Dual targeting of the androgen receptor and PI3K/AKT/mTOR pathways in prostate cancer models improves antitumor efficacy and promotes cell apoptosis.

Prostate cancer is a frequent malignancy in older men and has a very high 5-year survival rate if diagnosed early. The prognosis is much less promising if the tumor has already spread outside the prostate gland. Targeted treatments mainly aim at blocking androgen receptor (AR) signaling and initially show good efficacy. However, tumor progression due to AR-dependent and AR-independent mechanisms is often observed after some time, and novel treatment strategies are urgently needed. Dysregulation of the PI3K/AKT/mTOR pathway in advanced prostate cancer and its implication in treatment resistance has been reported. We compared the impact of PI3K/AKT/mTOR pathway inhibitors with different selectivity profiles on in vitro cell proliferation and on caspase 3/7 activation as a marker for apoptosis induction, and observed the strongest effects in the androgen-sensitive prostate cancer cell lines VCaP and LNCaP. Combination treatment with the AR inhibitor darolutamide led to enhanced apoptosis in these cell lines, the effects being most pronounced upon cotreatment with the pan-PI3K inhibitor copanlisib. A subsequent transcriptomic analysis performed in VCaP cells revealed that combining darolutamide with copanlisib impacted gene expression much more than individual treatment. A comprehensive reversal of the androgen response and the mTORC1 transcriptional programs as well as a marked induction of DNA damage was observed. Next, an in vivo efficacy study was performed using the androgen-sensitive patient-derived prostate cancer (PDX) model LuCaP 35 and a superior efficacy was observed after the combined treatment with copanlisib and darolutamide. Importantly, immunohistochemistry analysis of these treated tumors showed increased apoptosis, as revealed by elevated levels of cleaved caspase 3 and Bcl-2-binding component 3 (BBC3). In conclusion, these data demonstrate that concurrent blockade of the PI3K/AKT/mTOR and AR pathways has superior antitumor efficacy and induces apoptosis in androgen-sensitive prostate cancer cell lines and PDX models.

Pan-PI3K inhibition with copanlisib overcomes Treg- and M2-TAM-mediated immune suppression and promotes anti-tumor immune responses.

The PI3K pathway is one of the most frequently altered signaling pathways in human cancer. In addition to its function in cancer cells, PI3K plays a complex role in modulating anti-tumor immune responses upon immune checkpoint inhibition (ICI). Here, we evaluated the effects of the pan-Class I PI3K inhibitor copanlisib on different immune cell types in vitro and on tumor growth and immune cell infiltration in syngeneic murine cancer models. Intermittent treatment with copanlisib resulted in a strong in vivo anti-tumor efficacy, increased tumor infiltration of activated T cells and macrophages, and increased CD8+ T cell/regulatory T cell and M1/M2 macrophage ratios. The strong in vivo efficacy was at least partially due to immunomodulatory activity of copanlisib, as in vitro these murine cancer cells were resistant to PI3K inhibition. Furthermore, the combination of copanlisib with the ICI antibody anti-PD-1 demonstrated enhanced anti-tumor efficacy in both ICI-sensitive and insensitive syngeneic mouse tumor models. Importantly, in an ICI-sensitive model, combination therapy resulted in complete remission and prevention of tumor recurrence. Thus, the combination of ICIs with PI3K inhibition by intermittently dosed copanlisib represents a promising new strategy to increase sensitivity to ICI therapies and to treat human solid cancers.

T cell-mediated elimination of cancer cells by blocking CEACAM6-CEACAM1 interaction.

Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), a cell surface receptor, is expressed on normal epithelial tissue and highly expressed in cancers of high unmet medical need, such as non-small cell lung, pancreatic, and colorectal cancer. CEACAM receptors undergo homo- and heterophilic interactions thereby regulating normal tissue homeostasis and angiogenesis, and in cancer, tumor invasion and metastasis. CEACAM6 expression on malignant plasma cells inhibits antitumor activity of T cells, and we hypothesize a similar function on epithelial cancer cells. The interactions between CEACAM6 and its suggested partner CEACAM1 on T cells were studied. A humanized CEACAM6-blocking antibody, BAY 1834942, was developed and characterized for its immunomodulating effects in co-culture experiments with T cells and solid cancer cells and in comparison to antibodies targeting the immune checkpoints programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1), and T cell immunoglobulin mucin-3 (TIM-3). The immunosuppressive activity of CEACAM6 was mediated by binding to CEACAM1 expressed by activated tumor-specific T cells. BAY 1834942 increased cytokine secretion by T cells and T cell-mediated killing of cancer cells. The in vitro efficacy of BAY 1834942 correlated with the degree of CEACAM6 expression on cancer cells, suggesting potential in guiding patient selection. BAY 1834942 was equally or more efficacious compared to blockade of PD-L1, and at least an additive efficacy was observed in combination with anti-PD-1 or anti-TIM-3 antibodies, suggesting an efficacy independent of the PD-1/PD-L1 axis. In summary, CEACAM6 blockade by BAY 1834942 reactivates the antitumor response of T cells. This warrants clinical evaluation.

