The novel JAK inhibitor AZD1480 blocks STAT3 and FGFR3 signaling, resulting in suppression of human myeloma cell growth and survival

Drugs and cytokines

AZD1480 was provided by AstraZeneca (Waltham, MA). For in vitro experiments, AZD1480 was dissolved in 100% DMSO to prepare a 10 mM stock and stored at -20°C. For in vivo experiments, AZD1480 was formulated daily in purified, sterile water supplemented with 0.5% Hypromellose and 0.1% Tween 80. Doxorubicin was provided by Sigma and melphalan was obtained from a pharmacy; both drugs were dissolved in RPMI-1640 medium to prepare mM range stocks and stored at 4°C or -20°C, respectively. IL-6 (R&D Systems) was reconstituted in sterile 1× PBS containing 0.1% BSA to prepare a 10 μg/mL stock and stored at -20°C. NF449 and JAK2 inhibitor IV were purchased from Calbiochem (San Diego, CA); NF007 and FGFR inhibitor were purchased from Tocris Bioscience (Ellisville, MO).

Cell lines and cell culture conditions

Human myeloma U266, RPMI 8226, MM1.S, IM-9, NCI-H929 cell lines as well as bone marrow stromal cells were obtained from the American Type Culture Collection (Manassas, VA). The OPM-2 cells were purchased from the European Collection of Cell Culture (ECACC). The Kms.11 and Kms.18 cells were a gift from Dr. P. L. Bergsagel. Cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum and 50 units/mL penicillin and streptomycin at 37°C in an atmosphere of 5% CO₂ and passaged twice a week.

Isolation of CD138⁺ cells from primary PBMCs and BMMCs

Bone marrow (BM) aspirates were collected from 4 patients with multiple myeloma and peripheral blood (PB) samples were collected from 5 healthy donors. BM mononuclear cells (BMMCs) and PB mononuclear cells (PBMCs) were isolated with Ficoll-Hypaque sedimentation (Sigma-Aldrich, St. Louis, MO) and enriched for CD138-positive cells by immunomagnetic nanoparticles positive selection method using the EasySep kit (StemCell Technologies, Vancouver, British Columbia, Canada). The yield of MM cells was high (>95%). Viability of the MM-cell enriched fractions was 99%. All donors and patients had given informed consent for sample acquisition as a part of a protocol approved by the local Institutional Review Board.

Cocultures with BMSCs

Bone marrow stromal layers were established by plating HS.5 cells at a density of 250 000 cells/well in 12-well plates for 24 h. For experiments to calculate the percentage of tumor cell death, Kms.11 cells were first labeled with 5 μM CellTrace™ CFSE (Molecular Probes, Eugene, OR), resuspended in serum-free medium and then applied to the wells containing the HS.5 stromal cells layer at a concentration of 250 000 cells/mL. After 24 h of coculture, AZD1480 was added to the coculture media. Following incubation for 48 h with AZD1480, Kms.11 cells were separated from the stromal layer by carefully pipetting twice with ice-cold PBS. Tumor cells were stained with DAPI and analyzed using a flow cytometer with excitation and emission wavelengths appropriate for fluorescein and DAPI. The % cell death was calculated based on all the DAPI-positive cells after gating on CFSE-positive Kms.11 cells.

Analysis of primary cells by DIMSCAN

Primary cells were cultured at a density of 25 000 cells/well in 96-well plates with RPMI-1640 medium containing 10% fetal bovine serum and 50 units/mL penicillin and streptomycin, and treated with different doses of AZD1480 up to 48 h. To determine the cytotoxicity of AZD1480, cells were assessed by the fluorescence-based DIMSCAN, which uses digital imaging microscopy to quantify viable cells that selectively accumulate fluorescein diacetate.

Cell viability assays

Cells were seeded in 96-well plates at a density of 10 000 cells/well. After 24, 48 or 72 h, cell viability was determined by assaying with MTS [(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay (Promega, Madison, WI). The MTS assay was performed according to instructions from the supplier. Absorbance was measured at 490 nm with a Chameleon plate reader (Bioscan, Washington DC).

Apoptosis assay by flow cytometry

Untreated and drug-treated cells were stained with Annexin V and Propidium Iodide (PI) using Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences Pharmingen, San Diego, CA). The percentage of apoptotic and nonviable cells was determined by flow cytometry. At least 50 000 cells were collected with a CyAn ADP Violet (Dako) cytometer and calculated using the Summit software (Dako). Percent apoptosis was calculated based on all the Annexin V-positive plus the Annexin V/PI-positive cells. The % loss of cell viability was calculated considering all the Annexin V-positive plus the PI-positive and the Annexin V/PI-positive cells.

