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Background Vitamin D3 (VitD) deficiency is linked to increased incidence and worse survival in bladder cancer (BCa). In addition to cystectomy, patients are treated with cisplatin-based chemotherapy, however 30%-50% of patients do not benefit from this treatment. The effects of VitD deficiency on response to chemotherapy remain unknown. Methods To test effects of VitD supplementation on the response to cisplatin we analyzed patient serum VitD levels and correlated that with survival. In vivo, VitD deficient mice were treated with cisplatin, with or without pretreatment with the active VitD metabolite, 1,25 dihydroxyvitamin D3 (1,25D3 ). Lastly, using BCa cell lines, T24 and RT-112, the mechanism of action of 1,25D3 and cisplatin combination treatment was determined by apoptosis assays, as well as western blot and RT-PCR. Results In this study, we determined that low serum 25 hydroxyvitamin D3 (25D3 ) levels was significantly associated with worse response to cisplatin. Pretreating deficient mice with 1,25D3 , reduced tumor volume compared to cisplatin monotherapy. In vitro, 1,25D3 pretreatment increased the apoptotic response to cisplatin. 1,25D3 pretreatment increased expression of TAp73 and its pro-apoptotic targets, in a VDR dependent manner. VDR and its transcriptional targets were induced after 1,25D3 treatment and further increased after the combination of 1,25D3 and cisplatin in a TAp73 dependent manner. Conclusions Our data suggest that VitD deficiency could be a biomarker for poor response to cisplatin, and pretreating with VitD can increase the apoptotic response to cisplatin through VDR and TAp73 signaling crosstalk.

Advanced lung cancer patients that present with activating mutations in the epidermal growth factor receptor (EGFR) are treated with EGFR tyrosine kinase inhibitors (EGFR TKIs). While initially effective, all patients eventually develop therapeutic resistance and experience disease progression. Strategies to prevent EGFR TKI resistance are needed to improve patient outcomes. We chronically exposed H1975 cells (EGFR-L858R/T790M) to EGFR TKI osimertinib to study emergence of resistance. H1975 cells that were expanded under drug treatment (designated H1975OR) acquired an EMT phenotype and were re-sensitized to EGFR TKI upon prolonged drug withdrawal. These features led us to classify H1975OR as a model of drug tolerance rather than a model of stable drug resistance. Bulk RNA-sequencing revealed significant dysregulation of chromatin modifying genes in H1975OR. Bromodomain containing protein 9 (BRD9) was among the set of significantly upregulated chromatin regulators and was selected for further investigation as a mediator of drug tolerance. Although BRD9 is known to control stemness and EMT, its role in promoting EMT-associated TKI resistance is unknown. To test the contribution of BRD9 to EGFR TKI tolerance, pharmacological inhibition of BRD9 by the selective inhibitor, I-BRD9 significantly increased sensitivity to TKI in models with EMT phenotypes (IC50 reduced 3-4 fold). In contrast, I-BRD9 did not affect TKI sensitivity in two models where EGFR TKI resistance was genetically fixed. To gain mechanistic insights, H1975OR cells were treated with osimertinib +/-I-BRD9 and subjected to RNA-sequencing. Combination of osimertinib with I-BRD9 resulted in a significant decrease in EMT-related genes, including MMP9, Zeb2, PDGFRb and IL6. Further, use of I-BRD9 in these models diminished the mesenchymal phenotype, as measured in cell invasion assays. Genetic knockdown of BRD9 via shRNA phenocopied effects of I-BRD9 treatment, supporting BRD9 as the therapeutic target of I-BRD9 in our cell line models. To further establish a role for BRD9 in the emergence of drug tolerant cells, we exposed treatment naïve H1975 cells to EGFR TKI ± I-BRD9. I-BRD9 significantly decreased the size of the EGFR TKI tolerant population. In time course studies, I-BRD9 also delayed onset of TKI resistance. In conclusion, our data identifies BRD9 as a novel mediator of EMT-associated EGFR-TKI tolerance in EGFR-mutant lung cancer. Our data further implicates BRD9 inhibition as a novel strategy to delay the emergence of drug tolerant cells that eventually give rise to stable drug resistance. This work was supported by the Roswell Park Alliance Foundation and National Cancer Institute (NCI) grant P30CA016056 involving the use of Roswell Park Comprehensive Cancer Center’s Genomics and Bioinformatics Shared Resources. Citation Format: Hannah Calkins, Tatiana Shaurova, David W. Goodrich, Mukund Seshadri, Candace S. Johnson, Pamela A. Hershberger. BRD9 inhibition overcomes epithelial to mesenchymal transition (EMT)-associated tyrosine kinase inhibitor (TKI) tolerance in epidermal growth factor receptor (EGFR) mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1093.

EGFR tyrosine kinase inhibitors (EGFR TKIs) are the standard of care treatment for patients with EGFR-mutant lung adenocarcinoma (LUAD). Although initially effective, EGFR TKIs are not curative. Disease inevitably relapses due to acquired drug resistance. We hypothesized that vitamin D metabolites could be used with EGFR TKIs to prevent therapeutic failure. To test this idea, we investigated the link between serum 25-hydroxyvitamin D3 (25(OH)D3) and progression-free survival (PFS) in patients with EGFR-mutant LUAD that received EGFR TKIs (erlotinib n = 20 and afatinib n = 1). Patients who were 25(OH)D3-sufficient experienced significantly longer benefit from EGFR TKI therapy (mean 14.5 months) than those with 25(OH)D3 insufficiency (mean 10.6 months, p = 0.026). In contrast, 25(OH)D3 had no prognostic value in patients with KRAS-mutant LUAD that received cytotoxic chemotherapy. To gain mechanistic insights, we tested 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) activity in vitro. 1,25(OH)2D3 promoted epithelial differentiation and restored EGFR TKI sensitivity in models of EGFR TKI resistance that were associated with epithelial&ndash, mesenchymal transition (EMT). 1,25(OH)2D3 was ineffective in a non-EMT model of resistance. We conclude that vitamin D sufficiency portends increased PFS among EGFR-mutant LUAD patients that receive EGFR TKIs, and that vitamin D signaling maintains drug efficacy in this specific patient subset by opposing EMT.

Despite the recognized link between aging and cancer, most preclinical studies in experimental tumor models are conducted with 6- to 8-wk-old rodents. The goal of the present study was to examine the impact of age on tumor incidence, growth, and microenvironmental characteristics in mouse models of head and neck squamous cell carcinoma (HNSCC). Experimental studies were conducted with the 4-nitroquinoline-oxide (4NQO) oral carcinogenesis model and orthotopic FaDu HNSCC xenografts, established in young (7 to 12 wk of age) and old (65 to 70 wk of age) female C57BL/6 mice ( n = 44; 4NQO model) and severe combined immunodeficient mice ( n = 13; HNSCC xenografts). Noninvasive whole body magnetic resonance imaging revealed increased subcutaneous and visceral fat in aging animals of both strains. On histologic examination, a higher incidence ( P < 0.001) of severe dysplasia/invasive squamous cell carcinoma was observed in old mice (92%) as compared with young mice (69%). Old C57BL/6 mice exposed to 4NQO exhibited increased incidence of oral and extraoral (peritoneal masses) neoplasms (42%) versus their young counterparts ( P < 0.05). The incidence of extraoral neoplasms was significantly lower (16%) in the younger cohort. Interestingly, no difference in growth rate and oxygen saturation was observed between orthotopic FaDu xenografts established in old and young severe combined immunodeficient mice. Our observations suggest that host age may have an impact on the growth kinetics and progression of HNSCC in the immunocompetent 4NQO model. Further investigation into the impact of aging on tumor response to preventive and therapeutic intervention is warranted.