Hepatocyte Growth Factor Receptor overexpression predicts reduced survival but its targeting is not effective in unselected HNSCC patients.

BACKGROUND: MET has emerged as target in head and neck squamous cell carcinoma (HNSCC). However, clinical data on MET inhibition in HNSCC are limited. METHODS: HNSCC biopsies and cell lines were tested for MET activity. The response of cell lines to BAY-853474 was tested in proliferation assays. The prognostic value of MET expression was also analyzed. RESULTS: HNSCC cell lines do not respond to MET inhibition. MET-dependent gastric cancer cell lines have much higher levels of MET expression and phosphorylation than HNSCC cell lines. Clinical samples of HNSCC contain much less MET than responsive models. CONCLUSIONS: No clinical response to MET inhibitors in monotherapy may be expected in unselected cases of HNSCC. Only selected patients with MET amplifications should be treated with MET inhibitors. Patients with increased MET immunoreactivity have shorter overall survival. MET might be useful as marker for the detection of patients with more aggressive types of HNSCC.

Preclinical Efficacy of a PSMA-Targeted Thorium-227 Conjugate (PSMA-TTC), a Targeted Alpha Therapy for Prostate Cancer.

PURPOSE: Prostate-specific membrane antigen (PSMA) is an attractive target for radionuclide therapy of metastatic castration-resistant prostate cancer (mCRPC). PSMA-targeted alpha therapy (TAT) has shown early signs of activity in patients with prostate cancer refractory to beta radiation. We describe a novel, antibody-based TAT, the PSMA-targeted thorium-227 conjugate PSMA-TTC (BAY 2315497) consisting of the alpha-particle emitter thorium-227 complexed by a 3,2-HOPO chelator covalently linked to a fully human PSMA-targeting antibody. EXPERIMENTAL DESIGN: PSMA-TTC was characterized for affinity, mode of action, and cytotoxic activity in vitro. Biodistribution, pharmacokinetics, and antitumor efficacy were investigated in vivo using cell line and patient-derived xenograft (PDX) models of prostate cancer. RESULTS: PSMA-TTC was selectively internalized into PSMA-positive cells and potently induced DNA damage, cell-cycle arrest, and apoptosis in vitro. Decrease in cell viability was observed dependent on the cellular PSMA expression levels. In vivo, PSMA-TTC showed strong antitumor efficacy with T/C values of 0.01 to 0.31 after a single injection at 300 to 500 kBq/kg in subcutaneous cell line and PDX models, including models resistant to standard-of-care drugs such as enzalutamide. Furthermore, inhibition of both cancer and cancer-induced abnormal bone growth was observed in a model mimicking prostate cancer metastasized to bone. Specific tumor uptake and efficacy were demonstrated using various PSMA-TTC doses and dosing schedules. Induction of DNA double-strand breaks was identified as a key mode of action for PSMA-TTC both in vitro and in vivo. CONCLUSIONS: The strong preclinical antitumor activity of PSMA-TTC supports its clinical evaluation, and a phase I trial is ongoing in mCRPC patients (NCT03724747).

Circulating endothelial cells as biomarker for cardiovascular diseases.

