uicc
global cancer control
IJC International Journal of Cancer
BIIB021, a synthetic Hsp90 inhibitor, has broad application against tumors with acquired multidrug resistance
Hong Zhang, Laura Neely, Karen Lundgren, Yong-Ching Yang, Rachel Lough, Noel Timple and Francis Burrows
Biogen Idec, 5200 Research Place, San Diego, CA
17-AAG, the first-generation clinical Hsp90 inhibitor, exhibits promising antitumor activity in clinical studies, but is limited by poor solubility and hepatotoxicity. To pursue compounds with better biopharmaceutical properties, we have developed a series of fully synthetic orally bioavailable inhibitors of Hsp90. Here, we report that 17-AAG and other ansamycin derivatives are inactive in P-gp and/or MRP-1 expressing cell lines and sensitivity could be restored by coadministration of P-gp or MRP inhibitors. In contrast, the synthetic Hsp90 inhibitor, BIIB021 was active in these models. Accordingly, BIIB021 was considerably more active than 17-AAG against adrenocortical carcinoma, a tumor that naturally expresses P-gp, both in vitro and in vivo. This efflux pump-mediated resistance is manifested in both cytotoxicity assays and measurements of target inhibition, such as client protein degradation. Other than this, the cytotoxic activity of BIIB021 was also not influenced by loss of NQO1 or Bcl-2 overexpression, molecular lesions that do not prevent client loss but are nonetheless associated with reduced cell killing by 17-AAG. Our results indicate that the activity of 17-AAG and other ansamycins may be curtailed in tumors that have upregulated efflux pumps or antiapoptotic proteins or other genetic alterations. These data indicate that the new generation of synthetic anti-Hsp90 drugs, exemplified by BIIB021 that is currently undergoing Phase II testing, may have broader application against tumors with acquired multidrug resistance or tumors located in organs protected by MDR proteins, such as the adrenal glands, brain and testis.
Hsp90 is an attractive target in cancer therapy due to its crit- ical role in mediating the maturation and stability of a variety of cancer-associated proteins, including p53, Src, Her-2, IGFR, VEGFR2, steroid receptors, AKT, Raf and Bcr-abl. Many Hsp90 inhibitors in clinical development, including KOS-953 (tanespimycin), IPI-504 (retaspimycin) and KOS- 1022 (DMAG), are all semisynthetic geldanamycin derivatives and are structurally closely related to 17-AAG.1,2 Previous studies have shown that the antitumor activity of 17-AAG and ansamycins are significantly curtailed by the expression of multidrug resistance (MDR) proteins, a major contributor to drug resistance commonly observed in heavily pretreated cancer patients.3,4 This raised the possibility that increased MDR expression in these patients may limit the response of the patients to 17-AAG and its analogues.
MDR is a significant obstacle in cancer therapy. It is mediated by a family of ATP-binding cassette (ABC) pro- teins, which function as efflux pumps. Two major members in this family are P-glycoprotein (P-gp) and MRP1 (MDR- related protein 1), whose expression are found to be elevated in a variety of cancers.3,5 Expression of MDR proteins confers
Key words: Hsp90, Hsp90 inhibitor, BIIB021, MDR, P-gp DOI: 10.1002/ijc.24825
History: Received 12 Mar 2009; Accepted 30 Jul 2009; Online 12 Aug 2009
Correspondence to: Hong Zhang, Biogen Idec, 5200 Research Place, San Diego, CA, USA. Fax: (858) 795-9661,
E-mail: Hong.zhang@biogenidec.com
resistance to a broad range of structurally and functionally unrelated chemotherapeutic agents and molecularly targeted drugs.4,6,7 P-gp not only elicits drug resistance at the cellular level, but was also found to alter the pharmacokinetics of vari- ous drugs and was associated with poor bioavailability.8-10 Moreover, chronic exposure to drugs, which are P-gp sub- strates, induces the expression of the MDR pump and results in acquired resistance of the tumor.11-13
Here, we examined the dependency of the potency of syn- thetic Hsp90 inhibitors on MDR expression and other molec- ular targets as described and compared it with that of 17- AAG and its derivatives. As expected, 17-AAG and DMAG are both P-gp substrates whose client protein effects and cy- totoxic potency were dramatically reduced in P-gp overex- pressing cell lines. Co-administration of P-gp or MRP inhibi- tors significantly increased the potency of 17-AAG. Unlike ansamycins, BIIB021 was relatively independent of MDR expression and was active against P-gp and/or MRP-1- expressing cell lines. This was also confirmed in a naturally P-gp expressing adrenocortical carcinoma cell line, in which BIIB021 was superior to 17-AAG both in vitro and in vivo. Interestingly, although 17-AAG was a preferred substrate for P-gp, it exhibited no inhibitory activity against P-gp, unlike many other P-gp substrates. Furthermore, BIIB021 outper- formed 17-AAG in other situations, such as NQO1 deletion or Bcl-2 overexpression, which was associated with reduced cell killing by 17-AAG.14-16 These data indicate that the new generation of synthetic anti-Hsp90 drug, such as BIIB021, that is currently under Phase II investigation, may have
broader application against tumors expressing MDR proteins or other genetic lesions.
