CYP2C19 and 3A4 Dominate Metabolic Clearance and Bioactivation of Terbinafine Based on Computational and Experimental Approaches.

Lamisil (terbinafine) is an effective, widely prescribed antifungal drug that causes rare idiosyncratic hepatotoxicity. The proposed toxic mechanism involves a reactive metabolite, 6,6-dimethyl-2-hepten-4-ynal (TBF-A), formed through three N-dealkylation pathways. We were the first to characterize them using in vitro studies with human liver microsomes and modeling approaches, yet knowledge of the individual enzymes catalyzing reactions remained unknown. Herein, we employed experimental and computational tools to assess terbinafine metabolism by specific cytochrome P450 isozymes. In vitro inhibitor phenotyping studies revealed six isozymes were involved in one or more N-dealkylation pathways. CYP2C19 and 3A4 contributed to all pathways, and so, we targeted them for steady-state analyses with recombinant isozymes. N-Dealkylation yielding TBF-A directly was catalyzed by CYP2C19 and 3A4 similarly. Nevertheless, CYP2C19 was more efficient than CYP3A4 at N-demethylation and other steps leading to TBF-A. Unlike microsomal reactions, N-denaphthylation was surprisingly efficient for CYP2C19 and 3A4, which was validated by controls. CYP2C19 was the most efficient among all reactions. Nonetheless, CYP3A4 was more selective at steps leading to TBF-A, making it more effective in terbinafine bioactivation based on metabolic split ratios for competing pathways. Model predictions did not extrapolate to quantitative kinetic constants, yet some results for CYP3A4 and CYP2C19 agreed qualitatively with preferred reaction steps and pathways. Clinical data on drug interactions support the CYP3A4 role in terbinafine metabolism, while CYP2C19 remains understudied. Taken together, knowledge of P450s responsible for terbinafine metabolism and TBF-A formation provides a foundation for investigating and mitigating the impact of P450 variations in toxic risks posed to patients.

Comprehensive kinetic and modeling analyses revealed CYP2C9 and 3A4 determine terbinafine metabolic clearance and bioactivation.

Terbinafine N-dealkylation pathways result in formation of 6,6-dimethyl-2-hepten-4-ynal (TBF-A), a reactive allylic aldehyde, that may initiate idiosyncratic drug-induced liver toxicity. Previously, we reported on the importance of CYP2C19 and 3A4 as major contributors to TBF-A formation. In this study, we expanded on those efforts to assess individual contributions of CYP1A2, 2B6, 2C8, 2C9, and 2D6 in terbinafine metabolism. The combined knowledge gained from these studies allowed us to scale the relative roles of the P450 isozymes in hepatic clearance of terbinafine including pathways leading to TBF-A, and hence, provide a foundation for assessing their significance in terbinafine-induced hepatotoxicity. We used in vitro terbinafine reactions with recombinant P450s to measure kinetics for multiple metabolic pathways and calculated contributions of all individual P450 isozymes to in vivo hepatic clearance for the average human adult. The findings confirmed that CYP3A4 was a major contributor (at least 30% total metabolism) to all three of the possible N-dealkylation pathways; however, CYP2C9, and not CYP2C19, played a critical role in terbinafine metabolism and even exceeded CYP3A4 contributions for terbinafine N-demethylation. A combination of their metabolic capacities accounted for at least 80% of the conversion of terbinafine to TBF-A, while CYP1A2, 2B6, 2C8, and 2D6 made minor contributions. Computational approaches provide a more rapid, less resource-intensive strategy for assessing metabolism, and thus, we additionally predicted terbinafine metabolism using deep neural network models for individual P450 isozymes. Cytochrome P450 isozyme models accurately predicted the likelihood for terbinafine N-demethylation, but overestimated the likelihood for a minor N-denaphthylation pathway. Moreover, the models were not able to differentiate the varying roles of the individual P450 isozymes for specific reactions with this particular drug. Taken together, the significance of CYP2C9 and 3A4 and to a lesser extent, CYP2C19, in terbinafine metabolism is consistent with reported drug interactions. This finding suggests that variations in individual P450 contributions due to other factors like polymorphisms may similarly contribute to terbinafine-related adverse health outcomes. Nevertheless, the impact of their metabolic capacities on formation of reactive TBF-A and consequent idiosyncratic hepatotoxicity will be mitigated by competing detoxification pathways, TBF-A decay, and TBF-A adduction to glutathione that remain understudied.

