Ram Ganapathi

Redefining the relevance of established cancer cell lines to the study of mechanisms of clinical anti-cancer drug resistance

Although in vitro models have been a cornerstone of anti-cancer drug development, their direct applicability to clinical cancer research has been uncertain. Using a state-of-the-art Taqman-based quantitative RT-PCR assay, we investigated the multidrug resistance (MDR) transcriptome of six cancer types, in established cancer cell lines (grown in monolayer, 3D scaffold, or in xenograft) and clinical samples, either containing >75% tumor cells or microdissected. The MDR transcriptome was determined a priori based on an extensive curation of the literature published during the last three decades, which led to the enumeration of 380 genes. No correlation was found between clinical samples and established cancer cell lines. As expected, we found up-regulation of genes that would facilitate survival across all cultured cancer cell lines evaluated. More troubling, however, were data showing that all of the cell lines, grown either in vitro or in vivo, bear more resemblance to each other, regardless of the tissue of origin, than to the clinical samples they are supposed to model. Although cultured cells can be used to study many aspects of cancer biology and response of cells to drugs, this study emphasizes the necessity for new in vitro cancer models and the use of primary tumor models in which gene expression can be manipulated and small molecules tested in a setting that more closely mimics the in vivo cancer microenvironment so as to avoid radical changes in gene expression profiles brought on by extended periods of cell culture.

von Hippel-Lindau Gene Status and Response to Vascular Endothelial Growth Factor Targeted Therapy for Metastatic Clear Cell Renal Cell Carcinoma

PURPOSE The von Hippel-Lindau (VHL) gene is often inactivated (by mutation or promoter hypermethylation) in renal cell carcinoma but the relation to therapeutic outcome is unclear. MATERIALS AND METHODS Patients with metastatic clear cell renal cell carcinoma with available baseline tumor samples who received vascular endothelial growth factor targeted therapy were included in analysis. Patient characteristics, VHL gene status and clinical outcome were documented. Our primary end point was to test for response rate in relation to VHL inactivation. Progression-free survival and overall survival in relation to VHL status were investigated as secondary end points. RESULTS A total of 123 patients were evaluable. Response rate, median progression-free survival and median overall survival were 37% (95% CI 28-46), 10.8 (95% CI 7.7-14.8) and 29.8 (CI not estimable) months, respectively. Patients with VHL inactivation had a response rate of 41% vs 31% for those with wild-type VHL (p = 0.34). Patients with loss of function mutations (frameshift, nonsense, splice and in-frame deletions/insertions) had a 52% response rate vs 31% with wild-type VHL (p = 0.04). On multivariate analysis the presence of a loss of function mutation remained an independent prognostic factor associated with improved response. Progression-free survival and overall survival were not significantly different based on VHL status. CONCLUSIONS To our knowledge this is the largest analysis investigating the impact of VHL inactivation on the outcome of vascular endothelial growth factor targeted agents in metastatic renal cell carcinoma. We did not find a statistically significant increase in response to vascular endothelial growth factor targeted agents in patients with VHL inactivation. Loss of function mutations identified a population of patients with a greater response. Investigation of downstream markers is under way.

Clinical modulation of doxorubicin resistance by the calmodulin-inhibitor, trifluoperazine: a phase I/II trial.

Drug resistance to chemotherapy agents such as doxorubicin appears to be an important cause of therapeutic failure in cancer treatment. Based on preclinical information demonstrating that the phenothiazine calmodulin-inhibitor trifluoperazine can enhance retention and cytotoxicity of doxorubicin in resistant cells, a phase I/II trial of the combination was performed to determine the maximally tolerated dose (MTD) of trifluoperazine that could be administered with doxorubicin. Patients with intrinsic (no previous response) and acquired (previous response with relapse) doxorubicin resistance were eligible. Doxorubicin was administered as a 96-hour continuous infusion (60 mg/m2) on days 2 through 5. Trifluoperazine was administered in divided doses orally on days 1 through 6, with dose escalation from 20 to 100 mg/d. Thirty-six patients were evaluable. The MTD of trifluoperazine was 60 mg/d, with dose-limiting toxicity being extrapyramidal side effects. No alteration of doxorubicin toxicity was observed. Seven of the 36 patients responded (one complete response [CR], six partial responses [PR]), with seven of 21 patients having acquired resistance, and zero of 15 with intrinsic resistance demonstrating responses. Doxorubicin plasma levels were not affected by trifluoperazine, and the maximal trifluoperazine plasma levels achieved were 129.83 ng/mL. This trial demonstrates the combination of trifluoperazine and doxorubicin is well tolerated, and the schedule recommended for phase II trials is doxorubicin, 60 mg/m2 (continuous infusion) days 2 through 5, and trifluoperazine, 15 mg four times per day orally days 1 through 6. Continued investigation of this combination is indicated for patients with acquired doxorubicin resistance.

