Targeted treatment of refractory primitive neuroectodermal tumor arising from an immature teratoma with crizotinib leading to a sustained response.

Here we present a case of metastatic PNET which arose from an immature teratoma that was refractory to standard Ewing sarcoma chemotherapy. This PNET was determined to have elevated levels of ALK protein via IHC. The patient was treated with crizotinib on a palliative basis with a sustained response.

Establishment and characterization of patient-derived xenograft of a rare pediatric anaplastic pleomorphic xanthoastrocytoma (PXA) bearing a CDC42SE2-BRAF fusion.

Pleomorphic xanthoastrocytoma (PXA) is a rare subset of primary pediatric glioma with 70% 5-year disease free survival. However, up to 20% of cases present with local recurrence and malignant transformation into more aggressive type anaplastic PXA (AXPA) or glioblastoma. The understanding of disease etiology and mechanisms driving PXA and APXA are limited, and there is no standard of care. Therefore, development of relevant preclinical models to investigate molecular underpinnings of disease and to guide novel therapeutic approaches are of interest. Here, for the first time we established, and characterized a patient-derived xenograft (PDX) from a leptomeningeal spread of a patient with recurrent APXA bearing a novel CDC42SE2-BRAF fusion. An integrated -omics analysis was conducted to assess model fidelity of the genomic, transcriptomic, and proteomic/phosphoproteomic landscapes. A stable xenoline was derived directly from the patient recurrent tumor and maintained in 2D and 3D culture systems. Conserved histology features between the PDX and matched APXA specimen were maintained through serial passages. Whole exome sequencing (WES) demonstrated a high degree of conservation in the genomic landscape between PDX and matched human tumor, including small variants (Pearson's r = 0.794-0.839) and tumor mutational burden (~ 3 mutations/MB). Large chromosomal variations including chromosomal gains and losses were preserved in PDX. Notably, chromosomal gain in chromosomes 4-9, 17 and 18 and loss in the short arm of chromosome 9 associated with homozygous 9p21.3 deletion involving CDKN2A/B locus were identified in both patient tumor and PDX sample. Moreover, chromosomal rearrangement involving 7q34 fusion; CDC42SE-BRAF t (5;7) (q31.1, q34) (5:130,721,239, 7:140,482,820) was identified in the PDX tumor, xenoline and matched human tumor. Transcriptomic profile of the patient's tumor was retained in PDX (Pearson r = 0.88) and in xenoline (Pearson r = 0.63) as well as preservation of enriched signaling pathways (FDR Adjusted P < 0.05) including MAPK, EGFR and PI3K/AKT pathways. The multi-omics data of (WES, transcriptome, and reverse phase protein array (RPPA) was integrated to deduce potential actionable pathways for treatment (FDR < 0.05) including KEGG01521, KEGG05202, and KEGG05200. Both xenoline and PDX were resistant to the MEK inhibitors trametinib or mirdametinib at clinically relevant doses, recapitulating the patient's resistance to such treatment in the clinic. This set of APXA models will serve as a preclinical resource for developing novel therapeutic regimens for rare anaplastic PXAs and pediatric high-grade gliomas bearing BRAF fusions.

Integrative Multi-OMICs Identifies Therapeutic Response Biomarkers and Confirms Fidelity of Clinically Annotated, Serially Passaged Patient-Derived Xenografts Established from Primary and Metastatic Pediatric and AYA Solid Tumors.

Establishment of clinically annotated, molecularly characterized, patient-derived xenografts (PDXs) from treatment-naïve and pretreated patients provides a platform to test precision genomics-guided therapies. An integrated multi-OMICS pipeline was developed to identify cancer-associated pathways and evaluate stability of molecular signatures in a panel of pediatric and AYA PDXs following serial passaging in mice. Original solid tumor samples and their corresponding PDXs were evaluated by whole-genome sequencing, RNA-seq, immunoblotting, pathway enrichment analyses, and the drug−gene interaction database to identify as well as cross-validate actionable targets in patients with sarcomas or Wilms tumors. While some divergence between original tumor and the respective PDX was evident, majority of alterations were not functionally impactful, and oncogenic pathway activation was maintained following serial passaging. CDK4/6 and BETs were prioritized as biomarkers of therapeutic response in osteosarcoma PDXs with pertinent molecular signatures. Inhibition of CDK4/6 or BETs decreased osteosarcoma PDX growth (two-way ANOVA, p < 0.05) confirming mechanistic involvement in growth. Linking patient treatment history with molecular and efficacy data in PDX will provide a strong rationale for targeted therapy and improve our understanding of which therapy is most beneficial in patients at diagnosis and in those already exposed to therapy.

Systems Biology Approach Identifies Prognostic Signatures of Poor Overall Survival and Guides the Prioritization of Novel BET-CHK1 Combination Therapy for Osteosarcoma.