Serum Levels of Hepatocyte Growth Factor and CD40 Ligand Predict Radiation-Induced Liver Injury.

BACKGROUND: Declining liver function is a concerning side effect associated with radiation therapy. Biomarkers of liver toxicity would be useful in personalizing therapy. METHODS: As part of two prospective clinical trials examining adaptive radiation therapy, we collected serum samples from patients receiving liver radiation. We performed a screen of 22 cytokines using a multiplex assay then used ELISA to quantify the cytokines of greatest interest. Subjects were split into screening and validation cohorts. Toxicity was defined as an increase in Child-Pugh score of 2 points or greater within 6 months. Logistic regression models were used to estimate the relationship between our toxicity endpoint and serum cytokine concentrations. RESULTS: Our initial screen (46 subjects, 11 events) identified hepatocyte growth factor (HGF), CD40L (CD154), and eotaxin (CCL11) as potentially predictive of toxicity. We then tested these markers in an expanded patient cohort (104 subjects, 18 events) with a batch correction due to varying age of the samples which confirmed that high HGF and low CD40L were associated with a subsequent decline in liver function following radiation therapy. Multivariate analysis factoring in baseline Child-Pugh score and mean liver radiation dose demonstrated that HGF and CD40L were potentially predictive of toxicity (HGF OR 4.3, P = .009; CD40L OR 0.5 P = .06). Additionally, higher than median baseline HGF levels (1.4 ng/ml) were significantly associated with decreased survival following liver radiation (27.1 vs 14.5 months, P = .03). CONCLUSIONS: Our study identifies high HGF and low CD40L as potential markers of liver toxicity following radiation therapy.

Glutaminase inhibitor CB-839 increases radiation sensitivity of lung tumor cells and human lung tumor xenografts in mice.

PURPOSE: The purpose of this study was to translate our in vitro therapy approach to an in vivo model. Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Studying lymph-node aspirates containing malignant lung tumor cells showed a strong correlation between glutamine consumption and glutathione (GSH) excretion. Subsequent experiments with A549 and H460 lung tumor cell lines provided additional evidence for glutamine's role in driving synthesis and excretion of GSH. Using stable-isotope-labeled glutamine as a tracer metabolite, we demonstrated that the glutamate group in GSH is directly derived from glutamine, linking glutamine utilization intimately to GSH syntheses. MATERIALS AND METHODS: To understand the possible mechanistic link between glutamine consumption and GSH excretion, we studied GSH metabolism in more detail. Inhibition of glutaminase (GLS) with BPTES, a GLS-specific inhibitor, effectively abolished GSH synthesis and excretion. Since our previous work, several novel GLS inhibitors became available and we report herein effects of CB-839 in A427, H460 and A549 lung tumor cells and human lungtumor xenografts in mice. RESULTS: Inhibition of GLS markedly reduced cell viability, producing ED50 values for inhibition of colony formation of 9, 27 and 217 nM in A427, A549 and H460, respectively. Inhibition of GLS is accompanied by ∼30% increased response to radiation, suggesting an important role of glutamine-derived GSH in protecting tumor cells against radiation-induced injury. In subsequent mouse xenografts, short-term CB-839 treatments reduced serum GSH by >50% and increased response to radiotherapy of H460-derived tumor xenografts by 30%. CONCLUSION: The results support the proposed mechanistic link between GLS activity and GSH synthesis and suggest that GLS inhibitors are effective radiosensitizers.

Dendritic cells pulsed with keyhole limpet hemocyanin and cryopreserved maintain anti-tumor activity in a murine melanoma model.

We compared viability, phenotype, in vitro function and therapeutic efficacy of murine unpulsed-dendritic cells (-DC), DC pulsed with keyhole limpet hemocyanin (KLH-DC) and cryopreserved KLH-DC (C-KLH-DC). Mean viability (%+/-SE) of unpulsed-DC, KLH-DC and C-KLH-DC was 93.6+/-0.9, 93.9+/-0.8 and 87.4+/-1.6, respectively. Pulsing DC with KLH did not induce maturation or affect in vitro function. Cryopreservation of KLH-DC reduced MHC I, CD80 and CD86 expression, endocytic capacity and allogeneic splenocyte stimulatory capacity. Intratumoral (i.t.) vaccination of mice bearing s.c. D5 melanoma with unpulsed-DC, KLH-DC or C-KLH-DC elicited comparable anti-tumor immune responses and inhibited tumor growth to the same extent. Combining radiotherapy with i.t. unpulsed-DC, KLH-DC or C-KLH-DC administration enhanced induction of anti-tumor immune responses and inhibition of tumor growth to a similar degree. Cryopreservation of KLH-DC slightly reduces viability, expression of co-stimulatory cell surface markers and in vitro function; however, in vivo anti-tumor activity is fully maintained with or without radiotherapy.

