Functional Genomic Identification of Predictors of Sensitivity and Mechanisms of Resistance to Multivalent Second-Generation TRAIL-R2 Agonists.

Multivalent second-generation TRAIL-R2 agonists are currently in late preclinical development and early clinical trials. Herein, we use a representative second-generation agent, MEDI3039, to address two major clinical challenges facing these agents: lack of predictive biomarkers to enable patient selection and emergence of resistance. Genome-wide CRISPR knockout screens were notable for the lack of resistance mechanisms beyond the canonical TRAIL-R2 pathway (caspase-8, FADD, BID) as well as p53 and BAX in TP53 wild-type models, whereas a CRISPR activatory screen identified cell death inhibitors MCL-1 and BCL-XL as mechanisms to suppress MEDI3039-induced cell death. High-throughput drug screening failed to identify genomic alterations associated with response to MEDI3039; however, transcriptomics analysis revealed striking association between MEDI3039 sensitivity and expression of core components of the extrinsic apoptotic pathway, most notably its main apoptotic effector caspase-8 in solid tumor cell lines. Further analyses of colorectal cell lines and patient-derived xenografts identified caspase-8 expression ratio to its endogenous regulator FLIP(L) as predictive of sensitivity to MEDI3039 in several major solid tumor types and a further subset indicated by caspase-8:MCL-1 ratio. Subsequent MEDI3039 combination screening of TRAIL-R2, caspase-8, FADD, and BID knockout models with 60 compounds with varying mechanisms of action identified two inhibitor of apoptosis proteins (IAP) that exhibited strong synergy with MEDI3039 that could reverse resistance only in BID-deleted models. In summary, we identify the ratios of caspase-8:FLIP(L) and caspase-8:MCL-1 as potential predictive biomarkers for second-generation TRAIL-R2 agonists and loss of key effectors such as FADD and caspase-8 as likely drivers of clinical resistance in solid tumors.

Comparative mutational landscape analysis of patient-derived tumour xenografts.

BACKGROUND: Screening of patients for cancer-driving mutations is now used for cancer prognosis, remission scoring and treatment selection. Although recently emerged targeted next-generation sequencing-based approaches offer promising diagnostic capabilities, there are still limitations. There is a pressing clinical need for a well-validated, rapid, cost-effective mutation profiling system in patient specimens. Given their speed and cost-effectiveness, quantitative PCR mutation detection techniques are well suited for the clinical environment. The qBiomarker mutation PCR array has high sensitivity and shorter turnaround times compared with other methods. However, a direct comparison with existing viable alternatives are required to assess its true potential and limitations. METHODS: In this study, we evaluated a panel of 117 patient-derived tumour xenografts by the qBiomarker array and compared with other methods for mutation detection, including Ion AmpliSeq sequencing, whole-exome sequencing and droplet digital PCR. RESULTS: Our broad analysis demonstrates that the qBiomarker's performance is on par with that of other labour-intensive and expensive methods of cancer mutation detection of frequently altered cancer-associated genes, and provides a foundation for supporting its consideration as an option for molecular diagnostics. CONCLUSIONS: This large-scale direct comparison and validation of currently available mutation detection approaches is extremely relevant for the current scenario of precision medicine and will lead to informed choice of screening methodologies, especially in lower budget conditions or time frame limitations.

Patient-derived xenografts for individualized care in advanced sarcoma.

BACKGROUND: Patients with advanced, metastatic sarcoma have a poor prognosis, and the overall benefit from the few standard-of-care therapeutics available is small. The rarity of this tumor, combined with the wide range of subtypes, leads to difficulties in conducting clinical trials. The authors previously reported the outcome of patients with a variety of common solid tumors who received treatment with drug regimens that were first tested in patient-derived xenografts using a proprietary method ("TumorGrafts"). METHODS: Tumors resected from 29 patients with sarcoma were implanted into immunodeficient mice to identify drug targets and drugs for clinical use. The results of drug sensitivity testing in the TumorGrafts were used to personalize cancer treatment. RESULTS: Of 29 implanted tumors, 22 (76%) successfully engrafted, permitting the identification of treatment regimens for these patients. Although 6 patients died before the completion of TumorGraft testing, a correlation between TumorGraft results and clinical outcome was observed in 13 of 16 (81%) of the remaining individuals. No patients progressed during the TumorGraft-predicted therapy. CONCLUSIONS: The current data support the use of the personalized TumorGraft model as an investigational platform for therapeutic decision-making that can guide treatment for rare tumors such as sarcomas. A randomized phase 3 trial versus physician's choice is warranted.