BACKGROUND: Endothelial dysfunction is involved in several cardiovascular diseases. Elevated levels of circulating endothelial cells (CECs) and low levels of endothelial progenitor cells (EPCs) have been described in different cardiovascular conditions, suggesting their potential use as diagnostic biomarkers for endothelial dysfunction. Compared to typical peripheral blood leukocyte subsets, CECs and EPCs occur at very low frequency. The reliable identification and characterization of CECs and EPCs is a prerequisite for their clinical use, however, a validated method to this purpose is still missing but a key for rare cell events. OBJECTIVES: To establish a validated flow cytometric procedure in order to quantify CECs and EPCs in human whole blood. METHODS: In the establishment phase, the assay sensitivity, robustness, and the sample storage conditions were optimized as prerequisite for clinical use. In a second phase, CECs and EPCs were analyzed in heart failure with preserved (HFpEF) and reduced (HFrEF) ejection fraction, in arterial hypertension (aHT), and in diabetic nephropathy (DN) in comparison to age-matched healthy controls. RESULTS: The quantification procedure for CECs and EPCs showed high sensitivity and reproducibility. CEC values resulted significantly increased in patients with DN and HFpEF in comparison to healthy controls. CEC quantification showed a diagnostic sensitivity of 90% and a sensitivity of 68.0%, 70.4%, and 66.7% for DN, HFpEF, and aHT, respectively. CONCLUSION: A robust and precise assay to quantify CECs and EPCs in pre-clinical and clinical studies has been established. CEC counts resulted to be a good diagnostic biomarker for DN and HFpEF.

Inhibition of BUB1 Kinase by BAY 1816032 Sensitizes Tumor Cells toward Taxanes, ATR, and PARP Inhibitors In Vitro and In Vivo.

PURPOSE: The catalytic function of BUB1 is required for chromosome arm resolution and positioning of the chromosomal passenger complex for resolution of spindle attachment errors and plays only a minor role in spindle assembly checkpoint activation. Here, we present the identification and preclinical pharmacologic profile of the first BUB1 kinase inhibitor with good bioavailability. EXPERIMENTAL DESIGN: The Bayer compound library was screened for BUB1 kinase inhibitors and medicinal chemistry efforts to improve target affinity and physicochemical and pharmacokinetic parameters resulting in the identification of BAY 1816032 were performed. BAY 1816032 was characterized for kinase selectivity, inhibition of BUB1 signaling, and inhibition of tumor cell proliferation alone and in combination with taxanes, ATR, and PARP inhibitors. Effects on tumor growth in vivo were evaluated using human triple-negative breast xenograft models. RESULTS: The highly selective compound BAY 1816032 showed long target residence time and induced chromosome mis-segregation upon combination with low concentrations of paclitaxel. It was synergistic or additive in combination with paclitaxel or docetaxel, as well as with ATR or PARP inhibitors in cellular assays. Tumor xenograft studies demonstrated a strong and statistically significant reduction of tumor size and excellent tolerability upon combination of BAY 1816032 with paclitaxel or olaparib as compared with the respective monotherapies. CONCLUSIONS: Our findings suggest clinical proof-of-concept studies evaluating BAY 1816032 in combination with taxanes or PARP inhibitors to enhance their efficacy and potentially overcome resistance.

EGFR overexpression is not common in patients with head and neck cancer. Cell lines are not representative for the clinical situation in this indication.

BACKGROUND: Based on expression data, Epidermal Growth Factor Receptor (EGFR) emerged as therapeutic target in Head and Neck Cancer but clinical efficacy of EGFR inhibitors was very limited. We reinvestigated the EGFR expression and activation status necessary for response in cell lines and compared that to clinical samples. METHODS: Clinical samples of head and neck squamous cell carcinoma (HNSCC, n=63), mostly from late stage (IV) and poorly or undifferentiated character and cultured cell lines (n=14) were tested by immunohistochemistry (IHC) (n=55) and sandwich immunoassays (n=63) for expression and phosphorylation of EGFR (Tyrosine-1173). Response of 14 different HNSCC cell lines to Erlotinib was tested in proliferation assays. RESULTS: Most HNSCC cell lines respond to Erlotinib. EGFR is phosphorylated in these cell lines. Resistant cell lines display very low level EGFR expression and phosphorylation. EGFR activity in clinical samples is significantly below that observed in cell lines. In clinical samples, EGFR is not overexpressed on the single cellular level. We show similar levels of EGFR expression in growing keratinocytes and tumor cells. CONCLUSIONS: Cell lines are not representative of the clinical situation in HNSCC. Larger studies should investigate whether patient subgroups with activating EGFR mutations or overexpression can be identified.

Isolation of circulating tumor cells from pancreatic cancer by automated filtration.