Recent studies highlight the importance of the RB1 tumor suppressor as a target for cancer therapy. Canonically, RB1 regulates cell cycle progression and represents the downstream target for cyclin-dependent kinase (CDK) 4/6 inhibitors that are in clinical use. However, newly discovered features of the RB1 pathway suggest new therapeutic strategies to counter resistance and improve precision medicine. These therapeutic strategies include deepening cell cycle exit with CDK4/6 inhibitor combinations, selectively targeting tumors that have lost RB1, and expanding therapeutic index by mitigating therapy-associated adverse effects. In addition, RB1 impacts immunological features of tumors and the microenvironment that can enhance sensitivity to immunotherapy. Lastly, RB1 specifies epigenetically determined cell lineage states that are disrupted during therapy resistance and could be re-installed through the direct use of epigenetic therapies. Thus, new opportunities are emerging to improve cancer therapy by exploiting the RB1 pathway.

Radiation therapy (RT) is an integral component of the standard of care for patients with head and neck squamous cell carcinoma (HNSCC). While advances in radiation delivery methods have led to improved outcomes, radioresistance poses a significant therapeutic challenge and a major cause of poor prognosis in this patient population. To date, a majority of studies investigating mechanisms of radioresistance have employed in vitro monolayer cell culture models that lack the heterogeneity and cell-extracellular matrix interactions. To overcome this limitation, we established and credentialed a panel of 3D patient-derived organoid (PDO) models of HNSCC to identify molecular signatures and signaling pathways that contribute to radiation resistance. A total of 7 PDOs were established from surgical HNSCC specimens and credentialed using a combination of histologic/immunohistochemical evaluation (H&E, cytokeratin, p16), genotyping (HPV), whole exome sequencing (WES) and RNA sequencing analysis. Radiation response of the PDO panel was also examined and assessed for concordance with patient response and PDOs were ranked based on their radiosensitivity and bioinformatic analysis was performed on radiosensitive and radioresistance PDOs to identify molecular pathways associated with resistance. All PDOs retained the histology, p16/HPV status and mutational landscape (p53, HLA-A) of donor patient tissues. Radiation response of PDOs showed excellent (100%) concordance with patient responses (n=5 with history of radiation treatment). RNA-seq revealed identified pathways associated with epithelial mesenchymal transition (IGFBP2, TIMP1, TIMP2, LAMA3, LOXL1), NOTCH signaling (NOTCH1, HES1, FZD1, FZD5, DTX1, NOTCH3, FZD7), E2F targets and G2M checkpoint signaling (MCM2, MCM7, MCM4, PCNA) and the p53 pathway (SFN, NDRG1, ALOX15B). Collectively, our findings illustrate the translational utility of PDOs as a robust platform in identifying potential therapeutic targets that could overcome radiation resistance in HNSCC. Ongoing studies are evaluating the potential of PDOs to serve as a screening platform to identify radiosensitizers through repurposing of FDA-approved drugs to facilitate rapid clinical translation. Citation Format: Vui King Vincent-Chong, Christian Gluck, Pamela A. Hershberger, Satrajit Sinha, Mukund Seshadri. Deciphering radiation resistance in head and neck cancer using patient derived organoids [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-061.

Patient-derived organoids (PDOs) are considered to be a cost-effective, high-throughput preclinical platform for studying cancer biology and evaluating novel therapeutics. While PDOs have been established and characterized for breast, prostate, colon, and pancreatic cancers, PDO models of head and neck squamous cell carcinomas (HNSCC) are limited. To address this paucity, in the present study, we established a panel of PDO models of HNSCC. Five HNSCC samples (3 larynx, 1 tongue, 1 parotid) were procured from donor patients following informed consent and processed to establish organoids. Histologic (H&E) and immunohistochemical evaluation (p16, cytokeratin) was performed along with assessment of PDO response to the alkylating agent, cisplatin, and two tyrosine kinase inhibitors, erlotinib and afatinib. Histologic analyses of established organoids revealed nested cells of squamous cell carcinoma with high N/C ratio similar to donor surgical tumor tissue. Immunohistochemistry revealed strong pan cytokeratin and absence of p16 staining in all samples. Paired PDO and PDX models also successfully retained the squamous histology. Dose-response curves revealed substantial variation in therapeutic profiles of the three agents across the organoid panel. While the IC50 of cisplatin was comparable across the larynx organoids, 2 larynx organoids were sensitive to the two TKIs while one was resistant, exhibiting ~5-fold higher IC50 values. The tongue organoid was relatively sensitive to all agents while the parotid SCC organoid was sensitive to EGFR TKIs but relatively resistant to cisplatin. Collectively, these results demonstrate the ability of organoid models to recapitulate the histologic architecture and response heterogeneity of human HNSCC. Investigation into the molecular profiles of our PDO panel is under way in our laboratory and will enable further development of organoids as a robust platform for evaluation of precision therapeutics against HNSCC. Citation Format: Vincent-Chong Vui King, Erin C. Tracy, Heinz Baumann, Pamela A Hershberger, Mukund Seshadri. Patient-derived organoids recapitulate response heterogeneity in head and neck cancer [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr A22.

Epidemiologic studies implicate vitamin D status as a factor that influences growth of EGFR mutant lung cancers. However, laboratory based evidence of the biological effect of vitamin D in this disease is lacking. To fill this knowledge gap, we determined vitamin D receptor (VDR) expression in human lung tumors using a tissue microarray constructed of lung cancer cases from never-smokers (where EGFR gene mutations are prevalent). Nuclear VDR was detected in 19/19 EGFR mutant tumors. Expression tended to be higher in tumors with EGFR exon 19 deletions than those with EGFR L858R mutations. To study anti-proliferative activity and signaling, EGFR mutant lung cancer cells were treated with the circulating metabolite of vitamin D, 25-hydroxyvitamin D3 (25D3). 25D3 inhibited clonogenic growth in a dose-dependent manner. CYP27B1 encodes the 1α-hydroxylase (1αOHase) that converts 25D3 to the active metabolite, 1,25-dihydroxyvitamin D3 (1,25D3). Studies employing VDR siRNA, CYP27B1 zinc finger nucleases, and pharmacologic inhibitors of the vitamin D pathway indicate that 25D3 regulates gene expression in a VDR-dependent manner but does not strictly require 1αOHase-mediated conversion of 25D3 to 1,25D3. To determine the effects of modulating serum 25D3 levels on growth of EGFR mutant lung tumor xenografts, mice were fed diets containing 100 or 10,000 IU vitamin D3/kg. High dietary vitamin D3 intake resulted in elevated serum 25D3 and significant inhibition of tumor growth. No toxic effects of supplementation were observed. These results identify EGFR mutant lung cancer as a vitamin D-responsive disease and diet-derived 25D3 as a direct VDR agonist and therapeutic agent.