Tyrosine phosphorylation of the p21 cyclin-dependent kinase inhibitor facilitates the development of proneural glioma.

Phosphorylation of Tyr-88/Tyr-89 in the 3(10) helix of p27 reduces its cyclin-dependent kinase (CDK) inhibitory activity. This modification does not affect the interaction of p27 with cyclin-CDK complexes but does interfere with van der Waals and hydrogen bond contacts between p27 and amino acids in the catalytic cleft of the CDK. Thus, it had been suggested that phosphorylation of this site could switch the tumor-suppressive CDK inhibitory activity to an oncogenic activity. Here, we examined this hypothesis in the RCAS-PDGF-HA/nestin-TvA proneural glioma mouse model, in which p21 facilitates accumulation of nuclear cyclin D1-CDK4 and promotes tumor development. In these tumor cells, approximately one-third of the p21 is phosphorylated at Tyr-76 in the 3(10) helix. Mutation of this residue to glutamate reduced inhibitory activity in vitro. Mutation of this residue to phenylalanine reduced the tumor-promoting activity of p21 in the animal model, whereas glutamate or alanine substitution allowed tumor formation. Consequently, we conclude that tyrosine phosphorylation contributes to the conversion of CDK inhibitors from tumor-suppressive roles to oncogenic roles.

Cyclin D1 and cdk4 mediate development of neurologically destructive oligodendroglioma.

Although the molecular changes that characterize gliomas have been studied, the pathogenesis of tumor development remains unclear. p21 contributes to gliomagenesis by stabilizing cyclin D1-cdk4 kinase complexes, suggesting that cyclin D1 and cdk4 may also be required for glial tumor development. In this study, we used a mouse model to attempt to confirm this hypothesis, finding that cyclin D1 and cdk4 played active roles in not only the tumor but also the tumor microenvironment. Loss of cdk4 blocked tumor development, but loss of cyclin D1 did not prevent gliomas from developing. Instead, loss of cyclin D1 impeded progression to higher stages of malignancy. Enforcing expression of cyclin D1 was insufficient to correct the progression defect observed in cyclin D1-deficient animals. In contrast, restoration of cdk4 in the cdk4-deficient animals restored cell proliferation and tumor formation, although at lower tumor grades. Notably, the failure of tumors in the cyclin D1- and cdk4-deficient animals to progress to higher grades was correlated with a failure to fully activate microglia in the tumor microenvironment. Moreover, when platelet-derived growth factor-transformed glial cells were engrafted orthotopically into the mice, the tumors that formed progressed to high grades in wild-type mice but not cyclin D1-deficient animals. Together, our findings establish that the cyclin D1-cdk4 axis is not only critical in glial tumor cells but also in stromal-derived cells in the surrounding tumor microenvironment that are vital to sustain tumor outgrowth.

Hdm2- and proteasome-dependent turnover limits p21 accumulation during S phase.

Double-strand DNA breaks detected in different phases of the cell cycle induce molecularly distinct checkpoints downstream of the ATM kinase. p53 is known to induce arrest of cells in G 1 and occasionally G 2 phase but not S phase following ionizing radiation, a time at which the MRN complex and cdc25-dependent mechanisms induce arrest. Our understanding of how cell cycle phase modulates pathway choice and the reasons certain pathways might be favored at different times is limited. In this report, we examined how cell cycle phase affects the activation of the p53 checkpoint and its ability to induce accumulation of the cdk2 inhibitor p21. Using flow cytometric tools and centrifugal elutriation, we found that the p53 response to ionizing radiation is largely intact in all phases of the cell cycle; however, the accumulation of p21 protein is limited to the G 1 and G 2 phase of the cell cycle because of the activity of a proteasome-dependent p21 turnover pathway in S-phase cells. We found that the turnover of p21 was independent of the SCF (skp2) E3 ligase but could be inhibited, at least in part, by reducing hdm2, although this depended on the cell type studied. Our results suggest that there are several redundant pathways active in S-phase cells that can prevent the accumulation of p21.

Immunohistochemical assessment of signal transduction and cell-cycle networks in neural tumors.

