Mutations of BRCA2 in canine mammary tumors and their targeting potential in clinical therapy.

Dogs develop cancer spontaneously with age, with breed-specific risk underlying differences in genetics. Mammary tumors are reported as the most frequent neoplasia in intact female dogs. Their high prevalence in certain breeds suggests a genetic component, as it is the case in human familial breast cancer, distinctly in BRCA2-associated cancers. However, the molecular genetics of BRCA2 in the pathogenesis of canine cancer are still under investigation.Genetic variations of canine BRCA2 comprised single nucleotide polymorphisms, insertions and deletions. The BRCA2 level has been shown to be reduced in tumor gland samples, suggesting that low expression of BRCA2 is contributing to mammary tumor development in dogs. Additionally, specific variations of the BRCA2 gene affect RAD51 binding strength, critically damage the BRCA2-RAD51 binding and further provoke a defective repair. In humans, preclinical and clinical data revealed a synthetic lethality interaction between BRCA2 mutations and PARP inhibition. PARP inhibitors are successfully used to increase chemo- and radiotherapy sensitivity, although they are also associated with numerous side effects and acquired resistance. Cancer treatment of canine patients could benefit from increased chemo- and radiosensitivity, as their cancer therapy protocols usually include only low doses of drugs or radiation. Early investigations show tolerability of iniparib in dogs. PARP inhibitors also imply higher therapy costs and consequently are less likely to be accepted by pet owners.We summarized the current evidence of canine BRCA2 gene alterations and their association with mammary tumors. Mutations in the canine BRCA2 gene have the potential to be exploited in clinical therapy through the usage of PARP inhibitors. However, further investigations are needed before introducing PARP inhibitors in veterinary clinical practice.

Dynamic In Vivo Profiling of DNA Damage and Repair after Radiotherapy Using Canine Patients as a Model.

Time resolved data of DNA damage and repair after radiotherapy elucidates the relation between damage, repair, and cell survival. While well characterized in vitro, little is known about the time-course of DNA damage response in tumors sampled from individual patients. Kinetics of DNA damage after radiotherapy was assessed in eight dogs using repeated in vivo samples of tumor and co-irradiated normal tissue analyzed with comet assay and phosphorylated H2AX (γH2AX) immunohistochemistry. In vivo results were then compared (in silico) with a dynamic mathematical model for DNA damage formation and repair. Maximum %DNA in tail was observed at 15-60 min after irradiation, with a rapid decrease. Time-courses of γH2AX-foci paralleled these findings with a small time delay and were not influenced by covariates. The evolutionary parameter search based on %DNA in tail revealed a good fit of the DNA repair model to in vivo data for pooled sarcoma time-courses, but fits for individual sarcoma time-courses suffer from the heterogeneous nature of the in vivo data. It was possible to follow dynamics of comet tail intensity and γH2AX-foci during a course of radiation using a minimally invasive approach. DNA repair can be quantitatively investigated as time-courses of individual patients by integrating this resulting data into a dynamic mathematical model.

Vitamin C is dispensable for oxygen sensing in vivo.

Prolyl-4-hydroxylation is necessary for proper structural assembly of collagens and oxygen-dependent protein stability of hypoxia-inducible transcription factors (HIFs). In vitro function of HIF prolyl-4-hydroxylase domain (PHD) enzymes requires oxygen and 2-oxoglutarate as cosubstrates with iron(II) and vitamin C serving as cofactors. Although vitamin C deficiency is known to cause the collagen-disassembly disease scurvy, it is unclear whether cellular oxygen sensing is similarly affected. Here, we report that vitamin C-deprived Gulo(-/-) knockout mice show normal HIF-dependent gene expression. The systemic response of Gulo(-/-) animals to inspiratory hypoxia, as measured by plasma erythropoietin levels, was similar to that of animals supplemented with vitamin C. Hypoxic HIF induction was also essentially normal under serum- and vitamin C-free cell-culture conditions, suggesting that vitamin C is not required for oxygen sensing in vivo. Glutathione was found to fully substitute for vitamin C requirement of all 3 PHD isoforms in vitro. Consistently, glutathione also reduced HIF-1α protein levels, transactivation activity, and endogenous target gene expression in cells exposed to CoCl(2). A Cys201Ser mutation in PHD2 increased basal hydroxylation rates and conferred resistance to oxidative damage in vitro, suggesting that this surface-accessible PHD2 cysteine residue is a target of antioxidative protection by vitamin C and glutathione.