The chemopreventive actions of vitamin D were examined in the N-nitroso-tris-chloroethylurea (NTCU) mouse model, a progressive model of lung squamous cell carcinoma (SCC). SWR/J mice were fed a deficient diet (D) containing no vitamin D3, a sufficient diet (S) containing 2,000 IU/kg vitamin D3, or the same diets in combination with the active metabolite of vitamin D, calcitriol (C; 80 μg/kg, weekly). The percentage (%) of the mucosal surface of large airways occupied by dysplastic lesions was determined in mice after treatment with a total dose of 15 or 25 μmol NTCU (N). After treatment with 15 μmol NTCU, the percentages of the surface of large airways containing high-grade dysplastic (HGD) lesions were vitamin D–deficient + NTCU (DN), 22.7% [P < 0.05 compared with vitamin D–sufficient +NTCU (SN)]; DN + C, 12.3%; SN, 8.7%; and SN + C, 6.6%. The extent of HGD increased with NTCU dose in the DN group. Proliferation, assessed by Ki-67 labeling, increased upon NTCU treatment. The highest Ki-67 labeling index was seen in the DN group. As compared with SN mice, DN mice exhibited a three-fold increase (P < 0.005) in circulating white blood cells (WBC), a 20% (P < 0.05) increase in IL6 levels, and a four-fold (P < 0.005) increase in WBC in bronchial lavages. Thus, vitamin D repletion reduces the progression of premalignant lesions, proliferation, and inflammation, and may thereby suppress development of lung SCC. Further investigations of the chemopreventive effects of vitamin D in lung SCC are warranted. Cancer Prev Res; 8(10); 895–904. ©2015 AACR.

Benzaldehyde dimethane sulfonate (BEN, DMS612, NSC281612) is a bifunctional alkylating agent currently in clinical trials. We previously characterized the degradation products of BEN in plasma and blood. The conversion of BEN to its carboxylic acid analogue (BA) in whole blood, but not plasma, suggests that an enzyme in RBCs may be responsible for this conversion. BEN conversion to BA was observed in renal carcinoma cells and appeared to correlate with IC₅₀. To better understand the pharmacology of BEN, we aimed to evaluate the metabolism and enzymes potentially responsible for the conversion of BEN to BA.Human red blood cells (RBC) were used to characterize kinetics and susceptibility to enzyme-specific inhibitors. Recombinant enzymes were used to confirm metabolism of BEN to BA. Analytes were quantitated with established LC-MS/MS methods.Average apparent Vmax and Km were 68 ng/mL min(-1) [10% RBC](-1) and 373 ng/mL, respectively. The conversion of BEN to BA in RBC was not inhibited by carbon monoxide, nitrogen gas, or menadione, an inhibitor of aldehyde oxidase. The conversion was inhibited by disulfiram, an inhibitor of ALDH. Of available ALDH isoforms ALDH1A1, ALDH3A1, ALDH2, and ALDH5A1, only ALDH1A1 converted BEN to BA.The activating conversion of BEN to BA is mediated not by CYP450 enzymes or aldehyde oxidase, but by ALDH1A1. This enzyme, a potential stem cell marker, may be a candidate biomarker for clinical activity of BEN.

Pharmacologic inhibition of DNA repair may increase the efficacy of many cytotoxic cancer agents. Inhibitors of DNA repair enzymes including APE1, ATM, ATR, DNA-PK and PARP have been developed and the PARP inhibitor olaparib is the first-in-class approved in Europe and the USA for the treatment of advanced BRCA-mutated ovarian cancer. Sensitive pharmacodynamic (PD) biomarkers are needed to further evaluate the efficacy of inhibitors of DNA repair enzymes in clinical trials. ATM is a protein kinase that mediates cell-cycle checkpoint activation and DNA double-strand break repair. ATM kinase activation at DNA double-strand breaks (DSBs) is associated with intermolecular autophosphorylation on serine-1981. Exquisite sensitivity and high stoichiometry as well as facile extraction suggest that ATM serine-1981 phosphorylation may be a highly dynamic PD biomarker for both ATM kinase inhibitors and radiation- and chemotherapy-induced DSBs. Here we report the pre-clinical analytical validation and fit-for-purpose biomarker method validation of a quasi-quantitative dual multiplexed immunoblot method to simultaneously analyze ATM and H2AX phosphorylation in human peripheral blood mononuclear cells (PBMCs). We explore the dynamics of these phosphorylations in PBMCs exposed to chemotherapeutic agents and DNA repair inhibitors in vitro, and show that ATM serine-1981 phosphorylation is increased in PBMCs in sarcoma patients treated with DNA damaging chemotherapy.

Veliparib (ABT-888) is an oral PARP inhibitor expected to increase gemcitabine activity. This phase I determined the maximal tolerable dose (MTD), dose-limiting toxicities (DLT), antitumor activity, pharmacokinetics (PK), and pharmacodynamics (PD) of veliparib combined with gemcitabine.Patients with advanced solid tumors received veliparib (10-40-mg PO BID) on chemotherapy weeks with gemcitabine 500-750-mg/mEleven patients were enrolled on the 28-day schedule. The 28-day schedule was considered intolerable and amended to a 21-day schedule, with 20 patients enrolled. Grade ≥ 3 adverse events were myelosuppression-related. The MTD was determined to be 750-mg/mGemcitabine at 750-mg/m

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) exerts anti-proliferative activity by binding to the vitamin D receptor (VDR) and regulating gene expression. We previously reported that non-small cell lung cancer (NSCLC) cells which harbor epidermal growth factor receptor (EGFR) mutations display elevated VDR expression (VDRhigh) and are vitamin D-sensitive. Conversely, those with K-ras mutations are VDRlow and vitamin D-refractory. Because EGFR mutations are found predominately in NSCLC cells with an epithelial phenotype and K-ras mutations are more common in cells with a mesenchymal phenotype, we investigated the relationship between vitamin D signaling capacity and the epithelial mesenchymal transition (EMT). Using NSCLC cell lines and publically available lung cancer cell line microarray data, we identified a relationship between VDR expression, 1,25(OH)2D3 sensitivity, and EMT phenotype. Further, we discovered that 1,25(OH)2D3 induces E-cadherin and decreases EMT-related molecules SNAIL, ZEB1, and vimentin in NSCLC cells. 1,25(OH)2D3-mediated changes in gene expression are associated with a significant decrease in cell migration and maintenance of epithelial morphology. These data indicate that 1,25(OH)2D3 opposes EMT in NSCLC cells. Because EMT is associated with increased migration, invasion, and chemoresistance, our data imply that 1,25(OH)2D3 may prevent lung cancer progression in a molecularly defined subset of NSCLC patients.