Material and Methods
Synthesis of Hsp90 inhibitors
17-AAG and BIIB021 were synthesized as previously described.17,18
Cell and reagents
Cell lines were obtained from ATCC or NCI unless otherwise indicated. Both MES-SA/DX5 and NCI/ADR-RES are known to express P-gp at high level. H295R is an adrenocortical car- cinoma that expresses P-gp naturally. Antibodies used were progesterone receptor (PR) (Novocastra, UK), PI3-Kinase p85 (Upstate Biotechnology, NY), AKT and CDK4 (Santa Cruz Biotechnology Inc., CA), P-gp (MRK16) (Research Diagnostic, MA). Doxorubincin, paclitaxel and rhodamine 123 were from Sigma, MO. Cyclosporin A (CsA) and MK571 were from Calbiochem.
MTS assay
Cells were seeded in 96-well plates at 2,000 cells/well in a final volume of 200 ul and were treated with increasing con- centrations of 17-AAG, BIIB021 or their analogs 24 hr later. In the combination treatment with either CsA or MK571, CsA and MK571 were added at fixed concentration as indi- cated, whereas the other compound was serially diluted. Fol- lowing drug addition, the plates were incubated for 120 hr before assay. Viable cell number was determined using the Celltiter-96 Aqueous cell-proliferation assay (Promega). The value of the background absorbance at 490 nm (A490) of wells not containing cells was subtracted. Percentage of viable cells = (A490 of treated sample/A490 untreated cells) × 100. The IC50 was defined as the concentration that reduced the viable cell number by 50%.
Flow cytometric analysis of P-gp expression
Cells were grown up in 24-well plates, rinsed with PBS, tryp- sinized and added to the wells of a round-bottomed 96 well plate, pelleted at 2,500 rpm for 10 sec, washed once in PBS containing 0.2 % BSA and 0.2% NaN3 (BA buffer), and the cell pellets were resuspended in 100 ul P-gp antibody (MRK16) and diluted in BA buffer at final concentration of 25 µg/ml. IgG antibody was used as background controls. Cells were incubated for 30 min at room temperature, washed twice and further incubated with 100 ul 0.5 mg/ml (50µg) FITC-conjugated goat anti-mouse antibody. After extensive washing, cells were resuspended in 500-ul BA buffer before analysis on a FACSCalibur™M flow cytometer equipped with Argon-ion laser that emits 15 mW of 488 nm light for excitation of the FITC fluorochrome.
Western blotting
Cell pellets were prepared in lysis buffer (10 mM HEPES, 42 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 1% Triton X-100, freshly supplemented with 1x protease inhibitor cocktail from Pierce). The lysate was quan- tified by BCA assay (Pierce) and normalized. Equal amounts of protein were loaded onto 4-12% Tris-glycine gels and subsequently transferred onto PVDF membranes. The mem- branes were blocked in 5% TBST, and primary antibodies were added and incubated at room temperature for 1 hr with shaking. The blots were washed extensively in TBST before secondary antibodies were added for overnight incubation at 4℃ with gentle shaking. The blots were again washed exten- sively and developed with SuperSignal West Femto substrate (Pierce).
