Erlichman, Charles

Abstract Despite studies that show the antitumor activity of Hsp90 inhibitors, such as geldanamycin (GA) and its derivative 17-allylamino-demethoxygeldanamycin (17-AAG), recent reports indicate that these inhibitors lack significant single-agent clinical activity. Resistance to Hsp90 inhibitors has been previously linked to expression of P-glycoprotein (P-gp) and the multidrug resistant (MDR) phenotype. However, the stress response induced by GA treatment can also cause resistance to Hsp90-targeted therapy. Therefore, we chose to further investigate the relative importance of P-gp and the stress response in 17-AAG resistance. Colony-forming assays revealed that high expression of P-gp could increase the 17-AAG IC50 6-fold in cells transfected with P-gp compared with parent cells. A549 cells selected for resistance to GA overexpressed P-gp, but verapamil did not reverse the resistance. These cells also overexpressed Hsp27, and Hsp70 was induced with 17-AAG treatment. When the GA and 17-AAG resistant cells were transfected with Hsp27 and/or Hsp70 small interfering RNA (siRNA), the 17-AAG IC50 decreased 10-fold compared with control transfected cells. Transfection with siRNA directed against Hsp27, Hsp70, or Hsp27 and Hsp70 also increased sensitivity to EC78, a purine scaffold-based Hsp90 inhibitor that is not a P-gp substrate. We conclude that P-gp may contribute, in part, to resistance to 17-AAG, but induction of stress response proteins, such as Hsp27 and Hsp70, by Hsp90-targeted therapy plays a larger role. Taken together, our results indicate that targeting of Hsp27 and Hsp70 should be exploited to increase the clinical efficacy of Hsp90-directed therapy. [Cancer Res 2008;68(18):7419–27]

Abstract We have reported previously the activity of the insulin-like growth factor-I (IGF-IR)/insulin receptor (InsR) inhibitor, BMS-554417, in breast and ovarian cancer cell lines. Further studies indicated treatment of OV202 ovarian cancer cells with BMS-554417 increased phosphorylation of HER-2. In addition, treatment with the pan-HER inhibitor, BMS-599626, resulted in increased phosphorylation of IGF-IR, suggesting a reciprocal cross-talk mechanism. In a panel of five ovarian cancer cell lines, simultaneous treatment with the IGF-IR/InsR inhibitor, BMS-536924 and BMS-599626, resulted in a synergistic antiproliferative effect. Furthermore, combination therapy decreased AKT and extracellular signal-regulated kinase activation and increased biochemical and nuclear morphologic changes consistent with apoptosis compared with either agent alone. In response to treatment with BMS-536924, increased expression and activation of various members of the HER family of receptors were seen in all five ovarian cancer cell lines, suggesting that inhibition of IGF-IR/InsR results in adaptive up-regulation of the HER pathway. Using MCF-7 breast cancer cell variants that overexpressed HER-1 or HER-2, we then tested the hypothesis that HER receptor expression is sufficient to confer resistance to IGF-IR-targeted therapy. In the presence of activating ligands epidermal growth factor or heregulin, respectively, MCF-7 cells expressing HER-1 or HER-2 were resistant to BMS-536924 as determined in a proliferation and clonogenic assay. These data suggested that simultaneous treatment with inhibitors of the IGF-I and HER family of receptors may be an effective strategy for clinical investigations of IGF-IR inhibitors in breast and ovarian cancer and that targeting HER-1 and HER-2 may overcome clinical resistance to IGF-IR inhibitors. [Mol Cancer Ther 2008;7(9):2589–98]

Abstract Purpose: To determine the maximum tolerated dose (MTD), characterize the principal toxicities, and assess the pharmacokinetics of EKB-569, an oral selective irreversible inhibitor of the epidermal growth factor receptor tyrosine kinase, in combination with capecitabine in patients with advanced colorectal cancer. Experimental Design: Patients were treated with EKB-569 daily for 21 days and capecitabine twice daily for 14 days of a 21-day cycle. The dose levels of EKB-569 (mg/day) and capecitabine (mg/m2 twice daily) assessed were 25/750, 50/750, 50/1,000 and 75/1,000. An expanded cohort was enrolled at the MTD to better study toxicity and efficacy. Samples of plasma were collected to characterize the pharmacokinetics of the agents. Treatment efficacy was assessed every other cycle. Results: A total of 37 patients, the majority of whom had prior chemotherapy, received a total of 163 cycles of treatment. Twenty patients were treated at the MTD, 50 mg EKB-569, daily and 1,000 mg/m2 capecitabine twice daily. Dose-limiting toxicities were diarrhea and rash. No patients had complete or partial responses but 48% had stable disease. The conversion of capecitabine to 5-fluorouracil was higher for the combination of EKB-569 and capecitabine (321 ± 151 ng*h/mL) than for capecitabine alone (176 ± 62 ng*hours/mL; P = 0.0037). Conclusion: In advanced colorectal cancer, 50 mg EKB-569 daily can be safely combined with 1,000 mg/m2 capecitabine twice a day. A statistically significant increase in plasma levels of 5-fluorouracil for the combination of EKB-569 and capecitabine may be due to the single-dose versus multiple-dose exposure difference, variability in exposure or a potential drug interaction.