Western blot analysis

Cells were washed with ice-cold PBS containing 0.1 mM sodium orthovanadate and total proteins were isolated using RIPA lysis buffer, which included protease inhibitors (leupeptin, antipain, and aprotinin), 0.5 mM PMSF and 0.2 mM sodium orthovanadate. Protein amounts were quantified using the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of proteins were loaded onto a SDS-PAGE gel, transferred onto nitrocellulose membrane, and probed with the indicated antibody: rabbit polyclonal anti-phospho-JAK2 (Chemicon International); rabbit polyclonal anti-STAT3, anti-phospho-STAT3 (Tyr705), anti-phospho-STAT3 (Ser727), anti-p44/42 MAPK and anti-phospho-p44/42 MAPK (Cell Signaling Technology, Danvers, MA); mouse monoclonal anti-c-Myc, rabbit polyclonal anti-Mcl-1, rabbit polyclonal anti-Cyclin-D2 (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal Bcl-xL antibody (Calbiochem); and mouse monoclonal β-actin antibody (Sigma-Aldrich, St Louis, MO). Membranes were then washed, reprobed with appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences, Buckinghamshire, UK), and developed with SuperSignal chemiluminescent substrate (Pierce Biotechnology, Rockford, IL).

Protein immunoprecipitation

Cell lysates (250 μg protein) were incubated with JAK1, JAK2 antibodies (Cell Signaling Technology, Danvers, MA) or FGFR3 antibody (Novus Biologicals, Littleton, CO) overnight at 4°C. To this mixture, washed protein A beads were added and incubated for 1 h at 4°C. Next, the immunoprecipitates were washed five times with the lysis buffer and proteins were eluted with the SDS sample buffer, loaded on 12% SDS-PAGE gels and analyzed by Western blotting analysis using phospho-JAK1, phospho-JAK2 antibodies (Cell Signaling Technology) or phospho-Tyr antibody (Millipore, Temecula, CA).

Kinase assay

Cell-free kinase assays were performed as previously described(19). Briefly, assays were carried-out in 50 μl of kinase buffer (60 mM HEPES-NaOH pH 7.5, 3 mM MgCl₂, 3 mM MnCl₂, 3 μM Na₃VO₄), for 30 minutes at 30°C in the presence of 2.5 μg of polyethylene glycol, 10 μM ATP, 200 ng of recombinant STAT1 (Prolias, Rockville, MD) as a substrate, 200 ng of recombinant kinase, and inhibitors added directly to the kinase reaction. Recombinant FGFR3, JAK2 and JAK3 were purchased from SignalChem (Richmond, Canada). The following antibodies were used: STAT1, P-STAT1 (Y701), JAK2, JAK3 (Cell Signaling Technology, Beverly, MA); FGFR3 (Santa Cruz Biotechnology, Santa Cruz, CA); 4G10 (Upstate Biotechnology, Lake Placid, NY).

Transfection

Transfections of Kms.11 cells were performed with the Nucleofector Kit C, program X-005 (Amaxa Biosystems, Gaithersburg, MD). siRNAs (200 nM) and plasmids (2 μg) were used in each transfection with 2 million cells. The 27mer dicer substrate STAT3 siRNA was designed using the City of Hope siRNA Site Selector/Duplex-End Energy Difference Calculator(20, 21). Cy3-labeled STAT3 siRNA was synthesized in the City of Hope DNA/RNA synthesis laboratory. The Cy3-labeled 27mer dicer substrates FGFR3 and negative control siRNA were obtained from IDT (Coralville, IA). Twenty-four hours after transfection with siRNAs, Cy3-positive cells were sorted with MoFlo MLS sorter (Beckman Coulter, Fort Collins, CO) and calculated with the software Summit version 4.3. The Cy3 label was excited by 514-nm laser and the signal was detected with a 600/30-nm bandpass filter. Constitutive activated STAT3 expression plasmid vector, pRC/CMV-STAT3c-Flag was a generous gift from J. Darnell(22). For establishing the stable STAT3c-expressing cells, plasmids pRC/CMV-vector and pRC/CMV-STAT3c-Flag were transected into Kms.11 cells. Twenty-four hours after transfection, 0.5 mg/ml G418 (Invitrogen) was added for selection. Resistant pools of cells were characterized by western blot and maintained in medium containing 0.2 mg/ml G418. Kms.11 cells were also transiently transfected with the pRK7 vectors carrying FLAG-tagged wild-type and Y373C-FGFR3 constructs, which were described previously(19). The vector containing no insert was pcDNA3.

