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.

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<0.0001 for both).

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<0.0001, ANOVA) but not in Ly18 (p=0.5472, ANOVA).

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).