Clear cell tubulopapillary renal cell carcinoma: A study of 36 distinctive low-grade epithelial tumors of the kidney

Recently several low-grade renal cell tumors, distinct from those recognized by the 2004 World Health Organization classification of renal tumors, have been described. These tumors had similar clinicopathologic features, being low-stage tumors with cystic, tubuloacinar, and/or papillary architecture. The tumor cells were low grade with variable amounts of clear cytoplasm that was positive for cytokeratin 7 (CK7), but negative for CD10. Genetic changes characteristic of clear cell or papillary renal cell carcinoma were not seen in these tumors. We investigated the morphologic, immunohistochemical, and genetic features of 36 additional tumors. Immunohistochemistry was carried out for CK7, carbonic anhydrase 9, α-methylacyl-CoA racemase, CD10, TFE-3, and desmin. Interphase fluorescence in situ hybridization was carried out with centromeric probes for chromosomes 3, 7, 17, and a subtelomeric probe for 3p25. Sequencing of von Hippel-Lindau gene and analysis of the methylation status of the promoter region was also carried out in 2 tumors. Thirty-six tumors from 33 patients (mean age: 60.4 , range: 26 to 88; 17 men and 16 women) were studied. Three patients had bilateral tumors and 1 patient had von Hippel-Lindau disease. Follow-up was available in 60% (20/33) of the patients for a mean of 27.4 (range 1 to 85) months. No patient had evidence of the disease after surgery except for the patient with von Hippel-Lindau disease, who was alive with stable disease in the contralateral kidney. All 36 tumors were small (mean size 2.4 cm; range 0.9 to 4.5 cm) and low stage (pT1). The majority was cystic and had prominent fibrous capsule and stroma. The tumors were composed of variable amount of cysts, papillae, tubules, acini, and solid nests. The most characteristic histologic features were branching tubules and acini and anastomosing clear cell ribbons with low-grade nuclei. All tumors were strongly positive for CK7 and variably positive for CA9, but largely negative for CD10, and negative for α-methylacyl-CoA racemase and TFE-3. All but 1 tumor had no gains of chromosomes 7 and 17 and deletion of 3p. Only 1 tumor had low copy number gains of chromosomes 7 and 17. VHL gene mutation and promoter methylation were negative in 2 tumors analyzed. We show that these tumors, which we term as “clear cell tubulopapillary renal cell carcinoma,” constitute a unique subtype in the spectrum of renal epithelial neoplasia based on their characteristic morphologic and immunohistochemical features.

Association of VEGF and VEGFR2 single nucleotide polymorphisms with hypertension and clinical outcome in metastatic clear cell renal cell carcinoma patients treated with sunitinib

Biomarkers that predict response or toxicity to antiangiogenic therapy are sought to favorably inform the risk/benefit ratio. This study evaluated the association of vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2) genetic polymorphisms with the development of hypertension (HTN) and clinical outcome in metastatic clear cell renal cell carcinoma (MCCRCC) patients treated with sunitinib.

A phase I dose-escalation study of Tivantinib (ARQ 197) in adult patients with metastatic solid tumors.

