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Dysregulation of innate immune signaling is a hallmark of hematologic malignancies. Recent therapeutic efforts to subvert aberrant innate immune signaling in MDS and AML have focused on the kinase IRAK4. IRAK4 inhibitors have achieved promising, though moderate, responses in pre-clinical studies and in clinical trials for MDS and AML. The reasons underlying the limited responses to IRAK4 inhibitors remain unknown. Here, we reveal that inhibiting IRAK4 in leukemic cells elicits functional complementation and compensation by its paralog, IRAK1. Using genetic approaches, we demonstrate that co-targeting IRAK1 and IRAK4 is required to suppress leukemic stem/progenitor cell (LSPC) function and induce differentiation in cell lines and patient-derived cells. While IRAK1 and IRAK4 are presumed to function primarily downstream of the proximal adapter MyD88, we found that complimentary and compensatory IRAK1 and IRAK4 dependencies in MDS/AML occur via non-canonical MyD88-independent pathways. Genomic and proteomic analyses revealed that IRAK1 and IRAK4 preserve the undifferentiated state of MDS/AML LSPCs by coordinating a network of pathways, including ones that converge on the PRC2 complex and JAK-STAT signaling. To translate these findings, we implemented a structure-based design of a potent and selective dual IRAK1 and IRAK4 inhibitor KME-2780. MDS/AML cell lines and patient-derived samples showed significant suppression of LSPCs in vitro and in xenograft studies when treated with KME-2780 as compared to selective IRAK4 inhibitors. Our results provide a mechanistic basis and rationale for co-targeting IRAK1 and IRAK4 for the treatment of cancers, including MDS/AML.

Metabolomics analyses suggest changes in amino acid abundance, particularly l-arginine (L-ARG), occur in patients with tuberculosis. Immune cells require L-ARG to fuel effector functions following infection. We have previously described an L-ARG synthesis pathway in immune cells; however, its role in APCs has yet to be uncovered. Using a coculture system with mycobacterial-specific CD4+ T cells, we show APC L-ARG synthesis supported T cell viability and proliferation, and activated T cells contained APC-derived L-ARG. We hypothesize that APCs supply L-ARG to support T cell activation under nutrient-limiting conditions. This work expands the current model of APC–T cell interactions and provides insight into the effects of nutrient availability in immune cells.

Dysregulation of innate immune signaling pathways is implicated in various hematologic malignancies. However, these pathways have not been systematically examined in acute myeloid leukemia (AML). We report that AML hematopoietic stem and progenitor cells (HSPCs) exhibit a high frequency of dysregulated innate immune-related and inflammatory pathways, referred to as oncogenic immune signaling states. Through gene expression analyses and functional studies in human AML cell lines and patient-derived samples, we found that the ubiquitin-conjugating enzyme UBE2N is required for leukemic cell function in vitro and in vivo by maintaining oncogenic immune signaling states. It is known that the enzyme function of UBE2N can be inhibited by interfering with thioester formation between ubiquitin and the active site. We performed in silico structure-based and cellular-based screens and identified two related small-molecule inhibitors UC-764864/65 that targeted UBE2N at its active site. Using these small-molecule inhibitors as chemical probes, we further revealed the therapeutic efficacy of interfering with UBE2N function. This resulted in the blocking of ubiquitination of innate immune- and inflammatory-related substrates in human AML cell lines. Inhibition of UBE2N function disrupted oncogenic immune signaling by promoting cell death of leukemic HSPCs while sparing normal HSPCs in vitro. Moreover, baseline oncogenic immune signaling states in leukemic cells derived from discrete subsets of patients with AML exhibited a selective dependency on UBE2N function in vitro and in vivo. Our study reveals that interfering with UBE2N abrogates leukemic HSPC function and underscores the dependency of AML cells on UBE2N-dependent oncogenic immune signaling states.

Myelodysplastic syndrome (MDS) is a heterogeneous myeloid malignancy characterized by blood cell morphological dysplasia, ineffective clonal hematopoiesis, and risk of transformation to secondary acute myeloid leukemia (sAML). A number of genetic abnormalities have been identified in MDS and sAML, but sensitive sequencing methods can detect these mutations in nearly all healthy individuals by 60 years of age. To discover novel cellular pathways that accelerate MDS and sAML, we performed a CRISPR/Cas9 screen in the human MDS-L cell line. We report here that loss of the F-Box protein FBXO11, a component of the SCF ubiquitin ligase complex, confers cytokine independent growth to MDS-L cells, suggesting a tumor suppressor role for FBXO11 in myeloid malignancies. Putative FBXO11 substrates are enriched for proteins with functions in RNA metabolism and, of note, spliceosome mutations that are commonly found in MDS/sAML are rare in patients with low FBXO11 expression. We also reveal that loss of FBXO11 leads to significant changes in transcriptional pathways influencing leukocyte proliferation, differentiation, and apoptosis. Last, we find that FBXO11 expression is reduced in patients with secondary AML. We conclude that loss of FBXO11 is a mechanism for disease transformation of MDS into AML, and may represent a future therapeutic target.

Advances in genetics and sequencing have identified a plethora of disease-associated and disease-causing genetic alterations. To determine causality between genetics and disease, accurate models for molecular dissection are required; however, the rapid expansion of transcriptional populations identified through single-cell analyses presents a major challenge for accurate comparisons between mutant and wild-type cells. Here we generate mouse models of human severe congenital neutropenia (SCN) using patient-derived mutations in the GFI1 transcription factor. To determine the effects of SCN mutations, we generated single-cell references for granulopoietic genomic states with linked epitopes1, aligned mutant cells to their wild-type equivalents and identified differentially expressed genes and epigenetic loci. We find that GFI1-target genes are altered sequentially, as cells go through successive states of differentiation. These insights facilitated the genetic rescue of granulocytic specification but not post-commitment defects in innate immune effector function, and underscore the importance of evaluating the effects of mutations and therapy within each relevant cell state. Mouse models of severe congenital neutropenia using patient-derived mutations in the GFI1 locus are used to determine the mechanisms by which the disease progresses.

