High In Vitro and In Vivo Activity of BI-847325, a Dual MEK/Aurora Kinase Inhibitor, in Human Solid and Hematologic Cancer Models.

BI-847325 is an ATP-competitive inhibitor of MEK/Aurora kinases with the potential to treat a wide range of cancers. In a panel of 294 human tumor cell lines in vitro, BI-847325 was found to be a highly selective inhibitor that was active in the submicromolar range. The most sensitive cancer types were acute lymphocytic and myelocytic leukemia, melanomas, bladder, colorectal, and mammary cancers. BI-847325 showed a broader range of activity than the MEK inhibitor GDC-0623. The high efficacy of BI-847325 was associated with but not limited to cell lines with oncogenic mutations in NRAS, BRAF, and MAP2K1.The high antiproliferative activity of BI-847325 was validated in vivo using subcutaneous xenograft models. After oral administration of 80 and 40 mg/kg once weekly for 3 or 4 weeks, BI-847325 was highly active in four of five colorectal, two of two gastric, two of two mammary, and one of one pancreatic cancer models (test/control < 25%), and tumor regressions were observed in five of 11 cancer models. The treatment was well tolerated with no relevant lethality or body weight changes. In combination with capecitabine, BI-847325 displayed synergism over single-agent therapies, leading to complete remission in the triple-negative mammary model MAXFTN 401, partial regression in the colon model CXF 1103, and stasis in the gastric models GXA 3011 and GXA 3023. In conclusion, dual MEK/Aurora kinase inhibition shows remarkable potential for treating multiple types of hematologic and solid tumors. The combination with capecitabine was synergistic in colorectal, gastric, and mammary cancer. SIGNIFICANCE: We report the preclinical evaluation of BI-847325, a MEK/Aurora kinase inhibitor. Our data demonstrate that BI-847325 has potent antitumor activity in a broad range of human solid and hematologic cancer models in vitro and in vivo and is well tolerated in animal models. It also shows synergistic effect when combined with capecitabine. These findings provide a strong rationale for further development of BI-847325 as a potential therapeutic for patients with cancer.

Elevated MACC1 Expression in Colorectal Cancer Is Driven by Chromosomal Instability and Is Associated with Molecular Subtype and Worse Patient Survival.

Metastasis-Associated in Colon Cancer 1 (MACC1) is a strong prognostic biomarker inducing proliferation, migration, invasiveness, and metastasis of cancer cells. The context of MACC1 dysregulation in cancers is, however, still poorly understood. Here, we investigated whether chromosomal instability and somatic copy number alterations (SCNA) frequently occurring in CRC contribute to MACC1 dysregulation, with prognostic and predictive impacts. Using the Oncotrack and Charité CRC cohorts of CRC patients, we showed that elevated MACC1 mRNA expression was tightly dependent on increased MACC1 gene SCNA and was associated with metastasis and shorter metastasis free survival. Deep analysis of the COAD-READ TCGA cohort revealed elevated MACC1 expression due to SCNA for advanced tumors exhibiting high chromosomal instability (CIN), and predominantly classified as CMS2 and CMS4 transcriptomic subtypes. For that cohort, we validated that elevated MACC1 mRNA expression correlated with reduced disease-free and overall survival. In conclusion, this study gives insights into the context of MACC1 expression in CRC. Increased MACC1 expression is largely driven by CIN, SCNA gains, and molecular subtypes, potentially determining the molecular risk for metastasis that might serve as a basis for patient-tailored treatment decisions.

Pharmacogenomics characterization of the MDM2 inhibitor MI-773 reveals candidate tumours and predictive biomarkers.

MI-773 is a recently developed small-molecule inhibitor of the mouse double minute 2 (MDM2) proto-oncogene. Preclinical data on the anti-tumour activity of MI-773 are limited and indicate that tumour cell lines (CLs) with mutated TP53 are more resistant to MI-773 than wild type TP53. Here, we explored the compound's therapeutic potential in vitro using a panel of 274 annotated CLs derived from a diversity of tumours. MI-773 exhibited a pronounced selectivity and moderate potency, with anti-tumour activity in the sub-micromolar range in about 15% of the CLs. The most sensitive tumour types were melanoma, sarcoma, renal and gastric cancers, leukaemia, and lymphoma. A COMPARE analysis showed that the profile of MI-773 was similar to that of Nutlin-3a, the first potent inhibitor of p53-MDM2 interactions, and, in addition, had a superior potency. In contrast, it poorly correlates with profiles of compounds targeting the p53 pathway with another mechanism of action. OMICS analyses confirmed that MI-773 was primarily active in CLs with wild type TP53. In silico biomarker investigations revealed that the TP53 mutation status plus the aggregated expression levels of 11 genes involved in the p53 signalling pathway predicted sensitivity or resistance of CLs to inhibitors of p53-MDM2 interactions reliably. The results obtained for MI-773 could help to refine the selection of cancer patients for therapy.