Comparative proteomic analysis of radiation-induced changes in mouse lung: fibrosis-sensitive and -resistant strains.

To determine whether comparative proteomics could detect differential protein expression after lung irradiation in two mouse strains with different radiation responses, lung proteins were subjected to two-dimensional orthogonal liquid-phase separations, with chromatofocusing in the first dimension and nonporous silica reverse-phase high-performance liquid chromatography (NPS-RP-HPLC) in the second. Five weeks after 12 Gy whole-lung irradiation, 15 and 31 proteins had significantly altered expression levels in C3H/HeJ (less likely to develop lung fibrosis) and C57BL/6J mice (more likely to develop lung fibrosis), respectively. These proteins were analyzed by HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) and identified by matching sequences in a peptide database. The proteins are associated with redox, energy consumption, glycolysis, or chromatin/ RNA structure formation. Five of the six redox-related proteins, including superoxide dismutase 1 (SOD1), cytochrome c oxidase, glutamate dehydrogenase, biliverdin reductase, peroxiredoxin and carbonyl reductase, were down-regulated in the irradiated C57BL/6J mice, whereas SOD1, sulfurtransferase and carbonyl reductase increased in the irradiated C3H/ HeJ mice. Thus decreased antioxidant proteins in the irradiated C57BL/6J mice may be correlated with increased early lung toxicity. Changes in SOD1 and 8-hydroxydeoxy-guanosine (8-OHdG, an oxidative stress marker) were further confirmed by immunohistochemistry and/or Western blot analysis. These data suggest that a proteomics approach has the potential to detect protein changes relevant to early lung toxicity after irradiation.

Effect of epidermal growth factor receptor inhibitor class in the treatment of head and neck cancer with concurrent radiochemotherapy in vivo.

PURPOSE: To optimally integrate epidermal growth factor receptor (EGFR) inhibitors into the clinical treatment of head and neck cancer, two important questions must be answered: (a) does EGFR inhibition add to the effects of radiochemotherapy, and (b) if so, which method of inhibiting EGFR is superior (an EGFR antibody versus a small molecule tyrosine kinase inhibitor)? We designed an in vivo study to address these questions. EXPERIMENTAL DESIGN: Nude mice with UMSCC-1 head and neck cancer xenografts received either single, double, or triple agent therapy with an EGFR inhibitor (either cetuximab or gefitinib), gemcitabine, and/or radiation for 3 weeks. Tumor volumes and animal weights were measured for up to 15 weeks. Immunoblotting and immunofluorescent staining were done on tumors treated with either cetuximab or gefitinib alone. RESULTS: The addition of an EGFR inhibitor significantly delayed the tumor volume doubling time, from a median of 40 days with radiochemotherapy (gemcitabine and radiation) alone, to 106 days with cetuximab and 66 days with gefitinib (both P < 0.005). Cetuximab resulted in significantly less weight loss than gefitinib. Immunoblot analysis and immunofluorescent staining of tumors show that although levels of phosphorylated AKT and extracellular signal-regulated kinase were decreased similarly in response to cetuximab or gefitinib, cetuximab caused prolonged suppression of pEGFR, pSTAT3, and Bcl(XL) compared with gefitinib. CONCLUSIONS: EGFR inhibition, particularly with cetuximab, improves the effectiveness of radiochemotherapy in this model of head and neck cancer. The correlation of response with prolonged suppression of EGFR, STAT3, and Bcl(XL) offers the possibility that these may be candidate biomarkers for response.

Synergistic effects of gemcitabine and gefitinib in the treatment of head and neck carcinoma.