It is now widely recognized that the isolation of circulating tumor cells based on cell surface markers might be hindered by variability in their protein expression. Especially in pancreatic cancer, isolation based only on EpCAM expression has produced very diverse results. Methods that are independent of surface markers and therefore independent of phenotypical changes in the circulating cells might increase CTC recovery also in pancreatic cancer. We compared an EpCAM-dependent (IsoFlux) and a size-dependent (automated Siemens Healthineers filtration device) isolation method for the enrichment of pancreatic cancer CTCs. The recovery rate of the filtration based approach is dramatically superior to the EpCAM-dependent approach especially for cells with low EpCAM-expression (filtration: 52%, EpCAM-dependent: 1%). As storage and shipment of clinical samples is important for centralized analyses, we also evaluated the use of frozen diagnostic leukapheresis (DLA) as source for isolating CTCs and subsequent genetic analysis such as KRAS mutation detection analysis. Using frozen DLA samples of pancreatic cancer patients we detected CTCs in 42% of the samples by automated filtration.

Preclinical Antitumor Efficacy of BAY 1129980-a Novel Auristatin-Based Anti-C4.4A (LYPD3) Antibody-Drug Conjugate for the Treatment of Non-Small Cell Lung Cancer.

C4.4A (LYPD3) has been identified as a cancer- and metastasis-associated internalizing cell surface protein that is expressed in non-small cell lung cancer (NSCLC), with particularly high prevalence in the squamous cell carcinoma (SCC) subtype. With the exception of skin keratinocytes and esophageal endothelial cells, C4.4A expression is scarce in normal tissues, presenting an opportunity to selectively treat cancers with a C4.4A-directed antibody-drug conjugate (ADC). We have generated BAY 1129980 (C4.4A-ADC), an ADC consisting of a fully human C4.4A-targeting mAb conjugated to a novel, highly potent derivative of the microtubule-disrupting cytotoxic drug auristatin via a noncleavable alkyl hydrazide linker. In vitro, C4.4A-ADC demonstrated potent antiproliferative efficacy in cell lines endogenously expressing C4.4A and inhibited proliferation of C4.4A-transfected A549 lung cancer cells showing selectivity compared with a nontargeted control ADC. In vivo, C4.4A-ADC was efficacious in human NSCLC cell line (NCI-H292 and NCI-H322) and patient-derived xenograft (PDX) models (Lu7064, Lu7126, Lu7433, and Lu7466). C4.4A expression level correlated with in vivo efficacy, the most responsive being the models with C4.4A expression in over 50% of the cells. In the NCI-H292 NSCLC model, C4.4A-ADC demonstrated equal or superior efficacy compared to cisplatin, paclitaxel, and vinorelbine. Furthermore, an additive antitumor efficacy in combination with cisplatin was observed. Finally, a repeated dosing with C4.4A-ADC was well tolerated without changing the sensitivity to the treatment. Taken together, C4.4A-ADC is a promising therapeutic candidate for the treatment of NSCLC and other cancers expressing C4.4A. A phase I study (NCT02134197) with the C4.4A-ADC BAY 1129980 is currently ongoing. Mol Cancer Ther; 16(5); 893-904. ©2017 AACR.

Endometriosis Leads to an Increased Trefoil Factor 3 Concentration in the Peritoneal Cavity but Does Not Alter Systemic Levels.

This study analyzed whether trefoil factor 3 (TFF3) is locally elevated and correlated with common biomarkers and inflammatory processes in endometriosis. Peritoneal fluid (PF) was obtained from 50 women and serum from 124 women with or without endometriosis. Experimental endometriosis was induced in female C57BL/6 mice by syngeneic transplantation of uterine tissue to the abdominal wall. Levels of TFF3 in PF of women with endometriosis were significantly increased ( P < .05) and correlated with local levels of known biomarkers for endometriosis: cancer antigen (CA) 125, CA-19-9, interleukin 8, monocyte chemotactic protein 1, and matrix metalloproteinase 7. Serum levels of TFF3 in women were significantly influenced by the menstrual cycle but were independent from disease state. In mice, local TFF3 levels were significantly elevated in early endometriosis (up to 4 weeks after transplantation, P < .001) and corresponded to increases in spleen weight as marker for systemic inflammation. This study provides the first evidence that TFF3 is locally elevated in the peritoneal cavity in endometriosis and might play a role in disease pathogenesis and its associated inflammatory processes. Furthermore, the results show that TFF3 is regulated through the menstrual cycle. With respect to animal models, syngeneic mouse model does reflect local TFF3 upregulation in the peritoneal cavity affected by endometriosis.