Evaluation of molecular subtypes and clonal selection during establishment of patient-derived tumor xenografts from gastric adenocarcinoma.

Patient-derived xenografts (PDX) have emerged as an important translational research tool for understanding tumor biology and enabling drug efficacy testing. They are established by transfer of patient tumor into immune compromised mice with the intent of using them as Avatars; operating under the assumption that they closely resemble patient tumors. In this study, we established 27 PDX from 100 resected gastric cancers and studied their fidelity in histological and molecular subtypes. We show that the established PDX preserved histology and molecular subtypes of parental tumors. However, in depth investigation of the entire cohort revealed that not all histological and molecular subtypes are established. Also, for the established PDX models, genetic changes are selected at early passages and rare subclones can emerge in PDX. This study highlights the importance of considering the molecular and evolutionary characteristics of PDX for a proper use of such models, particularly for Avatar trials.

Author Correction: Evaluation of molecular subtypes and clonal selection during establishment of patient-derived tumor xenografts from gastric adenocarcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Induction of Acquired Resistance towards EGFR Inhibitor Gefitinib in a Patient-Derived Xenograft Model of Non-Small Cell Lung Cancer and Subsequent Molecular Characterization.

In up to 30% of non-small cell lung cancer (NSCLC) patients, the oncogenic driver of tumor growth is a constitutively activated epidermal growth factor receptor (EGFR). Although these patients gain great benefit from treatment with EGFR tyrosine kinase inhibitors, the development of resistance is inevitable. To model the emergence of drug resistance, an EGFR-driven, patient-derived xenograft (PDX) NSCLC model was treated continuously with Gefitinib in vivo. Over a period of more than three months, three separate clones developed and were subsequently analyzed: Whole exome sequencing and reverse phase protein arrays (RPPAs) were performed to identify the mechanism of resistance. In total, 13 genes were identified, which were mutated in all three resistant lines. Amongst them the mutations in NOMO2, ARHGEF5 and SMTNL2 were predicted as deleterious. The 53 mutated genes specific for at least two of the resistant lines were mainly involved in cell cycle activities or the Fanconi anemia pathway. On a protein level, total EGFR, total Axl, phospho-NFκB, and phospho-Stat1 were upregulated. Stat1, Stat3, MEK1/2, and NFκB displayed enhanced activation in the resistant clones determined by the phosphorylated vs. total protein ratio. In summary, we developed an NSCLC PDX line modelling possible escape mechanism under EGFR treatment. We identified three genes that have not been described before to be involved in an acquired EGFR resistance. Further functional studies are needed to decipher the underlying pathway regulation.

Patient derived renal cell carcinoma xenografts exhibit distinct sensitivity patterns in response to antiangiogenic therapy and constitute a suitable tool for biomarker development.

Systemic treatment is necessary for one third of patients with renal cell carcinoma. No valid biomarker is currently available to tailor personalized therapy. In this study we established a representative panel of patient derived xenograft (PDX) mouse models from patients with renal cell carcinomas and determined serum levels of high mobility group B1 (HMGB1) protein under treatment with sunitinib, pazopanib, sorafenib, axitinib, temsirolimus and bevacizumab. Serum HMGB1 levels were significantly higher in a subset of the PDX collection, which exhibited slower tumor growth during subsequent passages than tumors with low HMGB1 serum levels. Pre-treatment PDX serum HMGB1 levels also correlated with response to systemic treatment: PDX models with high HMGB1 levels predicted response to bevacizumab. Taken together, we provide for the first time evidence that the damage associated molecular pattern biomarker HMGB1 can predict response to systemic treatment with bevacizumab. Our data support the future evaluation of HMGB1 as a predictive biomarker for bevacizumab sensitivity in patients with renal cell carcinoma.

Anti-tumor activity of the TGF-ß receptor kinase inhibitor galunisertib (LY2157299 monohydrate) in patient-derived tumor xenografts.