Although the combination of gemcitabine and radiation produces a high frequency of complete responses in the treatment of locally advanced head and neck cancer, substantial toxicity suggests that an improvement in the therapeutic index is required. The purpose of this study was to determine if gefitinib could improve the efficacy of gemcitabine and if drug schedule is important. We hypothesized that gemcitabine followed by gefitinib would be superior to the opposite order because of both cell cycle and growth factor signaling interactions. Using UMSCC-1 cells in vitro, we confirmed that gefitinib arrested cells in G(1) and suppressed phospho-epidermal growth factor receptor (p(Y845)EGFR) and that gemcitabine arrested cells in S phase and stimulated p(Y845)EGFR. The schedule of gemcitabine followed by gefitinib caused arrest of cells in S phase. Gefitinib suppressed gemcitabine-mediated p(Y845)EGFR stimulation. This schedule caused decreased p(S473)AKT, increased poly(ADP-ribose) polymerase cleavage, and increased apoptosis compared with gemcitabine alone. The schedule of gefitinib followed by gemcitabine also caused suppression of p(Y845)EGFR but arrested cells in G(1). This schedule in which gefitinib was used first was associated with stable levels of p(S473)AKT and minimal poly(ADP-ribose) polymerase cleavage and apoptosis. These results were reflected in experiments in nude mice bearing UMSCC-1 xenografts, in which there was greater tumor regression and apoptosis when animals received gemcitabine followed by gefitinib during the first week of therapy. These findings suggest that the schedule of gemcitabine followed by gefitinib may increase the therapeutic index over gemcitabine alone and, combined with clinical data, encourage exploration of combination of gemcitabine, EGFR inhibitors, and radiation.

Ataxia telangiectasia mutated down-regulates phospho-extracellular signal-regulated kinase 1/2 via activation of MKP-1 in response to radiation.

Ataxia telangiectasia mutated (ATM) kinase plays a crucial role in the cellular response to DNA damage and in radiation resistance. Although much effort has focused on the relationship between ATM and other nuclear signal transducers, little is known about interactions between ATM and mitogenic signaling pathways. In this study, we show a novel relationship between ATM kinase and extracellular signal-regulated kinase 1/2 (ERK1/2), a key mitogenic stimulator. Activation of ATM by radiation down-regulates phospho-ERK1/2 and its downstream signaling via increased expression of mitogen-activated protein kinase phosphatase MKP-1 in both cell culture and tumor models. This dephosphorylation of ERK1/2 is independent of epidermal growth factor receptor (EGFR) activity and is associated with radioresistance. These findings show a new function for ATM in the control of mitogenic pathways affecting cell signaling and emphasize the key role of ATM in coordinating the cellular response to DNA damage.

Lamisil (terbinafine) toxicity: Determining pathways to bioactivation through computational and experimental approaches.

Lamisil (terbinafine) may cause idiosyncratic liver toxicity through a proposed toxicological mechanism involving the reactive metabolite 6,6-dimethyl-2-hepten-4-ynal (TBF-A). TBF-A toxicological relevance remains unclear due to a lack of identification of pathways leading to and competing with TBF-A formation. We resolved this knowledge gap by combining computational modeling and experimental kinetics of in vitro hepatic N-dealkylation of terbinafine. A deep learning model of N-dealkylation predicted a high probability for N-demethylation to yield desmethyl-terbinafine followed by N-dealkylation to TBF-A and marginal contributions from other possible pathways. We carried out steady-state kinetic experiments with pooled human liver microsomes that relied on development of labeling methods to expand metabolite characterization. Those efforts revealed high levels of TBF-A formation and first order decay during metabolic reactions; actual TBF-A levels would then reflect the balance between those processes as well as reflect the impact of stabilizing adduction with glutathione and other biological molecules. Modeling predictions and experimental studies agreed on the significance of N-demethylation and insignificance of N-denaphthylation in terbinafine metabolism, yet differed on importance of direct TBF-A formation. Under steady-state conditions, the direct pathway was the most important source of the reactive metabolite with a Vmax/Km of 4.0 pmol/min/mg protein/µM in contrast to model predictions. Nevertheless, previous studies show that therapeutic dosing leads to accumulation of desmethyl-terbinafine in plasma, which means that likely sources for TBF-A would draw from metabolism of both the major metabolite and parent drug based on our modeling and experimental studies. Through this combination of novel modeling and experimental approaches, we are the first to identify pathways leading to generation of TBF-A for assessing its role in idiosyncratic adverse drug interactions.

Radiotherapy potentiates the therapeutic efficacy of intratumoral dendritic cell administration.