Background Cetuximab has demonstrated improved efficacy in combination with chemotherapy and radiotherapy. We evaluated the integration of cetuximab in the combined modality treatment of stage III non-small cell lung cancer (NSCLC). Methods Patients with surgically unresectable stage IIIA or IIIB NSCLC were treated with chest radiotherapy, 73.5 Gy (with lung and tissue heterogeneity corrections) in 35 fractions/7 weeks, once daily (63 Gy without heterogeneity corrections). Cetuximab was given weekly during radiotherapy and continued during consolidation therapy with carboplatin and paclitaxel up to a maximum of 26 weekly doses. The primary endpoint was overall survival. Baseline tumor tissue was analyzed for EGFR by fluorescence in situ hybridization (FISH). Results Forty patients were enrolled in this phase II study. The median overall survival was 19.4 months and the median progression-free survival 9.3 months. The best overall response rate in 31 evaluable patients was 67%. No grade 3 or 4 esophagitis was observed. Three patients experienced grade 3 rash; 16 patients (69%) developed grade 3/4 neutropenia during consolidation therapy. One patient died of pneumonitis, possibly related to cetuximab. EGFR gene copy number on baseline tumor tissues, analyzed by FISH, was not predictive of efficacy outcomes. Conclusions The addition of cetuximab to chest radiotherapy and consolidation chemotherapy was tolerated well and had modest efficacy in stage III NSCLC. Taken together with the lower incidence of esophagitis, our results support evaluation of targeted agents instead of chemotherapy with concurrent radiotherapy in this setting.

We observed a 53% response rate in non-small cell lung cancer (NSCLC) patients treated with vorinostat plus paclitaxel/carboplatin in a Phase I trial. Studies were undertaken to investigate the mechanism (s) underlying this activity. Growth inhibition was assessed in NSCLC cells by MTT assay after 72 hr of continuous drug exposure. Vorinostat (1 microM) inhibited growth by: 17% +/- 7% in A549, 28% +/- 6% in 128-88T, 39% +/- 8% in Calu1 and 41% +/- 7% in 201T cells. Vorinostat addition to carboplatin or paclitaxel led to significantly greater growth inhibition than chemotherapy alone in all 4 cell lines. Vorinostat (1 microM) synergistically increased the growth inhibitory effects of carboplatin/paclitaxel in 128-88T cells. When colony formation was measured after drug withdrawal, vorinostat significantly increased the effects of carboplatin but not paclitaxel. The % colony formation was control 100%; 1 microM vorinostat, 83% +/- 10%; 5 microM carboplatin, 41% +/- 11%; carboplatin/vorinostat, 8% +/- 4%; 2 nM paclitaxel, 53% +/- 11%; paclitaxel/vorinostat, 46% +/- 21%. In A549 and 128-88T, vorinostat potentiated carboplatin induction of gamma-H2AX (a DNA damage marker) and increased alpha-tubulin acetylation (a marker for stabilized mictrotubules). In A549, combination of vorinostat with paclitaxel resulted in a synergistic increase in alpha-tubulin acetylation, which reversed upon drug washout. We conclude that vorinostat interacts favorably with carboplatin and paclitaxel in NSCLC cells, which may explain the provocative response observed in our clinical trial. This likely involves a vorinostat-mediated irreversible increase in DNA damage in the case of carboplatin and a reversible increase in microtubule stability in the case of paclitaxel.

We have isolated from human breast cancers several mutations in the Helix 12 component of activation function 2 (AF-2) in the estrogen receptor alpha (ERalpha). We used a novel approach to detect changes in the hormone-binding domain of ERalpha, based on the evidence that antiestrogens, such as 4-hydroxytamoxifen (ZOHT) and ICI 182,780, block the function of ERalpha by binding and folding the AF-2 transcriptional domain in a way that inhibits its association with coactivator proteins. We have identified a Helix 12 mutation, M543V, which leads to greater ERalpha transcription with ZOHT and other antiestrogens (including 1,1-dichloro-2,2,3-triarylcyclopropanes, DTACs) than with 17-beta estradiol (E2). We also found an independent mutation at the same position, M543I, which did not show this inverted ligand phenotype. In comparison to further Helix 12 mutations made in vitro, it appears that relative hydrophobicity of the amino acid side chains on the inner face of Helix 12 is key to maintaining the transcriptionally active, agonist conformation with bound E2. This active conformation can be induced, resulting in increased transcription, by adding excess p160 coactivator AIB1 in transcriptional assays with E2-bound receptors, while the ZOHT-bound receptors were not further activated by AIB1. Other experiments show that the cross talk between ERalpha and AP-1 protein from AP-1-binding sites is not dependent on Helix 12 integrity. We show that two alleles containing a proline substitution in Helix 12 that inactivate AF-2 function of ERalpha at EREs have little negative effect on function through AP-1 elements, supporting a prominent role for the N-terminal AF-1 of ERalpha in AP-1/ERalpha transcriptional cross talk.

1Alpha,25-dihydroxyvitamin D3 (1,25D3) displays potent antiproliferative activity in a variety of tumor model systems and is currently under investigation in clinical trials in cancer. Studies were initiated to explore its potential in nonsmall cell lung cancer (NSCLC), as effective approaches to the treatment of that disease are needed. In evaluating factors that may affect activity in NSCLC, the authors found that CYP24 (25-hydroxyvitamin D3-24-hydroxylase), the enzyme that catabolizes 1,25D3, is frequently expressed in NSCLC cell lines but not in the nontumorigenic bronchial epithelial cell line, Beas2B. CYP24 expression by RT-PCR was also detected in 10/18 primary lung tumors but in only 1/11 normal lung tissue specimens. Tumor-specific CYP24 upregulation was confirmed at the protein level via immunoblot analysis of patient-matched normal lung tissue and lung tumor extracts. Enzymatically active CYP24 is expected to desensitize NSCLC cells to 1,25D3. The authors therefore implemented a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for 1,25D3 and its CYP24-generated metabolites to determine whether NSCLC cells express active enzyme. Analysis of NSCLC cell cultures revealed time-dependent loss of 1,25D3 coincident with the appearance of CYP24-generated metabolites. MK-24(S)-S(O)(NH)-Ph-1, a specific inhibitor of CYP24, slowed the loss of 1,25D3 and increased 1,25D3 half-life. Furthermore, combination of 1,25D3 with MK-24(S)-S(O)(NH)-Ph-1 resulted in a significant decrease in the concentration of 1,25D3 required to achieve maximum growth inhibition in NSCLC cells. These data suggest that increased CYP24 expression in lung tumors restricts 1,25D3 activity and support the preclinical evaluation of CYP24 inhibitors for lung cancer treatment.