MDR function analysis
Cells (1.0 × 10°) were washed, resuspended in 1 ml of se- rum-free DMEM medium and incubated with rhodamine 123 and compounds at the indicated concentrations for 1 hr at 37℃. The cells were then washed twice with serum-free medium and resuspended again in 1 ml serum-free medium to incubate at 37℃. After 2 hr of culture, the cells were washed twice with PBS, resuspended in 500-ul BA buffer and analyzed by flow cytometry using a FACScan by measuring events in the FL1 (rhodamine 123) channel.
Tumor model
Six to 8-week-old athymic female mice were obtained from Harlan, (Indianapolis, IN). NCI H295 tumor fragments (~2 mm3) were inoculated subcutaneously. Mice with established tumors (50-200 mm3) were randomized into groups of 7 for study. BIIB021 was administered orally (p.o.) at 120 mg/kg and 17-AAG was administered intraperitoneally (i.p.) at 60 mg/kg. Animals were dosed 5 days per week (Monday through Friday) for 4 weeks. Animals were weighed each dosing day, and the tumors were measured twice per week. The compounds and vehicle controls were administered to mice by exact individual animal body weight on each day of treatment. Tumor dimensions were measured using calipers and tumor volumes are calculated using the equation for an ellipsoid sphere 0.5 (1 x w2) = mm3, where I and w refer to the larger and smaller dimensions collected at each measurement.
Results
Overexpression of P-gp is associated with resistance to 17-AAG but not to the synthetic Hsp90 inhibitor BIIB021
17-AAG has been reported to be highly antiproliferative in a majority of tumor cell lines. However, some tumor lines were found to be extremely resistant, with IC50 increased up to several hundred-fold over the majority of cell lines. To study the mechanism of resistance to 17-AAG and to investigate if this phenomenon applied to other Hsp90 clinical candidates,
a
17-AAG
BIIB021
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- MES SA (low P-gp)
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| IC50 (nM) | |||
|---|---|---|---|
| Compound | MES-SA | MES-SA Dx5 | IC50 Ratio |
| 17-AAG | 9.5+6.1 | 3200±590 | 337 |
| 17-DMAG | 2.1±1.1 | >10000 | >4800 |
| Doxorubicin | 77±24 | 5500±1200 | 71 |
| Paclitaxel | 0.7±0.5 | 2450±640 | 3500 |
| BIIB021 | 175+25 | 190±45 | 1.1 |
| EC129 | 76 ±34 | 75 ±28 | 1.0 |
| MES-SA Dx5 NCI-ADR Res | |||||||
|---|---|---|---|---|---|---|---|
| IC50 (nM) | IC50 (nM) | ||||||
| w/o CsA | +1ug/ml CsA | +3ug/ml CsA | w/o CsA | +1ug/ml CsA | +3ug/ml CsA | ||
| 17AAG | 4000 ±620 | 1800 ±300 | 23 ±11 | 17-AAG | 4600±760 | 2200±450 | 57±23 |
| BIIB021 | 175 ±25 | 180 ±30 | 185 +25 | BIIB021 | 150±34 | 170±40 | 190±35 |
Figure 1. Comparison of natural and synthetic Hsp90 inhibitors in P-gp-overexpressing cell lines. (a) Chemical structures of 17-AAG and BIIB021 were shown. Both molecules were tested for cytotoxic activity by MTS assay in uterine sarcoma cell lines, expressing low or high levels of P-gp. (b) Synthetic Hsp90 inhibitors, including BIIB021 and its analog EC129 were tested for their P-gp dependency by MTS assay in the paired uterine sarcoma cell lines. 17-AAG, DMAG and a few other P-gp substrates were used as controls. IC50 was defined as the concentration of drug required to inhibit cell growth by 50%. (c) Co-administration with increasing amounts of cyclosporin A, a P-gp inhibitor significantly improved the cytotoxicity of 17-AAG in 2 high-P-gp expressing cell lines, MES-SA DX5 and NCI-ADR/RES. The potency of BIIB021 was not changed.