Congenital erythrocytosis associated with gain-of-function HIF2A gene mutations and erythropoietin levels in the normal range.

Hypoxia-inducible factor 2α (HIF-2α) plays a pivotal role in the balancing of oxygen requirements throughout the body. The protein is a transcription factor that modulates the expression of a wide array of genes and, in turn, controls several key processes including energy metabolism, erythropoiesis and angiogenesis. We describe here the identification of two cases of familial erythrocytosis associated with heterozygous HIF2A missense mutations, namely Ile533Val and Gly537Arg. Ile533Val is a novel mutation and represents the genetic HIF2A change nearest to Pro-531, the primary hydroxyl acceptor residue, so far identified. The Gly537Arg missense mutation has already been described in familial erythrocytosis. However, our patient is the only described case of a de novo HIF2A mutation associated with the development of congenital polycythemia. Functional in vivo studies, based on exogenous expression of hybrid HIF-2α transcription factors, indicated that these genetic alterations lead to the stabilization of HIF-2α protein. All the identified polycythemic subjects with HIF2A mutations show serum erythropoietin in the normal range, independently of the hematocrit values and phlebotomy frequency. The erythroid precursors obtained from the peripheral blood of patients showed an altered phenotype, including an increased rate of growth and a modified expression of some HIF-2α target genes. These results suggest the novel proposal that polycythemia observed in subjects with HIF2A mutations might also be due to primary changes in hematopoietic cells and not only secondary to increased erythropoietin levels.

Triple combination of lomustine, temozolomide and irradiation reduces canine glioma cell survival in vitro.

BACKGROUND: Combined chemoradiation offers a promising therapeutic strategy for dogs with glioma. The alkylating agents temozolomide (TMZ) and lomustine (CCNU) penetrate the blood-brain barrier, and doses for dogs are established. Whether such combinations are clinically advantageous remains to be explored together with tumour-specific markers. OBJECTIVE: To investigate if triple combination of lomustine, temozolomide and irradiation reduces canine glioma cell survival in vitro. METHODS: We evaluated the sensitising effect of CCNU alone and in combination with TMZ-irradiation in canine glioma J3T-BG cells and long-term drug-exposed subclones by using clonogenic survival and proliferation assays. Bisulphite-SEQ and Western Blot were used to investigate molecular alterations. RESULTS: TMZ (200 µM) or CCNU alone (5 µM) reduced the irradiated survival fraction (4 Gy) from 60% to 38% (p = 0.0074) and 26% (p = 0.0002), respectively. The double-drug combination reduced the irradiated survival fraction (4 Gy) more potently to 12% (p < 0.0001). After long-term drug exposure, both subclones show higher IC50 values against CCNU and TMZ. For CCNU-resistant cells, both, single-drug CCNU (p = 0.0006) and TMZ (p = 0.0326) treatment combined with irradiation (4 Gy) remained effective. The double-drug-irradiation combination reduced the cell survival by 86% (p < 0.0001), compared to 92% in the parental (nonresistant) cell line. For TMZ-resistant cells, only the double-drug combination with irradiation (4 Gy) reduced the cell survival by 88% (p = 0.0057) while single-drug treatment lost efficacy. Chemoresistant cell lines demonstrated higher P-gp expression while MGMT-methylation profile analysis showed a general high methylation level in the parental and long-term treated cell lines. CONCLUSIONS: Our findings indicate that combining CCNU with TMZ-irradiation significantly reduces canine glioma cell survival. Such a combination could overcome current challenges of therapeutic resistance to improve overall patient survival.