ATAD2 is an epigenetic reader of newly synthesized histone marks during DNA replication.

ATAD2 (ATPase family AAA domain-containing protein 2) is a chromatin regulator harboring an AAA+ ATPase domain and a bromodomain, previously proposed to function as an oncogenic transcription co-factor. Here we suggest that ATAD2 is also required for DNA replication. ATAD2 is co-expressed with genes involved in DNA replication in various cancer types and predominantly expressed in S phase cells where it localized on nascent chromatin (replication sites). Our extensive biochemical and cellular analyses revealed that ATAD2 is recruited to replication sites through a direct interaction with di-acetylated histone H4 at K5 and K12, indicative of newly synthesized histones during replication-coupled chromatin reassembly. Similar to ATAD2-depletion, ectopic expression of ATAD2 mutants that are deficient in binding to these di-acetylation marks resulted in reduced DNA replication and impaired loading of PCNA onto chromatin, suggesting relevance of ATAD2 in DNA replication. Taken together, our data show a novel function of ATAD2 in cancer and for the first time identify a reader of newly synthesized histone di-acetylation-marks during replication.

Detection of KRAS Mutations in Circulating Tumor DNA by Digital PCR in Early Stages of Pancreatic Cancer.

BACKGROUND: Since surgical removal remains the only cure for pancreatic cancer, early detection is of utmost importance. Circulating biomarkers have potential as diagnostic tool for pancreatic cancer, which typically causes clinical symptoms only in advanced stage. Because of their high prevalence in pancreatic cancer, KRAS proto-oncogene, GTPase [KRAS (previous name: Kirsten rat sarcoma viral oncogene homolog)] mutations may be used to identify tumor-derived circulating plasma DNA. Here we tested the diagnostic sensitivity of chip based digital PCR for the detection of KRAS mutations in circulating tumor DNA (ctDNA) in early stage pancreatic cancer. METHODS: We analyzed matched plasma (2 mL) and tumor samples from 50 patients with pancreatic cancer. Early stages (I and II) were predominant (41/50) in this cohort. DNA was extracted from tumor and plasma samples and tested for the common codon 12 mutations G12D, G12V, and G12C by chip-based digital PCR. RESULTS: We identified KRAS mutations in 72% of the tumors. 44% of the tumors were positive for G12D, 20% for G12V, and 10% for G12C. One tumor was positive for G12D and G12V. Analysis of the mutations in matched plasma samples revealed detection rates of 36% for G12D, 50% for G12V, and 0% for G12C. The detection appeared to be correlated with total number of tumor cells in the primary tumor. No KRAS mutations were detected in 20 samples of healthy control plasma. CONCLUSIONS: Our results support further evaluation of tumor specific mutations as early diagnostic biomarkers using plasma samples as liquid biopsy.

Heterogeneous PSMA expression on circulating tumor cells: a potential basis for stratification and monitoring of PSMA-directed therapies in prostate cancer.

The prostate specific membrane antigen (PSMA) is the only clinically validated marker for therapeutic decisions in prostate cancer (PC). Characterization of circulating tumor cells (CTCs) obtained from the peripheral blood of PC patients might provide an alternative to tissue biopsies called "liquid biopsy". The aim of this study was to develop a reliable assay for the determination of PSMA on CTCs. PSMA expression was analyzed on tissue samples (cohort one, n = 75) and CTCs from metastatic PC patients (cohort two, n = 29). Specific signals for the expression of PSMA could be seen for different prostate cancer cell line cells (PC3, LaPC4, 22Rv1, and LNCaP) by Western blot, immunohistochemistry (IHC), immunocytochemistry (ICC), and FACS. PSMA expression was found to be significantly increased in patients with higher Gleason grade (p = 0.0011) and metastases in lymph nodes (p = 0.0000085) or bone (p = 0.0020) (cohort one). In cohort two, CTCs were detectable in 20 out of 29 samples (69 %, range from 1 - 1000 cells). Twelve out of 20 CTC-positive patients showed PSMA-positive CTCs (67 %, score 1+ to 3+). We found intra-patient heterogeneity regarding the PSMA status between CTCs and the corresponding primary tumors. The results of our study could help to address the question whether treatment decisions based on CTC PSMA profiling will lead to a measurable benefit in clinical outcome for prostate cancer patients in the near future.