It has been suggested that miR-144 is pro-atherosclerotic via effects on reverse cholesterol transportation targeting the ATP binding cassette protein. This study used proteomic analysis to identify additional cardiovascular targets of miR-144, and subsequently examined the role of a newly identified regulator of atherosclerotic burden in miR-144 knockout mice receiving a high fat diet. To identify affected secretory proteins, miR-144 treated endothelial cell culture medium was subjected to proteomic analysis including two-dimensional gel separation, trypsin digestion, and nanospray liquid chromatography coupled to tandem mass spectrometry. We identified 5 gel spots representing 19 proteins that changed consistently across the biological replicates. One of these spots, was identified as vimentin. Atherosclerosis was induced in miR-144 knockout mice by high fat diet and vascular lesions were quantified by Oil Red-O staining of the serial sectioned aortic root and from en-face views of the aortic tree. Unexpectedly, high fat diet induced extensive atherosclerosis in miR-144 knockout mice and was accompanied by severe fatty liver disease compared with wild type littermates. Vimentin levels were reduced by miR-144 and increased by antagomiR-144 in cultured cardiac endothelial cells. Compared with wild type, ablation of the miR-144/451 cluster increased plasma vimentin, while vimentin levels were decreased in control mice injected with synthetic miR-144. Furthermore, increased vimentin expression was prominent in the commissural regions of the aortic root which are highly susceptible to atherosclerotic plaque formation. We conclude that miR-144 maybe a potential regulator of the development of atherosclerosis via changes in vimentin signaling.

Cocaine addiction remains a serious problem lacking an effective pharmacological treatment. Thus, we have developed a high-affinity anti-cocaine monoclonal antibody (mAb), h2E2, for the treatment of cocaine use disorders. We show that selective tryptophan (Trp) oxidation by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) resulted in a loss of high-affinity binding of cocaine to this mAb. The newly developed use of excess methionine (Met) to protect mAb met residues from AAPH oxidation did not substantially attenuate the effects of oxidation on cocaine binding but greatly decreased the modification of met residues in the mAb. Similar large decreases in ligand affinity (5000-10,000-fold) upon oxidation were observed using cocaine and two cocaine metabolites, cocaethylene and benzoylecgonine, which also bind with nanomolar affinity to this h2E2 mAb. The decrease in binding affinity was accompanied by a decrease of approximately 50% in Trp fluorescence, and increases in mAb 310 to 370 nm absorbance were consistent with the presence of oxidized forms of Trp. Finally, mass spectral analysis of peptides derived from control and AAPH-oxidized mAb indicated that excess free met did effectively protect mAb met residues from oxidation, and that AAPH-oxidized mAb heavy-chain Trp33 and light-chain Trp91 residues are important for cocaine binding, consistent with a recently derived h2E2 Fab fragment crystal structure containing bound benzoylecgonine. Thus, protection of the anti-cocaine h2E2 mAb from Trp oxidation prior to its clinical administration is critical for its proposed therapeutic use in the treatment of cocaine use disorders.

Pulmonary alveolar microlithiasis (PAM) is an autosomal recessive lung disease caused by a deficiency in the pulmonary epithelial Npt2b sodium-phosphate co-transporter that results in accumulation of phosphate and formation of hydroxyapatite microliths in the alveolar space. The single cell transcriptomic analysis of a PAM lung explant showing a robust osteoclast gene signature in alveolar monocytes and the finding that calcium phosphate microliths contain a rich protein and lipid matrix that includes bone resorbing osteoclast enzymes suggested a role for osteoclast-like cells in the defense against microliths. While investigating the mechanisms of microlith clearance, we found that Npt2b modulates pulmonary phosphate homeostasis through effects on alternative phosphate transporter activity and alveolar osteoprotegerin, and that microliths induce osteoclast formation and activity in a receptor activator of nuclear factor-κB ligand (RANKL) and dietary phosphate dependent manner. This work reveals that Npt2b and pulmonary osteoclast-like cells play key roles in pulmonary homeostasis and suggest potential new therapeutic targets for the treatment of lung disease.

Granulocyte colony stimulating factor receptor (G-CSFR) plays an important role in the production of neutrophil granulocytes. Mutated G-CSFRs have been directly associated with two distinct malignant phenotypes in patients, e.g. acute myeloid leukemia (AML) and chronic neutrophilic leukemia (CNL). However, the signaling mechanism of the mutated G-CSFRs is not well understood. Here, we present a comprehensive SILAC-based quantitative phosphoserine and phosphothreonine dataset of the normal and mutated G-CSFRs signaling using the BaF3 cell-line-based in vitro model system. High pH reversed phase concatenation and Titanium Dioxide Spin Tip column were utilized to increase the dynamic range and detection of the phosphoproteome of G-CSFRs. The dataset was further analyzed using several computational tools to validate the quality of the dataset. Overall, this dataset is the first global phosphoproteomics analysis of both normal and disease-associated-mutant G-CSFRs. We anticipate that this dataset will have a strong potential to decipher the phospho-signaling differences between the normal and malignant G-CSFR biology with therapeutic implications. The phosphoproteomic dataset is available via the PRIDE partner repository., Design Type(s)protein physical property analysis objective • replicate design • factorial designMeasurement Type(s)protein expression profilingTechnology Type(s)liquid chromatography-tandem mass spectrometryFactor Type(s)biological replicate • experimental conditionSample Characteristic(s)Mus musculus • BA/F3 cell Machine-accessible metadata file describing the reported data (ISA-Tab format)

Monoclonal antibodies and derived fragments are used extensively both experimentally and therapeutically. Thorough characterization of such antibodies is necessary and includes assessment of their thermal and storage stabilities. Thus, assessment of the underlying conformational stabilities of the antibodies is also important. We recently documented that non-reducing SDS-PAGE can be used to assess both monoclonal and polyclonal IgG domain thermal unfolding in SDS. Utilizing this same h2E2 anti-cocaine mAb, in this study we generated and analyzed various mAb antibody fragments to delineate the structural domains of the antibody responsible for the observed discrete bands following various heating protocols and analysis by non-reducing SDS-PAGE. Previously, these domain unfolding transitions and gel bands were hypothesized to stem from known mAb structural domains based on the relative thermal stability of those CH2, CH3, and Fab domains in the absence of SDS, as measured by differential scanning calorimetry. In this study, we generated and analyzed F(ab’)2, Fab, and Fc fragments, as well as a mAb consisting of only heavy chains, and examined the thermally induced domain unfolding in each of these fragments by non-reducing SDS-PAGE. The results were interpreted and integrated to generate an improved model of thermal unfolding for the mAb IgG in SDS. These results and the model presented should be generally applicable to many monoclonal and polyclonal antibodies and allow novel comparisons of conformational stabilities between chemically or genetically modified versions of a given antibody. Such modified antibodies and antibody drug conjugates are commonly utilized and important for experimental and therapeutic applications., Graphical abstract fx1, Highlights • mAb F(ab’)2 fragments exhibit multiple unfolded states in non-reducing SDS-PAGE. • Fab and Fc mAb fragments do not exhibit similar multiple unfolded state bands. • Previous mAb domain unfolding pathway in SDS is revised based on fragment analyses. • A heavy chain only mAb variant is detected and exhibits multiple unfolded states. • These results are likely relevant to analyses of many monoclonal and polyclonal Abs.