Estrogen receptor (ER) agonists and antagonists elicit distinct responses in non–small cell lung cancer (NSCLC) cells. To determine how such responses are generated, the expression of ERα, ERβ, and ER coregulators in human lung fibroblasts and human NSCLC cell lines was evaluated by immunoblot. Ligand-dependent estrogenic responses in NSCLC cells are probably generated via ERβ and the p160 coactivator GRIP1/TIF2, because expression of these proteins was detected, but not full-length ERα or the p160 coactivator SRC-1. ERβ and GRIP1/TIF2 are shown to interact in vitro in a ligand-dependent manner and thus may form functional transcription complexes in NSCLC cells. Furthermore, the capacity of ER ligands to regulate gene expression in NSCLC cells was explored using gene miniarrays. Expression profiles were examined after treatment with ER agonist 17-β-estradiol (E2), the pure ER antagonist ICI 182,780 (fulvestrant, Faslodex), or epidermal growth factor, which served as a positive control for an alternative growth stimulus. E-cadherin and inhibitor of differentiation 2 were differentially regulated by E2 versus ICI 182,780 in 201T and 273T NSCLC cell lines. Epidermal growth factor also stimulated proliferation of these cells but had no effect on expression of E-cadherin and inhibitor of differentiation 2, suggesting they are specific targets of ER signaling. These data show that NSCLC cells respond to estrogens/antiestrogens by altering endogenous gene expression and support a model in which ICI 182,780 reduces proliferation of NSCLC cells via its ability to disrupt ER signaling. ICI 182,780 may therefore have therapeutic benefit in NSCLC.

Calcitriol or 1,25-dihydroxycholecalciferol (vitamin D) is classically known for its effects on bone and mineral metabolism. Epidemiological data suggest that low vitamin D levels increase the risk and mortality from prostate cancer. Calcitriol is also a potent anti-proliferative agent in a wide variety of malignant cell types including prostate cancer cells. In prostate model systems (PC-3, LNCaP, DU145, MLL) calcitriol has significant anti-tumor activity in vitro and in vivo. Calcitriol’s effects are associated with an increase in cell cycle arrest, apoptosis, differentiation and in the modulation of growth factor receptors. Calcitriol induces asignificant Go/Gii arrest and modulates p21Waf1/Cip1 and p27Kip1, the cyclin dependent kinase inhibitors. Calcitriol induces PARP cleavage, increases the bax/bcl-2 ratio, reduces levels of phosphorylated mitogen-activated protein kinases (P-MAPKs, P-Erk-1/2) and phosphorylated Akt (P-Akt), induces caspase-dependent MEK cleavage and up-regula tion of MEKK-1, all potential markers of the apoptotic pathway. Glucocorticoids potentiate the anti-tumor effect of calcitriol and decrease calcitriol-induced hypercalcemia. In combination with calcitriol, dexamethasone results in a significant time- and dose-dependent increase in VDR protein and an enhanced apoptotic response as compared to calcitriol alone. Calcitriol can also significantly increase cytotoxic drug-mediated anti-tumor efficacy. As a result, phase I and II trials of calcitriol either alone or in combination with the carboplatin, paclitaxel, or dexamethasone have been initiated in patients with androgen-dependent and -independent prostate cancer and advanced cancer. Patients were evaluated for toxicity, maximum tolerated dose (MTD), schedule effects, and PSA response. Data from these studies indicate that high-dose calcitriol is feasible on an intermittent schedule, the MTD is still being delineated and dexamethasone or paclitaxel appear to ameliorate toxicity. Studies continue to define the MTD of calcitriol which can be safely administered on this intermittent schedule either alone or with other agents and to evaluate the mechanisms of calcitriol effects in prostate cancer.

Activation of the epidermal growth factor receptor (EGFR) pathway is an early event in head and neck carcinogenesis. As a result, targeting EGFR for chemoprevention of head and neck squamous cell carcinomas (HNSCC) has received considerable attention. In the present study, we examined the impact of 1,25(OH)2D3, the active metabolite of the nutritional supplement vitamin D on the chemopreventive efficacy of the EGFR inhibitor, erlotinib, against HNSCC. Experimental studies were conducted in patient-derived xenografts (PDX) and the 4-nitroquinoline-1-oxide (4NQO) carcinogen-induced model of HNSCC. Short-term treatment (4 weeks) of PDX-bearing mice with 1,25(OH)2D3 and erlotinib resulted in significant inhibition of tumor growth. Noninvasive MRI enabled longitudinal monitoring of disease progression in the 4NQO model with 100% of control animals showing evidence of neoplastic lesions by 24 weeks. Among the experimental groups, animals treated with the combination regimen showed the greatest reduction in tumor incidence and volume (P < 0.05). Combination treatment was well tolerated and was not associated with any significant change in body weight. Histopathologic assessment revealed a significant reduction in the degree of dysplasia with combination treatment. Immunoblot analysis of whole tongue extracts showed downregulation of phospho-EGFR and phospho-Akt with the combination regimen. These results highlight the potential of 1,25(OH)2D3 to augment the efficacy of erlotinib against HNSCC. Further optimization of schedule and sequence of this combination regimen along with investigation into the activity of less calcemic analogues or dietary vitamin D is essential to fully realize the potential of this approach. Cancer Prev Res; 8(9); 765–76. ©2015 AACR.

Background. Despite remarkable initial therapeutic responses, most patients develop resistance to the first generation of EGFR tyrosine kinase inhibitors (TKIs), erlotinib and gefitinib, within one year of treatment initiation. Epithelial-mesenchymal transition (EMT) is one of the key mechanisms of resistance to EGFR TKIs. We recently demonstrated that vitamin D promotes epithelial phenotype in NSCLC cells. We hypothesized that we could prevent/overcome EMT-driven resistance by combining vitamin D with EGFR TKIs. Methods. EGFR-mutant, erlotinib sensitive HCC827 cells were treated with: vehicle control, 100nM 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), 1ng/ml TGF-β, or 1,25(OH)2D3+TGF-β for 14 days. Expression of EMT markers was determined by q-RT PCR and immunoblot. Sensitivity to erlotinib was analyzed by MTT assay. For the in-vivo study, HCC 827 tumor xenografts were established in vitamin-D deficient mice. Mice were stratified based on tumor volume to receive diets containing 25 IU vitamin D3/kg (deficient) or 10,000 IU vitamin D3/kg (supplemented). Erlotinib treatment (12.5 mg/kg) was initiated two weeks after the diet switch and administered 5 days per week for the duration of the study. Tumors were collected two weeks after the diet switch (baseline), 48 hours after the erlotinib initiation (acute response) and upon treatment failure (outgrowth). Results. Vitamin D attenuated expression of mesenchymal markers in the presence of TGF-β in-vitro and opposed TGF-β driven resistance to erlotinib. Erlotinib EC50 values were 9.85 nM, 122 nM and 3.83 nM for vehicle, TGF-β, and 1,25(OH)2D3+TGF-β treated cells, respectively. In-vivo, vitamin D supplementation enhanced acute response to erlotinib. We documented a greater decrease in tumor volume after 48h of erlotinib initiation (1.1% of baseline ±19.3% in vitamin D deficient diet and 25.9% ±29.5% in supplemented diet). Greater efficacy was accompanied by increased suppression of STAT3 phosphorylation and decreased vimentin expression in the vitamin D supplemented group. Although we observed a trend towards a longer progression-free survival in animals maintained on vitamin D supplemented diet (8 vs 5.5 weeks), statistical significance was not achieved. The outgrowth tumors in both groups maintained their epithelial phenotype but exhibited a significant increase in the expression of AXL tyrosine kinase. However, outgrowth tumors from the vitamin D supplemented group had lower AXL expression than those from the vitamin D deficient group. Conclusion. Vitamin D supports epithelial phenotype in-vitro and enhances acute response to erlotinib in-vivo, possibly, by suppressing STAT3 signaling. Vitamin D supplementation also attenuates overexpression of AXL tyrosine kinase in erlotinib resistant tumors. The clinical implication of our work is that patients diagnosed with EGFR mutant NSCLC who receive erlotinib may benefit from vitamin D supplementation. Citation Format: Tatiana Shaurova, Suzanne F. Shoemaker, Pamela A. Hershberger. Vitamin D enhances erlotinib response in EGFR-mutant non-small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4674.

