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Author(s): Carlos Bernal-Mizrachi [1, 4]; Allison C. Gates [1, 4]; Sherry Weng [1]; Takuji Imamura [2]; Russell H. Knutsen [3]; Pascual DeSantis [1]; Trey Coleman [1]; R. Reid Townsend [1]; [...]

Recent advances in mass spectrometry (MS) have enabled extensive analysis of cancer proteomes. Here, we employed quantitative proteomics to profile protein expression across 24 breast cancer patient-derived xenograft (PDX) models. Integrated proteogenomic analysis shows positive correlation between expression measurements from transcriptomic and proteomic analyses; further, gene expression-based intrinsic subtypes are largely re-capitulated using non-stromal protein markers. Proteogenomic analysis also validates a number of predicted genomic targets in multiple receptor tyrosine kinases. However, several protein/phosphoprotein events such as overexpression of AKT proteins and ARAF, BRAF, HSP90AB1 phosphosites are not readily explainable by genomic analysis, suggesting that druggable translational and/or post-translational regulatory events may be uniquely diagnosed by MS. Drug treatment experiments targeting HER2 and components of the PI3K pathway supported proteogenomic response predictions in seven xenograft models. Our study demonstrates that MS-based proteomics can identify therapeutic targets and highlights the potential of PDX drug response evaluation to annotate MS-based pathway activities.

Somatic missense mutations in the phosphodegron domain of the MYC gene ( M YC Box I) are detected in the dominant clones of a subset of acute myeloid leukemia (AML) patients, but the mechanisms by which they contribute to AML are unknown. To unveil unique proprieties of MBI MYC mutant proteins, we systematically compared the cellular and molecular consequences of expressing similar oncogenic levels of wild type and MBI mutant MYC. We found that MBI MYC mutants can accelerate leukemia by driving unique transcriptional signatures in highly selected, myeloid progenitor subpopulations. Although these mutations increase MYC stability, they overall dampen MYC chromatin localization and lead to a cytoplasmic accumulation of the mutant proteins. This phenotype is coupled with increased translation of RNA binding proteins and nuclear export machinery, which results in altered RNA partitioning and accelerated decay of select transcripts encoding proapoptotic and proinflammatory genes. Heterozygous knockin mice harboring the germline MBI mutation Myc p.T73N exhibit cytoplasmic MYC localization, myeloid progenitors' expansion with similar transcriptional signatures to the overexpression model, and eventually develop hematological malignancies. This study uncovers that MBI MYC mutations alter MYC localization and disrupt mRNA subcellular distribution and turnover of select transcripts to accelerate tumor initiation and growth.

Phosphorylation of the cardiac Na V 1.5 channel pore-forming subunit is extensive and critical in modulating channel expression and function, yet the regulation of Na V 1.5 by phosphorylation of its accessory proteins remains elusive. Using a phosphoproteomic analysis of Na V channel complexes purified from mouse left ventricles, we identified nine phosphorylation sites on Fibroblast growth factor Homologous Factor 2 (FHF2). To determine the roles of phosphosites in regulating Na V 1.5, we developed two models from neonatal and adult mouse ventricular cardiomyocytes in which FHF2 expression is knockdown and rescued by WT, phosphosilent or phosphomimetic FHF2-VY. While the increased rates of closed-state and open-state inactivation of Na V channels induced by the FHF2 knockdown are completely restored by the FHF2-VY isoform in adult cardiomyocytes, sole a partial rescue is obtained in neonatal cardiomyocytes. The FHF2 knockdown also shifts the voltage-dependence of activation towards hyperpolarized potentials in neonatal cardiomyocytes, which is not rescued by FHF2-VY. Parallel investigations showed that the FHF2-VY isoform is predominant in adult cardiomyocytes, while expression of FHF2-VY and FHF2-A is comparable in neonatal cardiomyocytes. Similar to WT FHF2-VY, however, each FHF2-VY phosphomutant restores the Na V channel inactivation properties in both models, preventing identification of FHF2 phosphosite roles. FHF2 knockdown also increases the late Na + current in adult cardiomyocytes, which is restored similarly by WT and phosphosilent FHF2-VY. Together, our results demonstrate that ventricular FHF2 is highly phosphorylated, implicate differential roles for FHF2 in regulating neonatal and adult mouse ventricular Na V 1.5, and suggest that the regulation of Na V 1.5 by FHF2 phosphorylation is highly complex., Etoc Summary: Lesage et al . identify the phosphorylation sites of FHF2 from mouse left ventricular Na V 1.5 channel complexes. While no roles for FHF2 phosphosites could be recognized yet, the findings demonstrate differential FHF2-dependent regulation of neonatal and adult mouse ventricular Na V 1.5 channels.