Adjuvant Pembrolizumab versus IFNα2b or Ipilimumab in Resected High-Risk Melanoma.

We conducted a randomized phase III trial to evaluate whether adjuvant pembrolizumab for one year (647 patients) improved recurrence-free survival (RFS) or overall survival (OS) in comparison with high-dose IFNα-2b for one year or ipilimumab for up to three years (654 patients), the approved standard-of-care adjuvant immunotherapies at the time of enrollment for patients with high-risk resected melanoma. At a median follow-up of 47.5 months, pembrolizumab was associated with significantly longer RFS than prior standard-of-care adjuvant immunotherapies [HR, 0.77; 99.62% confidence interval (CI), 0.59-0.99; P = 0.002]. There was no statistically significant association with OS among all patients (HR, 0.82; 96.3% CI, 0.61-1.09; P = 0.15). Proportions of treatment-related adverse events of grades 3 to 5 were 19.5% with pembrolizumab, 71.2% with IFNα-2b, and 49.2% with ipilimumab. Therefore, adjuvant pembrolizumab significantly improved RFS but not OS compared with the prior standard-of-care immunotherapies for patients with high-risk resected melanoma. SIGNIFICANCE: Adjuvant PD-1 blockade therapy decreases the rates of recurrence, but not survival, in patients with surgically resectable melanoma, substituting the prior standard-of-care immunotherapies for this cancer. See related commentary by Smithy and Shoushtari, p. 599. This article is highlighted in the In This Issue feature, p. 587.

Modernizing Clinical Trial Eligibility Criteria: Recommendations of the ASCO-Friends of Cancer Research Washout Period and Concomitant Medication Work Group.

PURPOSE: Washout periods and concomitant medication exclusions are common in cancer clinical trial protocols. These exclusion criteria are often applied inconsistently and without evidence to justify their use. The authors sought to determine how washout period and concomitant medication allowances can be broadened to speed trial enrollment and improve the generalizability of trial data to a larger oncology practice population without compromising the safety of trial participants. EXPERIMENTAL DESIGN: A multistakeholder working group was convened to define problems associated with excessively long washout periods and exclusion of patients due to concomitant medications. The group performed a literature search and evaluated study data from the Pancreatic Cancer Action Network (PanCAN), Emory University School of Medicine (Atlanta, GA), and the FDA to understand recent approaches to these eligibility criteria. The group convened to develop consensus recommendations for broadened eligibility criteria. RESULTS: The data analysis found that exclusion criteria based on washout periods and concomitant medications are frequently inconsistent and lack scientific rationale. Scientific rationale for appropriate eligibility criteria are presented in the article; for washout periods, rationale is presented by treatment type. CONCLUSIONS: Arbitrary or blanket washout and concomitant medication exclusions should be eliminated. Where there is evidence to support them, clinically relevant washout periods and concomitant medication-related eligibility criteria may be included.See related commentary by Giantonio, p. 2369.

Pembrolizumab As Second-Line Therapy in Patients With Advanced Hepatocellular Carcinoma in KEYNOTE-240: A Randomized, Double-Blind, Phase III Trial.