such as DMAG (a 17-AAG analog) and BIIB021 (a fully syn- thetic Hsp90 inhibitor) (structures shown in Fig. 1a), we examined the cytotoxicity of these compounds in a pair of uterine sarcoma cell lines, MES-SA and MES-SA DX5 that differ in P-gp expression levels. Although 17-AAG was very active in parental MES-SA cells, with an IC50 in the low- nanomolar range, the IC50 was increased more than 300-fold in high-P-gp expressing MES-SA DX5 cells, indicating that expression of P-gp significantly reduced the activity of 17- AAG (Figs. 1a and 1b). To determine whether this extends to other ansamycin derivatives, we also examined the activity of DMAG, an analog of 17-AAG, under the same conditions. We found DMAG was even more sensitive to P-gp levels, and its IC50 was elevated about 5,000-fold from 2.1 nM (MES-SA) to more than 10,000 nM (MES-SA DX5). These changes were comparable to if not greater than that of classi- cal P-gp substrates, such as doxorubicin and taxol, whose IC50 climbed about 70 and 3,500-fold, respectively (Fig. 1b). By contrast, this did not occur with a new generation of syn- thetic Hsp90 inhibitors, represented by BIIB021 and its ana- log EC129, both of which were equally active in both cell lines (Fig. 1b and unpublished results). A similar result was
observed in another high-P-gp cell line, NCI/ADR-RES (Fig. 1c, right panel).
To further confirm this, we combined 17-AAG or BIIB021 with increasing concentrations of CsA, a known P- gp inhibitor9,19 in both MES-SA DX5 and NCI/ADR-RES cells. As shown in Figure 1c, the potency of 17-AAG was enhanced for 100-200-fold in both cell lines when concentra- tion of CsA reached 3 µg/ml. By contrast, the cytotoxicity of BIIB021 was not affected by the presence of CsA (Fig. 1c), indicating that BIIB021 could be more active in tumors where P-gp level is high.
The cytotoxicity of 17-AAG correlates with P-gp expression in vitro
To establish a relationship of P-gp expression with the antitu- mor activity of 17-AAG in vitro, we measured the P-gp levels in a variety of tumor cell lines by flow cytometry, using an anti-P-gp specific antibody, MRK 16. Among the 12 cell lines we tested, MES-SA DX5 and NCI/ADR-RES are the 2 exam- ples expressing highest levels of P-gp. H295R, a tumor cell line naturally expressing P-gp (Fig. 2a). To correlate the effect of P- gp expression on the cytotoxicity of 17-AAG and BIIB021, we
a
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2253.1
P-gp Level (MFI)
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H69
H82
H295R
MDA-468
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MES SA Dx5
NCI/ADR RES
OVCAR 4
RS11846
T47D
T98G
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P-gp level (MFI)
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IC50 (nM) of BIIB021
measured the IC50 of both compounds in all these cell lines and correlate it with P-gp levels. As shown in Figure 2b, a cor- relation between P-gp expression and the cytotoxic effect of 17-AAG was observed, confirming that the P-gp level influ- enced the antitumor activity of 17-AAG. By contrast, there was no such relationship apparent with BIIB021 (Fig. 2c).
MRP1 expression confers resistance to natural product Hsp90 inhibitors, but not to BIIB021
MRP1 is another major member of ABC family that mediates MDR in tumors. Many chemotherapeutic drugs, such as doxor- ubicin, etoposide and vincristine, are dual substrates of P-gp and MRP1. To examine whether MRP expression influences the activity of 17-AAG and BIIB021, we compared the IC50 of the 2 compounds in an additional pair of small cell lung cancer cell lines, NCI-H69 and NCI-H69AR, which differ in MRP1 levels.20 Figure 3 showed that the cytotoxicity of 17-AAG, DMAG and another Hsp90 inhibitor, radicicol, was markedly reduced in NCI-H69AR cells, which express high-MRP1. Addi- tion of a MRP1 specific inhibitor, MK571,21,22 restored the ac- tivity of these compounds to different extent. Again, the activity of BIIB021 was not affected by the presence of MRP1, nor was it changed by the addition of MK571 (Figs. 3a and 3b).