Animal model studies

Kms.11 cells (1×10⁷) were resuspended in RPMI medium and injected subcutaneously into the flank of four to six week old NOD/SCID IL2Rγ-null mice (Jackson Laboratories, Bar Harbor, ME). When tumors reached approximately 65 mm³, mice were divided into one control group and one treated group (10/group) which were dosed orally (p.o.) with the vehicle or AZD1480 (30 mg/kg), respectively. Mice were dosed twice a day for seven days a week. At the dose of AZD1480 indicated, no lethal toxicity or weight loss (greater than 10% body weight) was observed amongst treated animals. Tumors were measured every 3-4 days with vernier calipers, and tumor volumes were calculated by the following formula: 0.5 × (larger diameter) × (smaller diameter). All mice were maintained under specific pathogen-free conditions and were used in compliance with protocols approved by the local Institutional Animal Care and Use Committee.

Statistical analysis and software

The data shown represent mean values of at least three independent experiments and expressed as mean±SD. Statistical analysis was performed by the Student's t-test, using the statistical software GraphPad Prism 4. Statistical significance was set at a level of P<0.05. Four patients were selected based on the criteria that this is a sufficient sample size to rule out lack of drug action if there is consistent drug activity demonstrated in all samples (p<0.02). The Mann Whitney test was used to calculate the P value in Figure 1C and Figure 9. The statistical analysis in Figure 8A was performed with Two-way ANOVA. The statistical significance of the downregulation of Cyclin D2 in Figure 8B was calculated using the IDV values normalized to those of β-actin, which were determined by the densitometry software AlphaImager. The IC₅₀ values in Table 1 and the combination index (CI) values in Table 3 were calculated by the software Compusyn; the descriptions in Table 3 are based on the ranges of CI refined from those described by Chou(23).

AZD1480 inhibits proliferation and induces apoptosis of human myeloma cell lines

We investigated the effects of treatment with AZD1480 on a panel of human myeloma cell lines. We first determined the effect of AZD1480 on the proliferation of U266, Kms.11 and 8226 cells (Figure 1A). MTS assays showed that AZD1480 markedly inhibits the growth of U266, Kms.11 and 8226 cells in a time- and dose-dependent manner. The data also indicate that AZD1480 is more potent in U266 and Kms.11 cells than in 8226 cells for inhibiting proliferation. The 50% inhibitory concentration (IC₅₀) value for inhibition of proliferation at 48 h is approximately 2 μM for U266 cells and approximately 1 μM for Kms.11 cells; in the same cell lines the IC₅₀ at 72 h is approximately 1 μM and 0.5 μM, respectively. 8226 cells require higher concentrations of AZD1480 with an IC₅₀ at 72 h of approximately 3 μM. The IC₅₀ values were determined for a wider variety of myeloma cell lines. Table 1 shows that AZD1480 has broad efficacy, which correlates not only with the presence of activated STAT3 but also with the expression of FGFR3. Cells lacking both phospho-STAT3 and FGFR3 are less sensitive than cells possessing activated STAT3 or overexpressing FGFR3. The levels of FGFR3 and phospho-STAT3 detected by western blot analysis (Supplemental Figure 1) are in agreement with the references shown in Table 1.

The apoptotic effect of AZD1480 was determined in the two more sensitive cell lines, U266 and Kms.11 (Figure 1B). Exposure to AZD1480 induces apoptosis of both U266 and Kms.11 cells in a time- and dose-dependent manner. The IC₅₀ in terms of apoptosis at 72 h is approximately 1.5 μM for both U266 and Kms.11 cells. Representative flow cytometry plots for other cell lines are shown in Supplemental Figure 2, indicating that the apoptotic effects correlate with the IC₅₀ values. In contrast, no cytotoxic effects were observed in normal cells. Figure 1C shows that the viability of human peripheral blood mononuclear cells was not affected at concentrations in the IC₅₀ range for inducing apoptosis of Kms.11 cells.