PURPOSE: Pembrolizumab demonstrated antitumor activity and safety in the phase II KEYNOTE-224 trial in previously treated patients with advanced hepatocellular carcinoma (HCC). KEYNOTE-240 evaluated the efficacy and safety of pembrolizumab in this population. PATIENTS AND METHODS: This randomized, double-blind, phase III study was conducted at 119 medical centers in 27 countries. Eligible patients with advanced HCC, previously treated with sorafenib, were randomly assigned at a two-to-one ratio to receive pembrolizumab plus best supportive care (BSC) or placebo plus BSC. Primary end points were overall survival (OS) and progression-free survival (PFS; one-sided significance thresholds, P = .0174 [final analysis] and P = .002 [first interim analysis], respectively). Safety was assessed in all patients who received ≥ 1 dose of study drug. RESULTS: Between May 31, 2016, and November 23, 2017, 413 patients were randomly assigned. As of January 2, 2019, median follow-up was 13.8 months for pembrolizumab and 10.6 months for placebo. Median OS was 13.9 months (95% CI, 11.6 to 16.0 months) for pembrolizumab versus 10.6 months (95% CI, 8.3 to 13.5 months) for placebo (hazard ratio [HR], 0.781; 95% CI, 0.611 to 0.998; P = .0238). Median PFS for pembrolizumab was 3.0 months (95% CI, 2.8 to 4.1 months) versus 2.8 months (95% CI, 2.5 to 4.1 months) for placebo at the first interim analysis (HR, 0.775; 95% CI, 0.609 to 0.987; P = .0186) and 3.0 months (95% CI, 2.8 to 4.1 months) versus 2.8 months (95% CI, 1.6 to 3.0 months) at final analysis (HR, 0.718; 95% CI, 0.570 to 0.904; P = .0022). Grade 3 or higher adverse events occurred in 147 (52.7%) and 62 patients (46.3%) for pembrolizumab versus placebo; those that were treatment related occurred in 52 (18.6%) and 10 patients (7.5%), respectively. No hepatitis C or B flares were identified. CONCLUSION: In this study, OS and PFS did not reach statistical significance per specified criteria. The results are consistent with those of KEYNOTE-224, supporting a favorable risk-to-benefit ratio for pembrolizumab in this population.

Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial.

BACKGROUND: Immune checkpoint blockade therapy has shown promising results in patients with advanced hepatocellular carcinoma. We aimed to assess the efficacy and safety of pembrolizumab in this patient population. METHODS: KEYNOTE-224 is a non-randomised, multicentre, open-label, phase 2 trial that is set in 47 medical centres and hospitals across ten countries. Eligible patients had pathologically confirmed hepatocellular carcinoma; had previously been treated with sorafenib and were either intolerant to this treatment or showed radiographic progression of their disease after treatment; an Eastern Cooperative Oncology Group performance status of 0-1; adequate organ function, and were Child-Pugh class A. Participants received 200 mg pembrolizumab intravenously every 3 weeks for about 2 years or until disease progression, unacceptable toxicity, patient withdrawal, or investigator decision. The primary endpoint was objective response, defined as the proportion of patients with complete or partial response in all patients who received at least one dose of pembrolizumab, which was radiologically confirmed by use of the Response Evaluation Criteria in Solid Tumors version 1.1 by central review. Safety was also assessed in all treated patients. This trial is ongoing but closed to enrolment and is registered with ClinicalTrials.gov number NCT02702414. FINDINGS: Between June 7, 2016, and Feb 9, 2017, we screened 169 patients with advanced hepatocellular carcinoma, of whom 104 eligible patients were enrolled and treated. As of data cutoff on Feb 13, 2018, 17 (16%) patients were still receiving pembrolizumab. We recorded an objective response in 18 (17%; 95% CI 11-26) of 104 patients. The best overall responses were one (1%) complete and 17 (16%) partial responses; meanwhile, 46 (44%) patients had stable disease, 34 (33%) had progressive disease, and six (6%) patients who did not have a post-baseline assessment on the cutoff date were considered not to be assessable. Treatment-related adverse events occurred in 76 (73%) of 104 patients, which were serious in 16 (15%) patients. Grade 3 treatment-related events were reported in 25 (24%) of the 104 patients; the most common were increased aspartate aminotransferase concentration in seven (7%) patients, increased alanine aminotransferase concentration in four (4%) patients, and fatigue in four (4%) patients. One (1%) grade 4 treatment-related event of hyperbilirubinaemia occurred. One death associated with ulcerative oesophagitis was attributed to treatment. Immune-mediated hepatitis occurred in three (3%) patients, but there were no reported cases of viral flares. INTERPRETATION: Pembrolizumab was effective and tolerable in patients with advanced hepatocellular carcinoma who had previously been treated with sorafenib. These results indicate that pembrolizumab might be a treatment option for these patients. This drug is undergoing further assessment in two phase 3, randomised trials as a second-line treatment in patients with hepatocellular carcinoma. FUNDING: Merck & Co, Inc.

