High Expression and Persistent Activation of STAT1, 3 and 5 Predicts Vorinostat (Suberoylanilide Hydroxamic Acid, SAHA) Resistance in Cutaneous T-Cell Lymphoma Cells.

The hydroxamic acid vorinostat (SAHA) is an HDAC inhibitor that induces differentiation, growth arrest and/or apoptosis of malignant cells both in vitro and in vivo, and is currently being tested in the clinic for a variety of indications. In particular, vorinostat has demonstrated an overall response rate of approximately 30% in advanced cutaneous T-cell lymphoma (CTCL). The purpose of this study was to identify biomarkers that predict response to vorinostat in CTCL using preclinical model systems based upon gene expression profiling and pathogenesis of lymphoma. Herein we report the results of our evaluation of the STAT (signal transducer and activator of transcription) signaling pathway. The relative level of mRNA and protein expression as well as activation status of several members of this family have been evaluated in lymphoid cell lines with diverse vorinostat sensitivity (proliferation/viability IC₅₀ range: 0.3–14 mM). Among them, the more responsive CTCL cells lines HH and HUT102 exhibited an IC₅₀ ≃ 0.8 mM. These cells underwent apoptosis in response to vorinostat as assessed by TUNEL assay, in contrast to Hut78 and MJ cells (IC₅₀ > 2 mM) that did not show signs of DNA fragmentation upon incubation in vorinostat-containing media for 48 h. Our results indicate that in lymphoma cell lines elevated protein levels and persistent activation of STAT1, 3 and 5 correlate with resistance to vorinostat. Immunofluorescence microscopy studies revealed that STAT proteins preferentially localize to the nuclear compartment in cells with impaired vorinostat response, consistent with the expected distribution for this group of transcription factors in the active state. Simultaneous treatment with a pan-JAK inhibitor (JAK inhibitor I; Calbiochem) resulted in an additive antiproliferative effect consistent with a survival, antiapoptotic role for STAT proteins in the response to vorinostat treatment. Immunohistochemical analysis of STAT1 in skin biopsies isolated from CTCL patients (N = 49) enrolled in the vorinostat Phase IIb clinical trial showed that malignant T cells were positively stained in approximately half of the samples (21 positively stained; 25 negatively stained; 3 poor quality specimens), and in those cases a relationship between nuclear accumulation of STAT1 and lack of response to treatment exists (p=0.007<0.05; Fisher’s exact test). In conclusion, our results suggest that deregulation of STAT signaling plays a role in resistance to vorinostat in CTCL and strategies that block this pathway may improve response to vorinostat.