Elevated circulating Hsp70 levels are correlative for malignancies in different mammalian species.

Circulating Hsp70 levels were determined in feline and porcine cohorts using two different ELISA systems. These comparative animal models of larger organisms often reflect diseases, and especially malignant tumors, better than conventional rodent models. It is therefore essential to investigate the biology and utility of tumor biomarkers in animals such as cats and pigs. In this study, levels of free Hsp70 in the blood of cats with spontaneously occurring tumors were detected using a commercial Hsp70 ELISA (R&D Systems). Sub-analysis of different tumor groups revealed that animals with tumors of epithelial origin presented with significantly elevated circulating Hsp70 concentrations. In addition to free Hsp70 levels measured with the R&D Systems Hsp70 ELISA, levels of exosomal Hsp70 were determined using the compHsp70 ELISA in pigs. Both ELISA systems detected significantly elevated Hsp70 levels (R&D Systems: median 24.9 ng/mL; compHsp70: median 44.2 ng/mL) in the blood of a cohort of APC1311/+ pigs diagnosed with high-grade adenoma polyps, and the R&D Systems Hsp70 ELISA detected also elevated Hsp70 levels in animals with low-grade polyps. In contrast, in flTP53R167H pigs, suffering from malignant osteosarcoma, the compHsp70 ELISA (median 674.32 ng/mL), but not the R&D Systems Hsp70 ELISA (median 4.78 ng/mL), determined significantly elevated Hsp70 concentrations, indicating that in tumor-bearing animals, the dominant form of Hsp70 is of exosomal origin. Our data suggest that both ELISA systems are suitable for detecting free circulating Hsp70 levels in pigs with high-grade adenoma, but only the compHsp70 ELISA can measure elevated, tumor-derived exosomal Hsp70 levels in tumor-bearing animals.

Temozolomide is additive with cytotoxic effect of irradiation in canine glioma cell lines.

BACKGROUND: Similar to human glioblastoma patients, glial tumours in dogs have high treatment resistance and a guarded prognosis. In human medicine, the addition of temozolomide to radiotherapy leads to a favourable outcome in vivo as well as a higher antiproliferative effect on tumour cells in vitro. OBJECTIVES: The aim of the study was to determine the radio- and temozolomide-sensitivity of three canine glial tumour cell lines and to investigate a potential additive cytotoxic effect in combined treatment. Additionally, we wanted to detect the level of MGMT promoter methylation in these cell lines and to investigate a potential association between MGMT promoter methylation and treatment resistance. METHODS: Cells were treated with various concentrations of temozolomide and/or irradiated with 4 and 8 Gy. Radiosensitization by temozolomide was evaluated using proliferation assay and clonogenic assay, and MGMT DNA methylation was investigated using bisulfite next-generation sequencing. RESULTS: In all tested canine cell lines, clonogenicity was inhibited significantly in combined treatment compared to radiation alone. All canine glial cell lines tested in this study were found to have high methylation levels of MGMT promoter. CONCLUSIONS: Hence, an additive effect of combined treatment in MGMT negative canine glial tumour cell lines in vitro was detected. This motivates to further investigate the association between treatment resistance and MGMT, such as MGMT promoter methylation status.

The peptidyl prolyl cis/trans isomerase FKBP38 determines hypoxia-inducible transcription factor prolyl-4-hydroxylase PHD2 protein stability.

