Description: There is an urgent need to improve reproducibility and translatability of preclinical data to fully exploit opportunities for molecular therapeutics involving radiation and radiochemotherapy. For i n vitro research, the clonogenic assay remains the current state-of-the-art of preclinical assays, whereas newer moderate and high-throughput assays offer the potential for rapid initial screening. Studies of radiation response modification by molecularly targeted agents can be improved using more physiologic 3D culture models. Elucidating effects on the cancer stem cells (CSC, and CSC-like) and developing biomarkers for defining targets and measuring responses are also important. In vivo studies are necessary to confirm in vitro findings, further define mechanism of action, and address immunomodulation and treatment-induced modification of the microenvironment. Newer in vivo models include genetically engineered and patient-derived xenograft mouse models and spontaneously occurring cancers in domesticated animals. Selection of appropriate endpoints is important for in vivo studies; for example, regrowth delay measures bulk tumor killing, whereas local tumor control assesses effects on CSCs. The reliability of individual assays requires standardization of procedures and cross-laboratory validation. Radiation modifiers must be tested as part of clinical standard of care, which includes radiochemotherapy for most tumors. Radiation models are compatible with but also differ from those used for drug screening. Furthermore, the mechanism of a drug as a chemotherapeutic agent may be different from its interaction with radiation and/or radiochemotherapy. This provides an opportunity to expand the use of molecular-targeted agents. Clin Cancer Res; 22(13); 3138–47. ©2016 AACR .

Description: Purpose: To investigate the impact of hypoxia-induced gene expression and cancer stem cell (CSC) marker expression on outcome of postoperative cisplatin-based radiochemotherapy (PORT-C) in patients with locally advanced head and neck squamous cell carcinoma (HNSCC). Experimental Design: Expression of the CSC markers CD44, MET , and SLC3A2 , and hypoxia gene signatures were analyzed in the resected primary tumors using RT-PCR and nanoString technology in a multicenter retrospective cohort of 195 patients. CD44 protein expression was further analyzed in tissue microarrays. Primary endpoint was locoregional tumor control. Results: Univariate analysis showed that hypoxia-induced gene expression was significantly associated with a high risk of locoregional recurrence using the 15-gene signature ( P = 0.010) or the 26-gene signature ( P = 0.002). In multivariate analyses, in patients with HPV16 DNA–negative but not with HPV16 DNA–positive tumors the effect of hypoxia-induced genes on locoregional control was apparent (15-gene signature: HR 4.54, P = 0.006; 26-gene signature: HR 10.27, P = 0.024). Furthermore, MET, SLC3A2, CD44 , and CD44 protein showed an association with locoregional tumor control in multivariate analyses ( MET : HR 3.71, P = 0.016; SLC3A2 : HR 8.54, P = 0.037; CD44 : HR 3.36, P = 0.054; CD44 protein n/a because of no event in the CD44-negative group) in the HPV16 DNA–negative subgroup. Conclusions: We have shown for the first time that high hypoxia-induced gene expression and high CSC marker expression levels correlate with tumor recurrence after PORT-C in patients with HPV16 DNA–negative HNSCC. After validation in a currently ongoing prospective trial, these parameters may help to further stratify patients for individualized treatment de-escalation or intensification strategies. Clin Cancer Res; 22(11); 2639–49. ©2016 AACR .