RATIONALE: Among its many biological roles, fibroblast growth factor 2 (FGF2) protects the heart from dysfunction and damage associated with an ischemic attack. Our laboratory demonstrated that its protection against myocardial dysfunction occurs by the low molecular weight (LMW) isoform of FGF2, while the high molecular weight (HMW) isoforms are associated with a worsening in post-ischemic recovery of cardiac function. LMW FGF2-mediated cardioprotection is facilitated by activation of multiple kinases, including PKCalpha, PKCepsilon, and ERK, and inhibition of p38 and JNK. OBJECTIVE: Yet, the substrates of those kinases associated with LMW FGF2-induced cardioprotection against myocardial dysfunction remain to be elucidated. METHODS AND RESULTS: To identify substrates in LMW FGF2 improvement of post-ischemic cardiac function, mouse hearts expressing only LMW FGF2 were subjected to ischemia-reperfusion (I/R) injury and analyzed by a mass spectrometry (MS)-based quantitative phosphoproteomic strategy. MS analysis identified 50 phosphorylation sites from 7 sarcoendoplasmic reticulum (SR) proteins that were significantly altered in I/R-treated hearts only expressing LMW FGF2 compared to those hearts lacking FGF2. One of those phosphorylated SR proteins identified was phospholamban (PLB), which exhibited rapid, increased phosphorylation at Threonine-17 (Thr17) after I/R in hearts expressing only LMW FGF2; this was further validated using Single Reaction Monitoring-based MS workflow. To demonstrate a mechanistic role of phospho-Thr17 PLB in LMW FGF2-mediated cardioprotection, hearts only expressing LMW FGF2 and those expressing only LMW FGF2 with a mutant PLB lacking phosphorylatable Thr17 (Thr17Ala PLB) were subjected to I/R. Hearts only expressing LMW FGF2 showed significantly improved recovery of cardiac function following I/R (p

Glioblastoma multiforme (GBM), a common type of brain cancer, has a very poor prognosis. In general, viable GBM cells exhibit elevated phosphatidylserine (PS) on their membrane surface compared to healthy cells. We have developed a drug, saposin C-dioleoylphosphatidylserine (SapC-DOPS), that selectively targets cancer cells by honing in on this surface PS. To examine whether SapC-DOPS, a stable, blood&ndash, brain barrier-penetrable nanovesicle, could be an effective delivery system for precise targeted therapy of radiation, we iodinated several carbocyanine-based fluorescent reporters with either stable iodine (127I) or radioactive isotopes (125I and 131I). While all of the compounds, when incorporated into the SapC-DOPS delivery system, were taken up by human GBM cell lines, we chose the two that best accumulated in the cells (DiI (22,3) and DiD (16,16)). Pharmacokinetics were conducted with 125I-labeled compounds and indicated that DiI (22,3)-SapC-DOPS had a time to peak in the blood of 0.66 h and an elimination half-life of 8.4 h. These values were 4 h and 11.5 h, respectively, for DiD (16,16)-SapC-DOPS. Adult nude mice with GBM cells implanted in their brains were treated with 131I-DID (16,16)-SapC-DOPS. Mice receiving the radionuclide survived nearly 50% longer than the control groups. These data suggest a potential novel, personalized treatment for a devastating brain disease.

Clonal hematopoiesis (CH) is an aging-associated condition characterized by the clonal outgrowth of pre-leukemic cells that acquire specific mutations. Although individuals with CH are healthy, they are at an increased risk of developing myeloid malignancies, suggesting that additional alterations are needed for the transition from a pre-leukemia stage to frank leukemia. To identify signaling states that cooperate with pre-leukemic cells, we used an in vivo RNAi screening approach. One of the most prominent genes identified was the ubiquitin ligase TRAF6. Loss of TRAF6 in pre-leukemic cells results in overt myeloid leukemia and is associated with MYC-dependent stem cell signatures. TRAF6 is repressed in a subset of patients with myeloid malignancies, suggesting that subversion of TRAF6 signaling can lead to acute leukemia. Mechanistically, TRAF6 ubiquitinates MYC, an event that does not affect its protein stability but rather represses its functional activity by antagonizing an acetylation modification.

The overarching goal of this exploratory study is to link subcellular microdomain specific protein-protein interactomes with big data analytics. We isolated postsynaptic density-95 (PSD-95) complexes from four human brain regions and compared their protein interactomes using multiple bioinformatics techniques. We demonstrate that human brain regions have unique postsynaptic protein signatures that may be used to interrogate perturbagen databases. Assessment of our hippocampal signature using the iLINCS database yielded several compounds with recently characterized “off target” effects on protein-protein interactions in the posynaptic density compartment.

The recent advance in targeted label-free proteomics, SWATH-MS, can provide consistent protein detection and reproducible protein quantitation, which is a considerable advantage for biomarker study of urinary extracellular vesicles. We developed a SWATH-MS workflow with a curated spectral library of 1,145 targets. Application of the workflow across nine replicates of three sample types (exosome-like vesicles (ELVs), microvesicles (MVs) and urine proteins (UPs)) resulted in the quantitation of 888 proteins at FDR

N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic messenger RNAs (mRNAs) and is interpreted by its readers, such as YTH domain-containing proteins, to regulate mRNA fate. Here, we report the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3) as a distinct family of m6A readers that target thousands of mRNA transcripts through recognizing the consensus GG(m6A)C sequence. In contrast to the mRNA-decay-promoting function of YTH domain-containing family protein 2, IGF2BPs promote the stability and storage of their target mRNAs (for example, MYC) in an m6A-dependent manner under normal and stress conditions and therefore affect gene expression output. Moreover, the K homology domains of IGF2BPs are required for their recognition of m6A and are critical for their oncogenic functions. Thus, our work reveals a different facet of the m6A-reading process that promotes mRNA stability and translation, and highlights the functional importance of IGF2BPs as m6A readers in post-transcriptional gene regulation and cancer biology.

BRAF mutations occur in about 50% of melanoma patients. FDA approved BRAF and MEK inhibitors have improved the prognosis of patients with BRAF mutations. However, all responders develop resistance typically within one year of treatment. Recent observations demonstrate that BRAF inhibitors induce reactive oxygen species (ROS) in melanoma cells. A100, identified from a library screen, is a ROS-activated prodrug that self-cyclizes into a stable bicyclic ring and causes DNA double strand breaks. We proposed to examine if ROS activated therapy will inhibit tumor growth and evade resistance to BRAF inhibitors. In this study, the BRAF inhibitor dabrafenib was used to generate resistant cell lines (A375DR, SK-MEL-24DR and WM-115DR). Flow cytometry experiments showed that ROS levels are increased in these dabrafenib-resistant cells as compared to parental cells, assessed by both the H2DCFDA and MitoSOX assays. Furthermore, we observed that resistant cells had increased levels of the mitochondrial enzymes SOD2 and PRDX1, which function to reduce ROS levels in the mitochondria. We found that A100 sensitized the resistant melanoma cells to dabrafenib and induced DNA damage. Co-treatment of both A100 and dabrafenib significantly suppressed in vitro cell proliferation and three- dimensional (3D) matrigel growth. This study suggests that the combination of A100 with a BRAF inhibitor could be a potential strategy to treat melanoma patients with BRAF mutations.