Background: Tivantinib, an oral, non-ATP competitive, selective c-MET inhibitor, exhibited antitumor activity in preclinical models. This open-label, phase I, dose-escalation study evaluated the safety, tolerability, pharmacokinetics, and pharmacodynamics of tivantinib in patients with advanced or metastatic solid tumors refractory to standard therapy. Methods: Thirteen dose levels of tivantinib ranging from 10 to 360 mg twice a day were administered to patient cohorts in 21-day cycles (14 days on/7 days off); three active pharmaceutical ingredient forms of tivantinib (amorphous, crystalline A, and crystalline B) were also investigated. Treatment was continued until the occurrence of unacceptable toxicity, tumor progression, patient withdrawal, or death. Results: A total of 79 patients with advanced solid tumors were enrolled. A maximum tolerated dose was not determined. Tivantinib was well tolerated, with mild to moderate toxicities. Two patients discontinued the study drug due to treatment-emergent adverse events. Dose-limiting grade of 3 or more toxicities including leukopenia, neutropenia, thrombocytopenia, vomiting, and dehydration, were observed in 2 patients treated with tivantinib 360 mg twice a day. The rate of absorption of tivantinib peaked approximately 2 to 4 hours after initial dosing, followed by a linear decrease in plasma concentrations. Increases in tivantinib exposure were not dose proportional. There was significant interpatient pharmacokinetic variability; however the clinical safety of tivantinib seemed unaffected. Three patients (3.8%) achieved a partial response and 40 patients (50.6%) maintained stable disease for a median of 19.9 weeks. Conclusions: Tivantinib 360 mg twice a day was well tolerated in patients with refractory advanced solid tumors. The results of this trial warrant further clinical investigation. Clin Cancer Res; 17(24); 7754–64. ©2011 AACR.

A phase I clinical trial of a ribozyme-based angiogenesis inhibitor targeting vascular endothelial growth factor receptor-1 for patients with refractory solid tumors

Purpose: This study intended to determine the maximum tolerated dose, safety, pharmacokinetic variables, clinical response, and pharmacodynamic markers of daily s.c. administration of Angiozyme. Patients and Methods: Patients with refractory solid tumors were enrolled in a dose escalation and expanded cohort design. Dose escalation involved cohorts of patients at doses of 10, 30, 100, or 300 mg/m2/d for 29 days. A second component enrolled 15 additional patients at a daily dose of 100 mg/m2. Patients were eligible to continue on therapy until disease progression. Results: Thirty-one patients were enrolled and 28 were evaluable (range, 29–505 days; median, 89.5 days). A maximum tolerated dose was not defined by toxicity but rather by the maximal deliverable dose of 300 mg/m2/d. Grade 1 to 2 injection site reactions were the most common toxicities. One patient in the 300 mg/m2 group experienced a reversible grade 3 injection site reaction. Angiozyme showed dose-dependent plasma concentrations with good bioavailability. Surrogate markers showed Angiozyme localization in tumor biopsies and a significant increase in serum von Willebrand factor antigen, a marker for endothelial cell dysfunction. Although Angiozyme-reactive antibody production was noted for some patients, no antibody-related adverse events were noted. Seven of 28 (25%) evaluable patients had stable disease for ≥6 months, with the longest treatment duration of ≥16 months. Two patients (nasopharyngeal carcinoma and melanoma) showed minor responses. Conclusion: Angiozyme was well tolerated with satisfactory pharmacokinetic variables for daily s.c. dosing. Results have provided the basis for subsequent clinical trials of this first-of-class biologically targeted therapeutic.

HPC1/RNASEL Mediates Apoptosis of Prostate Cancer Cells Treated with 2′,5′-Oligoadenylates, Topoisomerase I Inhibitors, and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

