Acute erythroid leukemia (AEL) is characterized by distinct morphology, mutational spectrum, a lack of preclinical models and poor prognosis. Here, using multiplexed genome editing of mouse hematopoietic stem and progenitor cells and transplant assay, we developed preclinical models of AEL and non-erythroid acute leukemia and demonstrated the central role of mutational cooperativity in determining leukemia lineage. Different combination of mutations in Trp53, Bcor, Dnmt3a, Rb1 and Nfix resulted in the development of leukemia with erythroid phenotype, and were accompanied by the acquisition of alterations in signaling and transcription factor genes that recapitulate human AEL by cross-species genomic analysis. Clonal expansion during tumor evolution was driven by mutational co-occurrence, with clones harboring a higher number of founder and secondary lesions (e.g. mutations in signaling genes) showing greater evolutionary fitness. Mouse and human AEL exhibited deregulation of genes regulating erythroid development, notably Gata1, Klf1, and Nfe2, driven by the interaction of mutations of the epigenetic modifiers Dnmt3a and Tet2 that perturbed methylation and thus expression of lineage-specific transcription factors. The established mouse leukemias were used as platform for drug screening. Drug sensitivity was associated with the leukemia genotype, with the PARP inhibitor talazoparib and the demethylating agent decitabine efficacious in Trp53/Bcor mutant AEL, CDK7/9 inhibitors in Trp53/Bcor/Dnmt3a mutant AEL and gemcitabine and bromodomain inhibitors in NUP98-KDM5A leukemia. In conclusion, combinatorial genome editing has demonstrated the interplay of founding and secondary genetic alterations in phenotype and clonal evolution, epigenetic regulation of lineage-specific transcription factors and therapeutic tractability in erythroid leukemogenesis.

Pharmacokinetic, pharmacodynamic and pharmacogenomic studies of midazolam are currently being performed in critically ill children to find suitable dose regimens. Sensitive assays using small volumes of plasma are necessary to determine the concentrations of midazolam and its respective metabolites in pediatric studies. Midazolam is metabolized to hydroxylated midazolam isomers, which are present as free as well as the corresponding glucuronide conjugates. A high-performance liquid chromatographic method with tandem mass spectrometry has been developed and validated for the quantification of midazolam, and free and total 1-hydroxymidazolam and 4-hydroxymidazolam metabolites in small volumes of plasma. Cleanup consisted of 96-well µ-elution solid phase extraction (SPE). The analytes were separated by gradient elution using a C18 analytical column with a total run time of 5 min. Multiple reaction monitoring was employed using precursor to product ion transitions of m/z 326.2 →291.3 for midazolam, m/z 342.1 →203.0 for 1-hydroxymidazolam, m/z 342.1 →325.1 for 4-hydroxymidazolam and m/z 330.2 →295.3 for 2H4-midazolam (internal standard). Since authentic hydroxymidazolamglucuronide standards are not available, samples were hydrolyzed with β-glucuronidase under optimized conditions. Assay conditions were modified and optimized to provide appropriate recovery and stability because 4-hydroxymidazolam was very acid sensitive. Standard curves were linear from 0.5 to 1,000 ng/mL for all three analytes. Intra- and inter day accuracy and precision for quality control samples (2, 20, 200 and 800 ng/mL) were within 85–115% and 15% (coefficient of variation), respectively. Stability in plasma and extracts were sufficient under assay conditions. Plasma samples were processed and analyzed for midazolam, and free 1-hydroxymidazolam and 4-hydroxymidazolam metabolites. Plasma samples that were hydrolyzed with β-glucuronidase were processed and analyzed for midazolam, and total 1-hydroxymidazolam and 4-hydroxymidazolam metabolites under the same assay conditions. The difference in concentration between the total and free hydroxymidazolam metabolites provided an estimate of conjugated hydroxymidazolam metabolites. The combination of 96-well µ-elution SPE and LC-MS/MS allows reliable quantification of midazolam and its metabolites in small volumes of plasma for pediatric patients. This assay is currently being successfully utilized for analysis of samples from ongoing clinical trials.

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NGP1-01 (8-benzylamino-8, 11-oxapentacyclo [5.4.0.02, 6.03, 10.05, 9] undecane) is a heterocyclic cage compound with multifunctional calcium channel blocking activity that has been demonstrated to be neuroprotective in several neurodegenerative models. A sensitive internal standard LC-MS/MS method was developed and validated to quantify NGP1-01 in mouse serum. The internal standard (IS) was 8-phenylethyl-8, 11-oxapentacyclo [5.4.0.0(2, 6).0(3, 10).0(5, 9)] undecane. Sample preparation involved a protein precipitation procedure by addition of acetonitrile. Chromatographic separation was carried out on a Phenomenex Kinetex phenyl-hexyl column (100 x 2.1 mm, 2.6 μm) employing a gradient (45% isocratic 3 min, 45% to 95% linear gradient 6 min, 95% isocratic 3 min) of an elution mobile phase of 5 mM ammonium acetate in 100% acetonitrile mixing with an application mobile phase of 5 mM ammonium acetate in 2% acetonitrile. Detection was achieved by a QTrap 5500 mass spectrometer (AB Sciex) employing electrospray ionization in the positive mode with multiple-reaction-monitoring (MRM) for NGP1-01 (m/z 266 → 91) and IS (m/z 280 → 105). The method validation was carried out in accordance with Food and Drug Administration (FDA) guidelines. The method had a linear range of at least 0.5–50 ng/mL with a correlation coefficient 0.999. The intra-assay and inter-assay precisions (%CV) were equal to or within the range of 1.0 to 4.3% and the accuracies (% relative error) equal to or within −2.5% to 3.4%. The analyte was stable for at least 2 months at −20°C, for at least 8 h at room temperature and for at least three freeze thaw cycles. The extraction recovery was 94.9 to 105.0%, with a %CV ≤ 9.5%. The technique was found to be free of any matrix effects as determined by experiments involving five different lots of mouse serum. Cross-talk interferences were not present. Two different gradient slope chromatography runs were done on dosed mouse serum samples to assess a possible positive error in peak area determination from in-source fragmentation of metabolites generating the same MRM transitions as the parent drug or IS. No such interference was found in the NGP1-01 peak, while a minor interference was identified in the IS peak. The optimized method was applied to the measurement of NGP1-01 in serum of dosed mice.