PURPOSE: The transforming growth factor-beta (TGF-ß) signaling pathway is known to play a critical role in promoting tumor growth. Consequently, blocking this pathway has been found to inhibit tumor growth. In order to achieve an optimal anti-tumor effect, however, it remains to be established whether blocking the TGF-ß signaling pathway alone is sufficient, or whether the tumor microenvironment plays an additional, possibly synergistic, role. METHODS: To investigate the relevance of blocking TGF-ß signaling in tumor cells within the context of their respective tissue microenvironments, we treated a panel of patient-derived xenografts (PDX) with the selective TGF-ß receptor kinase inhibitor LY2157299 monohydrate (galunisertib) and assessed both the in vitro and in vivo effects. RESULTS: Galunisertib was found to inhibit the growth in an in vitro clonogenic assay in 6.3% (5/79) of the examined PDX. Evaluation of the expression profiles of a number of genes, representing both canonical and non-canonical TGF-ß signaling pathways, revealed that most PDX exhibited expression changes affecting TGF-ß downstream signaling. Next, we subjected 13 of the PDX to an in vivo assessment and, by doing so, observed distinct response patterns. These results suggest that, next to intrinsic, also extrinsic or microenvironmental factors can affect galunisertib response. pSMAD2 protein expression and TGF-ßRI mRNA expression levels were found to correlate with the in vivo galunisertib effects. CONCLUSIONS: From our data we conclude that intrinsic, tumor-dependent TGF-ß signaling does not fully explain the anti-tumor effect of galunisertib. Hence, in vivo xenograft models may be more appropriate than in vitro clonogenic assays to assess the anti-tumor activity of TGF-ß inhibitors such as galunisertib.

Prognostic value of PLAGL1-specific CpG site methylation in soft-tissue sarcomas.

Soft tissue sarcomas (STS) are rare, complex tumors with a poor prognosis. The identification of new prognostic biomarkers is needed to improve patient management. Our aim was to determine the methylation status of the 118 CpG sites in the PLAGL1 tumor-suppressor gene P1 CpG island promoter and study the potential prognostic impact of PLAGL1 promoter methylation CpG sites in STS. Training cohorts constituted of 28 undifferentiated sarcomas (US) and 35 leiomyosarcomas (LMS) were studied. PLAGL1 mRNA expression was investigated by microarray analysis and validated by RT-qPCR. Pyrosequencing was used to analyze quantitative methylation of the PLAGL1 promoter. Associations between global promoter or specific CpG site methylation and mRNA expression were evaluated using Pearson's product moment correlation coefficient. Cox univariate and multivariate proportional hazard models were used to assess the predictive power of CpG site methylation status. Sixteen CpG sites associated with PLAGL1 mRNA expression were identified in US and 6 in LMS. Statistical analyses revealed an association between CpG107 methylation status and both overall and metastasis-free survival in US, which was confirmed in a validation cohort of 37 US. The exhaustive study of P1 PLAGL1 promoter methylation identified a specific CpG site methylation correlated with mRNA expression, which was predictive for both metastasis-free and overall survival and may constitute the first US-specific biomarker. Such a biomarker may be relevant for identifying patients likely to derive greater benefit from treatment.

Correlating global gene regulation to angiogenesis in the developing chick extra-embryonic vascular system.

BACKGROUND: Formation of blood vessels requires the concerted regulation of an unknown number of genes in a spatial-, time- and dosage-dependent manner. Determining genes, which drive vascular maturation is crucial for the identification of new therapeutic targets against pathological angiogenesis. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] We accessed global gene regulation throughout maturation of the chick chorio-allantoic membrane (CAM), a highly vascularized tissue, using pan genomic microarrays. Seven percent of analyzed genes showed a significant change in expression (>2-fold, FDR<5%) with a peak occurring from E7 to E10, when key morphogenetic and angiogenic genes such as BMP4, SMO, HOXA3, EPAS1 and FGFR2 were upregulated, reflecting the state of an activated endothelium. At later stages, a general decrease in gene expression occurs, including genes encoding mitotic factors or angiogenic mediators such as CYR61, EPAS1, MDK and MYC. We identified putative human orthologs for 77% of significantly regulated genes and determined endothelial cell enrichment for 20% of the orthologs in silico. Vascular expression of several genes including ENC1, FSTL1, JAM2, LDB2, LIMS1, PARVB, PDE3A, PRCP, PTRF and ST6GAL1 was demonstrated by in situ hybridization. Up to 9% of the CAM genes were also overexpressed in human organs with related functions, such as placenta and lung or the thyroid. 21-66% of CAM genes enriched in endothelial cells were deregulated in several human cancer types (P<.0001). Interfering with PARVB (encoding parvin, beta) function profoundly changed human endothelial cell shape, motility and tubulogenesis, suggesting an important role of this gene in the angiogenic process. CONCLUSIONS/SIGNIFICANCE: Our study underlines the complexity of gene regulation in a highly vascularized organ during development. We identified a restricted number of novel genes enriched in the endothelium of different species and tissues, which may play crucial roles in normal and pathological angiogenesis.