Pembrolizumab Exposure-Response Assessments Challenged by Association of Cancer Cachexia and Catabolic Clearance.

PURPOSE: To investigate the relationship of pembrolizumab pharmacokinetics (PK) and overall survival (OS) in patients with advanced melanoma and non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: PK dependencies in OS were evaluated across three pembrolizumab studies of either 200 mg or 2 to 10 mg/kg every 3 weeks (Q3W). Kaplan-Meier plots of OS, stratified by dose, exposure, and baseline clearance (CL0), were assessed per indication and study. A Cox proportional hazards model was implemented to explore imbalances of typical prognostic factors in high/low NSCLC CL0 subgroups. RESULTS: A total of 1,453 subjects were included: 340 with pembrolizumab-treated melanoma, 804 with pembrolizumab-treated NSCLC, and 309 with docetaxel-treated NSCLC. OS was dose independent from 2 to 10 mg/kg for pembrolizumab-treated melanoma [HR = 0.98; 95% confidence interval (CI), 0.94-1.02] and NSCLC (HR = 0.98; 95% CI, 0.95-1.01); however, a strong CL0-OS association was identified for both cancer types (unadjusted melanoma HR = 2.56; 95% CI, 1.72-3.80 and NSCLC HR = 2.64; 95% CI, 1.94-3.57). Decreased OS in subjects with higher pembrolizumab CL0 paralleled disease severity markers associated with end-stage cancer anorexia-cachexia syndrome. Correction for baseline prognostic factors did not fully attenuate the CL0-OS association (multivariate-adjusted CL0 HR = 1.64; 95% CI, 1.06-2.52 for melanoma and HR = 1.88; 95% CI, 1.22-2.89 for NSCLC). CONCLUSIONS: These data support the lack of dose or exposure dependency in pembrolizumab OS for melanoma and NSCLC between 2 and 10 mg/kg. An association of pembrolizumab CL0 with OS potentially reflects catabolic activity as a marker of disease severity versus a direct PK-related impact of pembrolizumab on efficacy. Similar data from other trials suggest such patterns of exposure-response confounding may be a broader phenomenon generalizable to antineoplastic mAbs.See related commentary by Coss et al., p. 5787.

Pembrolizumab Utilization and Outcomes for Advanced Melanoma in US Community Oncology Practices.

The programmed death-1 inhibitor pembrolizumab has demonstrated efficacy and safety in clinical trials for treating advanced (unresectable/metastatic) melanoma. We investigated the real-world utilization of pembrolizumab and associated patient outcomes for advanced melanoma in US community oncology practices. This retrospective, observational study used deidentified data from electronic health records for adult patients with advanced melanoma who received pembrolizumab at The US Oncology Network sites from September 2014 through December 2015, with follow-up through September 2016. Patients enrolled in clinical trials were excluded. Overall survival (OS) and physician-stated progression-free survival (PFS) were analyzed from pembrolizumab initiation using Kaplan-Meier, and associations between pembrolizumab therapy and OS/PFS, using multivariable Cox regression. Of 168 patients studied, 110 (65%) were male; the median age was 66 years (range, 26-over 90). Pembrolizumab was prescribed as first-line, second-line, and third-line/later for 39 (23%), 87 (52%), and 42 (25%) patients, respectively. In total, 41 patients (24%) had brain metastases. At pembrolizumab initiation, 21/129 (16%) had Eastern Cooperative Oncology Group performance status (ECOG PS) >1; 51/116 (44%) had elevated lactate dehydrogenase. Median follow-up was 10.5 months (range, 0-25.1); median OS was 19.4 months (95% confidence interval, 14.0-not reached); median PFS was 4.2 months (95% confidence interval, 2.9-5.3). Brain metastases, ECOG PS>1, elevated lactate dehydrogenase, and third-line/later (vs. first-line) pembrolizumab were significant predictors (P<0.01) of decreased survival. Treatment-related toxicity was a discontinuation reason for 25% (29/117) of patients, and for 10 of these 29 patients (6% of the full-study cohort) treatment-related toxicity was the only reported reason. The real-world effectiveness and safety of pembrolizumab for advanced melanoma are consistent with clinical trial findings.