Because IL-6 has a prominent role in MM(9, 10) we evaluated whether AZD1480 suppresses the IL-6-induced proliferation and survival of myeloma cells that have been reported to be growth stimulated by IL-6(13, 24). The MTS assay (Figure 2A) showed that 0.5 μM AZD1480 at 48 h completely inhibits IL-6-induced cell proliferation in U266 cells and inhibits approximately 50% of IL-6-induced cell proliferation in Kms.11 cells. U266 and Kms.11 cells treated for 48 h with 2 μM AZD1480 compared with the untreated cells stimulated with IL-6 showed 70% and 50% cell proliferation inhibition, respectively. AZD1480 also inhibits the survival of both cell lines in the presence of IL-6, inducing 50% and 60% apoptosis at 2 μM at 48 h in U266 and Kms.11 cells compared with the untreated controls grown in presence of IL-6, respectively (Figure 2B). Therefore, the addition of IL-6, which induced proliferative responses in U266 and Kms.11 cells (20% and 30%, respectively), did not cause a significant shift in IC₅₀ for U266 cells and did not protect them from AZD1480-mediated inhibition of proliferation and survival. However, Kms.11 cells are less sensitive to AZD1480 in terms of inhibition of proliferation when cultured in the presence of IL-6 but still respond to the drug with IC₅₀ at 2 μM.

AZD1480 inhibits IL-6-inducible JAK2/STAT3 and MAPK signaling pathways in vitro

Because JAK family kinases are known to be activated by phosphorylation upon IL-6/gp130 engagement(25) we examined the effect of AZD1480 on IL-6-dependent JAK2 phosphorylation in myeloma cells pretreated with AZD1480 and then stimulated with IL-6. Figure 3A shows that IL-6-induced phosphorylation of JAK2 was inhibited at 0.25-0.5 μM and virtually abolished at 1 μM in both U266 and Kms.11 cells. The JAK1 phosphorylation was not inhibited at these doses.

It is well established that IL-6 activates distinct downstream signaling pathways: JAK/STAT3 and Ras/MAPK pathways(12, 26). We therefore investigated whether the blockade of IL-6-inducible activation of JAK2 by AZD1480 would prevent the phosphorylation of STAT3 and MAPK in myeloma cell lines pretreated with AZD1480 and then stimulated with IL-6. Figure 3B shows that the phosphorylation of both STAT3 and ERK1/2 induced by IL-6 in U266 and Kms.11 cells was reduced to basal levels at 0.5-1 μM and phosphorylation of STAT3 was abolished at 2 μM. We also demonstrated that AZD1480 inhibits constitutive tyrosine phosphorylation of STAT3 but not the serine phosphorylation (data not shown).

To better mimic the in vivo condition, U266 and Kms.11 cells were cultured for 16 h in the presence of IL-6 and then treated with AZD1480. In this setting, we observed a significant decrease of the level of STAT3 and MAPK phosphorylation at 4 h (Figure 3C) and 24 h post-treatment (data not shown). We also confirmed that 8226 cells lack constitutively activated STAT3, whereas IL-6 stimulated the tyrosine phosphorylation of STAT3 and the activation of MAPK, as previously described(27). AZD1480 suppressed the IL-6-induced phosphorylation of JAK2, STAT3 and MAPK even though phospho-JAK2 levels were downregulated at higher concentrations compared to those required to downregulate phospho-JAK2 levels in U266 and Kms.11 cells (data not shown). Therefore, the inhibition of IL-6-induced growth of myeloma cells by AZD1480 correlates with induction of apoptosis and decreased JAK2, STAT3 and MAPK phosphorylation.

Our observation that the IC₅₀ for AZD1480, in terms of inhibition of proliferation or survival, was higher than the concentration required to reduce STAT3 activation in myeloma cells may suggest that these cells may not be completely dependent on STAT3 for survival. Figure 3D demonstrates that Kms.11 cells depend on STAT3 for proliferation since downregulation of STAT3 by siRNA results in 50% inhibition of proliferation compared to cells transfected with control siRNA. Downregulation of STAT3 mRNA levels was confirmed by quantitative real-time PCR 72 hours post transfection (approximately 70% inhibition compared to control) (data not shown). To determine if STAT3 is involved in the response to AZD1480, we performed experiments in Kms.11 cells transfected with constitutive activated STAT3 (STAT3c) (Figure 3E). STAT3c rescues Kms.11 cells from AZD1480-mediated loss of viability, consistent with role of STAT3 in the biological effects mediated by AZD1480. Nevertheless, we do not exclude the possibility that other pathways may be involved in the response to AZD1480.