Abstract 17-Allylamino-demethoxygeldanamycin (17-AAG), currently in phase I and II clinical trials as an anticancer agent, binds to the ATP pocket of heat shock protein (Hsp90). This binding induces a cellular stress response that up-regulates many proteins including Hsp27, a member of the small heat shock protein family that has cytoprotective roles, including chaperoning of cellular proteins, regulation of apoptotic signaling, and modulation of oxidative stress. Therefore, we hypothesized that Hsp27 expression may affect cancer cell sensitivity to 17-AAG. In colony-forming assays, overexpression of Hsp27 increased cell resistance to 17-AAG whereas down-regulation of Hsp27 by siRNA increased sensitivity. Because Hsp27 is known to modulate levels of glutathione (GSH), we examined cellular levels of GSH and found that it was decreased in cells transfected with Hsp27 siRNA when compared with control siRNA. Treatment with buthionine sulfoximine, an inhibitor of GSH synthesis, also sensitized cells to 17-AAG. Conversely, treatment of Hsp27 siRNA–transfected cells with N-acetylcysteine, an antioxidant and GSH precursor, reversed their sensitivity to 17-AAG. A cell line selected for stable resistance to geldanamycin relative to parent cells showed increased Hsp27 expression. When these geldanamycin- and 17-AAG-resistant cells were transfected with Hsp27 siRNA, 17-AAG resistance was dramatically diminished. Our results suggest that Hsp27 up-regulation has a significant role in 17-AAG resistance, which may be mediated in part through GSH regulation. Clinical modulation of GSH may therefore enhance the efficacy of Hsp90-directed therapy. (Cancer Res 2006; 66(22): 10967-75)

Abstract Purpose: Rituximab is a chimeric antibody that induces B-cell apoptosis and recruits immune effector cells to mediate cell lysis. Interleukin-12 (IL-12) facilitates cytolytic responses by T cells and natural killer cells. This phase II study was done to determine the efficacy and toxicity of IL-12 in combination with rituximab in patients with B-cell non-Hodgkin's lymphoma (NHL). Experimental Design: Fifty-eight patients with histologically confirmed relapsed B-cell NHL were randomized to receive concurrent treatment with rituximab and IL-12 (arm A) or rituximab with subsequent treatment with IL-12 after documented nonresponse or progression after rituximab (arm B). Treatment consisted of 375 mg/m2 rituximab on days 1, 8, 15, and 22 and 300 ng/kg IL-12 given s.c. twice weekly starting on day 2 for arm A or upon progression for arm B. Results: The overall response rate was 37% (11 of 30) in arm A and 52% (13 of 25) in arm B. All of the responses seen in arm B occurred while patients received rituximab, and no responses occurred during treatment with subsequent IL-12. The median duration of response was 16 months for arm A and 12 months for arm B. Biopsy specimens were serially obtained in a subset of patients and showed that changes in gene expression were different when cells from the peripheral blood were compared with cells from lymph node biopsies. Conclusions: The concomitant use of IL-12 and rituximab had modest disease activity in patients with B-cell NHL, but the sequential administration of IL-12 after rituximab did not result in additional clinical responses.

Abstract Purpose: To determine the maximum tolerated dose (MTD), dose-limiting toxicity, and pharmacokinetics of 17-allylamino-demethoxy-geldanamycin (17-AAG) administered on days 1, 4, 8, and 11 every 21 days and to examine the effect of 17-AAG on the levels of chaperone and client proteins. Experimental Design: A phase I dose escalating trial in patients with advanced solid tumors was done. Toxicity and tumor responses were evaluated by standard criteria. Pharmacokinetics were done and level of target proteins was measured at various points during cycle one. Results: Thirteen patients were enrolled in the study. MTD was defined as 220 mg/m2. Dose-limiting toxicities were as follows: dehydration, diarrhea, hyperglycemia, and liver toxicity. At the MTD, the mean clearance of 17-AAG was 18.7 L/h/m2. There was a significant decrease in integrin-linked kinase at 6 hours after infusion on day 1 but not at 25 hours in peripheral blood mononuclear cells. Treatment with 17-AAG on day 1 significantly increased pretreatment levels of heat shock protein (HSP) 70 on day 4, which is consistent with the induction of a stress response. In vitro induction of a stress response and up-regulation of HSP70 resulted in an increased resistance to HSP90-targeted therapy in A549 cells. Conclusions: The MTD of 17-AAG on a twice-weekly schedule was 220 mg/m2. Treatment at this dose level resulted in significant changes of target proteins and also resulted in a prolonged increase in HSP70. This raises the possibility that HSP70 induction as part of the stress response may contribute to resistance to 17-AAG.