Anti-programmed-death-receptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: a randomised dose-comparison cohort of a phase 1 trial.

BACKGROUND: The anti-programmed-death-receptor-1 (PD-1) antibody pembrolizumab has shown potent antitumour activity at different doses and schedules in patients with melanoma. We compared the efficacy and safety of pembrolizumab at doses of 2 mg/kg and 10 mg/kg every 3 weeks in patients with ipilimumab-refractory advanced melanoma. METHODS: In an open-label, international, multicentre expansion cohort of a phase 1 trial, patients (aged ≥18 years) with advanced melanoma whose disease had progressed after at least two ipilimumab doses were randomly assigned with a computer-generated allocation schedule (1:1 final ratio) to intravenous pembrolizumab at 2 mg/kg every 3 weeks or 10 mg/kg every 3 weeks until disease progression, intolerable toxicity, or consent withdrawal. Primary endpoint was overall response rate (ORR) assessed with the Response Evaluation Criteria In Solid Tumors (RECIST, version 1.1) by independent central review. Analysis was done on the full-analysis set (all treated patients with measurable disease at baseline). This study is registered with ClinicalTrials.gov, number NCT01295827. FINDINGS: 173 patients received pembrolizumab 2 mg/kg (n=89) or 10 mg/kg (n=84). Median follow-up duration was 8 months. ORR was 26% at both doses--21 of 81 patients in the 2 mg/kg group and 20 of 76 in the 10 mg/kg group (difference 0%, 95% CI -14 to 13; p=0·96). Treatment was well tolerated, with similar safety profiles in the 2 mg/kg and 10 mg/kg groups and no drug-related deaths. The most common drug-related adverse events of any grade in the 2 mg/kg and 10 mg/kg groups were fatigue (29 [33%] vs 31 [37%]), pruritus (23 [26%] vs 16 [19%]), and rash (16 [18%] vs 15 [18%]). Grade 3 fatigue, reported in five (3%) patients in the 2 mg/kg pembrolizumab group, was the only drug-related grade 3 to 4 adverse event reported in more than one patient. INTERPRETATION: The results suggest that pembrolizumab at a dose of 2 mg/kg or 10 mg/kg every 3 weeks might be an effective treatment in patients for whom there are few effective treatment options. FUNDING: Merck Sharp and Dohme.

Tolerability, safety and pharmacokinetics of ridaforolimus in combination with bicalutamide in patients with asymptomatic, metastatic castration-resistant prostate cancer (CRPC).

PURPOSE: Recent data indicate that there is a significant cross-talk between the PI3K/Akt/mTOR and androgen receptor signaling pathways. We evaluated safety and tolerability as well as potential drug-drug interaction of ridaforolimus, a mammalian target of rapamycin (mTOR) inhibitor, when combined with the androgen receptor inhibitor bicalutamide in patients with asymptomatic, metastatic castration-resistant prostate cancer. PATIENTS AND METHODS: Patients were treated with the combination of ridaforolimus 30 mg/day for 5 consecutive days each week and bicalutamide 50 mg/day. Ridaforolimus pharmacokinetics was assessed with and without bicalutamide. RESULTS: Twelve patients were enrolled including 1 screen failure. Dose reductions were required in 7 patients. Three of the 11 patients experienced a dose-limited toxicity, 1 with Grade 3 hyperglycemia and 2 with Grade 2 stomatitis leading to <75 % of planned ridaforolimus dose during the first 35 days of study treatment. The pharmacokinetic results showed no differences in exposures to ridaforolimus with and without concomitant bicalutamide administration. CONCLUSIONS: Although there was no evidence of a clinically relevant pharmacological drug-drug interaction, the occurrence of dose-limiting toxicities in 3 of 11 evaluable patients at a reduced dose of ridaforolimus of 30 mg/day suggests that this combination may not be well suited for asymptomatic or minimally symptomatic prostate cancer patients.

Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma.

BACKGROUND: The programmed death 1 (PD-1) receptor is a negative regulator of T-cell effector mechanisms that limits immune responses against cancer. We tested the anti-PD-1 antibody lambrolizumab (previously known as MK-3475) in patients with advanced melanoma. METHODS: We administered lambrolizumab intravenously at a dose of 10 mg per kilogram of body weight every 2 or 3 weeks or 2 mg per kilogram every 3 weeks in patients with advanced melanoma, both those who had received prior treatment with the immune checkpoint inhibitor ipilimumab and those who had not. Tumor responses were assessed every 12 weeks. RESULTS: A total of 135 patients with advanced melanoma were treated. Common adverse events attributed to treatment were fatigue, rash, pruritus, and diarrhea; most of the adverse events were low grade. The confirmed response rate across all dose cohorts, evaluated by central radiologic review according to the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1, was 38% (95% confidence interval [CI], 25 to 44), with the highest confirmed response rate observed in the cohort that received 10 mg per kilogram every 2 weeks (52%; 95% CI, 38 to 66). The response rate did not differ significantly between patients who had received prior ipilimumab treatment and those who had not (confirmed response rate, 38% [95% CI, 23 to 55] and 37% [95% CI, 26 to 49], respectively). Responses were durable in the majority of patients (median follow-up, 11 months among patients who had a response); 81% of the patients who had a response (42 of 52) were still receiving treatment at the time of analysis in March 2013. The overall median progression-free survival among the 135 patients was longer than 7 months. CONCLUSIONS: In patients with advanced melanoma, including those who had had disease progression while they had been receiving ipilimumab, treatment with lambrolizumab resulted in a high rate of sustained tumor regression, with mainly grade 1 or 2 toxic effects. (Funded by Merck Sharp and Dohme; ClinicalTrials.gov number, NCT01295827.).

Voreloxin, a first-in-class anticancer quinolone derivative, in relapsed/refractory solid tumors: a report on two dosing schedules.

PURPOSE: Voreloxin, a novel replication-dependent DNA-damaging agent, intercalates DNA and inhibits topoisomerase II. Voreloxin induces site-selective DNA double-strand breaks and apoptosis. We report the phase 1 experience of voreloxin in patients with relapsed/refractory solid tumors, including dose-limiting toxicity (DLT), maximum-tolerated dose (MTD), pharmacokinetics, and clinical activity. EXPERIMENTAL DESIGN: Two dose-escalation studies evaluated voreloxin administered i.v. every 3 weeks (SPO-0001) or weekly for 3 weeks every 28 days (SPO-0002). In SPO-0001, patients were classified as heavily pretreated (HP) or minimally pretreated (MP) based on therapeutic history. RESULTS: In the SPO-0001 study, 41 patients (24 HP/17 MP) were treated in eight dose cohorts (3-75 mg/m(2)). At 60 mg/m(2), four HP patients experienced DLTs: grade 4 neutropenia (n = 3, one with fever) and grade 3 febrile neutropenia/pneumonia (n = 1). At 75 mg/m(2), two MP patients experienced DLTs: grade 4 neutropenia/thrombocytopenia (n = 1) or grade 2 oral thrush for >29 days (n = 1). Therefore, the MTD was 48 mg/m(2) (HP patients) and 60 mg/m(2) (MP patients). In the SPO-0002 study, 21 patients were treated in six dose cohorts (3-24 mg/m(2)). At 18 mg/m(2), two patients experienced DLTs: grade 3 neutropenia, one with pleural effusion (>14 days each). The MTD was 15 mg/m(2). Voreloxin exhibited low clearance (2 L/h/m(2)), a long terminal half-life (22 hours), and dose-proportional exposure. Overall, 31 of 62 patients had stable disease and 1 patient (ovarian cancer) had a partial response per Rustin criteria. CONCLUSIONS: Voreloxin showed an acceptable safety profile with clinical activity in patients with relapsed/refractory solid tumors. The MTD was schedule-dependent. Voreloxin is currently in clinical studies of ovarian cancer and acute myeloid leukemia.