Toll-like receptor (TLR) activation contributes to premalignant hematologic conditions, such as myelodysplastic syndromes (MDS). TRAF6, a TLR effector with ubiquitin (Ub) ligase activity, is overexpressed in MDS hematopoietic stem/progenitor cells (HSPCs). We found that TRAF6 overexpression in mouse HSPC results in impaired hematopoiesis and bone marrow failure. Using a global Ub screen, we identified hnRNPA1, an RNA-binding protein and auxiliary splicing factor, as a substrate of TRAF6. TRAF6 ubiquitination of hnRNPA1 regulated alternative splicing of Arhgap1, which resulted in activation of the GTP-binding Rho family protein Cdc42 and accounted for hematopoietic defects in TRAF6-expressing HSPCs. These results implicate Ub signaling in coordinating RNA processing by TLR pathways during an immune response and in premalignant hematologic diseases, such as MDS.

Objective: It has been suggested that miR-144 is pro-atherosclerotic via effects on reverse cholesterol transportation targeting the ATP binding cassette protein 1 (ABCA1). This study used proteomic analysis to identify additional cardiovascular targets of miR-144, and subsequently examined the role of a newly identified regulator of atherosclerotic burden in miR-144 KO mice receiving a high fat diet (HFD). Approach and Results: To identify affected secretory proteins, miR-144 treated endothelial cell culture medium was subjected to proteomic analysis including two-dimensional gel separation and nanospray liquid chromatography coupled to tandem mass spectrometry. We identified 5 gel spots representing 19 proteins that changed consistently across the biological replicates. One of these spots, was identified as vimentin. Atherosclerosis was induced in miR-144 KO mice by HFD and vascular lesions were quantified by Oil Red-O staining of the serial sectioned aortic root and from en-face views of the aortic tree. Unexpectedly, HFD induced extensive atherosclerosis in miR-144 KO mice in aortic root (WT 100% ± 22.71 vs miR-144 KO 256.88% ± 58.19, p < 0.05, n = 10) and arch areas (WT 100% ± 29.67 vs miR-144 KO 371.74% ± 73.48, p < 0.01, n = 10), and was accompanied by severe fatty liver disease compared with wild type littermates (WT). Vimentin levels were reduced by miR-144 and increased by antagomiR-144 in cultured cardiac endothelial cells without affecting vimentin mRNA levels. Compared with WT, ablation of the miR-144/451 cluster significantly increased plasma vimentin, while vimentin levels were decreased in control mice injected with synthetic miR-144 as double confirmed with Western blot and ELISA assays. Furthermore, increased vimentin expression was seen in the commissural regions of the aortic root (WT 100% ± 10.02 vs 164.19 ± 13.90, p < 0.01, n = 10) and collocated with other areas of aortic atherosclerosis. In contrast, neither liver ABCA1 nor circulating cholesterol levels were different between WT and miR-144/451 KO mice. Conclusion: We conclude that miR-144 is a potential regulator of the development of atherosclerosis via changes in vimentin signaling and both miR-144 and vimentin are potential therapeutic targets for atherosclerosis.

Purpose: Distinguishing urinary tract infection (UTI) from urinary tract colonization (UTC) in children with neurogenic bladders who require clean intermittent catheterization (CIC) is challenging. Our objective was to identify urinary proteins to distinguish UTI from UTC in CIC-dependent children that have potential to serve as objective markers of UTI. Experimental design: A total of 10 CIC-dependent children were included in the mass spectrometry analysis (UTI = 5, UTC = 5). Quantitative profiling of urine proteins with isobaric protein labeling was performed using tandem mass spectrometry. Candidate markers were normalized using a collective mixture of proteins from all samples. Relative quantitative abundance of proteins across all samples were compared. Proteins with >50% change in the average abundance were identified as proteins of interest, which were then measured using enzyme-linked immunosorbent assay (ELISA) in an additional 40 samples (no growth = 10, UTC = 15, UTI = 15). Results: Mass spectrometry revealed 8 differentially expressed proteins. Of these, apolipoprotein D, alpha-amylase 2B, non-secretory ribonuclease, CD44 antigen, and prosaposin were measurable by ELISA. Concentrations of both CD44 and prosaposin were significantly higher in UTI, with area under the curves (AUCs) of 0.72 and 0.78, respectively. Conclusion: Urinary CD44 and prosaposin are candidate markers that may assist with the diagnosis of UTI in CIC-dependent children.

Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab’)2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab’)2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications.

// Victor M. Blanco 1 , Zhengtao Chu 1, 2 , Kathleen LaSance 3 , Brian D. Gray 4 , Koon Yan Pak 4 , Therese Rider 5 , Kenneth D. Greis 5 , Xiaoyang Qi 1, 2 1 Division of Hematology-Oncology, Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA 2 Division of Human Genetics, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229, USA 3 Department of Radiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA 4 Molecular Targeting Technologies, Inc., West Chester, Pennsylvania 19380, USA 5 Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA Correspondence to: Xiaoyang Qi, email: xiaoyang.qi@uc.edu Keywords: glioblastoma, liposome, PET, optical imaging, SapC-DOPS Abbreviations: SapC, Saposin C; DOPS, dioleylphosphatidylserine; PS, phosphatidylserine Received: June 25, 2015 Accepted: March 28, 2016 Published: April 16, 2016 ABSTRACT Multimodal tumor imaging with targeted nanoparticles potentially offers both enhanced specificity and sensitivity, leading to more precise cancer diagnosis and monitoring. We describe the synthesis and characterization of phenol-substituted, lipophilic orange and far-red fluorescent dyes and a simple radioiodination procedure to generate a dual (optical and nuclear) imaging probe. MALDI-ToF analyses revealed high iodination efficiency of the lipophilic reporters, achieved by electrophilic aromatic substitution using the chloramide 1,3,4,6-tetrachloro-3α,6α-diphenyl glycoluril (Iodogen) as the oxidizing agent in an organic/aqueous co-solvent mixture. Upon conjugation of iodine-127 or iodine-124-labeled reporters to tumor-targeting SapC-DOPS nanovesicles, optical (fluorescent) and PET imaging was performed in mice bearing intracranial glioblastomas. In addition, tumor vs non-tumor (normal brain) uptake was compared using iodine-125. These data provide proof-of-principle for the potential value of SapC-DOPS for multimodal imaging of glioblastoma, the most aggressive primary brain tumor.