The heterodimeric hypoxia-inducible transcription factors (HIFs) are central regulators of the response to low oxygenation. HIF-alpha subunits are constitutively expressed but rapidly degraded under normoxic conditions. Oxygen-dependent hydroxylation of two conserved prolyl residues by prolyl-4-hydroxylase domain-containing enzymes (PHDs) targets HIF-alpha for proteasomal destruction. We identified the peptidyl prolyl cis/trans isomerase FK506-binding protein 38 (FKBP38) as a novel interactor of PHD2. Yeast two-hybrid, glutathione S-transferase pull-down, coimmunoprecipitation, colocalization, and mammalian two-hybrid studies confirmed specific FKBP38 interaction with PHD2, but not with PHD1 or PHD3. PHD2 and FKBP38 associated with their N-terminal regions, which contain no known interaction motifs. Neither FKBP38 mRNA nor protein levels were regulated under hypoxic conditions or after PHD inhibition, suggesting that FKBP38 is not a HIF/PHD target. Stable RNA interference-mediated depletion of FKBP38 resulted in increased PHD hydroxylation activity and decreased HIF protein levels and transcriptional activity. Reconstitution of FKBP38 expression abolished these effects, which were independent of the peptidyl prolyl cis/trans isomerase activity. Downregulation of FKBP38 did not affect PHD2 mRNA levels but prolonged PHD2 protein stability, suggesting that FKBP38 is involved in PHD2 protein regulation.

Role of HSP70 in response to (thermo)radiotherapy: analysis of gene expression in canine osteosarcoma cells by RNA-seq.

Pre-treatment of tumors with hyperthermia is often used to increase the efficacy of radiotherapy. One of the main proteins induced in response to hyperthermia is heat shock protein 70 (HSP70). The aim of our study was to investigate up- and down-regulated genes in response to (thermo)radiotherapy in HSP70 proficient and deficient canine osteosarcoma cell line (Abrams), and functional role of HSP70 in the mechanism of thermoradiosensitization. Cells were transfected with negative control siRNA or siRNA targeting HSP70 and treated with hyperthermia (HT), radiotherapy (RT), and thermoradiotherapy (HTRT). RNA sequencing was used to analyze gene expression. Hyperthermia and thermoradiotherapy, but not radiotherapy alone, induced differential gene expression. We identified genes differentially expressed only in HSP70 knockdown (thus HSP70-dependent) cells in response to hyperthermia and thermoradiotherapy. Interestingly, cell proliferation but not clonogenicity and apoptosis/necrosis was affected by the HSP70 knockdown in response to thermoradiotherapy. The results suggest that HSP70 regulates expression of specific genes in response to hyperthermia and thermoradiotherapy. Further investigations into the role of specific genes regulated in a HSP70-dependent manner in response to thermoradiotherapy could pave a way into new, combinatorial treatment options for (canine) osteosarcoma and other cancer types.

Methadone does not potentiate the effect of doxorubicin in canine tumour cell lines.

Opioid receptor activation was shown to enhance the efficacy of anti-neoplastic drugs in several human cancer cell lines. In these cell lines, doxorubicin increased the number of opioid receptors and methadone concurrently enhanced cellular doxorubicin uptake. Triggered through lay press and media, animal owners started to challenge veterinary oncologists with questions about methadone use in anti-cancer therapy. Especially in veterinary medicine, where side effects of chemotherapy are tolerated to a lesser extent and hence smaller doses are given, agents potentiating chemotherapeutic agents would be an optimal approach to treatment. Canine transitional cell carcinoma cells (TCC, K9TCC), canine osteosarcoma cells (OSA, Abrams) and canine hemangiosarcoma cells (HSA, DAL-4) were incubated with different combinations of methadone, buprenorphine and doxorubicin, in order to test inhibition of cell proliferation. Opioid receptor density was assessed with fluorescence-activated cell sorting in drug native and doxorubicin pretreated cells. In TCC and OSA cell lines opioid receptor density increased after doxorubicin pretreatment. In combination treatment, however, we did not find significant potentiation of doxorubicin's inhibitory effect on proliferation in these cell lines. Neither was there a significant increase of the effect of doxorubicin when the opioids were added 24 hr before doxorubicin. Hence, we could not confirm the hypothesis that opioids increase the anti-proliferative effect of the anti-neoplastic drug doxorubicin in any of these canine tumour cell lines. The lack of effect on a cellular level does not warrant a clinical approach to use opioids together with doxorubicin in dogs with cancer.