Formation of a unique end-to-end stacked pair of G-quadruplexes in the hTERT core promoter with implications for inhibition of telomerase by G-quadruplex-interactive ligands.

The hTERT core promoter contains a G-rich region of 12 consecutive G-tracts, embracing 3 Sp1 binding sites, and has the potential to form multiple G-quadruplexes. From the 12 runs of guanines, 9 putative hTERT G-quadruplex-forming sequences were selected to assay for G-quadruplex formation and stability using circular dichroism and a Taq polymerase stop assay. Results from biophysical and chemical assays demonstrate an approximate inverse correlation between total loop size and structure stability. Investigation of the full-length hTERT G-rich sequence using a Taq polymerase stop assay and dimethyl sulfate footprinting revealed the formation of a unique end-to-end stacked G-quadruplex structure from this sequence. This structure consists of an all parallel G-quadruplex, formed by four consecutive G-tracts, linked to another, atypical G-quadruplex, formed by two pairs of consecutive G-tracts separated by a 26-base loop. This 26-base loop likely forms a stable hairpin structure, which would explain the unexpected stability of this G-quadruplex. Significantly, the formation of this tandem G-quadruplex structure in the full-length sequence masks all three Sp1 binding sites, which is predicted to produce significant inhibition of hTERT promoter activity. Furthermore, our study implies that inhibition of telomerase activity by some G-quadruplex ligands is not only produced by targeting telomeric G-quadruplexes but also by stabilization of the hTERT promoter G-quadruplexes.

A novel G-quadruplex-forming GGA repeat region in the c-myb promoter is a critical regulator of promoter activity.

The c-myb promoter contains multiple GGA repeats beginning 17 bp downstream of the transcription initiation site. GGA repeats have been previously shown to form unusual DNA structures in solution. Results from chemical footprinting, circular dichroism and RNA and DNA polymerase arrest assays on oligonucleotides representing the GGA repeat region of the c-myb promoter demonstrate that the element is able to form tetrad:heptad:heptad:tetrad (T:H:H:T) G-quadruplex structures by stacking two tetrad:heptad G-quadruplexes formed by two of the three (GGA)(4) repeats. Deletion of one or two (GGA)(4) motifs destabilizes this secondary structure and increases c-myb promoter activity, indicating that the G-quadruplexes formed in the c-myb GGA repeat region may act as a negative regulator of the c-myb promoter. Complete deletion of the c-myb GGA repeat region abolishes c-myb promoter activity, indicating dual roles of the c-myb GGA repeat element as both a transcriptional repressor and an activator. Furthermore, we demonstrated that Myc-associated zinc finger protein (MAZ) represses c-myb promoter activity and binds to the c-myb T:H:H:T G-quadruplexes. Our findings show that the T:H:H:T G-quadruplex-forming region in the c-myb promoter is a critical cis-acting element and may repress c-myb promoter activity through MAZ interaction with G-quadruplexes in the c-myb promoter.

Mechanism of PNA transport to the nuclear compartment.

We evaluated the nuclear uptake of fluorescently labeled peptide nucleic acids and measured the binding of unlabeled peptide nucleic acids (PNAs) to the endogenous HER-2/neu promotor in digitonin-permeabilized SK-BR-3 cells. Fluorescently labeled PNAs readily enter the nucleus of digitonin-permeabilized cells, and binding to the chromosomal target sequence was detected with a bis-PNA. Nuclear uptake and target sequence binding were inhibited by N-ethylmaleimide (NEM) and GTPgammaS. We conclude that PNAs are transported into the nucleus through an energy-dependent process involving the nuclear pore complex.

