Fluorescent GTP analogues are utilized for an assortment of nucleic acid and protein characterization studies. Non-hydrolysable analogues such as GTPγS offer the advantage of keeping proteins in a GTP-bound conformation due to their resistance to hydrolysis into GDP. Two novel fluorescent GTPγS molecules were developed by linking fluorescein and tetramethylrhodamine to the γ-thiophosphate of GTPγS. Chemical and biological analysis of these two compounds revealed their successful synthesis and ability to bind to the nucleotide-binding site of tubulin. These two new fluorescent non-hydrolysable nucleotides offer new possibilities for biophysical and biochemical characterization of GTP-binding proteins.

Acute myeloid leukemia (AML) is a rare yet deadly cancer of the blood and bone marrow. Presently, induction chemotherapy with the DNA damaging drugs cytarabine (ARA-C) and idarubicin (IDA), known as 7 + 3, is the standard of care for most AML patients. However, 7 + 3 is a relatively ineffective therapy, particularly in older patients, and has serious therapy-related toxicities. Therefore, a diagnostic test to predict which patients will respond to 7 + 3 is a critical unmet medical need. We hypothesize that a threshold level of therapy-induced 7 + 3 drug-DNA adducts determines cytotoxicity and clinical response. We further hypothesize that in vitro exposure of AML cells to nontoxic diagnostic microdoses enables prediction of the ability of AML cells to achieve that threshold during treatment. Our test involves dosing cells with very low levels of ¹⁴C-labeled drug followed by DNA isolation and quantification of drug-DNA adducts via accelerator mass spectrometry. Here, we have shown proof of principle by correlating ARA-C- and DOX-DNA adduct levels with cellular IC₅₀ values of paired sensitive and resistant cancer cell lines and AML cell lines. Moreover, we have completed a pilot retrospective trial of diagnostic microdosing for 10 viably cryopreserved primary AML samples and observed higher ARA-C- and DOX-DNA adducts in the 7 + 3 responders than nonresponders. These initial results suggest that diagnostic microdosing may be a feasible and useful test for predicting patient response to 7 + 3 induction chemotherapy, leading to improved outcomes for AML patients and reduced treatment-related morbidity and mortality.

This rapid report focuses on the pharmacodynamic mechanism of the carboplatin/paclitaxel combination and correlates it with its cytotoxicity. Consistent with the synergistic to additive antitumor activity (the combination index ranging from 0.53 to 0.94), cells exposed to this combination had significantly increased carboplatin-DNA adduct formation when compared to that of carboplatin alone (450 ± 30 versus 320 ± 120 adducts per 10(8) nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 10(8) nucleotides per hour in carboplatin alone (p = 0.021). This rapid report provides the first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic.

Gemcitabine metabolites cause the termination of DNA replication and induction of apoptosis. We determined whether subtherapeutic "microdoses" of gemcitabine are incorporated into DNA at levels that correlate to drug cytotoxicity. A pair of nearly isogenic bladder cancer cell lines differing in resistance to several chemotherapy drugs were treated with various concentrations of ¹⁴C-labeled gemcitabine for 4-24 h. Drug incorporation into DNA was determined by accelerator mass spectrometry. A mechanistic analysis determined that RRM2, a DNA synthesis protein and a known resistance factor, substantially mediated gemcitabine toxicity. These results support gemcitabine levels in DNA as a potential biomarker of drug cytotoxicity.

Nanolipoprotein particles (NLPs) consist of a discoidal phospholipid lipid bilayer confined by an apolipoprotein belt. NLPs are a promising platform for a variety of biomedical applications due to their biocompatibility, size, definable composition, and amphipathic characteristics. However, poor serum stability hampers the use of NLPs for in vivo applications such as drug formulation. In this study, NLP stability was enhanced upon the incorporation and subsequent UV-mediated intermolecular cross-linking of photoactive DiynePC phospholipids in the lipid bilayer, forming cross-linked nanoparticles (X-NLPs). Both the concentration of DiynePC in the bilayer and UV exposure time significantly affected the resulting X-NLP stability in 100% serum, as assessed by size exclusion chromatography (SEC) of fluorescently labeled particles. Cross-linking did not significantly impact the size of X-NLPs as determined by dynamic light scattering and SEC. X-NLPs had essentially no degradation over 48 h in 100% serum, which is a drastic improvement compared to non-cross-linked NLPs (50% degradation by ∼10 min). X-NLPs had greater uptake into the human ATCC 5637 bladder cancer cell line compared to non-cross-linked particles, indicating their potential utility for targeted drug delivery. X-NLPs also exhibited enhanced stability following intravenous administration in mice. These results collectively support the potential utility of X-NLPs for a variety of in vivo applications.

ErbB (Erythroblastic Leukemia Viral Oncogene Homolog) receptor tyrosine kinases are critical for tissue development and maintenance, and frequently become oncogenic when mutated or overexpressed. In vitro analysis of ErbB receptor kinases can be difficult because of their large size and poor water solubility. Here we report improved production and assembly of the correctly folded full-length EGF receptor (EGFR) into nanolipoprotein particles (NLPs). NLPs are ~10 nm in diameter discoidal cell membrane mimics composed of apolipoproteins surrounding a lipid bilayer. NLPs containing EGFR were synthesized via incubation of baculovirus-produced recombinant EGFR with apolipoprotein and phosphoplipids under conditions that favor self-assembly. The resulting EGFR-NLPs were the correct size, formed dimers and multimers, had intrinsic autophosphorylation activity, and retained the ability to interact with EGFR-targeted ligands and inhibitors consistent with previously-published in vitro binding affinities. We anticipate rapid adoption of EGFR-NLPs for structural studies of full-length receptors and drug screening, as well as for the in vitro characterization of ErbB heterodimers and disease-relevant mutants.