Data-independent acquisition (DIA)-based proteomics has become increasingly complicated in recent years because of the vast number of workflows described, coupled with a lack of studies indicating a rational framework for selecting effective settings to use. To address this issue and provide a resource for the proteomics community, we compared 12 DIA methods that assay tryptic peptides using various mass-isolation windows. Our findings indicate that the most sensitive single injection LC-DIA method uses 6 m/z isolation windows to analyze the densely populated tryptic peptide range from 450 to 730 m/z, which allowed quantification of 4465 Escherichia coli peptides. In contrast, using the sequential windowed acquisition of all theoretical fragment-ions (SWATH) approach with 26 m/z isolation windows across the entire 400-1200 m/z range, allowed quantification of only 3309 peptides. This reduced sensitivity with 26 m/z windows is caused by an increase in co-eluting compounds with similar precursor values detected in the same tandem MS spectra, which lowers the signal-to-noise of peptide fragment-ion chromatograms and reduces the amount of low abundance peptides that can be quantified from 410 to 920 m/z. Above 920 m/z, more peptides were quantified with 26 m/z windows because of substantial peptide (13) C isotope distributions that parse peptide ions into separate isolation windows. Because reproducible quantification has been a long-standing aim of quantitative proteomics, and is a so-called trait of DIA, we sought to determine whether precursor-level chromatograms used in some methods rather than their fragment-level counterparts have similar precision. Our data show that extracted fragment-ion chromatograms are the reason DIA provides superior reproducibility. Copyright © 2015 John Wiley & Sons, Ltd.

PURPOSE: To evaluate cellular protein changes in response to treatment with an approved drug, ibrutinib, in cells expressing normal or mutated Granulocyte-Colony Stimulating Factor Receptor (G-CSFR). G-CSFR mutations are associated with some hematological malignancies. Previous studies showed the efficacy of ibrutinib (a Bruton’s tyrosine kinase inhibitor) in mutated G-CSFR leukemia models but did not addressed broader signaling mechanisms. EXPERIMENTAL DESIGN: A label-free quantitative proteomics workflow to evaluate the cellular effects of ibrutinib treatment was established. This included 3 biological replicates of normal and mutated G-CSFR expressed in a mouse progenitor cell (32D cell line) with and without ibrutinib treatment. RESULTS: The proteomics dataset showed about 1,000 unique proteins quantified with nearly 400 significant changes (p value < 0.05), suggesting a highly dynamic network of cellular signaling in response to ibrutinib. Importantly, the dataset was very robust with coefficients of variation (CVs) for quantitation at 13.0-20.4% resulting in dramatic patterns of protein differences among the groups. CONCLUSIONS AND CLINICAL RELEVANCE: This robust data set is available for further mining, hypothesis generation, and testing. A detailed understanding of the restructuring of the proteomics signaling cascades by ibrutinib in leukemia biology will provide new avenues to explore its use for other related malignancies.

SUMMARY TRAF-interacting protein with a forkhead-associated domain B (TIFAB) is implicated in myeloid malignancies with deletion of chromosome 5q. Employing a combination of proteomic and genetic approaches, we find that TIFAB regulates ubiquitin-specific peptidase 15 (USP15) ubiquitin hydrolase activity. Expression of TIFAB in hematopoietic stem/progenitor cells (HSPCs) permits USP15 signaling to substrates, including MDM2 and KEAP1, and mitigates p53 expression. Consequently, TIFAB-deficient HSPCs exhibit compromised USP15 signaling and are sensitized to hematopoietic stress by derepression of p53. In MLL-AF9 leukemia, deletion of TIFAB increases p53 signaling and correspondingly decreases leukemic cell function and development of leukemia. Restoring USP15 expression partially rescues the function of TIFAB-deficient MLL-AF9 cells. Conversely, elevated TIFAB represses p53, increases leukemic progenitor function, and correlates with MLL gene expression programs in leukemia patients. Our studies uncover a function of TIFAB as an effector of USP15 activity and rheostat of p53 signaling in stressed and malignant HSPCs., Graphical Abstract, In Brief Niederkorn et al. identify TIFAB as a critical node in hematopoietic cells under stressed and oncogenic cell states. Their studies indicate that deregulation of the TIFAB-USP15 complex, as observed in del(5q) myelodysplasia or MLL-rearranged leukemia, modulates p53 activity and has critical functional consequences for stressed and malignant hematopoietic cells.

OBJECTIVES: We used an unbiased proteomics approach to identify candidate urine biomarkers (CUBMs) predictive of LN chronicity and pursued their validation in a larger cohort. METHODS: In this cross-sectional pilot study, we selected urine collected at kidney biopsy from 20 children with varying levels of LN damage (discovery cohort) and performed proteomic analysis using isobaric tags for relative and absolute quantification (iTRAQ). We identified differentially excreted proteins based on degree of LN chronicity and sought to distinguish markers exhibiting different relative expression patterns using hierarchically clustered log10-normalized relative abundance data with linked and distinct functions by biological network analyses. For each CUBM, we performed specific ELISAs on urine from a validation cohort (n = 41) and analysis of variance to detect differences between LN chronicity, with LN activity adjustment. We evaluated for CUBM expression in LN biopsies with immunohistochemistry. RESULTS: iTRAQ detected 112 proteins in urine from the discovery cohort, 51 quantifiable in all replicates. Simple analysis of variance revealed four differentially expressed, chronicity-correlated proteins (P-values < 0.05). Further correlation and network analyses led to selection of seven CUBMs for LN chronicity. In the validation cohort, none of the CUBMs distinguished LN chronicity degree; however, urine SERPINA3 demonstrated a moderate positive correlation with LN histological activity. Immunohistochemistry further demonstrated SERPINA3 staining in proximal tubular epithelial and endothelial cells. CONCLUSION: We identified SERPINA3, a known inhibitor of neutrophil cathepsin G and angiotensin II production, as a potential urine biomarker to help quantify LN activity.