Because AZD1480 inhibits proliferation and induces apoptosis associated with inhibition of IL-6-inducible STAT3 phosphorylation, we investigated the effect of AZD1480 on direct or indirect STAT3 targets in MM that have key roles in cell-cycle regulation (Cyclin D2), proliferation (c-Myc) and survival (Mcl-1 and Bcl-xL)(11, 28). We first analyzed the levels of these proteins in U266 and Kms.11 cells cultured for 16 h in the presence of IL-6 and then treated with AZD1480. Figure 4A shows that IL-6 upregulates c-Myc, Cyclin D2 and Bcl-xL in U266 cells. Both c-Myc and Cyclin D2 were decreased in a dose-dependent manner by AZD1480 at 24 h, while Bcl-xL was decreased after 48 h by AZD1480 treatment. At 48 h post-treatment the inhibition of c-Myc and Cyclin-D2 was even more dramatic (Figure 4B). We did not observe IL-6-dependent upregulation of Mcl-1, which was downregulated by AZD1480. IL-6 upregulates Cyclin D2 also in Kms.11 cells and AZD1480 inhibits this upregulation in a dose-dependent manner (Figure 4A), even more so at 48 h post-treatment (Figure 4B). Cyclin D2 protein levels were decreased in U266 or Kms.11 cells treated for 24 h with AZD1480 without being precultured in the presence of IL-6 (data non shown). No change in Cyclin D2, or Mcl-1, or Bcl-xL was observed in 8226 cells prestimulated for 16 h with IL-6 and then treated with AZD1480 for 24 h (data not shown).

We then analyzed the protein levels of c-Myc, Mcl-1, Cyclin D2 and Bcl-xL in U266 and Kms.11 cells pretreated with AZD1480 and then stimulated with IL-6, demonstrating that AZD1480 inhibits the IL-6-dependent expression of c-Myc and Cyclin D2 in U266 and Kms.11, respectively (Figure 4C). These results suggest that AZD1480 could inhibit myeloma cell proliferation and survival through the down-regulation of c-Myc or Cyclin D2.

AZD1480 effects on myeloma cells cocultured in vitro with bone marrow stroma cells

The bone marrow stromal environment is a major factor in myeloma cell growth and resistance to chemotherapy(29, 30). We first investigated whether AZD1480 has cytotoxic effects in the human bone marrow stroma-derived HS.5 cell line. Figure 5A shows that the proliferation of HS.5 cells was not affected at concentrations in the IC₅₀ range for Kms.11 cells. We then tested the effect of the bone marrow environment on myeloma cell response to AZD1480. Figure 5B shows that Kms.11 cells cocultured with the bone marrow stromal cells HS.5 are not resistant to AZD1480, showing the same percentage of cell death compared to the tumor cells grown alone.

To determine if STAT3 and MAPK signaling pathways are downregulated by AZD1480 in myeloma cells cocultured with bone marrow stromal cells, we treated U266 and Kms.11 cell lines for 2 h after being cocultured for two days with HS.5 cells monolayer (Figure 5C). In this setting, we observed a significant decrease in the level of stroma-induced STAT3 phosphorylation in U266 cells, although stroma-induced phosphorylation of MAPK was minimal. In contrast, we did not observe any significant increase in stroma-induced phospho-JAK2/STAT3 levels in the Kms.11 cell line and AZD1480 had no apparent effect on JAK2/STAT3 phosphorylation in these cells; instead there was a decrease in phospho-MAPK levels, suggesting that stromal cells are modulating AZD1480 responses differently compared to cells cultured with IL-6.

AZD1480 inhibits both JAK2 and FGFR3 activity in vitro

Recurrent translocations have been identified in approximately 50% of primary MM patient samples; the most frequent of these translocations involves the receptor tyrosine kinase FGFR3(31, 32). Because Kms.11 cells possess translocated FGFR3 and AZD1480 has activity against FGFR1(18), combined with the finding that Kms.11 cocultured with bone marrow stromal cells are sensitive to AZD1480 without showing inhibition of phospho-STAT3, we hypothesized that AZD1480 may inhibit FGFR3 signaling in these settings. (Figure 5D) demonstrates that AZD1480 inhibits phosphorylation of FGFR3 in Kms.11 cells cocultured with HS.5 bone marrow stromal cells. These results suggest that inhibition of FGFR3 signaling is very likely driving sensitivity of Kms.11 cells, rather than JAK/STAT signaling, in these settings.