Description: 3,4-Methylenedioxymethamphetamine (MDMA) is a widely abused illicit drug that can cause severe and even fatal adverse effects. However, interest remains for its possible clinical applications in posttraumatic stress disorder and anxiety treatment. Preclinical studies to determine MDMA’s safety are needed. We evaluated MDMA’s pharmacokinetics and metabolism in male rats receiving 2.5, 5, and 10 mg/kg s.c. MDMA, and the associated pharmacodynamic consequences. Blood was collected via jugular catheter at 0, 0.5, 1, 2, 4, 6, 8, 16, and 24 hours, with simultaneous serotonin (5-HT) behavioral syndrome and core temperature monitoring. Plasma specimens were analyzed for MDMA and the metabolites (±)-3,4-dihydroxymethamphetamine (HHMA), (±)-4-hydroxy-3-methoxymethamphetamine (HMMA), and (±)-3,4-methylenedioxyamphetamine (MDA) by liquid chromatography–tandem mass spectrometry. After 2.5 mg/kg MDMA, mean MDMA C max was 164 ± 47.1 ng/ml, HHMA and HMMA were major metabolites, and 〈20% of MDMA was metabolized to MDA. After 5- and 10-mg/kg doses, MDMA areas under the curve (AUCs) were 3- and 10-fold greater than those after 2.5 mg/kg; HHMA and HMMA AUC values were relatively constant across doses; and MDA AUC values were greater than dose-proportional. Our data provide decisive in vivo evidence that MDMA and MDA display nonlinear accumulation via metabolic autoinhibition in the rat. Importantly, 5-HT syndrome severity correlated with MDMA concentrations ( r = 0.8083; P 〈 0.0001) and core temperature correlated with MDA concentrations ( r = 0.7595; P 〈 0.0001), suggesting that MDMA’s behavioral and hyperthermic effects may involve distinct mechanisms. Given key similarities between MDMA pharmacokinetics in rats and humans, data from rats can be useful when provided at clinically relevant doses.

Description: Purpose: The aim of this study was to identify and independently validate a novel gene signature predicting locoregional tumor control (LRC) for treatment individualization of patients with locally advanced HPV-negative head and neck squamous cell carcinomas (HNSCC) who are treated with postoperative radio(chemo)therapy (PORT-C). Experimental Design: Gene expression analyses were performed using NanoString technology on a multicenter training cohort of 130 patients and an independent validation cohort of 121 patients. The analyzed gene set was composed of genes with a previously reported association with radio(chemo)sensitivity or resistance to radio(chemo)therapy. Gene selection and model building were performed comparing several machine-learning algorithms. Results: We identified a 7-gene signature consisting of the three individual genes HILPDA, CD24, TCF3 , and one metagene combining the highly correlated genes SERPINE1, INHBA, P4HA2 , and ACTN1 . The 7-gene signature was used, in combination with clinical parameters, to fit a multivariable Cox model to the training data (concordance index, ci = 0.82), which was successfully validated (ci = 0.71). The signature showed improved performance compared with clinical parameters alone (ci = 0.66) and with a previously published model including hypoxia-associated genes and cancer stem cell markers (ci = 0.65). It was used to stratify patients into groups with low and high risk of recurrence, leading to significant differences in LRC in training and validation ( P 〈 0.001). Conclusions: We have identified and validated the first hypothesis-based gene signature for HPV-negative HNSCC treated by PORT-C including genes related to several radiobiological aspects. A prospective validation is planned in an ongoing prospective clinical trial before potential application in clinical trials for patient stratification. Clin Cancer Res; 24(6); 1364–74. ©2018 AACR .

Description: Purpose: Following cytotoxic therapy, 70% of patients with human papillomavirus (HPV)-positive oropharyngeal head and neck squamous cell carcinoma (HNSCC) are alive at 5 years compared with 30% of those with similar HPV-negative cancer. Loss of TGFβ signaling is a poorly studied consequence of HPV that could contribute to patient outcome by compromising DNA repair. Experimental Design: Human HNSCC cell lines ( n = 9), patient-derived xenografts ( n = 9), tissue microarray ( n = 194), TCGA expression data ( n = 279), and primary tumor specimens ( n = 10) were used to define the relationship between TGFβ competency, response to DNA damage, and type of DNA repair. Results: Analysis of HNSCC specimens in situ and in vitro showed that HPV associated with loss of TGFβ signaling that increased response to radiation or cisplatin. TGFβ suppressed miR-182, which inhibited both BRCA1, necessary for homologous recombination repair (HRR), and FOXO3, required for ATM kinase activity. TGFβ signaling blockade by either HPV or inhibitors released miR182 control, compromised HRR and increased response to PARP inhibition. Antagonizing miR-182 rescued the HRR deficit in HPV-positive cells. Loss of TGFβ signaling unexpectedly increased repair by error prone, alternative end-joining (alt-EJ). Conclusions: HPV-positive HNSCC cells are unresponsive to TGFβ. Abrogated TGFβ signaling compromises repair by HRR and increases reliance on alt-EJ, which provides a mechanistic basis for sensitivity to PARP inhibitors. The effect of HPV in HNSCC provides critical validation of TGFβ’s role in DNA repair proficiency and further raises the translational potential of TGFβ inhibitors in cancer therapy.