We examined whether radiotherapy (RT) could enhance the efficacy of dendritic cell (DC)-based immunotherapy of cancer. Mice bearing s.c. D5 melanoma or MCA 205 sarcoma tumors were treated with intratumoral (i.t.) injections of bone marrow-derived unpulsed DCs in combination with local fractionated tumor irradiation. DC administration alone slightly inhibited D5 tumor growth and had no effect on MCA 205. RT alone caused a modest inhibition of both tumors. DC administration combined with RT inhibited D5 and MCA 205 tumor growth in an additive and synergistic manner, respectively. In both tumor models, RT intensified the antitumor efficacy of DC administration independent of apoptosis or necrosis within the tumor mass. Combination treatment of i.t. DCs plus RT was superior to s.c. injections of tumor lysate-pulsed DCs plus interleukin 2 in inhibiting D5 tumor growth and prolonging survival of mice. Splenocytes from mice treated with i.t. DCs plus RT contained significantly more tumor-specific, IFN-gamma-secreting T cells compared with control groups. Moreover, adoptive transfer of these splenocytes mediated significant tumor regression in mice bearing established pulmonary metastases. Combined treatment followed by resection of residual s.c. tumor conferred protective immunity against a subsequent i.v. tumor challenge. Furthermore, i.t. DC plus RT treatment of s.c. tumor in mice bearing concomitant pulmonary metastases resulted in a significant reduction of lung tumors. i.t. DC administration combined with RT induces a potent local and systemic antitumor response in tumor-bearing mice. This novel regimen may be beneficial in the treatment of human cancers.

Wee1 Kinase Inhibitor AZD1775 Radiosensitizes Hepatocellular Carcinoma Regardless of TP53 Mutational Status Through Induction of Replication Stress.

PURPOSE: Wee1 kinase inhibitors are effective radiosensitizers in cells lacking a G1 checkpoint. In this study we examined the potential effect of Wee1 kinase inhibition on inducing replication stress in hepatocellular carcinoma (HCC). METHODS AND MATERIALS: Five independent datasets from the Oncomine database comparing gene expression in HCC compared to normal tissue were combined and specific markers associated with Wee1 sensitivity were analyzed. We then performed a series of in vitro experiments to study the effect of Wee1 inhibition on irradiated HCC cell lines with varying p53 mutational status. Clonogenic survival assays and flow cytometry using anti-γH2AX and phospho-histone H3 antibodies with propidium iodide were performed to study the effect of AZD1775 on survival, cell cycle, and DNA repair. Additionally, nucleoside enriched medium was used to examine the effect of altering nucleotide pools on Wee1 targeted radiation sensitization. RESULTS: Our analysis of the Oncomine database found high levels of CDK1 and other cell cycle regulators indicative of Wee1 sensitivity in HCC. In our in vitro experiments, treatment with AZD1775 radiosensitized and chemosensitized Hep3B, Huh7, and HepG2 cell lines and was associated with delayed resolution of γH2AX foci and the induction of pan-nuclear γH2AX staining. Wee1 inhibition attenuated radiation-induced G2 arrest in the Hep3B (TP53 null) and Huh7 (TP53 mutant) cell lines but not in the TP53 wild-type cell line HepG2. Supplementation with nucleosides reversed the radiation-sensitizing effect of AZD1775 and reduced the amount of cells with pan-nuclear γH2AX staining after radiation. CONCLUSIONS: Radiation sensitization with Wee1 inhibition occurs in cells regardless of their p53 mutational status. In this study we show for the first time that replication stress via the overconsumption of nucleotides plays an important role in AZD1775-induced radiation sensitization.

Sensitization of Pancreatic Cancers to Gemcitabine Chemoradiation by WEE1 Kinase Inhibition Depends on Homologous Recombination Repair.