Cytidine drugs, such as gemcitabine, undergo rapid catabolism and inactivation by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU), a potent CD inhibitor, has been applied preclinically and clinically as a modulator of cytidine analogue metabolism. However, THU is only 20% orally bioavailable, which limits its preclinical evaluation and clinical use. Therefore, we characterized THU pharmacokinetics after the administration to mice of the more lipophilic pro-drug triacetyl-THU (taTHU).Mice were dosed with 150 mg/kg taTHU i.v. or p.o. Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma and urine pharmacokinetic parameters were calculated non-compartmentally and compartmentally.taTHU did not inhibit CD. THU, after 150 mg/kg taTHU i.v., had a 235-min terminal half-life and produced plasma THU concentrations1 μg/mL, the concentration shown to inhibit CD, for 10 h. Renal excretion accounted for 40-55% of the i.v. taTHU dose, 6-12% of the p.o. taTHU dose. A two-compartment model of taTHU generating THU fitted the i.v. taTHU data best. taTHU, at 150 mg/kg p.o., produced a concentration versus time profile with a plateau of approximately 10 μg/mL from 0.5-2 h, followed by a decline with a 122-min half-life. Approximately 68% of i.v. taTHU is converted to THU. Approximately 30% of p.o. taTHU reaches the systemic circulation as THU.The availability of THU after p.o. taTHU is 30%, when compared to the 20% achieved with p.o. THU. These data will support the clinical studies of taTHU.

Lung cancer has long been thought of as a cancer that mainly affects men, but over the past several decades, because of the high increase in tobacco use by women, there has been a corresponding dramatic increase in lung cancer among women. Since 1998, lung cancer deaths in women have surpassed those caused by breast cancer in the United States. Annual lung cancer deaths among US women currently surpass those caused by breast, ovarian, and cervical cancers combined. Women are more likely than men to be diagnosed with adenocarcinoma and small cell carcinoma of the lung compared to squamous cell carcinoma, and never-smokers diagnosed with lung cancer are almost three times more likely to be female than male. These observations in the population, coupled to the findings that both estrogen receptors (ERs) and aromatase, the enzyme that synthesizes 17beta-estradiol, are expressed by lung tumors, suggest a role for female steroid hormones in control of lung cancer growth. Preclinical data and clinical data are increasingly emerging to support this concept, and to suggest that a local production of estrogen and expression of ERs occurs in lung tumors that arise in men as well as in women. An additional protein that recognizes 17beta-estradiol with high affinity, GPR30, also is expressed in lung tumors at high levels and may be responsible for some of the proliferation signals induced by estrogen.

1α,25-Dihydroxyvitamin D3 (1,25D3) exhibits antitumor activity in a variety of cancers including squamous cell carcinoma (SCC). Intrinsic resistance of SCC cells to cisplatin was observed and led to the investigation into whether 1,25D3 sensitizes SCC cells to cisplatin. Pretreatment with 1,25D3 followed by cisplatin enhanced growth inhibition in SCC cells compared with 1,25D3 alone as assessed by cytotoxicity and in vitro clonogenic assays. In addition, 1,25D3 sensitized SCC cells to cisplatin-mediated apoptosis. Treatment of tumor-bearing C3H mice with 1,25D3 before cisplatin reduced clonogenic survival using in vivo excision clonogenic assay. These results were not observed in a 1,25D3-resistant SCC variant, indicating the critical role of 1,25D3 in sensitizing SCC cells to cisplatin. Further, a marked decrease in fractional tumor volume was observed when SCC tumor-bearing mice were treated with 1,25D3 before cisplatin compared with either agent administered alone. Cisplatin has been shown to modulate p73 protein level in certain cancer cells. Our data showed that p73 level was not affected by cisplatin but increased by 1,25D3 in SCC cells. Knocking down p73 by small interfering RNA protected SCC cells against 1,25D3 and cisplatin-mediated clonogenic cell kill and apoptosis. Increasing p73 protein level by knocking down UFD2a, which mediates p73 degradation, promoted 1,25D3 and cisplatin-mediated clonogenic cell kill. These results suggest that 1,25D3 potentiates cisplatin antitumor activity in vitro and in vivo in a SCC model system possibly through p73 induction and apoptosis. The combination treatment may provide a more effective therapeutic regimen in cancer treatment. [Mol Cancer Ther 2008;7(9):3047–55]

Background Adenocarcinoma of the prostate is the most frequently diagnosed non-cutaneous cancer and the second leading cause of cancer-related deaths among men in the United States. The most successful therapies to date for this tumor have involved some form of androgen ablation. However, these therapies become ineffective as the tumor evolves to an androgen-insensitive state. Ceramide is a lipid second messenger that has been shown to mediate growth arrest or cell death when added exogenously to prostate cancer cells. As a first step toward understanding the events that lead to the transition of prostate cancer cells to an androgen-independent state, we considered investigating the effect of androgen ablation on endogenous ceramide levels in androgen-sensitive and androgen-insensitive prostate cancer cells. Methods To investigate the mechanisms of growth arrest/apoptosis in androgen-sensitive (LNCaP) and insensitive (DU-145, PC–3) cells, we used various methods including nonyl acridine orange (NAO) staining, propidium iodide (PI) staining/cell-cycle analysis, lipid analysis, and Western blotting assays. Results In this study, we demonstrate that androgen ablation drives G0/G1-phase cell-cycle arrest followed by progressive apoptosis in vitro, in LNCaP cells. Lipid analysis indicated an increase in C16 ceramide, which was generated via the de novo pathway as revealed by blockade of ceramide synthase by fumonisin B1. The addition of 5α-dihydrotestosterone (DHT) or fumonisin B1 rescued LNCaP cells from apoptosis induced by androgen ablation, and decreased levels of intracellular C16 ceramide. Neither apoptosis nor an increase in C16 ceramide was observed in androgen-independent cell lines following androgen ablation. Prostate 57: 66–79, 2003. © 2003 Wiley-Liss, Inc.

