Chemical Genetics Identify eIF2α Kinase Heme Regulated Inhibitor as Anti-Cancer Target

Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2·GTP·Met-tRNAi translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemia. The chemical modifiers of the eIF2·GTP·Met-tRNAi ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining if this complex can be pharmacologically targeted for therapeutic purposes. Using a cell based assay, we identified N,N’-diarylureas as novel inhibitors of the ternary complex abundance. Direct functional-genetics and biochemical evidence demonstrated that the N,N’-diarylureas activate heme regulated inhibitor kinase, thereby phosphorylate eIF2α and reduce abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N’-diarylureas are potent and specific tools for studying the role eIF2·GTP·Met-tRNAi ternary complex in the pathobiology of human disorders

Tumor suppression by small molecule inhibitors of translation initiation

Translation initiation factors are over-expressed and/or activated in many human cancers and may contribute to their genesis and/or progression. Removal of physiologic restraints on translation initiation causes malignant transformation. Conversely, restoration of physiological restrains on translation initiation reverts malignant phenotypes. Here, we extensively characterize the anti-cancer activity of two small molecule inhibitors of translation initiation: #1181, which targets the eIF2-GTP-Met-tRNAi ternary complex, and 4EGI-1, which targets the eIF4F complex. In vitro, both molecules inhibit translation initiation, abrogate preferentially translation of mRNAs coding for oncogenic proteins, and inhibit proliferation of human cancer cells. In vivo, both #1181 and 4EGI-1 strongly inhibit growth of human breast and melanoma cancer xenografts without any apparent macroscopic- or microscopic-toxicity. Mechanistically, #1181 phosphorylates eIF2α while 4EGI-1 disrupts eIF4G/eIF4E interaction in the tumors excised from mice treated with these agents. These data indicate that inhibition of translation initiation is a new paradigm in cancer therapy

A phase I/II trial of hydroxychloroquine in conjunction with radiation therapy and concurrent and adjuvant temozolomide in patients with newly diagnosed glioblastoma multiforme

Preclinical studies indicate autophagy inhibition with hydroxychloroquine (HCQ) can augment the efficacy of DNA-damaging therapy. The primary objective of this trial was to determine the maximum tolerated dose (MTD) and efficacy of HCQ in combination with radiation therapy (RT) and temozolomide (TMZ) for newly diagnosed glioblastoma (GB). A 3 + 3 phase I trial design followed by a noncomparative phase II study was conducted in GB patients after initial resection. Patients received HCQ (200 to 800 mg oral daily) with RT and concurrent and adjuvant TMZ. Quantitative electron microscopy and immunoblotting were used to assess changes in autophagic vacuoles (AVs) in peripheral blood mononuclear cells (PBMC). Population pharmacokinetic (PK) modeling enabled PK-pharmacodynamic correlations. Sixteen phase I subjects were evaluable for dose-limiting toxicities. At 800 mg HCQ/d, 3/3 subjects experienced Grade 3 and 4 neutropenia and thrombocytopenia, 1 with sepsis. HCQ 600 mg/d was found to be the MTD in this combination. The phase II cohort (n = 76) had a median survival of 15.6 mos with survival rates at 12, 18, and 24 mo of 70%, 36%, and 25%. PK analysis indicated dose-proportional exposure for HCQ. Significant therapy-associated increases in AV and LC3-II were observed in PBMC and correlated with higher HCQ exposure. These data establish that autophagy inhibition is achievable with HCQ, but dose-limiting toxicity prevented escalation to higher doses of HCQ. At HCQ 600 mg/d, autophagy inhibition was not consistently achieved in patients treated with this regimen, and no significant improvement in overall survival was observed. Therefore, a definitive test of the role of autophagy inhibition in the adjuvant setting for glioma patients awaits the development of lower-toxicity compounds that can achieve more consistent inhibition of autophagy than HCQ

Development and characterization of a novel C-terminal inhibitor of Hsp90 in androgen dependent and independent prostate cancer cells

Background: The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells.Methods: PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies.Results: KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model.Conclusions: Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.Peer reviewedBiochemistry and Molecular Biolog

Effect of phenytoin on celecoxib pharmacokinetics in patients with glioblastoma

Cyclooxygenase-2 (COX-2) expression has been linked to the prognosis, angiogenesis, and radiation sensitivity of many malignancies. Celecoxib, a selective COX-2 inhibitor, is predominantly eliminated by hepatic metabolism. This study was conducted to determine the effects of hepatic enzyme-inducing antiseizure drugs (EIASDs) on the pharmacokinetics of celecoxib. The safety of celecoxib administered with radiation for glioblastoma and the effect of the combined treatment on survival were also evaluated. Patients were stratified based on concomitant use of EIASDs. Celecoxib (400) mg was administered orally twice a day until tumor progression or dose-limiting toxicity. Standard radiation was administered without adjuvant chemotherapy. Sampling was performed to define the plasma concentration/time profile for the initial dose of celecoxib and steady-state trough concentrations. Thirty-five patients (22 +EIASD, 13 −EIASD) were enrolled. There were no significant differences in age, performance status, extent of surgery, or Mini Mental State Exam scores between the two cohorts. The treatment was well tolerated. All patients in the +EIASD arm were taking phenytoin. There were no significant differences in any celecoxib pharmacokinetic parameters between 15 +EIASD and 12 −EIASD patients. With 31 of 35 patients deceased, estimated median survival time for all patients was 12 months (+EIASD, 11.5 months; − EIASD, 16 months; p = 0.11). The pharmacokinetics of celecoxib is not significantly affected by the concomitant administration of phenytoin. Celecoxib administered during and after radiation is well tolerated. The potential difference in survival between the +EIASD and −EIASD groups deserves further evaluation