BIIB021 is more potent than 17-AAG against adrenal cortical carcinoma H295R
As both MES-SA DX5 and NCI/ADR-RES express extremely high levels of P-gp, they may not be fully representative of
a
| IC50(nM) | |||
|---|---|---|---|
| H69 | H69AR | Ratio | |
| 17-AAG | 1000 ±330 | 5900 ±1500 | 5.9 |
| 17-DMAG | 57.5±12 | 620 ±130 | 10.8 |
| Radicicol | 77.5 ±30 | 550 ±230 | 7.1 |
| BIIB021 | 255 ±85 | 175 ±43 | 0.7 |
b
| IC50 (nM) | |||
|---|---|---|---|
| - MK571 | + MK571 | Ratio | |
| 17-AAG | 7400 ±1300 | 3000 ±430 | 2.5 |
| 17-DMAG | 1000 ±320 | 110 ±20 | 9.1 |
| Radicicol | 600 ±150 | 130 ±55 | 4.6 |
| BIIB021 | 200 ±60 | 180 ±35 | 1.1 |
Figure 3. BIIB021 was not affected by MRP1 expression. (a) A variety of synthetic Hsp90 inhibitors were tested for their MRP1 dependency in paired SCLC cell lines, H69 and H69AR, expressing low and high level of MRP1, respectively. The activities of 17-AAG, DMAG and radicicol were significantly reduced in MRP1 overexpressing cell line, H69AR, whereas that of BIIB021 did not change. (b) Addition of 50 µM MK571, a MRP1-specific inhibitor dramatically increased the cytotoxic activity of 17-AAG, DMAG and radicicol in the high-MRP1 cell line, H69AR, but had no effect on the cytotoxicity of BIIB021 under the same conditions.
| IC50 (nM) | |
|---|---|
| Compound | H295R |
| 17-AAG | 710±110 |
| 17-DMAG | 670±70 |
| BIIB021 | 260±40 |
| EC129 | 210±54 |
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AKT
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17-AAG 60mg/kg qdx5
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the physiological condition, and so here, we studied 17-AAG, DMAG and the synthetic compounds in an unmodified adre- nocortical carcinoma cell line, H295R. The P-gp level in this cell line is about 8-9-fold less than that of MES-SA DX5 or NCI/ADR-RES, but is much higher than that of the other cell lines examined (Fig. 2). The IC50 of the compounds in this cell line is shown in Figure 4a. The moderate P-gp level in H295R increased the IC50 of 17-AAG/DMAG from the low-nanomolar range to ~700 nM, but not to the range as of MES-SA DX5 and NCI/ADR-RES cell lines. In contrast, BIIB021 again retained its activity and exhibited potency comparable to other cell lines. This result demonstrated that even moderate expres- sion of P-gp, which naturally occurs in human cancers, would markedly curtail the potency of 17-AAG and its derivatives, but not that of the synthetic compounds.
To explore whether the cytotoxic activity of 17-AAG and BIIB021 in H295R cells was reflected in differential degradation of client proteins, we monitored the changes of the expression of certain clients in this cell line by Western blot, following treatment with 17-AAG or BIIB021. As shown in Figure 4b, several client proteins, including PR, AKT and CDK4 were degraded in a dose-dependent manner by BIIB021. However, 17-AAG was not as effective and only exhibited tangible effect at high concentration. Hsp70 induction by both compounds showed a similar profile as that of the client proteins.
To determine how P-gp expression affects the antitumor activity of 17-AAG and BIIB021 in vivo, we performed an ef-
ficacy study with the 2 compounds in H295R xenograft-bear- ing mice. In this experiment, H295R tumors were grown sub- cutaneously and treated with vehicle, 17-AAG or BIIB021 at their respective maximum tolerated doses. Only BIIB021 showed significant suppression of growth of this naturally P- gp expressing tumor; 17-AAG was totally inactive in this sce- nario (Fig. 4c). This again emphasized the superiority of BIIB021 under high-P-gp expressions.
