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CBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.

Data dependent acquisition (DDA) and data independent acquisition (DIA) are traditionally separate experimental paradigms in bottom-up proteomics. In this work, we developed a strategy combining the two experimental methods into a single LC-MS/MS run. We call the novel strategy, data dependent-independent acquisition proteomics, or DDIA for short. Peptides identified by conventional and robust DDA identification workflow provide useful information for interrogation of DIA scans. Deep learning based LC-MS/MS property prediction tools, developed previously can be used repeatedly to produce spectral libraries facilitating DIA scan extraction. A complete DDIA data processing pipeline, including modules for iRT vs RT calibration curve generation, DIA extraction classifier training, FDR control has been developed. A key advantage of the DDIA method is that it requires minimal information for processing its data.GRAPHIC ABSTRACT

The balance between comprehensively analyzing the proteome and using valuable mass spectrometry time is a genuine challenge in the field of proteomics. Multidimensional fractionation strategies have significantly increased proteome coverage, but often at the cost of increased mass analysis time, despite advances in mass spectrometer acquisition rates. Recently, the Evosep One liquid chromatography system was shown to analyze peptide samples in a high throughput manner without sacrificing in depth proteomics coverage. We demonstrate incorporation of Evosep One technology into our multiplexing workflow for quantitative analysis of tandem mass tag (TMT)-labeled non-small cell lung carcinoma (NSCLC) patient-derived xenografts (PDXs). Using a 30 samples per day Evosep workflow, >12,000 proteins were identified in 48 hours of mass spectrometry time, which is comparable to the number of proteins identified by our conventional concatenated EASY-nLC workflow in 67.5 hours. Shorter Evosep gradient lengths reduced the number of protein identifications by 10%, while decreasing mass analysis time by 50%. Thus, our Evosep workflow enables quantitative analysis of multiplexed samples in less time without conceding depth of proteome coverage.

BackgroundGenomic profiling of patient tumors has linked somatic driver mutations to survival outcomes of non-small cell lung cancer (NSCLC) patients, especially for those receiving targeted therapies. However, it remains unclear whether specific non-driver mutations have any prognostic utility.MethodsWhole exomes and transcriptomes were measured from NSCLC xenograft models of patients with diverse clinical outcomes. Penalised regression analysis was performed to identify a set of 865 genes associated with patient survival. The number of somatic copy number aberrations, point mutations and associated expression changes within the 865 genes were used to stratify independent NSCLC patient populations, filtered for chemotherapy naive and early-stage. In-depth genomic analysis and functional testing was conducted on the genomic alterations to understand their effect on improving survival.ResultsHigh burden of somatic alterations are associated with longer disease-free survival (HR=0.153, P=1.48×10-4) in NSCLC patients. When somatic alterations burden was integrated with gene expression, we were able to predict prognosis in three independent patient datasets. Patients with high alteration burden could be further stratified based on the presence of immunogenic mutations, revealing another subgroup of patients with even better prognosis (85% with >5 years survival), and associated with cytotoxic T-cell expression. In addition, 95% of these 865 genes lack documented activity relevant to cancer, but are in pathways regulating cell proliferation, motility and immune response were implicated.ConclusionOur results demonstrate that non-driver somatic alterations may influence the outcome of cancer patients by increasing beneficial immune response and inhibiting processes associated to tumorigenesis.

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is an inherited cancer syndrome associated with a highly aggressive form of type 2 papillary renal cell carcinoma (PRCC). Germ line inactivating alterations in Fumarate Hydratase (FH) cause HLRCC, and result in elevated levels of reactive oxygen species (ROS). Recent work indicates that FH -/-PRCC cells have increased ABL1 activation, which promotes tumor growth, but how ABL1 is activated remained unclear. Oxidation can regulate protein-tyrosine phosphatase (PTP) catalytic activity; conceivably, ROS-catalyzed inactivation of an ABL-directed PTP might account for ABL1 activation in this malignancy. Previously, our group developed “q-oxPTPome,” a method that can globally monitor the oxidation of classical PTPs. We have now refined the q-oxPTPome approach, increasing its sensitivity by >10X. Applying q-oxPTPome to FH-deficient cell models shows that multiple PTPs are either highly oxidized (including PTPN12) or overexpressed. In general, highly oxidized PTPs were those that have relatively high sensitivity to exogenous H2O2. Most PTP oxidation in FH-deficient cells is reversible, although nearly 40% of PTPN13 is oxidized irreversibly to the sulfonic acid state. Using “substrate-trapping mutants”, we mapped PTPs to their putative substrates, and found that only PTPN12 could target ABL1. Furthermore, knockdown experiments identify PTPN12 as the major ABL1 phosphatase in HLRCC. Overall, our results show that ROS-induced PTPN12 oxidation accounts for ABL1 phosphorylation in HLRCC-associated PRCC, reveal a novel mechanism for inactivating a tumor suppressor gene product, and establish a direct link between pathological PTP oxidation and neoplastic disease.