Background Non-invasive biomarkers of lupus nephritis (LN) damage are needed to guide treatment decisions. Urinary proteomics has advanced as a tool for novel biomarker discovery in recent years. Specifically, isobaric tags for relative and absolute quantification (iTRAQ) is an advanced proteomics technique that quantifies and compares protein expression among samples by mass spectrometry in a single experiment. We used an unbiased proteomics approach to identify candidate urine biomarkers (CUBMs) predictive of LN chronicity and further pursued their validation in a larger cohort. Methods In this cross-sectional pilot study, we selected urine collected at kidney biopsy from 20 children with varying levels of LN damage (discovery cohort) and performed proteomic analysis using iTRAQ. We identified differentially excreted proteins based on degree of LN chronicity and sought to distinguish markers exhibiting different relative expression patterns using hierarchically-clustered log10-normalized relative abundance data with linked and distinct functions by biological network analyses. For each CUBM, we performed specific enzyme-linked immunosorbent assays (ELISAs) on urine from a validation cohort (n=41) and analysis of variance (ANOVA) to detect differences between LN chronicity, with LN activity adjustment. We evaluated for CUBM expression in LN biopsies with immunohistochemistry. Results iTRAQ detected 112 proteins from urine samples in the discovery cohort, 51 of which were quantifiable in all replicates. Simple ANOVA revealed four differentially expressed, chronicity-correlated proteins (p-values Conclusions Using advanced proteomic techniques followed by confirmation using specific ELISAs, we identified SERPINA3, a known inhibitor of neutrophil cathepsin G and angiotensin II production, as a potential urine biomarker to help quantify LN damage. SERPINA3 expression may be a protective mechanism from further kidney damage. Further validation of SERPINA3 as an LN damage biomarker in an independent cohort is needed to determine its ability to guide treatment and predict prognosis. Acknowledgements This study was supported by grants from the National Institutes of Health [P50 DK 096418, U01 AR065098, T32 AR069512, P30 AR070549] and a Lupus Foundation Career Development Award to Dr. Turnier. Mass spectrometry data were collected on a system funded through an NIH shared instrumentation grant (S10 RR027015–01; KD Greis-PI).

Granulocyte-colony stimulating factor receptor (G-CSFR) controls myeloid progenitor proliferation and differentiation to neutrophils. Mutations in CSF3R (encoding G-CSFR) have been reported in patients with chronic neutrophilic leukemia (CNL) and acute myeloid leukemia (AML); however, despite years of research, the malignant downstream signaling of the mutated G-CSFRs is not well understood. Here, we utilized a quantitative phospho-tyrosine analysis to generate a comprehensive signaling map of G-CSF induced tyrosine phosphorylation in the normal versus mutated (proximal: T618I and truncated: Q741x) G-CSFRs. Unbiased clustering and kinase enrichment analysis identified rapid induction of phospho-proteins associated with endocytosis by the wild-type G-CSFR only; while G-CSFR mutants showed abnormal kinetics of canonical STAT3, STAT5 and MAPK phosphorylation, and aberrant activation of Bruton’s Tyrosine Kinase (Btk). Mutant-G-CSFR-expressing cells displayed enhanced sensitivity (3 to 5-fold lower IC50) for Ibrutinib-based chemical inhibition of Btk. Primary murine progenitor cells from G-CSFR-Q741x knock-in mice validated activation of Btk by the mutant receptor and retrovirally-transduced human CD34+ umbilical cord blood cells expressing mutant receptors displayed enhanced sensitivity to Ibrutinib. A significantly lower clonogenic potential was displayed by both murine and human primary cells expressing mutated receptors upon ibrutinib treatment. Finally, a dramatic synergy was observed between ibrutinib and ruxolinitib at lower dose of the individual drug. Together, these data demonstrate the strength of unsupervised proteomics analyses in dissecting oncogenic pathways, and suggest repositioning Ibrutinib for therapy of myeloid leukemia bearing CSF3R mutations. Phospho-tyrosine data is available via ProteomeXchange with identifier PXD009662.

We designed ROS-activated cytotoxic agents (RACs) that are active against AML cancer cells. In this study, the mechanism of action and synergistic effects against cells coexpressing the AML oncogenes MLL-AF9 fusion and FLT3-ITD were investigated. One RAC (RAC1) had an IC50 value of 1.8 ± 0.3 µm, with ninefold greater selectivity for transformed cells compared to untransformed cells. Treatment induced DNA strand breaks, apoptosis, and cell cycle arrest. Proteomics and transcriptomics revealed enhanced expression of the pentose phosphate pathway, DNA repair, and pathways common to cell stress. Western blotting confirmed repair by homologous recombination. Importantly, RAC1 treatment was synergistic in combination with multiple pathway-targeting therapies in AML cells but less so in untransformed cells. Together, these results demonstrate that RAC1 can selectively target poor prognosis AML and that it does so by creating DNA double-strand breaks that require homologous recombination.

Phosphoproteomic analysis offers a unique view of cellular function and regulation in biological systems by providing global measures of a key cellular regulator in the form of protein phosphorylation. Understanding the phosphorylation changes between normal and diseased cells or tissues offers a window into the mechanism of disease and thus potential targets for therapeutic intervention. A key step in these studies is the enrichment of phosphorylated peptides that are typically separated and analyzed by using liquid chromatography mass spectrometry. The mesoporous titania beads/particles (e.g., Titansphere TiO2 beads from GL Sciences Inc., Japan) that are widely used for phosphopeptide enrichment are expensive and offer very limited opportunities for further performance improvement. Titiania nanotube arrays have shown promising characteristics for phosphopeptide separation. Here we report a proof-of-concept study to evaluate the efficacy of nanotubes on Ti-wire for phosphoproteomics research. We used titania nanotubes radially grown on titanium wires as well as the commercial beads to separate phosphopeptides generated from mouse liver complex tissue extracts. Our studies revealed that the nanotubes on metal wire provide comparable efficacy for enrichment of phophopeptides and offer an ease of use advantage versus mesoporous beads, thus having the potential to become a low cost and more practical material/methodology for phosphopeptide enrichment in biological studies.

SummaryGametogenesis is dependent on the expression of germline-specific genes. However, it remains unknown how the germline epigenome is distinctly established from that of somatic lineages. Here we show that genes commonly expressed in somatic lineages and spermatogenesis-progenitor cells undergo repression in a genome-wide manner in late stages of the male germline and identify underlying mechanisms. SCML2, a germline-specific subunit of a Polycomb repressive complex 1 (PRC1), establishes the unique epigenome of the male germline through two distinct antithetical mechanisms. SCML2 works with PRC1 and promotes RNF2-dependent ubiquitination of H2A, thereby marking somatic/progenitor genes on autosomes for repression. Paradoxically, SCML2 also prevents RNF2-dependent ubiquitination of H2A on sex chromosomes during meiosis, thereby enabling unique epigenetic programming of sex chromosomes for male reproduction. Our results reveal divergent mechanisms involving a shared regulator by which the male germline epigenome is distinguished from that of the soma and progenitor cells.