Description: Therapeutics that target the epidermal growth factor receptor (EGFR) can enhance the cytotoxic effects of ionizing radiation (IR). However, predictive genomic biomarkers of this radiosensitization have remained elusive. By screening 40 non–small cell lung cancer cell (NSCLC) lines, we established a surprising positive correlation between the presence of a KRAS mutation and radiosensitization by the EGFR inhibitors erlotinib and cetuximab. EGFR signaling in KRAS-mutant NSCLC cells promotes chromatin condensation in vitro and in vivo, thereby restricting the number of DNA double-strand breaks (DSB) produced by a given dose of IR. Chromatin condensation in interphase cells is characterized by an unexpected mitosis-like colocalization of serine 10 phosphorylation and lysine 9 trimethylation on histone H3. Aurora B promotes this process in a manner that is codependent upon EGFR and protein kinase C α (PKCα). PKCα, in addition to MEK/ERK signaling, is required for the suppression of DSB-inducible premature senescence by EGFR. Blockade of autophagy results in a mutant KRAS-dependent senescence-to-apoptosis switch in cancer cells treated with IR and erlotinib. In conclusion, we identify EGFR as a molecular target to overcome a novel mechanism of radioresistance in KRAS-mutant tumor cells, which stands in contrast to the unresponsiveness of KRAS-mutant cancers to EGFR-directed agents in monotherapy. Our findings may reposition EGFR-targeted agents for combination with DSB-inducing therapies in KRAS-mutant NSCLC. Cancer Res; 74(10); 2825–34. ©2014 AACR.

Description: Determining the structural elements that define substrates and inhibitors at the monoamine transporters is critical to elucidating the mechanisms underlying these disparate functions. In this study, we addressed this question directly by generating a series of N -substituted 3,4-methylenedioxyamphetamine analogs that differ only in the number of methyl substituents on the terminal amine group. Starting with 3,4-methylenedioxy- N -methylamphetamine, 3,4-methylenedioxy- N , N -dimethylamphetamine (MDDMA) and 3,4-methylenedioxy- N , N , N -trimethylamphetamine (MDTMA) were prepared. We evaluated the functional activities of the compounds at all three monoamine transporters in native brain tissue and cells expressing the transporters. In addition, we used ligand docking to generate models of the respective protein-ligand complexes, which allowed us to relate the experimental findings to available structural information. Our results suggest that the 3,4-methylenedioxyamphetamine analogs bind at the monoamine transporter orthosteric binding site by adopting one of two mutually exclusive binding modes. 3,4-methylenedioxyamphetamine and 3,4-methylenedioxy- N -methylamphetamine adopt a high-affinity binding mode consistent with a transportable substrate, whereas MDDMA and MDTMA adopt a low-affinity binding mode consistent with an inhibitor, in which the ligand orientation is inverted. Importantly, MDDMA can alternate between both binding modes, whereas MDTMA exclusively binds to the low-affinity mode. Our experimental results are consistent with the idea that the initial orientation of bound ligands is critical for subsequent interactions that lead to transporter conformational changes and substrate translocation.

Description: Radiotherapy is a curative treatment option in prostate cancer. Nevertheless, patients with high-risk prostate cancer are prone to relapse. Identification of the predictive biomarkers and molecular mechanisms of radioresistance bears promise to improve cancer therapies. In this study, we show that aldehyde dehydrogenase (ALDH) activity is indicative of radioresistant prostate progenitor cells with an enhanced DNA repair capacity and activation of epithelial–mesenchymal transition (EMT). Gene expression profiling of prostate cancer cells, their radioresistant derivatives, ALDH+ and ALDH− cell populations revealed the mechanisms, which link tumor progenitors to radioresistance, including activation of the WNT/β-catenin signaling pathway. We found that expression of the ALDH1A1 gene is regulated by the WNT signaling pathway and co-occurs with expression of β-catenin in prostate tumor specimens. Inhibition of the WNT pathway led to a decrease in ALDH+ tumor progenitor population and to radiosensitization of cancer cells. Taken together, our results indicate that ALDH+ cells contribute to tumor radioresistance and their molecular targeting may enhance the effectiveness of radiotherapy. Cancer Res; 75(7); 1482–94. ©2015 AACR.