To improve the efficacy of chemoradiation therapy for locally advanced pancreatic cancer and begin to establish patient selection criteria, we investigated the combination of the WEE1 inhibitor AZD1775 with gemcitabine-radiation in homologous recombination (HR) repair proficient and deficient pancreatic cancers. Sensitization to gemcitabine-radiation by AZD1775 was assessed in pancreatic cancer cells by clonogenic survival and in patient-derived xenografts by tumor growth. The contributions of HR repair inhibition and G2 checkpoint abrogation to sensitization were assessed by γH2AX, BRCA2 manipulation, and RAD51 focus formation and pHistone H3 flow cytometry, respectively. We found that AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type but not BRCA2 mutant pancreatic cancer cells. In all cells, AZD1775 caused inhibition of CDK1 phosphorylation and G2 checkpoint abrogation. However, sensitization by AZD1775 was associated with persistent γH2AX and inhibition of RAD51 focus formation. In HR-proficient (BRCA2 wild-type) or -deficient (BRAC2 null) isogenic cells, AZD1775 sensitized to gemcitabine-radiation in BRCA2 wild-type, but not in BRCA2 null cells, despite significant G2 checkpoint abrogation. In patient-derived pancreatic tumor xenografts, AZD1775 significantly inhibited tumor growth and impaired RAD51 focus formation in response to gemcitabine-radiation. In conclusion, WEE1 inhibition by AZD1775 is an effective strategy for sensitizing pancreatic cancers to gemcitabine chemoradiation. Although this sensitization is accompanied by inhibition of CDK1 phosphorylation and G2 checkpoint abrogation, this mechanism is not sufficient for sensitization. Our findings demonstrate that sensitization to chemoradiation by WEE1 inhibition results from inhibition of HR repair and suggest that patient tumors without underlying HR defects would benefit most from this therapy.

Extracellular expression of cytosine deaminase results in increased 5-FU production for enhanced enzyme/prodrug therapy.

BACKGROUND: The cytosine deaminase/5-fluorocytosine (CD/5-FC), strategy for cancer gene therapy shows considerable promise in experimental models but, because CD is a cytosolic enzyme, intracellular production of 5-fluorouracil (5-FU) causes the demise of the transduced cells before cytotoxic concentrations of' 5-FU can be achieved within the extracellular milieu. MATERIALS AND METHODS: A soluble secreted form of CD was constructed and evaluated compared to intracellular CD in vitro and in vivo. RESULTS: The secreted form of CD temporarily spared transduced cells and enhanced accumulation of extracellular 5-FU. Cytosolic CD produced rapid inhibition of thymidylate synthase and cell death before significant extracellular concentrations of 5-FU developed. Finally, tumors expressing the secreted form of CD had an improved response to 5-FC treatment compared to tumors expressing intracellular CD. CONCLUSION: Further evaluation of extracellular expression of CD for enzyme/prodrug therapy may provide improvements in this commonly studied gene therapy strategy.

Low dose rate radiosensitization of hepatocellular carcinoma in vitro and in patients.

Transarterial radioembolization (TARE) with (90)Y microspheres delivers low dose rate radiation (LDR) to intrahepatic tumors. In the current study, we examined clonogenic survival, DNA damage, and cell cycle distribution in hepatocellular carcinoma (HCC) cell lines treated with LDR in combination with varying doses and schedules of 5-fluorouracil (5-FU), gemcitabine, and sorafenib. Radiosensitization was seen with 1 to 3 µM 5-FU (enhancement ratio 2.2-13.9) and 30 to 100 nM gemcitabine (enhancement ratio 1.9-2.9) administered 24 hours before LDR (0.26 Gy/h to 4.2 Gy). Sorafenib radiosensitized only at high concentrations (3-10 µM) when administered after LDR. For a given radiation dose, greater enhancement was seen with LDR compared to standard dose rate therapy. Summarizing our clinical experience with low dose rate radiosensitization, 13 patients (5 with HCC, 8 with liver metastases) were treated a total of 16 times with TARE and concurrent gemcitabine. Six partial responses and one complete response were observed with a median time to local failure of 7.1 months for all patients and 9.9 months for patients with HCC. In summary, HCC is sensitized to LDR with clinically achievable concentrations of gemcitabine and 5-FU in vitro. Encouraging responses were seen in a small cohort of patients treated with TARE and concurrent gemcitabine. Future studies are needed to validate the safety and efficacy of this approach.

Combined inhibition of Wee1 and PARP1/2 for radiosensitization in pancreatic cancer.