Osteosarcoma (OS) patients exhibit poor overall survival, partly due to copy number variations (CNVs) resulting in dysregulated gene expression and therapeutic resistance. To identify actionable prognostic signatures of poor overall survival, we employed a systems biology approach using public databases to integrate CNVs, gene expression, and survival outcomes in pediatric, adolescent, and young adult OS patients. Chromosome 8 was a hotspot for poor prognostic signatures. The MYC-RAD21 copy number gain (8q24) correlated with increased gene expression and poor overall survival in 90% of the patients (n = 85). MYC and RAD21 play a role in replication-stress, which is a therapeutically actionable network. We prioritized replication-stress regulators, bromodomain and extra-terminal proteins (BETs), and CHK1, in order to test the hypothesis that the inhibition of BET + CHK1 in MYC-RAD21+ pediatric OS models would be efficacious and safe. We demonstrate that MYC-RAD21+ pediatric OS cell lines were sensitive to the inhibition of BET (BETi) and CHK1 (CHK1i) at clinically achievable concentrations. While the potentiation of CHK1i-mediated effects by BETi was BET-BRD4-dependent, MYC expression was BET-BRD4-independent. In MYC-RAD21+ pediatric OS xenografts, BETi + CHK1i significantly decreased tumor growth, increased survival, and was well tolerated. Therefore, targeting replication stress is a promising strategy to pursue as a therapeutic option for this devastating disease.

PAK1 phosphorylation of MEK1 regulates fibronectin-stimulated MAPK activation.

Activation of the Ras-MAPK signal transduction pathway is necessary for biological responses both to growth factors and ECM. Here, we provide evidence that phosphorylation of S298 of MAPK kinase 1 (MEK1) by p21-activated kinase (PAK) is a site of convergence for integrin and growth factor signaling. We find that adhesion to fibronectin induces PAK1-dependent phosphorylation of MEK1 on S298 and that this phosphorylation is necessary for efficient activation of MEK1 and subsequent MAPK activation. The rapid and efficient activation of MEK and phosphorylation on S298 induced by cell adhesion to fibronectin is influenced by FAK and Src signaling and is paralleled by localization of phospho-S298 MEK1 and phospho-MAPK staining in peripheral membrane-proximal adhesion structures. We propose that FAK/Src-dependent, PAK1-mediated phosphorylation of MEK1 on S298 is central to the organization and localization of active Raf-MEK1-MAPK signaling complexes, and that formation of such complexes contributes to the adhesion dependence of growth factor signaling to MAPK.

Aurora A-Selective Inhibitor LY3295668 Leads to Dominant Mitotic Arrest, Apoptosis in Cancer Cells, and Shows Potent Preclinical Antitumor Efficacy.

Although Aurora A, B, and C kinases share high sequence similarity, especially within the kinase domain, they function distinctly in cell-cycle progression. Aurora A depletion primarily leads to mitotic spindle formation defects and consequently prometaphase arrest, whereas Aurora B/C inactivation primarily induces polyploidy from cytokinesis failure. Aurora B/C inactivation phenotypes are also epistatic to those of Aurora A, such that the concomitant inactivation of Aurora A and B, or all Aurora isoforms by nonisoform-selective Aurora inhibitors, demonstrates the Aurora B/C-dominant cytokinesis failure and polyploidy phenotypes. Several Aurora inhibitors are in clinical trials for T/B-cell lymphoma, multiple myeloma, leukemia, lung, and breast cancers. Here, we describe an Aurora A-selective inhibitor, LY3295668, which potently inhibits Aurora autophosphorylation and its kinase activity in vitro and in vivo, persistently arrests cancer cells in mitosis, and induces more profound apoptosis than Aurora B or Aurora A/B dual inhibitors without Aurora B inhibition-associated cytokinesis failure and aneuploidy. LY3295668 inhibits the growth of a broad panel of cancer cell lines, including small-cell lung and breast cancer cells. It demonstrates significant efficacy in small-cell lung cancer xenograft and patient-derived tumor preclinical models as a single agent and in combination with standard-of-care agents. LY3295668, as a highly Aurora A-selective inhibitor, may represent a preferred approach to the current pan-Aurora inhibitors as a cancer therapeutic agent.

Parthenolide and sulindac cooperate to mediate growth suppression and inhibit the nuclear factor-kappa B pathway in pancreatic carcinoma cells.

Activation of the transcription factor nuclear factor-kappa B (NF-kappa B) has been implicated in pancreatic tumorigenesis. We evaluated the effect of a novel NF-kappa B inhibitor, parthenolide, a sesquiterpene lactone isolated from the herb feverfew, in three human pancreatic tumor cell lines (BxPC-3, PANC-1, and MIA PaCa-2). Parthenolide inhibited pancreatic cancer cell growth in a dose-dependent manner with substantial growth inhibition observed between 5 and 10 micromol/L parthenolide in all three cell lines. Parthenolide treatment also dose-dependently increased the amount of the NF-kappa B inhibitory protein, I kappa B-alpha, and decreased NF-kappa B DNA binding activity. We have previously shown that nonsteroidal anti-inflammatory drugs (NSAID) suppress the growth of pancreatic cancer cells. To determine whether inhibition of the NF-kappa B pathway by parthenolide could sensitize pancreatic cancer cells to NSAID inhibition, BxPC-3, PANC-1, and MIA PaCa-2 cells were treated with parthenolide and the NSAID sulindac, either alone or in combination. Treatment with the combination of parthenolide and sulindac inhibited cell growth synergistically in MIA PaCa-2 and BxPC-3 cells and additively in PANC-1 cells. In addition, treatment with the parthenolide/sulindac combination lowered the threshold for apoptosis. Increased levels of I kappa B-alpha protein were detected, especially in MIA PaCa-2 cells, after treatment with parthenolide and sulindac compared with each agent alone. Similarly, decreased NF-kappa B DNA binding and transcriptional activities were detected in cells treated with the combination compared with the single agents, demonstrating cooperative targeting of the NF-kappa B pathway. These data provide preclinical support for a combined chemotherapeutic approach with NF-kappa B inhibitors and NSAIDs for the treatment of pancreatic adenocarcinoma.