PNA-nitrogen mustard conjugates are effective suppressors of HER-2/neu and biological tools for recognition of PNA/DNA interactions.

Peptide nucleic acids (PNAs) are promising tools for gene regulation. One of the challenges of using PNAs as gene regulators is the need to optimize the efficiency of interaction with critical sequences of DNA. To improve the efficiency of binding between PNAs and the HER-2/neu promoter, mono- and bis-pyrimidine-rich PNAs were conjugated to a nitrogen mustard at either the amino or carboxy terminus. Gel shift analysis demonstrated that conjugation to an alkylating agent slowed PNA binding and favored PNA:DNA:DNA triplex helix formation while preserving a high binding affinity. Sites of DNA alkylation were visualized by piperidine cleavage and showed PNA binding first by Hoogsteen bond formation with the target duplex to form a stable PNA:DNA:DNA triplex structure which is later converted to a PNA:DNA:PNA triple helix by strand invasion and Watson-Crick base pairing by a second PNA molecule. In this way, PNA-directed DNA alkylation was used to deduce the mode of PNA binding. Transient transfection experiments demonstrated that the PNA-nitrogen mustard conjugates suppressed HER-2/neu expression by up to 80%. In comparison with an unmodified mono-PNA or a bis-PNA, these results indicate that the covalent adducts stabilized PNA binding in cells and suggest that the conjugation of PNAs to nitrogen mustards is a robust strategy for developing antigene PNA oligonucleotides to prevent transcription.

Evidence for the presence of a guanine quadruplex forming region within a polypurine tract of the hypoxia inducible factor 1alpha promoter.

The promoter of the hypoxia inducible factor 1 alpha (HIF-1alpha) gene has a polypurine/polypyrimidine tract (-65 to -85) overlapping or adjacent to several putative transcription factor binding sites, and we found that mutagenesis of this region diminished basal HIF-1alpha expression. Oligonucleotides representing this region of the HIF-1alpha promoter were analyzed by electrophoretic mobility shift, chemical probing, circular dichroism, and DNA polymerase arrest assays. The guanine-rich strand was found to form a parallel, unimolecular quadruplex in the presence of potassium that was further stabilized by two known quadruplex binding compounds, the cationic porphyrin TmPyP4 and the natural product telomestatin, while TmPyP2, a positional isomer of TmPyP4, did not stabilize quadruplex formation. These data suggest that a quadruplex structure may form in a region of the HIF-1alpha promoter that regulates basal HIF-1alpha expression.

Peptide nucleic acid conjugates: synthesis, properties and applications.

Artificial control of gene expression has great potential in the treatment of many human diseases, and peptide nucleic acids (PNAs) offer several potential advantages for silencing gene expression in mammalian cells. The pseudopeptide backbone of the PNA makes it resistant to enzymatic degradation, and PNAs bind complementary DNA and RNA with high affinity and specificity. PNAs are potentially leading agents for antigene and antisense therapeutics, but the application of PNAs in the in vivo setting is hampered by their poor intracellular delivery. This problem has been addressed by PNA conjugation to lipophilic moieties, peptides, and cell-specific receptor ligands. The biological activity of PNAs can also benefit from conjugation to DNA interactive compounds like intercalators and alkylators. Here we review the most interesting literature concerning PNA conjugation with small molecules, emphasizing synthetic approaches, properties and applications of the PNA conjugates.

Targeting and regulation of the HER-2/neu oncogene promoter with bis-peptide nucleic acids.