PURPOSE: While the addition of radiation to chemotherapy improves survival in patients with locally advanced pancreatic cancer, more effective therapies are urgently needed. Thus, we investigated the radiosensitizing efficacy of the novel drug combination of Wee1 and PARP1/2 inhibitors (AZD1775 and olaparib, respectively) in pancreatic cancer. EXPERIMENTAL DESIGN: Radiosensitization of AsPC-1 or MiaPaCa-2 human pancreatic cancer cells was assessed by clonogenic survival and tumor growth assays. Mechanistically, the effects of AZD1775, olaparib, and radiation on cell cycle, DNA damage (γH2AX), and homologous recombination repair (HRR) were determined. RESULTS: Treatment of AsPC-1 and MiaPaCa-2 cells with either AZD1775 or olaparib caused modest radiosensitization, whereas treatment with the combination significantly increased radiosensitization. Radiosensitization by the combination of AZD1775 and olaparib was associated with G2 checkpoint abrogation and persistent DNA damage. In addition, AZD1775 inhibited HRR activity and prevented radiation-induced Rad51 focus formation. Finally, in vivo, in MiaPaCa-2-derived xenografts, olaparib did not radiosensitize, whereas AZD1775 produced moderate, yet significant, radiosensitization (P < 0.05). Importantly, the combination of AZD1775 and olaparib produced highly significant radiosensitization (P < 0.0001) evidenced by a 13-day delay in tumor volume doubling (vs. radiation alone) and complete eradication of 20% of tumors. CONCLUSIONS: Taken together, these results demonstrate the efficacy of combined inhibition of Wee1 and PARP inhibitors for radiosensitizing pancreatic cancers and support the model that Wee1 inhibition sensitizes cells to PARP inhibitor-mediated radiosensitization through inhibition of HRR and abrogation of the G2 checkpoint, ultimately resulting in unrepaired, lethal DNA damage and radiosensitization. Clin Cancer Res; 20(19); 5085-96. ©2014 AACR.

Inhibition of protein phosphatase 2A radiosensitizes pancreatic cancers by modulating CDC25C/CDK1 and homologous recombination repair.

PURPOSE: To identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer and thus improve survival, we conducted an siRNA library screen in pancreatic cancer cells. We investigated PPP2R1A, a scaffolding subunit of protein phosphatase 2A (PP2A) as a lead radiosensitizing target. EXPERIMENTAL DESIGN: We determined the effect of PP2A inhibition by genetic (PPP2R1A siRNA) and pharmacologic (LB100, a small molecule entering phase I clinical trials) approaches on radiosensitization of Panc-1 and MiaPaCa-2 pancreatic cancer cells both in vitro and in vivo. RESULTS: PPP2R1A depletion by siRNA radiosensitized Panc-1 and MiaPaCa-2 cells, with radiation enhancement ratios of 1.4 (P < 0.05). Likewise, LB100 produced similar radiosensitization in pancreatic cancer cells, but minimal radiosensitization in normal small intestinal cells. Mechanistically, PPP2R1A siRNA or LB100 caused aberrant CDK1 activation, likely resulting from accumulation of the active forms of PLK1 (pPLK1 T210) and CDC25C (pCDC25C T130). Furthermore, LB100 inhibited radiation-induced Rad51 focus formation and homologous recombination repair (HRR), ultimately leading to persistent radiation-induced DNA damage, as reflected by γ-H2AX expression. Finally, we identified CDC25C as a key PP2A substrate involved in LB100-mediated radiosensitization as depletion of CDC25C partially reversed LB100-mediated radiosensitization. In a mouse xenograft model of human pancreatic cancer, LB100 produced significant radiosensitization with minimal weight loss. CONCLUSIONS: Collectively, our data show that PP2A inhibition radiosensitizes pancreatic cancer both in vitro and in vivo via activation of CDC25C/CDK1 and inhibition of HRR, and provide proof-of-concept evidence that PP2A is a promising target for the improvement of local therapy in pancreatic cancer.

Sensitization of pancreatic cancer to chemoradiation by the Chk1 inhibitor MK8776.

