Abstract
Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. Although many DLBCLs are responsive to CD20 antibody/chemotherapy regimens, a significant proportion of patients will still die of their disease. It is possible that targeting important lymphomagenic pathways could improve the potency and reduce the toxicity of future lymphoma therapy. One of the challenges of harnessing molecular targets for the therapy of DLBCL is the remarkable genetic and molecular heterogeneity of this disease. Along these lines, we found that Hsp90 is expressed in more than 90% of patients with DLBCL. Taken together with the fact that Hsp90 is a crucial component of a wide range of signaling processes that are important for cancer cell survival, we hypothesized that Hsp90 inhibition could be an effective strategy for heterogeneous tumors with multiple pathway aberrations such as DLBCL. However, the clinical translation of the available benzoquinone Hsp90 inhibitors has been challenged by their liver toxicity. We developed a non-quinone Hsp90 inhibitor PU-H71 with potent activity in pre-clinical models of DLBCL. When screened against a panel of 32 DLBCL cell lines, PU-H71 was among the most active drugs even compared to classical chemotherapy and new targeted drugs. Inhibition of Hsp90 by PU-H71 in DLBCL (OCI-Ly7, Farage and SUDHL4 cell lines) resulted in significant activation of apoptosis, as observed morphologically and biochemically by detecting the activation of caspase-3, 7 and the cleavage of PARP. Hsp90 inhibition by PU-H71 in DLBCL was associated with the destabilization and subsequent degradation of several onco-proteins such as Akt, c-Raf and IKK/Nemo, thus affecting the activity of many oncogenic pathways. To evaluate the in vivo anti-lymphoma effect of PU-H71, OCI-Ly7, Farage and SUDHL4 xenografted tumors were established in SCID mice. Mice received either 75mg/kg/day of PU-H71 or vehicle (water) (n=5 per treatment and per cell line). By day 10, the tumors in the PU-H71 treated mice were significantly smaller in volume than their respective controls, with 76%, 95% and 95% inhibition of tumor growth observed in Farage, OCI-Ly7 and SUDHL4 mice, respectively (p=0.002, p<0.0001and p=0.0002, respectively). The tumor weight at day 10 was also reduced by PU-H71, as were the serum levels of the DLBCL surrogate marker human B-2-microglobulin. There was also a significant increase in the survival time of the PU-H71 treated mice (n=15) compared to the control treated mice (n=15) (Kaplan-Meier survival curve, Cox’s F test p<0.0001). No toxicity was observed during treatment as evidenced by a lack of significant change in animal weight, fur appearance, appetite and posture. Furthermore, no visible internal organ damage was detected at sacrifice upon gross inspection and histological examination. Additional toxicity studies in normal mice which also included biochemical panels and CBC, confirmed these results. There were no abnormalities in liver enzymes levels nor was the appearance of gastrointestinal mucositis observed in PU-H71 treated mice, in contrast to previous reports with the benzoquinone Hsp90 inhibitors, 17-AAG and 17-DMAG. The serum and tissues of Farage xenografts were analyzed for PU-H71 concentration by HPLC-MS. Pharmacologically relevant doses of PUH71 were found retained in tumors even at 24 h post-administration (42.1 ug/g at 6 h, 27.6 ug/g at 12 h and 12 ug/g at 24 h). In contrast, the levels of PU-H71 in normal tissues rapidily dropped at 12 h post administration and were at all-times bellow the level reached in tumors (at 12 h the tumor concentration of PU-H71 was between 11.2 and 125.6 times higher than in normal tissues). This indicates that PU-H71 is preferentially retained in DLBCL compared to normal tissues, partly explaining the lack of toxicity observed at highly efficient therapeutic doses of PU-H71. Its preferential tumor retention could also be observed by PET in living animals using a radiolabeled PU-H71. Due to its potent anti-tumor activity and favorable toxicity profile, PU-H71 is undergoing late-stage IND evaluation and is scheduled to enter Phase I clinical evaluation in patients with lymphomas in 2009.
Mitochondrial perturbation reduces susceptibility to xenobiotics through altered efflux in Candida albicans