LY2606368 Causes Replication Catastrophe and Antitumor Effects through CHK1-Dependent Mechanisms.

CHK1 is a multifunctional protein kinase integral to both the cellular response to DNA damage and control of the number of active replication forks. CHK1 inhibitors are currently under investigation as chemopotentiating agents due to CHK1's role in establishing DNA damage checkpoints in the cell cycle. Here, we describe the characterization of a novel CHK1 inhibitor, LY2606368, which as a single agent causes double-stranded DNA breakage while simultaneously removing the protection of the DNA damage checkpoints. The action of LY2606368 is dependent upon inhibition of CHK1 and the corresponding increase in CDC25A activation of CDK2, which increases the number of replication forks while reducing their stability. Treatment of cells with LY2606368 results in the rapid appearance of TUNEL and pH2AX-positive double-stranded DNA breaks in the S-phase cell population. Loss of the CHK1-dependent DNA damage checkpoints permits cells with damaged DNA to proceed into early mitosis and die. The majority of treated mitotic nuclei consist of extensively fragmented chromosomes. Inhibition of apoptosis by the caspase inhibitor Z-VAD-FMK had no effect on chromosome fragmentation, indicating that LY2606368 causes replication catastrophe. Changes in the ratio of RPA2 to phosphorylated H2AX following LY2606368 treatment further support replication catastrophe as the mechanism of DNA damage. LY2606368 shows similar activity in xenograft tumor models, which results in significant tumor growth inhibition. LY2606368 is a potent representative of a novel class of drugs for the treatment of cancer that acts through replication catastrophe.

Insulin signaling inhibits the 5-HT2C receptor in choroid plexus via MAP kinase.

BACKGROUND: G protein-coupled receptors (GPCRs) interact with heterotrimeric GTP-binding proteins (G proteins) to modulate acute changes in intracellular messenger levels and ion channel activity. In contrast, long-term changes in cellular growth, proliferation and differentiation are often mediated by tyrosine kinase receptors and certain GPCRs by activation of mitogen-activated protein (MAP) kinases. Complex interactions occur between these signaling pathways, but the specific mechanisms of such regulatory events are not well-understood. In particular it is not clear whether GPCRs are modulated by tyrosine kinase receptor-MAP kinase pathways. RESULTS: Here we describe tyrosine kinase receptor regulation of a GPCR via MAP kinase. Insulin reduced the activity of the 5-HT2C receptor in choroid plexus cells which was blocked by the MAP kinase kinase (MEK) inhibitor, PD 098059. We demonstrate that the inhibitory effect of insulin and insulin-like growth factor type 1 (IGF-1) on the 5-HT2C receptor is dependent on tyrosine kinase, RAS and MAP kinase. The effect may be receptor-specific: insulin had no effect on another GPCR that shares the same G protein signaling pathway as the 5-HT2C receptor. This effect is also direct: activated MAP kinase mimicked the effect of insulin, and removing a putative MAP kinase site from the 5-HT2C receptor abolished the effect of insulin. CONCLUSION: These results show that insulin signaling can inhibit 5-HT2C receptor activity and suggest that MAP kinase may play a direct role in regulating the function of a specific GPCR.

Cyclo-oxygenase-2 expression in primary cancers of the lung and bladder compared to normal adjacent tissue.

Multiple observations in the laboratory and the clinical setting have linked expression of the enzyme cyclo-oxygenase-2 (COX-2) to carcinogenesis. The frequency and amount of COX-2 and Bcl-2 expression in primary lung and bladder cancer sites were detennined by immunoblot analysis of cell lysates prepared from frozen human tumor tissue and matched normal adjacent tissue. COX-2 protein was expressed statistically more frequently and at a higher level in primary adenocarcinomas and squamous cell carcinomas of the lung as well as transitional cell carcinomas of the bladder than in normal adjacent tissue. No correlation was observed between COX-2 and Bcl-2 expression in either the lung or bladder cancer specimens. Immunohistochemistry was also employed to localize COX-2 expression. In addition to expression in the malignant tissues, COX-2 was occasionally localized to the normal bronchial and transitional cell epithelia of the normal adjacent tissue. Detection of COX-2 in histologically normal appearing adjacent tissue suggests that COX-2 may be involved in early cellular changes leading to the development of lung and bladder cancer.