PURPOSE: The combination of radiation with chemotherapy is the most effective therapy for unresectable pancreatic cancer. To improve upon this regimen, we combined the selective Checkpoint kinase 1 (Chk1) inhibitor MK8776 with gemcitabine-based chemoradiation in preclinical pancreatic cancer models. EXPERIMENTAL DESIGN: We tested the ability of MK8776 to sensitize to gemcitabine-radiation in homologous recombination repair (HRR)-proficient and -deficient pancreatic cancer cells and assessed Rad51 focus formation. In vivo, we investigated the efficacy, tumor cell selectivity, and pharmacodynamic biomarkers of sensitization by MK8776. RESULTS: We found that MK8776 significantly sensitized HRR-proficient (AsPC-1, MiaPaCa-2, BxPC-3) but not -deficient (Capan-1) pancreatic cancer cells to gemcitabine-radiation and inhibited Rad51 focus formation in HRR-proficient cells. In vivo, MiaPaCa-2 xenografts were significantly sensitized to gemcitabine-radiation by MK8776 without significant weight loss or observable toxicity in the small intestine, the dose-limiting organ for chemoradiation therapy in pancreatic cancer. We also assessed pChk1 (S345), a pharmacodynamic biomarker of DNA damage in response to Chk1 inhibition in both tumor and small intestine and found that MK8776 combined with gemcitabine or gemcitabine-radiation produced a significantly greater increase in pChk1 (S345) in tumor relative to small intestine, suggesting greater DNA damage in tumor than in normal tissue. Furthermore, we demonstrated the utility of an ex vivo platform for assessment of pharmacodynamic biomarkers of Chk1 inhibition in pancreatic cancer. CONCLUSIONS: Together, our results suggest that MK8776 selectively sensitizes HRR-proficient pancreatic cancer cells and xenografts to gemcitabine-radiation and support the clinical investigation of MK8776 in combination with gemcitabine-radiation in locally advanced pancreatic cancer.

Baseline plasma proteomic analysis to identify biomarkers that predict radiation-induced lung toxicity in patients receiving radiation for non-small cell lung cancer.

PURPOSE: To identify new plasma proteomic markers before radiotherapy start to predict later grade ≥2 radiation-induced lung toxicity (RILT2). METHODS: Fifty-seven patients with non-small cell lung cancer received radiotherapy (RT) were eligible. Forty-eight patients with minimum follow-up of 1 year, nine with RILT2 with tumor stage matched to 39 without RILT2, were enrolled for this analysis. Platelet-poor plasma was obtained within 2 weeks before radiotherapy. The plasma proteomes were compared using a multiplexed quantitative proteomics approach involving ExacTag labeling, reverse-phase high-performance liquid chromatography, and nano liquid chromatography electrospray ionization tandem mass spectrometry. Z scores and Bonferroni-adjusted p values for the two-sample mean comparison were used to identify the differential protein expression between patients with and without RILT2. RESULTS: More than 200 proteins were identified and quantified. After excluding proteins that were not detected in at least 40% of the 48 patient samples, C4b-binding protein alpha chain and vitronectin had significantly higher (p < 0.001 and p = 0.02) expression levels in patients with RILT2 compared with patients without RILT2. These two proteins were validated by Western blot. Ingenuity pathway analysis revealed that they both play important roles in the inflammatory response and are associated with the known pathways of radiation-induced lung damage. CONCLUSIONS: This proteomic approach demonstrates new plasma protein biomarkers before treatment for future studies on RILT2 prediction.

Radiosensitization of head and neck squamous cell carcinoma by a SMAC-mimetic compound, SM-164, requires activation of caspases.

Chemoradiation is the treatment of choice for locally advanced head and neck squamous cell carcinoma (HNSCC). However, radioresistance, which contributes to local recurrence, remains a significant therapeutic problem. In this study, we characterized SM-164, a small second mitochondria-derived activator of caspase -mimetic compound that promotes degradation of cellular inhibitor of apoptosis-1(cIAP-1; also known as baculoviral IAP repeat-containing protein 2, BIRC2) and releases active caspases from the X-linked inhibitor of apoptosis inhibitory binding as a radiosensitizing agent in HNSCC cells. We found that SM-164 at nanomolar concentrations induced radiosensitization in some HNSCC cell lines in a manner dependent on intrinsic sensitivity to caspase activation and apoptosis induction. Blockage of caspase activation via short interfering RNA knockdown or a pan-caspase inhibitor, z-VAD-fmk, largely abrogated SM-164 radiosensitization. On the other hand, the resistant lines with a high level of Bcl-2 that blocks caspase activation and apoptosis induction became sensitive to radiation on Bcl-2 knockdown. Mechanistic studies revealed that SM-164 radiosensitization in sensitive cells was associated with NF-κB activation and TNFα secretion, followed by activation of caspase-8 and -9, leading to enhanced apoptosis. Finally, SM-164 also radiosensitized human tumor xenograft while causing minimal toxicity. Thus, SM-164 is a potent radiosensitizer via a mechanism involving caspase activation and holds promise for future clinical development as a novel class of radiosensitizer for the treatment of a subset of head and neck cancer patients.