Abstract
Background : HSP90 plays an important role in chaperoning key proteins implicated in malignant disease and is a promising therapeutic target. We now report the in vitro and in vivo activity of a novel HSP90 inhibitor of non-ansamycin, non-purine analogue class, KW-2478, (Kyowa Hakko Kirin) in B-cell malignancies including multiple myeloma (MM), B-cell lymphoma (BCL) and mantle cell lymphoma (MCL) cells, and in primary tumour cells from MM and BCL patients.
Procedures: The binding affinity of KW-2478 to HSP90 was examined using immobilised human HSP90a and a biotinylated HSP90 binding agent, radicicol (bRD). The effect of KW-2478 on cell viability, cell growth and apoptosis induction were evaluated in cell lines, with KW-2478 induced changes in major HSP90 client proteins studied by Western blotting analysis. The in vivo anti-tumour activity of KW-2478 was evaluated in a human MM xenograft mouse model,. Primary MM cells were studied using a co-culture system with the HS-5 bone marrow stromal cell line (BMSCs), while primary BCL samples were cultured on CHO cells stably transfected to produce CD40L.
Results: KW-2478 inhibited the binding of bRD to HSP90α in concentration-dependent manner with an IC50 value of 3.8 nM. KW-2478 clearly inhibited cancer cell growth in all cell lines, with EC50 values from 101–252 nM in BCL, 81.4–91.4 nM in MCL and 120–622 nM in MM. The drug also exhibited potent growth inhibitory activity in primary CLL (n=3) and NHL (n=2) cells with EC50 values of 40–170 nM and 200–400 nM, respectively. In 2 of 4 human primary myeloma cells, KW-2478 at 2 μM inhibited cell growth by at least 50%. The presence of BMSCs did not affect drug activity against primary MM cells and importantly there was little or no effect on cell number or viability of normal BMSCs at up to 20 μM KW-2478. Exposure of MM and BCL cell lines to KW-2478 for 24 hours resulted in the degradation of HSP90 client proteins (IGF-1Rβ and Raf-1) and the induction of HSP70. KW-2478 also induced PARP cleavage and dephosphorylated Erk1/2 in NCI-H929 cells. Further studies in selected cell lines showed that exposure to 1 μM KW-2478 or lower resulted in the depletion of p53 and Akt proteins, a reduction in nuclear NFKB, and the cleavage of caspase-3. In the NCI-H929 xenograft model, KW-2478 (qd×5, i.v.) showed a statistically significant suppression of tumour growth at the doses of 25, 50, 100 and 200 mg/kg. Moreover, tumour regressions were observed at doses of 100 and 200 mg/kg, with a significant decrease in serum M protein concentration at doses of 50, 100 and 200 mg/kg. No severe KW-2478 toxicity was observed as assessed by treatment-related mortality and body weight change.
Conclusions: The novel HSP90 inhibitor KW-2478 showed a potent anti-tumour activity both in vitro and in vivo, including activity in primary patient samples. The agent retained its activity in primary myeloma cells in the presence of BMSCs, suggesting that KW-2478 can overcome the protective effect of the bone marrow microenvironment. Additional pharmacokinetic and safety data support the further development of KW-2478 and the drug is currently undergoing clinical evaluation in a phase I trial.
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