The heat shock protein 90 inhibitor IPI-504 induces apoptosis of AKT-dependent diffuse large B-cell lymphomas

Abstract

Heat shock protein 90 (HSP90) is a molecular chaperone that stabilizes critical client proteins in multiple cancers. Gene expression profiling was utilized to characterize HSP90 isoform expression in primary human diffuse large B-cell lymphomas (DLBCLs). HSP90 alpha and beta isoforms were differentially expressed in subsets of tumours defined by their transcriptional profiles. Thereafter, we assessed the activity of the HSP90 inhibitor, IPI-504, in an extensive panel of DLBCL cell lines. IPI-504, which interacts with the conserved ATP-binding site in both HSP90 isoforms, inhibited proliferation and induced apoptosis in the majority of DLBCL cell lines at low micromolar concentrations. IPI-504-sensitive cell lines expressed high levels of the HSP90 client protein, pAKT, and exhibited dose-dependent decreases in pAKT levels following IPI-504 treatment and significantly reduced proliferation following AKT RNAi. Furthermore, the combination of low-dose (<1 micromol/l) IPI-504 and the AKT/Pi3K pathway inhibitor, LY24009, was synergistic in IPI-504-sensitive DLBCL cell lines. Low-dose IPI-504 was also synergistic with the chemotherapeutic agent, doxorubicin. The HSP90 inhibitor IPI-504 warrants further investigation in DLBCL alone and in combination with identified client protein inhibitors and active chemotherapeutic agents.

Figures

Fig. 1. HSP90 isoform expression in DLBCL subsets

(A) Relative HSP90 α and β transcript abundance in the DLBCL comprehensive clusters, Oxidative Phosphorylation “OxPhos”, B-cell receptor/proliferation “BCR”, and host response “HR”. Each column is a sample and each row is a gene probe. Color scale at bottom indicates relative expression and SDs from the mean. (B) Boxplots demonstrate the relative increased abundance of HSP90 α and β isoforms in OxPhos and BCR DLBCLs, respectively (p

Fig. 2. IPI-504-mediated cytotoxicity in DLBCL

Eight DLBCL cell lines with the lowest and highest EC50s (Table 1) were analyzed for IPI-504-mediated apoptosis, using 0, 0.5, 1.0 and 2.0 µM IPI-504 and annexin V/PI staining. A) IPI-504 sensitive DLBCLs. B) IPI-504 resistant DLBCLs.

Fig. 3. pAKT expression in IPI-504-treated DLBCLs

pAKT abundance in sensitive (A) and resistant (B) DLBCLs was evaluated at baseline and following treatment with 0.2 – 1.6 µM IPI-504. Total AKT and actin (control) were assessed in the same samples.

Fig. 4. Consequences of AKT1 and 2 RNAi in IPI-504 sensitive and resistant DLBCLs

A) AKT1 and 2 levels in IPI-504 sensitive and resistant cell lines. (DHL7 and LY18, respectively) following AKT1 or 2 RNAi or treatment with a scrambled control oligonucleotide. B) Cellular proliferation of DHL7 and LY18 following AKT1 or 2 RNAi or treatment with a scrambled control oligonucleotide. Following AKT1 or AKT2 RNAi, proliferation was significantly decreased in DHL7, compared to the negative control (p

Fig. 5. Combined treatment with IPI-504 and a PI3K inhibitor

The IPI-504-senstive cell lines DHL7 (A) and Karpas-422 (B) were treated with increasing doses of the PI3K inhibitor LY24009 (0, 0.1, 0.25, 0.5 and 1 µM) alone and in combination with increasing concentrations of IPI-504 (0, 0.1, 0.25, 0.5 and 1 µM). The combination of IPI-504 and LY24009 was synergistic through 1 µM IPI-504. Synergy was assessed using the method of Chou and Talalay(35) with Calcusyn Software.

Fig. 6. Combined treatment with IPI-504 and doxorubicin

The IPI-504-senstive cell lines DHL7 (A) and Karpas-422 (B) were treated with doxorubicin alone and in combination with increasing concentrations of IPI-504 (0, 0.1, 0.25, 0.5 and 1 µM). The combination of IPI-504 and doxorubicin was synergistic through 1 µM IPI-504.

Fig. 7. Induction of HSP70 by IPI-504 alone or in combination with doxorubicin

DLBCL cells (DHL7) were treated with increasing doses of IPI-504 (0.1, 0.25, 1µ M) alone or in combination with doxorubicin 25 ng/ml and evaluated thereafter for HSP70 abundance by western blot. Densitometric analysis of X-ray films was performed with ChemiImager 4400 software (Alpha Innotech, San Leandro, CA).