Abstract Purpose: Patients with advanced cancer received temsirolimus (Torisel, CCI-779), a novel inhibitor of mammalian target of rapamycin, i.v. once daily for 5 days every 2 weeks to determine the maximum tolerated dose, toxicity profile, pharmacokinetics, and preliminary antitumor efficacy. Experimental Design: Doses were escalated in successive cohorts of patients using a conventional phase I clinical trial design. Samples of whole blood and plasma were collected to determine the pharmacokinetics of temsirolimus and sirolimus, its principal metabolite. Results: Sixty-three patients were treated with temsirolimus (0.75-24 mg/m2/d). The most common drug-related toxicities were asthenia, mucositis, nausea, and cutaneous toxicity. The maximum tolerated dose was 15 mg/m2/d for patients with extensive prior treatment because, in the 19 mg/m2/d cohort, two patients had dose-limiting toxicities (one with grade 3 vomiting, diarrhea, and asthenia and one with elevated transaminases) and three patients required dose reductions. For minimally pretreated patients, in the 24 mg/m2/d cohort, one patient developed a dose-limiting toxicity of grade 3 stomatitis and two patients required dose reductions, establishing 19 mg/m2/d as the maximum acceptable dose. Immunologic studies did not show any consistent trend toward immunosuppression. Temsirolimus exposure increased with dose in a less than proportional manner. Terminal half-life was 13 to 25 hours. Sirolimus-to-temsirolimus exposure ratios were 0.6 to 1.8. A patient with non–small cell lung cancer achieved a confirmed partial response, which lasted for 12.7 months. Three patients had unconfirmed partial responses; two patients had stable disease for ≥24 weeks. Conclusion: Temsirolimus was generally well tolerated on this intermittent schedule. Encouraging preliminary antitumor activity was observed.

Purpose Epidermal growth factor receptor/human epidermal growth factor receptor 1 and ligand expression is common in biliary cancers (BILI) and may be associated with worse outcome. The primary objective of this study was to determine the proportion of patients with advanced BILI who were progression-free at 6 months. Methods Patients with either unresectable or metastatic disease were studied. Only one prior systemic or locoregional therapy was allowed. Erlotinib was administered continuously at a dose of 150 mg per day orally. Results Forty-two patients with BILI were enrolled. The median age was 67 years (range, 33 to 82 years). Fifty-two percent of patients had Eastern Cooperative Oncology Group performance status of 1. Fifty-seven percent of patients had received prior chemotherapy for advanced BILI. HER1/EGFR expression by immunohistochemistry in tumor cells was detected in 29 (81%) of the 36 assessable patients. Seven of the patients (17%; 95% CI, 7% to 31%) were progression free at 6 months. Three patients had partial response by Response Evaluation Criteria in Solid Tumors Group classification of duration 4, 4, and 14 months, respectively. All responding patients had mild (grade 1/2) skin rash and two patients had positive tumoral HER1/EGFR expression. Three patients (7%) had toxicity-related dose reductions of erlotinib due to grade 2/3 skin rash. Conclusion Results suggest a therapeutic benefit for EGFR blockade with erlotinib in patients with biliary cancer. Additional studies with erlotinib as a single agent and in combination with other targeted agents are warranted in this disease.

Purpose The maximum tolerated dose (MTD) and the dose-limiting toxicities of EKB-569, a selective, irreversible inhibitor of the epidermal growth factor receptor (EGFR), when administered orally once daily on an intermittent-dose schedule (14 days of a 28-day cycle) or on a continuous-dose schedule (each day of a 28-day cycle), were determined in patients with advanced solid tumors. Patients and Methods Planned dose escalation was 25, 50, 75, 125, 175, and 225 mg. Pharmacokinetic sampling was performed on days 1 and 14 for the intermittent-dose cohort and on days 1 and 15 for the continuous-dose cohort. Results Thirty patients received a median of two cycles (range, one to 10 cycles) in the intermittent-dose cohort; 29 patients received a median of three cycles (range, one to eight cycles) in the continuous-dose cohort. Dose-limiting toxicity was grade 3 diarrhea, and the MTD was 75 mg EKB-569 per day for both cohorts. Other common toxicities included rash, nausea, and asthenia. Exposure to EKB-569 was dose proportional. At the MTD, the mean ± standard deviation terminal half-life was 21.7 ± 4.2 hours and peak concentration increased 1.2-fold from day 1 to day 14/15. No major antitumor responses were observed. However, one patient with non–small-cell lung cancer and one with cutaneous squamous cell carcinoma had stable disease for 33 and 24 weeks, respectively. Conclusion The MTD of once-daily oral EKB-569 is 75 mg. The tolerable toxicity profile and long half-life of this irreversible EGFR inhibitor warrant its further evaluation as a single agent and in combination with other drugs.