Novel Mps1 Kinase Inhibitors with Potent Antitumor Activity.

Monopolar spindle 1 (Mps1) has been shown to function as the key kinase that activates the spindle assembly checkpoint (SAC) to secure proper distribution of chromosomes to daughter cells. Here, we report the structure and functional characterization of two novel selective Mps1 inhibitors, BAY 1161909 and BAY 1217389, derived from structurally distinct chemical classes. BAY 1161909 and BAY 1217389 inhibited Mps1 kinase activity with IC50 values below 10 nmol/L while showing an excellent selectivity profile. In cellular mechanistic assays, both Mps1 inhibitors abrogated nocodazole-induced SAC activity and induced premature exit from mitosis ("mitotic breakthrough"), resulting in multinuclearity and tumor cell death. Both compounds efficiently inhibited tumor cell proliferation in vitro (IC50 nmol/L range). In vivo, BAY 1161909 and BAY 1217389 achieved moderate efficacy in monotherapy in tumor xenograft studies. However, in line with its unique mode of action, when combined with paclitaxel, low doses of Mps1 inhibitor reduced paclitaxel-induced mitotic arrest by the weakening of SAC activity. As a result, combination therapy strongly improved efficacy over paclitaxel or Mps1 inhibitor monotreatment at the respective MTDs in a broad range of xenograft models, including those showing acquired or intrinsic paclitaxel resistance. Both Mps1 inhibitors showed good tolerability without adding toxicity to paclitaxel monotherapy. These preclinical findings validate the innovative concept of SAC abrogation for cancer therapy and justify clinical proof-of-concept studies evaluating the Mps1 inhibitors BAY 1161909 and BAY 1217389 in combination with antimitotic cancer drugs to enhance their efficacy and potentially overcome resistance. Mol Cancer Ther; 15(4); 583-92. ©2016 AACR.

Enumeration and Molecular Characterization of Tumor Cells in Lung Cancer Patients Using a Novel In Vivo Device for Capturing Circulating Tumor Cells.

PURPOSE: The use of circulating tumor cells (CTC) as "liquid biopsy" is limited by the very low yield of CTCs available for subsequent analyses. Most in vitro approaches rely on small sample volumes (5-10 mL). EXPERIMENTAL DESIGN: Here, we used a novel approach, the GILUPI CellCollector, which enables an in vivo isolation of CTCs from peripheral blood. In total, 50 lung cancer patients were screened in two subsequent device applications before and after therapy (n = 185 applications). RESULTS: By in vivo isolation, 58% (108/185) of the patients were positive for ≥1 CTC (median, 5 CTCs; range, 1-56 cells) as compared with 27% (23/84; range, 1-300 cells) using the FDA-cleared CellSearch system. Furthermore, we could show that treatment response during therapy was associated with significant decreases in CTC counts (P = 0.001). By dPCR, mutations in the KRAS and EGFR genes relevant for treatment decisions could be detected in CTCs captured by in vivo isolation and confirmed in the primary tumors of the same patients. CONCLUSIONS: In vivo isolation of CTCs overcomes blood volume limitations of other approaches, which might help to implement CTC-based "liquid biopsies" into clinical decision making. Clin Cancer Res; 22(9); 2197-206. ©2015 AACR.

Modular Assembly of Allosteric MEK Inhibitor Structural Elements Unravels Potency and Feedback-Modulation Handles.

Having recently identified a so-far unexplored area adjacent to the known binding site of allosteric mitogen-activated protein kinase kinase (MEK) inhibitors, we now report an extension of these studies by combining our new side chains with different MEK inhibitor cores in a modular manner. Replacement of the amide headgroup with inverse sulfonamides resulted in the identification of new MEK inhibitors with at least 10-fold higher cellular potency against K-Ras-mutated tumor cells. A selected inhibitor from this new series retained the favorable pharmacokinetic profile of its predecessor in rodent and non-rodent species and displayed significant in vivo efficacy at once-daily oral doses of 0.25-1 mg kg(-1) in a K-Ras-mutated xenograft model. The brain penetration potential of this analogue was significantly attenuated relative to PD325901. In a second series, the central fluorophenyl core was replaced by a pyridine moiety which gave rise to a similar boost in cellular potency. Most notably, analogues from this second series do not show MEK feedback phosphorylation in K-Ras-mutated A549 cells. Our results complement recent reports on the structural intricacies of MEK-Raf feedback interactions.