17-AAG shows no P-gp inhibitory activity
Numerous studies have shown that P-gp substrates usually possess P-gp suppressive activity when it reaches the concen- tration that saturates the export capability of the pump.3, 3,19 Because 17-AAG is a P-gp substrate, we sought to determine if 17-AAG could also inhibit the activity of the efflux pump. Here, we used a rhodamine 123 efflux functional assay. Rho- damine 123 is a fluorescent P-gp substrate, which is con- stantly pumped out of cells when P-gp is functioning. Inhibi- tion of P-gp retains rhodamine within cells and results in high fluorescence, which can be detected by fluorometer. We examined the rhodamine 123 level in MES-SA DX5 cells, in the presence or absence of 17-AAG or BIIB021. Regarding P- gp inhibition, CsA was used as a control. CsA (10 µM) sig- nificantly suppressed efflux of rhodamine 123 and increased the intracellular level of fluorescence (Fig. 5a). By contrast, neither 17-AAG nor BIIB021 showed any effect on rhoda- mine efflux even at concentration up to 50 uM, indicating
that neither of these compounds inhibited P-gp activity (Fig. 5a). It was not unanticipated for BIIB021 to behave as a non-P-gp inhibitor, as it was not a P-gp substrate. However,
a
MES-SA Dx5
50uM BIIB021
10μΜ CsA
50UM 17-AAG
CNTL
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the inability of 17-AAG to inhibit P-gp function was unex- pected, as most P-gp substrates are able to block P-gp activity at high levels.
One possibility for the lack of P-gp suppression by 17- AAG was due to the low-inhibitory potency of the com- pound against P-gp being insufficient to inhibit such high level of P-gp in MES-SA DX5 cells at 50 uM. To pursue this, we again used the naturally occurring P-gp expressing H295R cell line, whose P-gp level was about 9-fold less. In this study, CsA inhibited P-gp activity at concentrations lower than that required for MES-SA DX5 cells; however, 17- AAG remained inactive at concentrations up to 10 µM (Fig. 5b). This was not due to impermeability of the compound, as it was more active than CsA in H295R in cytotoxic studies (Fig. 4a and data not shown). Both results suggested that 17- AAG, although a P-gp substrate, did not possess P-gp inhibi- tory activity. Therefore, 17-AAG might be transported by P- gp in a distinct manner or that the magnitude of the influ- ence of P-gp on ansamycin activity may be related to the slow kinetics of action of these Hsp90 inhibitors in cells.
BIIB021 is active in 17-AAG resistant cell lines
It has been reported that some cell lines are particularly re- sistant to 17-AAG, but not other Hsp90 inhibitors, such as geldanamycin and radicicol.16,23,24 We therefore examined whether BIIB021 was active in those situations, including SCLC cell lines negative for NQO1 and BCL-2 overexpressing lymphoma cell lines. NQO1 encodes DT-diaphorase, which processes 17-AAG to 17-AAGH2, making it more potent against Hsp90. It was reported that abrogation of NQO1 in cells dramatically reduced the sensitivity to 17-AAG.14,16 Here, we examined the cytotoxicity of BIIB021 in 2 NQO1”
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SCLC (Rb-defective)
b Follicular Lymphoma (Bcl-2++)
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NCI-H69
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RS11846
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| IC50 (nM) | ||
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| Compound | H82 | NCI-H69 |
| 17-AAG | 1000±130 | 1000±330 |
| BIIB021 | 210+43 | 150+26 |
| IC50 (nM) | ||
|---|---|---|
| Compound | 697 | RS11846 |
| 17-AAG | 375±110 | 350±75 |
| BIIB021 | 82±27 | 79±11 |
Figure 6. BIIB021 is more potent than 17-AAG against NQ01-SCLC and Bcl-2+++ follicular lymphoma cell lines. Two NQ01 negative SCLC cell lines NCI-H82 and NCI-H69 (a) and 2 Bcl-2 overexpressing follicular lymphoma 697 and RS11846 (b) were examined for sensitivity to 17-AAG and BIIB021 by a 5-day MTS assay described previously. The IC50S for both compounds are listed. BIIB021 was more active than 17-AAG in both scenarios.