Introduction 25-hydroxyvitamin D3 (25(OH)D3) levels are associated with increased overall survival in early stage non-small cell lung cancer (NSCLC). Studies suggest that 25(OH)D3 is converted locally in tissues by CYP27B1 into the active metabolite, 1,25-dihydroxyvitamin D (1α,25(OH)2D3). Once produced, 1α,25(OH)2D3 exerts anti-tumor effects by binding to the vitamin D receptor (VDR) and regulating target gene expression. It is unknown whether CYP27B1 is necessary for NSCLC cells to respond to 25(OH)D3. We therefore tested the requirement for VDR and CYP27B1 in mediating the anti-tumor effects of 25(OH)D3 in NSCLC. Methods Immunoblot and qRT-PCR were used to evaluate VDR expression. Small interfering RNA was used to knock down the VDR. Colony formation assays and qRT-PCR were used to assess effects of 25(OH)D3 and 1α,25(OH)2D on growth and transcriptional responses. To study the requirement for CYP27B1, pharmacological inhibition was achieved using ketoconazole, and 1α,25(OH)2D3 production was measured by LC/MS-MS. A xenograft study was performed to determine the in vivo effect of modulating 25(OH)D3 levels. Results NSCLC cells expressing high levels of VDR (VDRhigh) are more responsive to vitamin D metabolites. In these cells, 25(OH)D3 and 1α,25(OH)2D3 induce target gene transcription and inhibit colony formation. These effects are not observed in VDRlow cells. When VDRhigh cells were transfected with VDR siRNA, the ability of 25(OH)D3 to induce target gene transcription was diminished, and no VDR protein was observed. VDRhigh cells were exposed to ketoconazole to determine if effects of 25(OH)D3 required conversion to 1α,25(OH)2D3. Ketoconazole effectively abrogated production of 1α,25(OH)2D3 from 25(OH)D3. However, VDR target gene expression remained induced by 25(OH)D3. Thus, NSCLC cells may not require CYP27B1 to promote vitamin D signaling. To more specifically inhibit CYP27B1, zinc finger nucleases (ZFNs) were used to create CYP27B1 knockout cells. The ZFNs have been transfected into cells, assays optimized, and positive clones are being expanded. Lastly, a mouse xenograft experiment was performed to determine if 25(OH)D3 promotes a decrease in tumor volume. Mice were fed diets containing 100, 1,000 or 10,000 IU/kg vitamin D3 prior to implantation of VDRhigh NSCLC cells. Mice fed the 10,000 IU/kg diet had a statistically significant decrease in tumor volume. Conclusions NSCLC cells expressing high levels of VDR display increased responsiveness to vitamin D metabolites. Although NSCLC cells express CYP27B1, our results demonstrate that the enzyme may not be required to achieve vitamin D signaling. Rather, 25(OH)D3 may act via the VDR to elicit an anti-tumor responses. The implication of these findings is that dietary supplementation to increase circulating 25(OH)D3 may be beneficial in a subset of NSCLC patients. Funding provided by NIH RO1 CA132844, training grant 5T32CA009072-37 and P30CA47904 Citation Format: Alissa R. Verone, Suzanne Shoemaker, Robert Parise, Jan H. Beumer, Pamela A. Hershberger. 25-hydroxyvitamin D3 induces vitamin D signaling independent of CYP27B1 in non-small cell lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4751. doi:10.1158/1538-7445.AM2014-4751

Progress has recently been made in identifying populations at risk for lung cancer using genetic, clinical and demographic information. The N-nitroso-tris-chloroethylurea (NTCU) mouse model, in which animals develop premalignant histopathology similar to that seen in humans, is used to examine the potential efficacy of chemoprevention agents to be utilized in at risk populations. We identified 25 µl of 40 mM NTCU /week as the optimal dosing regimen in SWR/J mice for chemoprevention studies. At this dose, topical treatments of NTCU induce predominantly low-grade dysplastic lesions by 15 weeks (w) and high-grade dysplastic (HGD) lesions by 25 w in the large airways. Additionally we found that NTCU stimulates a state of chronic inflammation associated with the development of dysplasias. Epidemiologic studies indicate that there is an inverse relationship between vitamin D and the risk and prognosis of lung cancer. Vitamin D acts through the vitamin D receptor to promote cellular differentiation and inhibit proliferation and inflammation. The effect of vitamin D on cancer progression was tested in the NTCU model, using dietary vitamin D3 (0 or 2000 IU/kg) alone and in combination with intraperitoneal injections of the active metabolite of vitamin D, calcitriol (80 ug/kg). Female mice were randomized to 6 treatment groups (n=15 mice/group/time point (15 and 25 w)). Disease was evaluated by enumerating the percentage of HGD lesions, per the total area in serial H&E sections of the lung. The percentage of HGD lesions in the large airways was reduced in the vitamin D sufficient mice (SN) (8.72%, P Furthermore, there was a significant increase in proliferation associated with increased HGD. Following 25 w of NTCU treatment there was a 2-fold increase in Ki-67 staining all groups compared all groups after 15 w of NTCU. However, the was 30% more Ki-67staining in the DN (P Citation Format: Sarah A. Mazzilli, Mary E. Reid, Paul N. Bogner, Kristopher Attwood, Pamela A. Hershberger, Donald L. Trump, Candace S. Johnson. Vitamin D sufficiency slows the progression of dysplasic lesions in the NTCU mouse model of lung squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 238. doi:10.1158/1538-7445.AM2014-238

7019 Background: C, an anti-EGFR monoclonal antibody, enhances the efficacy of both RT and CT. We evaluated a novel strategy of RT plus C followed by consolidation CT plus C for the treatment of st...

Introduction: TMZ is a non-classical alkylating agent that induces DNA damage leading to cytotoxicity, with modest clinical activity in metastatic melanoma. DNA repair modulation with PARP inhibitors enhances TMZ cytotoxicity in vitro and in vivo, and this strategy is being investigated in clinical trials. We hypothesized that HDAC inhibition with SAHA would lead to increased DNA damage and potentiate the effect of the PARP inhibitor MK-4827 on TMZ cytotoxicity. Methods: Human melanoma cell lines A375 and FEMX-1 were used. Cells were treated in the absence or presence of TMZ, SAHA, and MK-4827 at the indicated concentrations for 72 hr in 96-well plates. Cell viability was determined using MTS assays and normalized to vehicle-treated control cells. Drugs were used at the final concentrations indicated in Table 1. Results: MK-4827 at 10 nM increased the cytotoxicity of TMZ in FEMX-1 but not in A375 cells, however 100 and 200 nM MK-4827 moderately potentiated TMZ cytotoxicity in both cell lines. The combination of TMZ with 1 µM SAHA and 200 nM MK-4827 resulted in profound cytotoxicity at reduced doses of TMZ (0.2 mM in FEMX-1 and 1.0 mM in A375), much lower than TMZ alone or in combination with either SAHA or MK-4827. >80% cell kill was achieved in A375 and >95% in FEMX-1 near the IC50 concentration of TMZ and at clinically achievable concentrations. Conclusion: PARP inhibition offers a strategy to reverse melanoma TMZ resistance. The concomitant inhibition of HDAC with SAHA and of PARP with MK-4827 profoundly enhanced TMZ cytotoxicity at clinically achievable doses of all 3 agents. We are presently optimizing the dosing schedules of the 3 drugs in preclinical models and investigating the mechanism(s) underlying this potentiation. These findings support the investigation of the triple drug combination (TMZ+SAHA+MK-4827) for metastatic melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5499. doi:10.1158/1538-7445.AM2011-5499