Antigene oligonucleotides have the potential to regulate gene expression through site-specific DNA binding. However, in vivo applications have been hindered by inefficient cellular uptake, degradation, and strand displacement. Peptide nucleic acids (PNAs) address several of these problems, as they are resistant to degradation and bind DNA with high affinity. We designed two cationic pyrimidine bis-PNAs (cpy-PNAs) to target the polypurine tract of the HER-2/neu promoter and compared them to an unmodified phosphodiester triplex-forming oligonucleotide (TFO1) and a TFO-nitrogen mustard conjugate (TFO2). PNA1 contains a + 2 charge and bound two adjacent 9-bp target sequences with high affinity and specificity, but only at low pH. PNA2 contains a +5 charge and bound one 11-bp target with high affinity up to pH 7.4, but with lower specificity. The PNA:DNA:PNA triplex formed by these cpy-bis-PNAs presented a stable barrier to DNA polymerase extension. The cpy-bis-PNAs and the TFO-alkylator conjugate prevented HER-2/neu transcription in a reporter gene assay (TFO2 = PNA1 > PNA2 >> TFO1). Both PNAs and TFOs were effective at binding the target sequence in naked genomic DNA, but only the TFO-alkylator (TFO2) and the more cationic PNA (PNA2) were detected at the endogenous HER-2/neu promoter in permeabilized cells. This work demonstrates the potential for preventing HER-2/neu gene expression with cpy-bis-PNAs in tumor cells.

Synthesis and evaluation of a triplex-forming oligonucleotide-pyrrolobenzodiazepine conjugate.

In most cases, unmodified oligonucleotides designed as antigene molecules are incapable of binding to DNA with sufficient stability to prevent gene expression. To stabilize binding to a polypurine tract in the HER-2/neu promoter, a triplex forming oligonucleotide (TFO) was conjugated to a pyrrolo[1,4]benzodiazepine (PBD), desmethyltomaymycin, and site-specific DNA binding was evaluated. An activated ester of the PBD moiety was conjugated by an acylation reaction to a free primary amine on a 50-atom aliphatic linker at the 5' end of the TFO. This long aliphatic linker was designed to provide a bridge from the major groove binding site of the TFO to the minor groove binding site of the PBD. Triplex formation by the resulting TFO-PBD conjugate occurred more slowly and with a nearly 30-fold lower affinity compared to an unconjugated TFO. PBD binding to the triplex target was demonstrated by protection from restriction enzyme digestion, and covalent binding to the exocyclic amino group of guanine was inferred by substituting specific guanines with inosines. Although the binding of the TFO was less efficient, this report demonstrates that in principle, TFOs can be used to direct the binding of a PBD to specific location. Further optimization of TFO-PBD conjugate design, likely involving optimization of the linker and perhaps placing a PBD at both ends of the TFO, will be needed to make gene modification robust.

Renal cell carcinoma: rationale and development of therapeutic inhibitors of angiogenesis.

Inhibition of tumor angiogenesis is a promising therapeutic approach to treat cancer; translation of this concept into clinical practice requires an understanding of the molecular events that are responsible for the development of tumor vasculature. Renal cell carcinoma is characterized by the frequent loss of the von Hippel-Lindau tumor suppressor gene which results in the loss of one of the critical mechanisms for regulating the level of hypoxia inducible factor 1 and leads to the overproduction of vascular endothelial growth factor (VEGF) by the tumor cell. Therapeutic strategies to inhibit the function of these important pathways have been effective in preventing tumor angiogenesis in preclinical models of kidney cancer, and more recently, in the clinical setting. Strategies to treat renal cell carcinoma with agents that are designed to prevent angiogenesis have included interruption of the VEGF signaling pathway, mimics of endogenous angiogenesis inhibitors, prevention of destruction of the basement membrane, and direct inhibition of endothelial cells by a variety of agents with complex, novel, or undetermined mechanisms. Recent clinical studies of bevacizumab, the first anti-VEGF agent to be marketed for the treatment of cancer, have provided proof for the concept that these strategies can lead to tangible benefits for patients who have advanced renal cell carcinoma and likely will be applicable broadly to the treatment of cancer.