Background

Treatment with daunorubicin (DNR) in acute myeloid leukemia is moderately effective and associated with significant side effects, including cardiac toxicity. We recently developed a nanomicellar formulation of DNR that specifically targets acute myeloid leukemia stem cells.

Materials & methods

Pharmacokinetics analysis of free DNR, DNR in nanomicellar formulations was performed in Balb/c mice and Sprague-Dawley rats. Histochemical staining, caspase 3/7, troponin and creatine kinase MB isoenzyme were used to assess toxicity.

Results

Compared with free DNR, the nanomicellar formulations of DNR had less cardiotoxicity as evidenced by milder histopathological changes, lower caspase 3/7 activity in heart tissue (p = 0.002), lower plasma creatine kinase MB isoenzyme (p = 0.002) and troponin concentrations (p = 0.001) postinjection. The area under curve concentration of DNR in micelles increased by 31.9-fold in mice (p < 0.0001) and 22.0-fold higher in rats (p < 0.001).

Conclusion

Leukemia stem cell-targeting micelles dramatically change the pharmacokinetics and reduce the cardiac toxicity of DNR, which may enable improved DNR-based treatment of acute myeloid leukemia.

Purpose: Activation of the PI3K pathway occurs in over 40% of bladder urothelial cancers. The aim of this study is to determine the therapeutic potential, the underlying action, and the resistance mechanisms of drugs targeting the PI3K pathway.Experimental Design: Urothelial cancer cell lines and patient-derived xenografts (PDXs) were analyzed for alterations of the PI3K pathway and for their sensitivity to the small-molecule inhibitor pictilisib alone and in combination with cisplatin and/or gemcitabine. Potential predictive biomarkers for pictilisib were evaluated, and RNA sequencing was performed to explore drug resistance mechanisms.Results: The bladder cancer cell line TCCSUP, which harbors a PIK3CA E545K mutation, was sensitive to pictilisib compared to cell lines with wild-type PIK3CA Pictilisib exhibited stronger antitumor activity in bladder cancer PDX models with PI3KCA H1047R mutation or amplification than the control PDX model. Pictilisib synergized with cisplatin and/or gemcitabine in vitro, significantly delayed tumor growth, and prolonged survival compared with single-drug treatment in the PDX models. The phosphorylation of ribosomal protein S6 correlated with response to pictilisib both in vitro and in vivo, and could potentially serve as a biomarker to predict response to pictilisib. Pictilisib activated the compensatory MEK/ERK pathway that likely contributed to pictilisib resistance, which was reversed by cotreatment with the RAF inhibitor sorafenib. RNA sequencing of tumors resistant to treatment suggested that LSP1 downregulation correlated with drug resistance.Conclusions: These preclinical results provide new insights into the therapeutic potential of targeting the PI3K pathway for the treatment of bladder cancer. Clin Cancer Res; 23(21); 6580-91. ©2017 AACR.

FOLFOX is one of the most effective treatments for advanced colorectal cancer. However, cumulative oxaliplatin neurotoxicity often results in halting the therapy. Oxaliplatin functions predominantly via the formation of toxic covalent drug-DNA adducts. We hypothesize that oxaliplatin-DNA adduct levels formed in vivo in peripheral blood mononuclear cells (PBMC) are proportional to tumor shrinkage caused by FOLFOX therapy. We further hypothesize that adducts induced by subtherapeutic "diagnostic microdoses" are proportional to those induced by therapeutic doses and are also predictive of response to FOLFOX therapy. These hypotheses were tested in colorectal cancer cell lines and a pilot clinical study. Four colorectal cancer cell lines were cultured with therapeutically relevant (100 μmol/L) or diagnostic microdose (1 μmol/L) concentrations of [¹⁴C]oxaliplatin. The C-14 label enabled quantification of oxaliplatin-DNA adduct level with accelerator mass spectrometry (AMS). Oxaliplatin-DNA adduct formation was correlated with oxaliplatin cytotoxicity for each cell line as measured by the MTT viability assay. Six colorectal cancer patients received by intravenous route a diagnostic microdose containing [¹⁴C]oxaliplatin prior to treatment, as well as a second [¹⁴C]oxaliplatin dose during FOLFOX chemotherapy, termed a "therapeutic dose." Oxaliplatin-DNA adduct levels from PBMC correlated significantly to mean tumor volume change of evaluable target lesions (5 of the 6 patients had measurable disease). Oxaliplatin-DNA adduct levels were linearly proportional between microdose and therapeutically relevant concentrations in cell culture experiments and patient samples, as was plasma pharmacokinetics, indicating potential utility of diagnostic microdosing.

This review summarizes recent developments in radiocarbon tracer technology and applications. Technologies covered include accelerator mass spectrometry (AMS), including conversion of samples to graphite, and rapid combustion to carbon dioxide to enable direct liquid sample analysis, coupling to HPLC for real-time AMS analysis, and combined molecular mass spectrometry and AMS for analyte identification and quantitation. Laser-based alternatives, such as cavity ring down spectrometry, are emerging to enable lower cost, higher throughput measurements of biological samples. Applications covered include radiocarbon dating, use of environmental atomic bomb pulse radiocarbon content for cell and protein age determination and turnover studies, and carbon source identification. Low dose toxicology applications reviewed include studies of naphthalene-DNA adduct formation, benzo[a]pyrene pharmacokinetics in humans, and triclocarban exposure and risk assessment. Cancer-related studies covered include the use of radiocarbon-labeled cells for better defining mechanisms of metastasis and the use of drug-DNA adducts as predictive biomarkers of response to chemotherapy.