Holistic View on Cell Survival and DNA Damage: How Model-Based Data Analysis Supports Exploration of Dynamics in Biological Systems.

In this work, a method is established to calibrate a model that describes the basic dynamics of DNA damage and repair. The model can be used to extend planning for radiotherapy and hyperthermia in order to include the biological effects. In contrast to "syntactic" models (e.g., describing molecular kinetics), the model used here describes radiobiological semantics, resulting in a more powerful model but also in a far more challenging calibration. Model calibration is attempted from clonogenic assay data (doses of 0-6 Gy) and from time-resolved comet assay data obtained within 6 h after irradiation with 6 Gy. It is demonstrated that either of those two sources of information alone is insufficient for successful model calibration, and that both sources of information combined in a holistic approach are necessary to find viable model parameters. Approximate Bayesian computation (ABC) with simulated annealing is used for parameter search, revealing two aspects that are beneficial to resolving the calibration problem: (1) assessing posterior parameter distributions instead of point-estimates and (2) combining calibration runs from different assays by joining posterior distributions instead of running a single calibration run with a combined, computationally very expensive objective function.

Cell line-specific efficacy of thermoradiotherapy in human and canine cancer cells in vitro.

OBJECTIVE: Aims were to investigate sensitivity of various human and canine cancer cell lines to hyperthermia and the influence of particular treatment conditions, and to analyze the DNA-damage response and mode of cell death in cell line radiosensitized by hyperthermia. Additionally, we were interested in the involvement of HSP70 in radiosensitization. METHODS: Radiosensitization by hyperthermia was determined in a panel of human and canine cancer cell lines using clonogenic cell survival assay, as well as levels of heat shock proteins (HSPs) using immunoblotting. The influence of the hyperthermia-radiotherapy time gap, different temperatures and the order of treatments on clonogenicity of hyperthermia-sensitive A549 cells was investigated. Additionally, DNA damage and cell death were assessed by Comet assay and an apoptosis/necrosis assay. Further we induced transient knockdown in A549 cells to test HSP70's involvement in radiosensitization. RESULTS: Out of eight cell lines tested, only two (A549 and Abrams) showed significant decrease in clonogenic cell survival when pre-treated with hyperthermia at 42°C. Strong induction of HSP70 upon thermoradiotherapy (HT-RT) treatment was found in all cell lines. Transient knockdown of HSP70 in A549 cells did not result in decrease of clonogenic cell survival in response to HT-RT. CONCLUSION: Tumor cell-type, temperature and order of treatment play an important role in radiosensitization by hyperthermia. However, hyperthermia has limited potency to radiosensitize canine cancer cells grown in a 2D cell culture setting presented here. DNA damage and apoptosis/necrosis did not increase upon combined treatment and cytosolic levels of HSP70 appear not to play critical role in the radiosensitization of A549 cells.

Aberrant Lck Signal via CD28 Costimulation Augments Antigen-Specific Functionality and Tumor Control by Redirected T Cells with PD-1 Blockade in Humanized Mice.

