Abstract
Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease that is currently treated with a homogeneous approach based on empiric combination chemotherapy. To elucidate molecular differences in DLBCL subsets and identify more rational treatment targets, coordinate transcriptional profiling was used to define three groups of DLBCLs: “Oxidative Phosphorylation” (OxPhos), “B-cell Receptor/Proliferation” (BCR), and “Host Response” (HR). Of interest, the HSP90 α and β isoforms were differentially expressed in the DLBCL subsets; the stress-induced α isoform was more abundant in OxPhos tumors (p < .0001) whereas the constitutively expressed β isoform was increased in BCR DLBCLs (p < .0001). HSP90 α and β differ in the length of their N-termini but share a common ATP binding domain, the target site for recently developed HSP90 inhibitors, such as IPI-504 (Infinity Pharmaceuticals, Cambridge, MA). Although differential HSP90 isoform expression likely reflects underlying biological differences in the DLBCL subsets, the shared ATP binding domain suggests that both isoforms, and the majority of DLBCLs, may be effectively targeted with HSP90 inhibitors. For these reasons, we determined the IC50 and cytotoxic potential of IPI-504 in an extensive series of DLBCL cell lines. IPI-504 inhibited the proliferation of DLBCL cell lines at IC50s ranging from 0.04 μM in the most sensitive line to 12.67 μM in the most resistant line. Annexin V/PI staining revealed that IPI-504 was cytotoxic to most DLBCL cell lines at low micromolar doses; even cell lines that were relatively resistant to IPI-504 at lower doses (2 μM) underwent apoptosis with prolonged exposure to increased IPI-504 doses (72 hrs. and 8 μM). Cell lines that were most sensitive to IPI-504 expressed high levels of a major HSP90 client protein, pAKT, and exhibited a dose-dependent decrease in pAKT levels following IPI-504 treatment. For these reasons, we assessed the efficacy of IPI-504 in association with a PI3K/AKT pathway inhibitor, LY24009, in the DLBCL panel. Combined treatment with the HSP90 and PI3K inhibitors was highly synergistic (Method of Chou and Talalay; Calcusyn software, Biosoft, Ferguson, MO), inhibiting cellular proliferation by over 95% in evaluated DLBCL cell lines. In contrast, the combination of concurrent IPI-504 and an empiric chemotherapeutic agent, doxorubicin, was antagonistic, underscoring the importance of rational combination therapy. We conclude that: 1) HSP90 isoforms are differentially expressed in primary DLBCL subsets; and 2) inhibition of HSP90, via the conserved ATP binding domain, leads to dose-dependent apoptosis that is associated with depletion of pAKT in DLBCL cell lines. The HSP90 inhibitor IPI-504 possesses both single-agent efficacy as well as synergy when combined with PI3K inhibitors, suggesting that this rational targeted approach warrants further clinical investigation.
NEMO-Binding Domain Peptide Inhibits Constitutive NF-κB Activity and Reduces Tumor Burden in a Canine Model of Relapsed, Refractory Diffuse Large B-Cell Lymphoma