Preclinical evaluation of a novel c-Met inhibitor in a gastric cancer xenograft model using small animal PET.

PURPOSE: Here, we describe the efficacy of the novel small molecule c-Met inhibitor BAY 853474 in reducing tumor growth in the Hs746T gastric cancer xenograft model and tested the suitability of 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG) versus 3'-deoxy-3'-18F-fluorothymidine ([(18)F]FLT) for response monitoring in a gastric cancer xenograft mouse model using small animal PET. PROCEDURES: The c-Met inhibitor or vehicle control was administered orally at various doses in tumor-bearing mice. Glucose uptake and proliferation was measured using PET before, 48 and 96 h after the first treatment. The PET data were compared to data from tumor growth curves, autoradiography, Glut-1 and Ki-67 staining of tumor sections, and biochemical analysis of tissue probes, i.e., c-Met and ERK phosphorylation and cyclin D1 levels. RESULTS: BAY 853474 significantly reduces tumor growth. [(18)F]FDG uptake in Hs746T tumors was significantly reduced in the groups receiving the drug, compared with the control group. The [(18)F]FLT uptake in the tumor tissue was completely absent 96 h after treatment. Autoradiographic, immunohistochemical, and biochemical analyses confirmed the PET findings. Treatment with the c-Met inhibitor did not affect body weight or glucose levels, and no adverse effects were observed in the animals. CONCLUSION: These preclinical findings suggest that clinical PET imaging is a useful tool for early response monitoring in clinical studies.

Cancer therapy monitoring in xenografts by quantitative analysis of circulating tumor DNA.

CONTEXT: Circulating tumor DNA (ctDNA) is a promising biomarker in cancer. MATERIALS AND METHODS: We generated xenograft models of cancer and detected ctDNA in plasma by qRCR targeting human AluJ sequences. RESULTS: Our assay reached single cell sensitivity in vitro and a correlation between ctDNA amount and tumor size was observed in vivo. Treatment with a mitogen activated protein kinase kinase (MEK)-inhibitor (BAY 869766) reduced ctDNA levels. Using this assay, we also confirmed that high levels of cell-free DNA are found in cancer patients compared to healthy individuals. DISCUSSION AND CONCLUSION: We show that ctDNA may be useful biomarker for monitoring tumor growth and treatment response.

Circulating tumour cells escape from EpCAM-based detection due to epithelial-to-mesenchymal transition.

BACKGROUND: Circulating tumour cells (CTCs) have shown prognostic relevance in metastatic breast, prostate, colon and pancreatic cancer. For further development of CTCs as a biomarker, we compared the performance of different protocols for CTC detection in murine breast cancer xenograft models (MDA-MB-231, MDA-MB-468 and KPL-4). Blood samples were taken from tumour bearing animals (20 to 200 mm2) and analysed for CTCs using 1. an epithelial marker based enrichment method (AdnaTest), 2. an antibody independent technique, targeting human gene transcripts (qualitative PCR), and 3. an antibody-independent approach, targeting human DNA-sequences (quantitative PCR). Further, gene expression changes associated with epithelial-to-mesenchymal transition (EMT) were determined with an EMT-specific PCR assay. METHODS: We used the commercially available Adna Test, RT-PCR on human housekeeping genes and a PCR on AluJ sequences to detect CTCs in xenografts models. Phenotypic changes in CTCs were tested with the commercially available "Human Epithelial to Mesenchymal Transition RT-Profiler PCR Array". RESULTS: Although the AdnaTest detects as few as 1 tumour cell in 1 ml of mouse blood spiking experiments, no CTCs were detectable with this approach in vivo despite visible metastasis formation. The presence of CTCs could, however, be demonstrated by PCR targeting human transcripts or DNA-sequences - without epithelial pre-enrichment. The failure of CTC detection by the AdnaTest resulted from downregulation of EpCAM, whereas mesenchymal markers like Twist and EGFR were upregulated on CTCs. Such a change in the expression profile during metastatic spread of tumour cells has already been reported and was linked to a biological program termed epithelial-mesenchymal transition (EMT). CONCLUSIONS: The use of EpCAM-based enrichment techniques leads to the failure to detect CTC populations that have undergone EMT. Our findings may explain clinical results where low CTC numbers have been reported even in patients with late metastatic cancers. These results are a starting point for the identification of new markers for detection or capture of CTCs, including the mesenchymal-like subpopulations.