Purpose: Combination therapy of adoptively transferred redirected T cells and checkpoint inhibitors aims for higher response rates in tumors poorly responsive to immunotherapy like malignant pleural mesothelioma (MPM). Only most recently the issue of an optimally active chimeric antigen receptor (CAR) and the combination with checkpoint inhibitors is starting to be addressed.Experimental Design: Fibroblast activation protein (FAP)-specific CARs with different costimulatory domains, including CD28, Δ-CD28 (lacking lck binding moiety), or 4-1BB were established. CAR-T cells were characterized in vitro and antitumor efficacy was tested in vivo in a humanized mouse model in combination with PD-1 blockade. Finally, the Δ-CD28 CAR was tested clinically in a patient with MPM.Results: All the three CARs demonstrated FAP-specific functionality in vitro Gene expression data indicated a distinct activity profile for the Δ-CD28 CAR, including higher expression of genes involved in cell division, glycolysis, fatty acid oxidation, and oxidative phosphorylation. In vivo, only T cells expressing the Δ-CD28 CAR in combination with PD-1 blockade controlled tumor growth. When injected into the pleural effusion of a patient with MPM, the Δ-CD28 CAR could be detected for up to 21 days and showed functionality.Conclusions: Overall, anti-FAP-Δ-CD28/CD3ζ CAR T cells revealed superior in vitro functionality, better tumor control in combination with PD-1 blockade in humanized mice, and persistence up to 21 days in a patient with MPM. Therefore, further clinical investigation of this optimized CAR is warranted. Clin Cancer Res; 24(16); 3981-93. ©2018 AACR.

Regulated function of the prolyl-4-hydroxylase domain (PHD) oxygen sensor proteins.

Cellular oxygen is sensed by prolyl-4-hydroxylase domain (PHD) proteins that hydroxylate hypoxia-inducible factor (HIF) alpha subunits. Under normoxic conditions, hydroxylated HIFalpha is bound by the von Hippel-Lindau (pVHL) tumor suppressor, leading to ubiquitinylation and proteasomal degradation. Under hypoxic conditions, hydroxylation becomes reduced, leading to HIFalpha stabilization. The authors recently showed that changes in PHD abundance and activity can regulate HIFalpha stability under normoxic as well as under hypoxic conditions. Thus, the PHD oxygen sensors themselves represent effectors of cellular signalling pathways as well as potential drug targets. Here, a cell-free in vitro microtiter plate-based peptide hydroxylation assay was used to investigate the influence of ferrous iron, Krebs cycle intermediates, transition metals, and vitamin C and other antioxidants on the activity of purified PHD1 to 3. PHD activity depends not only on oxygen availability but is also regulated by iron, vitamin C, and Krebs cycle intermediates, suggesting a physiological relevance of their cellular concentrations. Copper but not iron, cobalt, or nickel salts catalyzed vitamin C oxidation. While vitamin C is essential for PHD activity in vitro, N-acetyl-L-cysteine had no effect, and gallic acid or n-propyl gallate efficiently inhibited the activity of all three PHDs, demonstrating different functions of these antioxidants.

The hypoxia-activated prodrug evofosfamide in combination with multiple regimens of radiotherapy.

The promising treatment combination of ionizing radiation (IR) with a hypoxia-activated prodrug (HAP) is based on biological cooperation. Here we investigated the hypoxia-activated prodrug evofosfamide in combination with different treatment regimens of IR against lung A549- and head&neck UT-SCC-14-derived tumor xenografts. DNA damage-related endpoints and clonogenic cell survival of A549 and UT-SCC-14 carcinoma cells were probed under normoxia and hypoxia.Evofosfamide (TH-302) induced DNA-damage and a dose-dependent antiproliferative response in A549 cells on cellular pretreatment under hypoxia, and supra-additively reduced clonogenic survival in combination with IR. Concomitant treatment of A549-derived tumor xenografts with evofosfamide and fractionated irradiation induced the strongest treatment response in comparison to the corresponding neoadjuvant and adjuvant regimens. Adjuvant evofosfamide was more potent than concomitant and neoadjuvant evofosfamide when combined with a single high dose of IR. Hypoxic UT-SCC-14 cells and tumor xenografts thereof were resistant to evofosfamide alone and in combination with IR, most probably due to reduced P450 oxidoreductase expression, which might act as major predictive determinant of sensitivity to HAPs.In conclusion, evofosfamide with IR is a potent combined treatment modality against hypoxic tumors. However, the efficacy and the therapeutic outcome of this combined treatment modality is, as indicated here in preclinical tumor models, dependent on scheduling parameters and tumor type, which is most probably related to the status of respective HAP-activating oxidoreductases. Further biomarker development is necessary for the launch of successful clinical trials.