The protein scaffold and signaling regulator p62 is important in critical cellular functions, including bone homeostasis, obesity, and cancer, because of its interactions with various signaling intermediaries. p62 is overexpressed in human cancers and is induced during cell transformation. Its genetic ablation inhibits lung tumorigenesis in vivo and cell proliferation in culture by regulating the TRAF6/NF-κB signaling cascade to control reactive oxygen species (ROS) production and apoptosis. Here we show that cdk1 phosphorylates p62 in vitro and in vivo at T269 and S272, which is necessary for the maintenance of appropriate cyclin B1 levels and the levels of cdk1 activity necessary to allow cells to properly enter and exit mitosis. The lack of cdk1-mediated phosphorylation of p62 leads to a faster exit from mitosis, which translates into enhanced cell proliferation and tumorigenesis in response to Ras-induced transformation. Therefore, p62 emerges as a node for the control of not only cell survival but also cell transit through mitosis.

DEK is an oncoprotein that is overexpressed in many forms of cancer and participates in numerous cellular pathways. Of these different pathways, relevant interacting partners and functions of DEK are well described in regard to the regulation of chromatin structure, epigenetic marks, and transcription. Most of this understanding was derived by investigating DNA-binding and chromatin processing capabilities of the oncoprotein. To facilitate the generation of mechanism-driven hypotheses regarding DEK activities in underexplored areas, we have developed the first DEK interactome model using tandem-affinity purification and mass spectrometry. With this approach, we identify IMPDH2, DDX21, and RPL7a as novel DEK binding partners, hinting at new roles for the oncogene in de novo nucleotide biosynthesis and ribosome formation. Additionally, a hydroxyurea-specific interaction with replication protein A (RPA) was observed, suggesting that a DEK-RPA complex may form in response to DNA replication fork stalling. Taken together, these findings highlight diverse activities for DEK across cellular pathways and support a model wherein this molecule performs a plethora of functions.

Nat. Immunol. 18, 236–245; published online 26 December 2016; corrected after print 23 January 2017 In the version of this article initially published online, the 16th author's surname was spelled incorrectly as 'Salamonis'. The correct spelling is 'Salomonis'. The error has been corrected in the PDF and HTML versions of this article.

The acid β-glucosidase (glucocerbrosidase (GCase)) binding sequence to LIMP-2 (lysosomal integral membrane protein 2), the receptor for intracellular GCase trafficking to the lysosome, has been identified. Heterologous expression of deletion constructs, the available GCase crystal structures, and binding and co-localization of identified peptides or mutant GCases were used to identify and characterize a highly conserved 11-amino acid sequence, DSPIIVDITKD, within human GCase. The binding to LIMP-2 is not dependent upon a single amino acid, but the interactions of GCase with LIMP-2 are heavily influenced by Asp399 and the di-isoleucines, Ile402 and Ile403. A single alanine substitution at any of these decreases GCase binding to LIMP-2 and alters its pH-dependent binding as well as diminishing the trafficking of GCase to the lysosome and significantly increasing GCase secretion. Enterovirus 71 also binds to LIMP-2 (also known as SCARB2) on the external surface of the plasma membrane. However, the LIMP-2/SCARB2 binding sequences for enterovirus 71 and GCase are not similar, indicating that LIMP-2/SCARB2 may have multiple or overlapping binding sites with differing specificities. These findings have therapeutic implications for the production of GCase and the distribution of this enzyme that is delivered to various organs.

Background The human brain is comprised of billions of neurons that form networks of connections within and between brain regions. These connections facilitate neuroplastic events that underlie learning and memory, critical aspects of cognitive function often perturbed in neuropsychiatric illnesses. Neuronal signaling is mediated by fast and slow transmission events, encompassing receptors, ligands, ions, enzymes, and other substrates. These elements are spatially arranged in subcellular microdomains, facilitating juxtaposition of proteins that coordinate various biological processes. For example, synaptic transmission is modulated via release of neurotransmitter into the synaptic cleft, where receptors are activated and the postsynaptic cell modulated via electrical and chemical signals. The pre- and postsynaptic compartments include highly specialized protein clusters, with elegant and complex regulatory mechanisms that traffic proteins to and from these zones. In particular, postsynaptic densities are microdomains comprised of about 1000 unique proteins that are interacting with one another via specialized multipotent scaffolding molecule. Postsynaptic density-95 (PSD-95) is a multipotent scaffolding, trafficking, and clustering protein that links glutamate receptors, signaling molecules, and other structural proteins at postsynaptic sites. More than 95% of PSD-95 expression is localized to excitatory synapses, and it is the most abundant scaffolding protein within the postsynaptic density. Mounting genetic, proteomic, and pharmacological evidence converges on alterations in the postsynaptic density of excitatory synapses in subjects with schizophrenia. Cognitive and negative symptoms associated with dysfunction of limbic circuitry, including working memory and motivation, are particularly implicated by this mechanism. To investigate excitatory postsynaptic protein hubs in schizophrenia, we assessed the PSD-95 protein interactome from brain tissue of subjects with schizophrenia and controls. Methods Human brain tissue from fifteen subjects with schizophrenia and fifteen control subjects from the DLPFC was processed for affinity purification of PSD-95 protein complexes. We confirmed PSD-95 capture and enrichment from each sample using Western blot analyses. Samples were then pooled by region and assessed by mass spectrometry for a quality control step. Pooled samples were run in triplicate. Go versus nogo was based on finding more than 500 unique peptides in each pooled sample from each region. Next, individual samples were run through our mass spectrometry protocol in triplicate. We then subtracted any non-specifically captured peptides identified by our IgG control studies that were performed in parallel to PSD-95 affinity purification. Data were normalized within each of the mass spec runs to the most intense PSD-95 peptide. This PSD-95 peptide used for normalization was the same in every sample. Peptides that were present in at least 2 of 3 technical replicates were carried forward and subjected to quantile normalization. Peptides missing in a technical replicate were replaced by imputation, and the dataset subjected to unsupervised clustering. We also performed a semisupervised clustering protocol, non negative matrix factoring (NMF). Consensus signatures of 200 peptides were identified for each brain region, and subjected to traditional and alternative bioinformatics analyses to identify pathways, processes and compounds associated with the signatures for each brain region. Results Preliminary analyses indicate changes in the PSD-95 interactome consistent with diminished NMDA receptor signaling complex activity in schizophrenia, with lower levels of NMDA-subtype glutamate receptor subunits, as well as protein kinases associated with postsynaptic signaling in this complex. Specific biological pathways and processes identified include metabolic and inflammatory pathways. We will also present proteomic signatures generated from this dataset, and interrogate the iLINCS perturbagen database to identify drugs and genes that simulate or reverse this signature. Discussion Our preliminary data suggest that NMDA receptor function is compromised in schizophrenia. Additional work is needed to see if this is an effect of antipsychotic medications. Our proteomic findings extend the NMDA receptor hypothesis beyond the transcriptome, highlighting an important new approach for assessing abnormalities of synapses in postmortem brain.