| Cell line | Cell type | 17-AAG IC50 (nM) | BIIB021 IC50 (nM) | Pgp | MRP | NQO1 | Bcl-2/Bcl-Xl |
|---|---|---|---|---|---|---|---|
| RS11846 | B cell lymphoma | 350 | 80 | + | - | + | +++++ |
| 697 | B cell lymphoma | 375 | 82 | - | - | + | ++++ |
| CWR22/RV-1 | Prostate | 370 | 85 | - | - | + | |
| H1963 | sclc | 390 | 49 | þ | |||
| NCI-H69 | sclc | 1,000 | 150 | - | - | - | +++ |
| NCI-H69AR | sclc | 5,900 | 350 | - | +++ | ||
| H146 | sclc | 380 | 110 | - | + | ++++ | |
| H82 | sclc | 1,000 | 210 | - | - | - | - |
| OVCAR-4 | Ovary adenocarcinoma | 230 | 275 | + | - | ++++ | |
| H295R | Adrenal | 710 | 260 | ++ | - | ||
| MES SA Dx5 | Uterine sarcoma | 3,200 | 200 | +++ | - | ||
| NCI-ADR-RES | Breast | 4,700 | 155 | +++ | - | ||
| T47D | Breast | 1,000 | 330 | - | - | +++ | |
| MDA468 | Breast | 450 | 180 | - | - | - | |
| IE8 | AML | 3,500 | 170 | - | - | ++ | |
| Nalm6 | AML | 7,250 | 960 | - | - | ++++ | |
| RS4ill | AML | 260 | 180 | - | - | +++ | |
| 17-AAG sensitive cells | Various (20 cell lines) | ~50 | ~200 | - | - | ++ | - |
The IC50 of 17-AAG and BIIB021 was measured in a panel of cell lines that are relatively resistant to 17-AAG.
SCLC cell lines, NCI-H69 and NCI-H82, and 2 BCL-2++ lym- phoma, 697 and RS11846, using 17-AAG as control. Figure 6 shows that BIIB021was more active than17-AAG in both cir- cumstances. Similar results were observed in the 2 Bcl-2-over- expressing lymphoma cell lines, suggesting that BIIB021 would be more effective against tumors with these genetic alterations. Based on this, the activity of 17-AAG and BIIB021 was com- pared in a broader group of cell lines, most of which are rela- tively resistant to 17-AAG. The results are summarized in Ta- ble 1. 17-AAG, which is active in many tumor cell lines and has an average IC50 of about 50 nM in 20 sensitive tumor lines, here exhibited high nanomolar up to more than 7 micromolar IC50. By comparison, BIIB021 was active in almost all of these cell lines with IC50 between 49 and 390 nM, except the AML cell line Nalm6. These results were comparable to the activities we got from the 17-AAG sensitive tumor cell lines, in which the average IC50 for BIIB021 was around 200 nM. All these data indicated that many factors, which affected the sensitivity of tumor cells to 17-AAG, would not be an issue for BIIB021; therefore, the synthetic compound, although innately a slightly weaker Hsp90 inhibitor than 17-AAG, could have a broader spectrum of therapeutic activity.
Discussion
17-AAG is a prototypical Hsp90 inhibitor that is currently being investigated in phase III clinical trials. Many preclinical studies have shown that 17-AAG has a low-nanomolar IC50 against a wide variety of tumor cell lines and was shown to be active in many types of tumor in vivo.25-27 Efficacy in
cancer patients has been observed with several type of tumors in 17-AAG clinical trials, and the therapeutic effect correlates with the degradation of key client proteins.28-30 These studies prove the concept and validate Hsp90 as an effective target for cancer therapy. Nonetheless, 17-AAG is difficult to for- mulate and has to be administered in vehicles containing ei- ther DMSO or cremophor via the IV route.31 This not only increased the toxicity of the compound, but also significantly affected the therapeutic efficacy in clinic.32,33 Numerous efforts have been made to address the issue, leading to water- soluble derivatives, such as DMAG and IPI-504, or lipid- based formulations, such as CNF1010. However, all these molecules still share other drawbacks of 17-AAG, such as high cost of manufacture and vulnerability to common mech- anisms of drug resistance.