To confirm the finding that AZD1480 inhibits FGFR3 signaling we examined the effect of AZD1480 on b-FGF-dependent FGFR3 phosphorylation in Kms.11 cells pretreated with AZD1480 and then stimulated with b-FGF. Figure 6A shows that b-FGF-induced phosphorylation of FGFR3 was inhibited in Kms.11 cells at 0.25 μM and virtually abolished at 1 μM, which are similar doses that inhibit IL-6-induced-phosphorylation of STAT3 in U266 and Kms.11 cells. We postulated that Kms.11 cells may be sensitive to downregulation of FGFR3 by siRNA. Figure 6B demonstrates that Kms.11 cells depend on FGFR3 for proliferation since downregulation of FGFR3 by siRNA results in 40% inhibition of proliferation compared to cells transfected with control siRNA. Downregulation of FGFR3 mRNA levels was confirmed by quantitative real-time PCR 72 hours post transfection (approximately 60% inhibition compared to control) (data not shown). To demonstrate if FGFR3 is involved in the response to AZD1480, we performed experiments in Kms.11 cells transfected with WT FGFR3 or mutant activated FGFR3 (Figure 6C). Both WT and activated FGFR3 rescue Kms.11 cells from AZD1480-mediated loss of viability, indicating that inhibition of FGFR3 may contribute to AZD1480 efficacy in Kms.11 cells. In vitro kinase assays (Figure 7) confirmed that AZ1480 inhibits both JAK2 and FGFR3 activity, consistent with role of JAK2/STAT3 and FGFR3 in the biological effects mediated by AZD1480. Nevertheless, we do not exclude the possibility that other pathways may be involved in the response to AZD1480. Figure 7 shows also that AZD1480 is effective against JAK2 and FGFR3 activity at lower doses than another JAK2 or FGFR3 inhibitor. Compared with a commercially available JAK2 inhibitor, AZD1480 is even more potent in terms of cytotoxicity (Supplemetal Figure 3).

AZD1480 blocks growth of Kms.11 xenografts associated with inhibition of JAK2/STAT3 and FGFR3

The presence of translocated FGFR3 in patients is associated with shorter duration of remission, higher relapse rate, and shorter survival(33). Thus, the Kms.11 cell line represents more aggressive disease, and for this reason we considered Kms.11 as a good model to study the effect of AZD1480 in vivo. In NOD/SCID IL2Rγ-null mice treated twice a day with 30 mg/kg AZD1480, we observed statistically significant tumor growth inhibition (Figure 7A). After only six days from the first treatment, we observed regression of the tumor in 80% of the mice for as much as 40% volume reduction compared with the initial tumor volume prior to the first treatment. The average tumor size of the treated group was approximately six and 12 times smaller than the average of tumor size of control vehicle group 6 and 12 days post treatment, respectively. Similar data were observed in a separate experiment with athymic nude mice (data not shown). Regression of the tumor was associated with complete inhibition of STAT3 and FGFR3 phosphorylation and modest inhibition of Cyclin D2 (approximately 50% reduction, P=0.0286) in tumors harvested 2 h after dosing (Figure 7B-C). However, we did not observe consistent inhibition of phospho-MAPK among tumors treated with AZD1480 (data not shown).

AZD1480 causes loss of viability of primary MM cells cultured in vitro and enhances the sensitivity to chemotherapy

We next determined whether AZD1480 treatment could also be active against primary plasma cells isolated from bone marrow samples of multiple myeloma patients. Table 2 indicates that all 4 patient samples analyzed responded to the AZD1480 treatment with an increase in the percentage of nonviable cells in a dose-dependent manner. Importantly, one of these samples was treated with AZD1480 in the presence of bone marrow stromal cells and responded even better than when it was treated in the absence of stroma. In contrast, no cytotoxic effects were observed in 5 normal CD138⁺ samples at the same doses (Figure 9).

The bone marrow stromal environment is a major factor in myeloma cell resistance to chemotherapeutic agents such as dexamethasone(34), melphalan and mitoxantrone(30, 35). We tested the effect of the bone marrow environment on myeloma cell response to AZD1480 in combination with doxorubicin and melphalan in terms of induction of cell death (data not shown). Coculturing of Kms.11 cells with human bone marrow stromal HS-5 cells does not protect the myeloma cells to doxorubicin compared with the controls that were not cocultured. Protection from melphalan treatment was also not observed. AZD1480 potentiated the cell death induced by doxorubicin and melphalan in Kms.11 grown alone and even more if cocultured with HS.5 cells. This enhanced response with combination treatment represents an additive to synergistic response (Table 2).