae described with other HSP90 inhibitors. We observed downregulated expression of AKT, MEK, p-MEK, STAT3, p-STAT3, IL-6Rα, p65, and pp65 and upregulation of HSP27 and HSP70. The downregulation of multiple proteins simultaneously by NVP-HSP990 accentuates the ability to disrupt multiple crucial multiple myeloma pathways (JAK/STAT, PI3K, NF-κβ, and RAS/ERK). NF-κβ has been implicated in many aspects of oncogenesis due to its ability to promote antiapoptotic gene expression and it is considered a prime mediator of tumor cell drug resistance (22, 23). Thus, the ability of NVP-HSP990 and other HSP90 inhibitors to impede NF-κβ signaling would be a potential mechanism underlying the wide-ranging synergistic capacity seen with the combination of HSP90 inhibition and other therapeutics. Furthermore, the capacity of NVP-HSP990 to interfere with both the JAK/STAT and PI3K/AKT signaling pathways via the downregulation of STAT3 and AKT expression, respectively, provides the potential for NVP-HSP990 to disrupt key BMME-mediated prosurvival signals for primary multiple myeloma cells. Although this possibility requires further evaluation our preliminary demonstration that NVP-HSP990 can induce killing of primary multiple myeloma cells in an autologous marrow coculture is consistent with this.

Kinase suppressor of RAS (KSR) is a multiprotein scaffold complex that contains HSP90 and is postulated to modulate RAS-induced MAPK signaling (24). Under resting conditions the complex is retained in the cytoplasm but upon stimulation translocates to the cell membrane to facilitate downstream signaling activation. Sacks and colleagues (25) have shown that in the absence of functional KSR that MEK-ERK binds with high avidity to a golgi membrane protein SEF, thus effectively preventing nuclear translocation of activated ERK and subsequent transcriptional activation. We have shown that following treatment with NVP-HSP990 p-MEK translocates from the cytoplasmic cellular fraction to the cellular membrane fraction and postulate that this would be consistent with NVP-HSP990–induced KSR disruption thus favoring binding of MEK to SEF. Therefore, the disruption of chaperone-client protein complexes subsequent to inhibition of HSP90 and the consequent degradation of the client proteins can perturb multiple signaling pathways thus directly inducing tumor cell death and/or resensitizing drug-resistant tumor cells to alternative therapeutics.

HSP90 inhibitors have been used in clinical trials in breast cancer (26), prostate cancer (27), melanoma (28), lung cancer (29), colon cancer (30), and renal cancer (31) with promising clinical activities. Moreover, preclinical studies in multiple myeloma and other hematologic malignancies such as lymphoma and leukemia have shown synergistic effects with HSP90 inhibition when combined with a telomerase inhibitor (GRN163L; ref. 32), imatinib (33), HDAC inhibition (LBH589; ref. 34), and a Chk1 inhibitor (UCN-01; ref. 35). Currently a number of clinical trials involving HSP90 inhibitors at various phases are being conducted in multiple myeloma (36). Richardson and colleagues (37) have successfully evaluated the efficacy of tanespimycin, a HSP90 inhibitor, in patients with relapsed or relapsed and refractory multiple myeloma and found that the inhibitor was generally well tolerated and achieved therapeutic plasma concentrations as well as target inhibition. Moreover, a phase 1/2 study evaluating the combination of tanespimycin and bortezomib led by Richardson and colleagues (38) showed antitumor activity in heavily pretreated multiple myeloma patients. Unfortunately, Bristol–Myers Squibb has had to halt further development of tanespimycin in clinic due to difficulties with synthesizing stable drug in adequate amounts and in accordance with GMP. Thus, the need to evaluate other compounds in this drug class is of additional importance. Another phase 1/2 study in subjects with relapsed and/or refractory multiple myeloma involving a nonpurine inhibitor of HSP90 (KW-2478) reported that KW-2478 in combination with bortezomib was well tolerated and the PK profiles were comparable with each agent when administered alone. The recommended doses for both compounds were 175 mg/m2 (KW-2478) and 1.3 mg/m2 (bortezomib; ref. 39). In our study, we found synergism when the orally bioavailable HSP90 inhibitor NVP-HSP990 was combined with either the DNA methyltransferase inhibitor azacytidine or the proteasome inhibitor bortezomib but the time course of anti-multiple myeloma activity when combined with the former would suggest a nonepigenetic mechanism of anti-multiple myeloma activity as we have recently described (40). The synergistic killing of the HMCL NCI H929, OCI-MY1, and U266 was not schedule dependent, however, when tested against primary multiple myeloma cells there was clear superiority when the cells were pretreated with azacytidine or bortezomib before NVP-HSP990 exposure. Again highlighting the potentially critical role that drug scheduling may play in the optimal use of novel anti-multiple myeloma therapeutics (41).

The overexpression of cytoprotective HSP has been described in hematologic malignancies (42) and solid tumors (43) and may contribute to drug resistance and a poorer prognosis (44). Furthermore, the silencing of HSP27 or HSP70 has been shown to increase sensitivity to chemotherapeutic agents such as dexamethasone (45) and doxorubicin (46). Upregulation of both HSP27 and HSP70 protein expression was observed following NVP-HSP990 treatment and theoretically could abrogate NVP-HSP990–induced killing of multiple myeloma cells. Therefore, the inhibition of HSP27 or HSP70 via a siRNA such as OGX-427 (HSP27 silencer; ref. 47) or a small molecule HSP70 inhibitor, respectively, in combination with HSP990 may enhance cell killing and promote sensitivity to other chemotherapeutic regimens.

In conclusion, this report provides a basis for assessing NVP-HSP990 further for the treatment of multiple myeloma and shows synergy when NVP-HSP990 is used in combination with either azacytidine or bortezomib. The capacity for NVP-HSP990 to induce apoptosis and cell-cycle arrest plus the ability to modulate multiple cell survival pathways in multiple myeloma cells shows the important potential therapeutic benefits of this inhibitor. The ideal strategy for the use of HSP90 inhibitors in multiple myeloma remains to be defined but the oral bioavailability of NVP-HSP990 will facilitate greater flexibility in dosing and drug scheduling that in turn may have important implications in terms of tolerability, duration, and success of therapy.

No potential conflicts of interest were disclosed.

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.

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