Many types of cancer express relatively high levels of P- glycoprotein, a major type of MDR protein. These include colon, kidney, adrenocortical and hepatocellular cancers, whereas breast, lung, neuroblastoma and other tumors express P-gp at intermediate levels.3,5 Some cancers also express other MDR family members, such as MRP1 and BCRP.34,35 In many cases, untreated tumors, which do not express MDR proteins, could be induced to express P-gp and other MDR proteins to a high level upon treatment with drugs that are P-gp substrates,11-13 including chemotherapeu- tic drugs and molecularly targeted compounds. Thus, many tumors overexpress P-gp or related proteins, either innately or after initial treatment, which leads to diminished respon- siveness to subsequent treatment.
Here, we showed clearly that 17-AAG is a MDR substrate. The compound exhibited no activity in cells expressing high levels of P-gp or MRP1, even though it was active in the con- genic parental cells. This was also confirmed by the fact that the activity of 17-AAG could be significantly enhanced in those cells by combination with either P-gp or MRP1 inhibi- tors. Unlike most P-gp substrates, which are also P-gp inhibi- tors, 17-AAG showed no inhibitory activity against P-gp, implying that even if 17-AAG was given at high concentra- tions, it would not be able to overcome the P-gp effect and therefore would continue to be effluxed from the cell. This property could significantly limit the antitumor activity of 17-AAG, especially in patients who have been heavily pre- treated with chemotherapeutic drugs or in those who have cancers located in adrenal glands, brain and testis, which normally contain high levels of P-gp or other MDR proteins. In addition, a recent report has shown that 17-AAG can itself induce P-gp expression, suggesting that the drug could become increasingly less effective during treatment due to the increase of P-gp level.36
These limitations of 17-AAG and its derivatives stimulated efforts to design synthetic Hsp90 inhibitors, which possess better pharmaceutical properties. BIIB021 was the first fully synthetic small molecule Hsp90 inhibitor to enter clinical tri- als. It has improved biopharmaceutical properties, is easy to formulate and orally bioavailable. BIIB021 binds to Hsp90 with high affinity and dramatically inhibits tumor growth in a variety of xenograft models.37 In phase I clinical trials, BIIB021 was well tolerated and exhibited antitumor activity. Here, we show that, unlike 17-AAG and its derivatives,
BIIB021 exhibits no MDR dependency. It has strong antipro- liferative activity in either engineered or natural P-gp and MRP1 overexpressing cells. All these characteristics make BIIB021 a potentially superior Hsp90 inhibitor in the situa- tion where P-gp is expressed, which is able to overcome the MDR barrier that commonly undermines cancer therapy.
17-AAG is relatively inactive under certain circumstances, such as loss of NQO1 or overexpression of the antiapoptotic Bcl-2 family members.15 Because NQO1 encodes for an enzyme which converts 17-AAG into a more active form, 17- AAGH2, it is not surprising that the presence of NQO1 will make cells more sensitive to 17-AAG. This does not apply to other Hsp90 inhibitors, including its most close analog DMAG, as the molecular group at the 17-position where the reaction occurs is different. Obviously, this would not be expected to affect the activity of BIIB021, as we confirmed in this study. It is unclear how Bcl-2 expression impacts the ac- tivity of 17-AAG, but not that of BIIB021. It may be related to the pharmacodynamic or kinetic properties of the com- pound itself. We have noted that, at equivalent pharmaceuti- cal concentrations, BIIB021 shows prolonged inhibition of certain client proteins. Whether this accounts for the better activity of BIIB021 in Bcl-2 overexpressing lymphomas requires further investigation.
Overall, our studies indicate that BIIB021 out performs 17-AAG in several situations, including MDR and Bcl-2 over- expression, which are commonly encountered in current can- cer therapy. This could limit the application of ansamycin- derived drugs and suggests that BIIB021 might be a better choice in those scenarios.
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