Phase I and pharmacokinetic study of karenitecin in patients with recurrent malignant gliomas

Karenitecin is a highly lipophilic camptothecin analogue with a lactone ring that is relatively resistant to inactivating hydrolysis under physiologic conditions. This phase I clinical trial was conducted to determine the maximum tolerated dose (MTD) of karenitecin in adults with recurrent malignant glioma (MG), to describe the effects of enzyme-inducing antiseizure drugs (EIASDs) on its pharmacokinetics, and to obtain preliminary evidence of activity. Karenitecin was administered intravenously over 60 min daily for 5 consecutive days every 3 weeks to adults with recurrent MG who had no more than one prior chemotherapy regimen. The continual reassessment method was used to escalate doses, beginning at 1.0 mg/m2/day, in patients stratified by EIASD use. Treatment was continued until disease progression or treatment-related dose-limiting toxicity (DLT). Plasma pharmacokinetics was determined for the first daily dose of karenitecin. Thirty-two patients (median age, 52 years; median KPS score, 90) were accrued. Seventy-eight percent had glioblastoma, and 22% had anaplastic glioma. DLT was reversible neutropenia or thrombocytopenia. The MTD was 2.0 mg/m2 in +EIASD patients and 1.5 mg/m2 in −EIASD patients. The mean (±SD) total body clearance of karenitecin was 15.9 ± 9.6 liters/h/m2 in +EIASD patients and 10.2 ± 3.5 liters/h/m2 in −EIASD patients (p = 0.02). No objective responses were observed in 11 patients treated at or above the MTD. The total body clearance of karenitecin is significantly enhanced by the concurrent administration of EIASDs. This schedule of karenitecin, a novel lipophilic camptothecin analogue, has little activity in recurrent MG

Efficacy and toxicity of the antisense oligonucleotide aprinocarsen directed against protein kinase C-α delivered as a 21-day continuous intravenous infusion in patients with recurrent high-grade astrocytomas1, 2

Protein kinase C alpha (PKC-α) is a cytoplasmic serine threonine kinase involved in regulating cell differentiation and proliferation. Aprinocarsen is an antisense oligonucleotide against PKC-α that reduces PKC-α in human cell lines and inhibits a human glioblastoma tumor cell line in athymic mice. In this phase 2 study, aprinocarsen was administered to patients with recurrent high-grade gliomas by continuous intravenous infusion (2.0 mg/kg/day for 21 days per month). Twenty-one patients entered this trial. Their median age was 46 years (range, 28–68 years), median Karnofsky performance status was 80 (range, 60–100), median tumor volume was 58 cm3 (range, 16–254 cm3), and histology included glioblastoma multiforme (n = 16), anaplastic oligodendroglioma (n = 4), and anaplastic astrocytoma (n = 1). The number of prior chemotherapy regimens included none (n = 3), one (n = 10), and two (n = 8). No tumor responses were observed. Patients on this therapy rapidly developed symptoms of increased intracranial pressure with increased edema, enhancement, and mass effect on neuroimaging. The median time to progression was 36 days, and median survival was 3.4 months. The observed toxicities were mild, reversible, and uncommon (grade 3 thrombocytopenia [n = 3] and grade 4 AST [n = 1]), and no coagulopathy or CNS bleeding resulted from this therapy. Plasma concentrations of aprinocarsen during the infusion exhibited significant interpatient variability (mean = 1.06 μg/ml; range, 0.34–6.08 μg/ml). This is the first study to use an antisense oligonucleotide or a specific PKC-α inhibitor in patients with high-grade gliomas. No clinical benefit was seen. The rapid deterioration seen in these patients could result from tumor growth or an effect of aprinocarsen on blood-brain barrier integrity

Chemical genetics identify eIF2α kinase heme-regulated inhibitor as an anticancer target

Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2–GTP–tRNAi Met translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemias. The chemical modifiers of the eIF2–GTP–tRNAi Met ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining whether this complex can be pharmacologically targeted for therapeutic purposes. Using a cell-based assay, we identified N,N9-diarylureas as unique inhibitors of ternary complex accumulation. Direct functional-genetic and biochemical evidence demonstrated that the N,N9-diarylureas activate heme-regulated inhibitor kinase, thereby phosphorylating eIF2a and reducing the abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N9-diarylureas are potent and specific tools for studying the role of eIF2–GTP–tRNAi Met ternary complex in the pathobiology of human disorders.</p