Introduction Cytidine drugs, such as gemcitabine, undergo rapid, inactivating catabolism by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU), a potent CD inhibitor, has been applied preclinically and clinically as a modulator of cytidine drug metabolism. However, p.o. THU is at most 20% bioavailable (Beumer et al.), which limits its preclinical evaluation and clinical use. Therefore, the more lipophilic prodrug triacetyl THU (taTHU) was developed. We characterized THU pharmacokinetics after administration of taTHU to mice. Methods Mice were dosed 150 mg/kg taTHU i.v. or p.o. Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma and urine pharmacokinetic parameters and p.o. bioavailability were calculated non-compartmentally and compartmentally. We assessed taTHU inhibition of metabolism of gemcitabine by recombinant CD. ResultsTHU parameterstaTHU i.v. 150 mg/kgtaTHU p.o. 150 mg/kgCmax (µg/mL)10611.8Tmax (min)560Half-life (min)235122AUC0-inf (mg/mL*min)7.553.36Vd/F (L/kg)4.495.26Cl/F (mL/min/kg)13.229.8F-THUi.v. (%)68.030.3% dose in 0-16 h urine405.6% dose in 0-24 h urine5512 THU, after 150 mg/kg taTHU i.v., had a 235 min terminal half-life and produced plasma THU concentrations >1 µg/mL, the concentration shown to inhibit CD, for 10 h. A multi-compartment model fit the data best. 150 mg/kg p.o. taTHU produced a concentration versus time profile with a plateau of approximately 10 µg/mL from 0.5-2 h, followed by a decline with a 122 min half-life. Approximately 68% of i.v. taTHU was converted to THU. Approximately 30% of p.o. taTHU reached the systemic circulation as THU. After i.v dosing, renal excretion accounted for 40-55% of the taTHU dose, 26-35% as THU and an additional 14-19% as acetylated forms of THU. After p.o. dosing, the renal excretion percentages were 6-12%, 5.2-11%, and 0.42-0.71%, respectively. taTHU did not inhibit recombinant CD. Conclusion The availability of THU after p.o. taTHU in mice is 30%, as compared to the 20% achieved after p.o. THU. Clinical studies are warranted to evaluate availability of THU from taTHU in humans. Support: N01-CM07106, N01-CM52202, P30-CA47904 from the NCI and P41-EB001978. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4549.

Introduction: We previously reported that the vitamin D3 catabolizing enzyme CYP24 is over-expressed in non-small cell lung cancer (NSCLC) cells, where it decreases the growth inhibitory effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Studies were conducted to establish the clinical relevance of this finding and the mechanism for CYP24 over-expression in NSCLC cells. Methods: CYP24 mRNA expression was quantified by real-time PCR in patient-matched histologically normal lung tissue and primary lung tumors. To elucidate factors that control CYP24 expression, a panel of NSCLC cell lines that exhibit a 70-fold variation in CYP24 mRNA levels under basal growth conditions was used (relative mRNA expression H292 =1.0; 201T = 7.0; 128.88T = 68; A549 =69). CYP24 gene copy number was assessed by fluorescence in situ hybridization. CYP24 promoter activity was measured following transfection of NSCLC cells with either a wildtype promoter-luciferase reporter plasmid or plasmids harboring CYP24 promoter mutations. Transcription factor binding to CYP24 promoter elements was evaluated by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift (EMSA) assays. Results: CYP24 was significantly over-expressed in tumors (p = 0.0017), with 53% of NSCLC cases showing a minimum of 5-fold over-expression. CYP24 gene copy number was comparable among the NSCLC cell lines used. When NSCLC cells were transfected with a wildtype CYP24 promoter-luciferase reporter plasmid, significant differences in promoter activity were observed that paralleled endogenous CYP24 transcript levels. ChIP assays revealed that the CCAAT-enhancer binding protein C/EBPβ binds to the endogenous CYP24 promoter under basal growth conditions in 128.88T cells. To establish a role for C/EBP proteins in regulating CYP24 transcription, we introduced mutations into the C/EBP binding site within the CYP24 promoter. The mutations disrupted C/EBP binding and significantly reduced basal promoter activity in 128.88T and A549 cells. EMSAs that employed a consensus C/EBP site as probe demonstrated that C/EBP binding increases with CYP24 promoter activity: nuclear extracts from untreated H292 and 201T cells contained low levels of C/EBP binding activity, whereas 128.88T and A549 extracts contained high levels. Conclusions: CYP24 mRNA is frequently over-expressed in primary lung tumors. A C/EBPβ-mediated transcriptional control mechanism contributes to the increased expression of CYP24 in NSCLC cells. Signaling pathways that increase C/EBPβ expression/activation may contribute to lung cancer by upregulating CYP24 and allowing neoplastic cells to bypass the antiproliferative effects of 1,25(OH)2D3. This work was supported by the University of Pittsburgh Center for Environmental Oncology, the Hillman Foundation, the Pittsburgh Foundation and R01 CA132844. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4973.

10567 Background: Vorinostat, a Histone Deacetylase (HDAC) inhibitor is a novel targeted antineoplastic agent with promising activity when combined with carboplatin-paclitaxel against NSCLC. The exact molecular mechanism underlying its growth inhibitory and apoptotic effects is not well understood. We investigated the influence of p53 gene status on the interaction of vorinostat and carboplatin (a DNA targeting agent) in various NSCLC cell lines. Methods: NSCLC cells with wild type p53 (A549, 128.88T), mutant p53 (201T) and p53 null phenotype (Calu-1) were used. Cytotoxicity induced by serial dilution of carboplatin in the presence and absence of a fixed dose (1μM) of vorinostat was assessed by MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay. In a separate experiment, cells were also transiently transfected with a p21 promoter-luciferase reporter construct to assess p53 activation following a 24 h exposure to vorinostat, carboplatin, or vorinostat/Carboplatin combination. Luciferase activity was quantified by luminometry and corrected for total protein. Results: Vorinostat displayed single agent activity in each cell line, with greater growth inhibition observed in the p53 mutant and null cells. Synergistic interactions between carboplatin and vorinostat were observed in p53 wild type cells and the IC50 for carboplatin was reduced 3- to 5-fold. In contrast, the interaction between vorinostat and carboplatin was additive or less than additive in p53 mutant and p53 null cells. Vorinostat also increased expression of the p21 reporter construct in each of the cell lines. Conclusions: Vorinostat regulates p21 gene expression and elicits anti-tumor activity in NSCLC cells independent of their p53 status. Vorinostat potentiates carboplatin-induced cytotoxicity in NSCLC with wild type p53 but not p53 deficient cells, suggesting involvement of a p53 dependent pathway. The addition of vorinostat may allow for a reduction in standard dose of carboplatin with improvement in overall therapeutic index. A phase II/III clinical trial is in progress to evaluate vorinostat in combination with carboplatin-based regimen in advanced NSCLC. Support: CA099168–01, ASCO Foundation CDA No significant financial relationships to disclose.