The ability to detect transient changes in molecular networks lies at the heart of cancer biology research. This is especially apparent during tumorigenesis, where initiating mutations typically affect mitogens and cell-cycle molecules such as PDGF or retinoblastoma protein (Rb). One of the primary consequences of such processes is the inappropriate stimulation of downstream targets, normally through posttranslational modification. Immunohistochemistry (IHC) provides an important tool for assessing such changes in situ, permitting different aspects of tumor biology to be examined as a tissue undergoes transformation. Nevertheless, this can be difficult to achieve, particularly in complex environments like the brain. Here, we provide the automated methodology we have employed for the successful detection of phosphorylation of S6 ribosomal protein (S6-RP) and the retinoblastoma protein (Rb) in response to PDGF stimulation in a mouse model of glial brain tumor development.

A novel magnetic bead-based assay with high sensitivity and selectivity for analysis of telomerase in exfoliated cells from patients with bladder and colon cancer.

Telomerase activity is elevated in more than 85% of cancer cells and absent in most of the normal cells and thus represents a potential cancer biomarker. We report its measurement in colon and bladder cancer cells captured using antibody-coated magnetic beads. The cells are lysed and telomerase activity is detected using a biosensor assay that employs an oligonucleotide containing the telomerase recognition sequence also covalently coupled to magnetic beads. Telomerase activity is measured by the incorporation of multiple biotinylated nucleotides at the 3'-end of the oligonucleotide strands during elongation which are then reacted with streptavidin-conjugated horseradish peroxidase. A luminescent signal is generated when hydrogen peroxidase is added in the presence of luminol and a signal enhancer. LOD experiments confirm sensitivity down to ten cancer cell equivalents. The telomerase assay reliably identified patient samples considered by an independent pathological review to contain cancer cells. Samples from normal healthy volunteers were all telomerase negative. The assay, which is amenable to automation, demonstrated high sensitivity and specificity in a small clinical cohort, making it of potential benefit as a first line assay for detection and monitoring of colon and bladder cancer.

Expression of stress response protein glucose regulated protein-78 mediated by c-Myb.

Glucose regulated protein-78, GRP78 has been implicated in the protection of tumor cells from cytotoxic damage and apoptosis. When protein profiles of colon cell lines were investigated we found remarkably high GRP78 expression in two cell lines. These cell lines express elevated levels of the transcription factor c-Myb due to genomic amplification of the c-myb locus and we hypothesized that c-Myb regulates GRP78 expression in colon cancer cells. The promoters of human and murine GRP78 and the related family member GRP94 were examined and potential c-Myb binding sites were identified and characterized. DNA binding studies with recombinant c-Myb and nuclear extracts together with ChIP assays on colon cell lines validated these sites. Endogenous GRP78 expression was further induced in these colon cells in response to Thapsigargin treatment, a potent inducer of the unfolded protein response. Transactivation studies with the human GRP78 promoter in colon cell lines showed reporter activity was dependent upon the presence of a conserved c-Myb binding site independent of sequences associated with the unfolded protein response. Finally, over-expression of c-Myb induced the endogenous GRP78 gene. These data suggest that amplification of c-myb in tumor cells may lead to robust GRP78 gene induction, which may in turn assist cells in survival under conditions of oxygen deprivation and nutrient stress.

Colon epithelial cell differentiation is inhibited by constitutive c-myb expression or mutant APC plus activated RAS.

Blocked differentiation is a hallmark of cancer cells and the restoration of differentiation programs in vivo is an actively pursued clinical aim. Understanding the key regulators of cyto-differentiation may focus therapies on molecules that reactivate this process. c-myb expression declines rapidly when human colon cancer epithelial cells are induced to differentiate with the physiologically relevant short-chain fatty acid, sodium butyrate. These cells show increased expression of alkaline phosphatase and cytokeratin 8. Similarly, murine Immorto-epithelial cells derived from wild-type colon cells also show c-myb mRNA declines when induced to differentiate with sodium butyrate. Immorto-cells harboring a single APC mutation are indistinguishable from wild-type cells with regard to differentiation, while addition of activated RAS alone markedly enhances differentiation. In marked contrast, complete differentiation arrest occurs when both APC and RAS are mutated. Expression of MybER, a 4-hydroxytamoxifen-activatable form of c-Myb, blocks differentiation in wildtype and APC mutant Immorto-cell lines as well as LIM1215 human colon carcinoma cells. These data identify two pathways of oncogenic change that lead to retarded epithelial cell differentiation, one involving the presence of a single APC mutation in conjunction with activated RAS or alternatively constitutive c-myb expression.