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.

Characterization of the Stereoselective Disposition of Bupropion and Its Metabolites in Rat Plasma and Brain.

BACKGROUND AND OBJECTIVES: Bupropion is an atypical antidepressant and smoking cessation aid; its use is associated with wide intersubject variability in efficacy and safety. Knowledge of the brain pharmacokinetics of bupropion and its pharmacologically active metabolites is considered important for understanding the cause-effect relationships driving this variability. METHODS: Brain concentrations from rats administered a 10 mg/kg subcutaneous dose of racemic bupropion were analyzed using a stereoselective LC/MS-MS method. A 2 mg/kg dose of (S,S)-hydroxybupropion, which has comparable pharmacologic potency to bupropion, was administered to a separate group of rats. Plasma exposure and unbound concentrations in both matrices from companion equilibrium dialysis experiments were determined to assess potential carrier-mediated transport at the blood-brain barrier. RESULTS: Exposures to unbound forms of bupropion enantiomers were similar in plasma; this was also true in brain. This trend held for reductive diastereomer metabolite pairs in the two matrices. Unbound (R,R)-hydroxybupropion exposure was 1.5-fold higher than (S,S)-hydroxybupropion exposure in plasma and brain following bupropion administration. Unbound concentration ratios (Kp,uu) of a given molecular form decreased over time: between 4 and 6 h, these were < 1 for the two bupropion enantiomers, and they were ~ 1 for metabolites that formed. Administration of preformed (S,S)-hydroxybupropion also demonstrated a declining Kp,uu. CONCLUSIONS: The temporal shift in Kp,uu among the different molecular forms provides evidence regarding the operation of carrier-mediated transport and/or within-brain metabolism of bupropion, and, thereby, fresh insight regarding the causes of intersubject variability in the safety and efficacy of bupropion therapy.

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.

Reproducibility of total choline/water ratios in mouse U87MG xenograft tumors by 1H-MRS.

PURPOSE: To evaluate the reproducibility of the measurement of the total choline-to-water ratio, and the effect of repositioning the subject between scans, using (1) H-magnetic resonance spectroscopy in a mouse U87MG xenograft model. MATERIALS AND METHODS: In vivo single-voxel MR spectra at 7T from xenograft tumors were obtained using both a water-suppressed and a nonwater-suppressed point-resolved spectroscopy (PRESS) sequence. Reproducibility of the total choline/water ratio was evaluated under the conditions of immediate rescan with no change in position of the animal or voxel, immediate reposition, and reposition after 1 or 7 days. RESULTS: Total choline-to-water ratios in U87MG tumor xenografts averaged ≈0.018 across all of the groups. The average percent difference between the two scans in each condition was always less than ≈3.0%, and the coefficient of variation was always less than ≈12%. Bias was unrelated to the testing condition and relatively negligible in magnitude (<3%). Due to heteroscedasticity in the ratios, the limits of agreement were calculated after log transformation of the data and ranged from ≈12% when animals were maintained in the same position and immediately rescanned to ≈52% when the two scans were 7 days apart. CONCLUSION: The total choline-to-water ratio provides a reproducible measure of choline-containing metabolites in subcutaneous U87MG xenograft tumors in mice.