The hereditary prostate cancer 1 (HPC1) allele maps to the RNASEL gene encoding a protein (RNase L) implicated in the antiviral activity of interferons. To investigate the possible role of RNase L in apoptosis of prostate cancer cells, we decreased levels of RNase L by severalfold in the DU145 human prostate cancer cell line through the stable expression of a small interfering RNA (siRNA). Control cells expressed siRNA with three mismatched nucleotides to the RNase L sequence. Cells deficient in RNase L, but not the control cells, were highly resistant to apoptosis by the RNase L activator, 2′,5′-oligoadenylate (2-5A). Surprisingly, the RNase L-deficient cells were also highly resistant to apoptosis by combination treatments with a topoisomerase (Topo) I inhibitor (camptothecin, topotecan, or SN-38) and tumor necrosis factor-related apoptosis-inducing ligand [TRAIL (Apo2L)]. In contrast, cells expressing siRNA to the RNase L inhibitor RLI (HP68) showed enhanced apoptosis in response to Topo I inhibitor alone or in combination with TRAIL. An inhibitor of c-Jun NH2-terminal kinases reduced apoptosis induced by treatment with either 2-5A or the combination of camptothecin and TRAIL, thus implicating c-Jun NH2-terminal kinase in the apoptotic signaling pathway. Furthermore, prostate cancer cells were sensitive to apoptosis from the combination of 2-5A with either TRAIL or Topo I inhibitor, whereas normal prostate epithelial cells were partially resistant to apoptosis. These findings indicate that RNase L integrates and amplifies apoptotic signals generated during treatment of prostate cancer cells with 2-5A, Topo I inhibitors, and TRAIL.

Correlation between potency of calmodulin inhibitors and effects on cellular levels and cytotoxic activity of doxorubicin (adriamycin) in resistant P388 mouse leukemia cells.

The relationship between potency of phenothiazine and naphthalene-sulfonamide calmodulin inhibitors and their effects on cellular levels and cytotoxic activity of doxorubicin was evaluated using the doxorubicin-sensitive and greater than 100-fold doxorubicin-resistant P388 mouse leukemia model system. In cytotoxicity studies using cell counts based on proliferation following a 24-hr drug exposure and in survival based on colony formation in soft-agar after a 2-hr drug exposure, the calmodulin inhibitors significantly enhanced the cytotoxic effects of doxorubicin in the resistant but not parent-sensitive P388 cells. However, survival in soft-agar (based on colony formation) following long-term drug exposure (approximately 120 hr) revealed that the cytotoxic effects of doxorubicin were significantly increased by the calmodulin inhibitors in both sensitive and resistant P388 cells. Laser flow cytometry studies on single-cell doxorubicin levels indicated that treatment with doxorubicin in the presence of trifluoperazine had no effect on drug levels in sensitive cells but significantly enhanced cellular accumulation and retention of doxorubicin in resistant cells. Furthermore, unlike treatment with doxorubicin alone, in the presence of trifluoperazine, heterogeneity in cellular drug levels in the resistant P388 cells was not observed. Among the various calmodulin inhibitors effective in enhancing cellular levels and cytotoxic effects of doxorubicin in the resistant P388 cells, chlorpromazine was approximately two-fold less potent than trifluoperazine or prochlorperazine and only N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide but not N-(4-aminobutyl)-2-naphthalenesulfonamide was active.

Roles of NF-κB and 26 S Proteasome in Apoptotic Cell Death Induced by Topoisomerase I and II Poisons in Human Nonsmall Cell Lung Carcinoma

Activation of signaling pathways after DNA damage induced by topoisomerase (topo) poisons can lead to cell death by apoptosis. Treatment of human nonsmall cell lung carcinoma (NSCLC-3 or NSCLC-5) cells with the topo I poison SN-38 or the topo II poison etoposide (VP-16) leads to activation of NF-κB before induction of apoptosis. Inhibiting the degradation of IκBα by pretreatment with the proteasome inhibitor MG-132 significantly inhibited NF-κB activation and apoptosis but not DNA damage induced by SN-38 or VP-16. Transfection of NSCLC-3 or NSCLC-5 cells with dominant negative mutant IκBα (mIκBα) inhibited SN-38 or VP-16 induced transcription and DNA binding activity of NF-κB without altering drug-induced apoptosis. Regulation of apoptosis by mitochondrial release of cytochrome c and activation of pro-caspase 9 followed by cleavage of poly(ADP-ribose) polymerase by effector caspases 3 and 7 was similar in neo and mIκBα cells treated with SN-38 or VP-16. In contrast to pretreatment with MG-132, exposure to MG-132 after SN-38 or VP-16 treatment of neo or mIκBα cells decreased cell cycle arrest in the S/G2 + M fraction and enhanced apoptosis compared with drug alone. In summary, apoptosis induced by topoisomerase poisons in NSCLC cells is not mediated by NF-κB but can be manipulated by proteasome inhibitors.