The novel microtubule targeting agent BAL101553 in combination with radiotherapy in treatment-refractory tumor models.

BACKGROUND AND PURPOSE: Resistance to microtubule targeting agents (MTA) represents a major drawback in successful cancer therapy with MTAs. Here we investigated the combined treatment modality of the novel MTA BAL101553 in combination with radiotherapy in paclitaxel and epothilone-resistant tumor models. MATERIAL AND METHODS: Multiple regimens of BAL101553, or its active moiety BAL27862 for in vitro experiments, were probed in combination with radiotherapy in P-glycoprotein-overexpressing, human colon adenocarcinoma cells (SW480) and in tubulin-mutated human NSCLC cells (A549EpoB40) and tumors thereof. RESULTS: BAL27862 reduced the proliferative activity of SW480 and A549EpoB40 tumor cells with similar potency as in A549 wildtype cells. Combined treatment of BAL27862 with ionizing radiation in vitro resulted in an additive reduction of clonogenicity. Moreover, treatment of paclitaxel- and epothilone-resistant tumors with fractionated irradiation and different regimens of BAL101553 (a single i.v. bolus vs. oral daily) suppressed tumor growth and resulted in an extended additive tumor growth delay with strong reduction of tumor proliferation and mean tumor vessel density. CONCLUSIONS: BAL101553 is a promising alternative in taxane- and epothilone-refractory tumors as part of a combined treatment modality with ionizing radiation. Its potent antitumor effect is not only tumor cell-directed but also targets the tumor microenvironment.

Combined treatment strategies for microtubule stabilizing agent-resistant tumors.

BACKGROUND: Resistance to microtubule-stabilizing agents is a major hurdle for successful cancer therapy. We investigated combined treatment of microtubule-stabilizing agents (MSAs) with inhibitors of angiogenesis to overcome MSA resistance. METHODS: Treatment regimens of clinically relevant MSAs (patupilone and paclitaxel) and antiangiogenic agents (everolimus and bevacizumab) were investigated in genetically defined MSA-resistant lung (A549EpoB40) and colon adenocarcinoma (SW480) tumor xenografts in nude mice (CD1-Foxn1, ICRnu; 5-14 per group). Tumor growth delays were calculated by Kaplan-Meier analysis with Holm-Sidak tests. All statistical tests were two-sided. RESULTS: Inhibition of mTOR-kinase by everolimus only minimally reduced the proliferative activity of ß tubulin-mutated lung adenocarcinoma cells alone and in combination with the MSA patupilone, but everolimus inhibited expression and secretion of vascular endothelial growth factor (VEGF) from these cells. mTOR-kinase inhibition strongly sensitized tumor xenografts derived from these otherwise MSA-resistant tumor cells to patupilone. Tumors treated with the combined modality of everolimus and patupilone had statistically significantly reduced tumor volume and stronger tumor growth delay (16.2 ± 1.01 days) than control- (7.7 ± 0.3 days, P = .004), patupilone- (10 ± 0.97 days, P = .009), and everolimus-treated (10.6 ± 1.4 days, P = .014) tumors. A combined treatment modality with bevacizumab also resensitized this MSA-refractory tumor model to patupilone. Treatment combination also strongly reduced microvessel density, corroborating the relevance of VEGF targeting for the known antivasculature-directed potency of MSA alone in MSA-sensitive tumor models. Resensitization to MSAs was also probed in P glycoprotein-overexpressing SW480-derived tumor xenografts. Different bevacizumab regimens also sensitized this otherwise-resistant tumor model to clinically relevant MSA paclitaxel. CONCLUSIONS: A treatment combination of MSAs with antiangiogenic agents is potent to overcome tumor cell-linked MSA resistance and should be considered as strategy for MSA-refractory tumor entities.