Individuals with clonal hematopoiesis of indeterminant potential (CHIP) are healthy, however they are at an increased risk of developing hematopoietic malignancies. The most frequent mutations in CHIP target DNMT3A and TET2, also are observed in acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), and myelodysplastic syndromes (MDS). These findings indicate that additional alterations are needed for the transition from a pre-leukemic stage to frank leukemia, although the identity of such molecular events remains uncharacterized. To identify cellular states that cooperate with Tet2 loss, we used in vivo RNAi screening and identified the ubiquitin ligase TRAF6 required for malignant transformation of pre-leukemic TET2-deficient hematopoietic stem/progenitor cell (HSPC). Importantly, TRAF6 expression is significantly reduced in 25-50% of AML and MPN patients as compared to healthy controls. Furthermore, TET2 mutations are more strongly correlated with lower expression of TRAF6 as compared to patients with higher TRAF6 expression in certain subsets of AML. To evaluate the consequences of TRAF6 deletion on TET2-deficienct pre-leukemic cells, we generated mice in which TRAF6 and TET2 are conditionally deleted in hematopoietic cells (VavCre;Traf6fl/fl;Tet2fl/fl[DKO]). Traf6KO mice developed a lethal phenotype with signs of MPN, including lymphopenia, neutrophilia, and increased hemoglobin levels; however, this disease was not transplantable. In striking contrast, deletion of TRAF6 in the context of TET2-deficient HSPC resulted in a rapid, penetrant, aggressive, and transplantable MPN/AML. To firmly establish that TRAF6 exhibits tumor suppressor functions, we determined whether physiological levels of TRAF6 overexpression could prevent malignant transformation. Overexpression of TRAF6 in FLT3-ITD mice inhibited malignant myeloid cell expansion in FLT3-ITD mice, and rescued the survival of the animals. To uncover the molecular basis of TRAF6's tumor suppressor function, we performed gene expression profiling and proteomic characterization of TRAF6 ubiquitination substrates in leukemic cells. RNA-sequencing of HSPC revealed that deletion of TRAF6 resulted in a significant overexpression of MYC regulated genes in pre-leukemic HSPC. In support of these findings, the proteomic screen along with extensive in vitro validation experiments identified MYC as a substrate of TRAF6. Unlike the majority of reported ubiquitin-dependent post-translational modifications of MYC, we found that ubiquitination of MYC on Lysine (K) 148 by TRAF6 does not affect its protein stability but rather antagonizes acetylation of MYC on the same lysine and thus suppresses MYC oncogenic activity. We extended these observations to investigate whether inflammatory signaling via Toll-like receptors (TLRs) can antagonize MYC function and suppress leukemic cells. Stimulation of TLRs on leukemic cells resulted in TRAF6-dependent ubiquitination of MYC at K148, which coincided with repositioning of MYC off of its target gene promoters and enhancers, and ultimately in the suppression of leukemic cell viability. Our results demonstrate that TRAF6 functions as a tumor suppressor via its ubiquitination activity that antagonizes K148 acetylation leading to a decrease of MYC transcriptional activity without affecting its protein abundance. Our findings identify TRAF6 as a novel, context-dependent tumor suppressor in myeloid neoplasms, and suggest that innate immune signaling via TLR/TRAF6 could explain why some of the clonal hematopoiesis patients develop AML and others do not. Disclosures Lowe: Blueprint Medicines: Consultancy, Equity Ownership; PMV Pharmaceuticals: Consultancy, Equity Ownership; Petra Pharmaceuticals: Consultancy, Equity Ownership; Constellation Pharma: Consultancy, Equity Ownership; Mirimus: Consultancy, Equity Ownership; ORIC pharmaceuticals: Consultancy, Equity Ownership; Faeth Therapeutics: Consultancy, Equity Ownership. Starczynowski:Kurome Therapeutics: Consultancy.

SynopsisObjective The objective of this work was to identify whether low levels of redox metals such as copper will accelerate damage to hair on exposure to UV irradiation and whether this damage can be prevented. Methods The methods used were proteomics to measure the protein damage via protein loss after different periods of exposure and mass spectroscopy methods to identify specific marker peptides that are specifically created by this type of damage. Results In this work, we have developed new insights into the mechanism of UV damage using these proteomic methods. A marker fragment in the hair protein loss extract was identified (m/z = 1279) that is unique to UV exposure and increases with time of UV exposure. We have also identified for the first time in hair the role of exogenous copper in increasing UV damage both in terms of total protein degradation and also increased formation of the marker fragment and proposed a mechanism of action. It has been demonstrated that shampoo treatment containing a chelant such as N,N'-ethylenediamine disuccinic acid (EDDS) reduced copper accumulation in hair. Conclusion This work provides evidence for the role of copper in UV-induced damage to hair and strategies to reduce copper levels in hair using a chelant such as EDDS.

Signaling of bone morphogenetic protein (BMP) ligands is antagonized by a number of extracellular proteins, including noggin, follistatin and members of the DAN (differential screening selected gene abberative in neuroblastoma) family. Structural studies on the DAN family member sclerostin (a weak BMP antagonist) have previously revealed that the protein is monomeric and consists of an eight-membered cystine knot motif with a fold similar to transforming growth factor-β ligands. In contrast to sclerostin, certain DAN family antagonists, including protein related to DAN and cerberus (PRDC), have an unpaired cysteine that is thought to function in covalent dimer assembly (analogous to transforming growth factor-β ligands). Through a combination of biophysical and biochemical studies, we determined that PRDC forms biologically active dimers that potently inhibit BMP ligands. Furthermore, we showed that PRDC dimers, surprisingly, are not covalently linked, as mutation of the unpaired cysteine does not inhibit dimer formation or biological activity. We further demonstrated that the noncovalent PRDC dimers are highly stable under both denaturing and reducing conditions. This study was extended to the founding family member DAN, which also forms noncovalent dimers that are highly stable. These results demonstrate that certain DAN family members can form both monomers and noncovalent dimers, implying that biological activity of DAN family members might be linked to their oligomeric state.

Granulocyte colony-stimulating factor is a hematopoietic cytokine that stimulates neutrophil production and hematopoietic stem cell mobilization by initiating the dimerization of homodimeric granulocyte colony-stimulating factor receptor. Different mutations of CSF3R have been linked to a unique spectrum of myeloid disorders and related malignancies. Myeloid disorders caused by the CSF3R mutations include severe congenital neutropenia, chronic neutrophilic leukemia, and atypical chronic myeloid leukemia. In this review, we provide an analysis of granulocyte colony-stimulating factor receptor, various mutations, and their roles in the severe congenital neutropenia, chronic neutrophilic leukemia, and malignant transformation, as well as the clinical implications and some perspective on approaches that could expand our knowledge with respect to the normal signaling mechanisms and those associated with mutations in the receptor.