Dysregulated innate immune signaling cooperates with RUNX1 mutations to transform an MDS-like disease to AML.

Dysregulated innate immune signaling is linked to preleukemic conditions and myeloid malignancies. However, it is unknown whether sustained innate immune signaling contributes to malignant transformation. Here we show that cell-intrinsic innate immune signaling driven by miR-146a deletion (miR-146aKO), a commonly deleted gene in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), cooperates with mutant RUNX1 (RUNX1mut) to initially induce marrow failure and features of MDS. However, miR-146aKO hematopoietic stem and/or progenitor cells (HSPCs) expressing RUNX1mut eventually progress to a fatal AML. miR-146aKO HSPCs exhaust during serial transplantation, while expression of RUNX1mut restored their hematopoietic cell function. Thus, HSPCs exhibiting dysregulated innate immune signaling require a second hit to develop AML. Inhibiting the dysregulated innate immune pathways with a TRAF6-UBE2N inhibitor suppressed leukemic miR-146aKO/RUNX1mut HSPCs, highlighting the necessity of TRAF6-dependent cell-intrinsic innate immune signaling in initiating and maintaining AML. These findings underscore the critical role of dysregulated cell-intrinsic innate immune signaling in driving preleukemic cells toward AML progression.

Prenatal SARS-CoV-2 infection alters postpartum human milk-derived extracellular vesicles.

Human milk-derived extracellular vesicles (HMEVs) are crucial functional components in breast milk, contributing to infant health and development. Maternal conditions could affect HMEV cargos; however, the impact of SARS-CoV-2 infection on HMEVs remains unknown. This study evaluated the influence of SARS-CoV-2 infection during pregnancy on postpartum HMEV molecules. Milk samples (9 prenatal SARS-CoV-2 vs. 9 controls) were retrieved from the IMPRINT birth cohort. After defatting and casein micelle disaggregation, 1 mL milk was subjected to a sequential process of centrifugation, ultrafiltration, and qEV-size exclusion chromatography. Particle and protein characterizations were performed following the MISEV2018 guidelines. EV lysates were analyzed through proteomics and miRNA sequencing, while the intact EVs were biotinylated for surfaceomic analysis. Multi-Omics was employed to predict HMEV functions associated with prenatal SARS-CoV-2 infection. Demographic data between the prenatal SARS-CoV-2 and control groups were similar. The median duration from maternal SARS-CoV-2 test positivity to milk collection was 3 months (range: 1-6 months). Transmission electron microscopy showed the cup-shaped nanoparticles. Nanoparticle tracking analysis demonstrated particle diameters of <200 nm and yields of >1e11 particles from 1 mL milk. Western immunoblots detected ALIX, CD9 and HSP70, supporting the presence of HMEVs in the isolates. Thousands of HMEV cargos and hundreds of surface proteins were identified and compared. Multi-Omics predicted that mothers with prenatal SARS-CoV-2 infection produced HMEVs with enhanced functionalities involving metabolic reprogramming and mucosal tissue development, while mitigating inflammation and lower EV transmigration potential. Our findings suggest that SARS-CoV-2 infection during pregnancy boosts mucosal site-specific functions of HMEVs, potentially protecting infants against viral infections. Further prospective studies should be pursued to reevaluate the short- and long-term benefits of breastfeeding in the post-COVID era.

Paralog-specific signaling by IRAK1/4 maintains MyD88-independent functions in MDS/AML.

Dysregulation of innate immune signaling is a hallmark of hematologic malignancies. Recent therapeutic efforts to subvert aberrant innate immune signaling in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) have focused on the kinase IRAK4. IRAK4 inhibitors have achieved promising, though moderate, responses in preclinical studies and clinical trials for MDS and AML. The reasons underlying the limited responses to IRAK4 inhibitors remain unknown. In this study, we reveal that inhibiting IRAK4 in leukemic cells elicits functional complementation and compensation by its paralog, IRAK1. Using genetic approaches, we demonstrate that cotargeting IRAK1 and IRAK4 is required to suppress leukemic stem/progenitor cell (LSPC) function and induce differentiation in cell lines and patient-derived cells. Although IRAK1 and IRAK4 are presumed to function primarily downstream of the proximal adapter MyD88, we found that complementary and compensatory IRAK1 and IRAK4 dependencies in MDS/AML occur via noncanonical 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 polycomb repressive complex 2 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 xenograft and in vitro studies when treated with KME-2780 as compared with selective IRAK4 inhibitors. Our results provide a mechanistic basis and rationale for cotargeting IRAK1 and IRAK4 for the treatment of cancers, including MDS/AML.

Repression of TRIM13 by chromatin assembly factor CHAF1B is critical for AML development.

Acute myeloid leukemia (AML) is an aggressive blood cancer that stems from the rapid expansion of immature leukemic blasts in the bone marrow. Mutations in epigenetic factors represent the largest category of genetic drivers of AML. The chromatin assembly factor CHAF1B is a master epigenetic regulator of transcription associated with self-renewal and the undifferentiated state of AML blasts. Upregulation of CHAF1B, as observed in almost all AML samples, promotes leukemic progression by repressing the transcription of differentiation factors and tumor suppressors. However, the specific factors regulated by CHAF1B and their contributions to leukemogenesis are unstudied. We analyzed RNA sequencing data from mouse MLL-AF9 leukemic cells and bone marrow aspirates, representing a diverse collection of pediatric AML samples and identified the E3 ubiquitin ligase TRIM13 as a target of CHAF1B-mediated transcriptional repression associated with leukemogenesis. We found that CHAF1B binds the promoter of TRIM13, resulting in its transcriptional repression. In turn, TRIM13 suppresses self-renewal of leukemic cells by promoting pernicious entry into the cell cycle through its nuclear localization and catalytic ubiquitination of cell cycle-promoting protein, CCNA1. Overexpression of TRIM13 initially prompted a proliferative burst in AML cells, which was followed by exhaustion, whereas loss of total TRIM13 or deletion of its catalytic domain enhanced leukemogenesis in AML cell lines and patient-derived xenografts. These data suggest that CHAF1B promotes leukemic development, in part, by repressing TRIM13 expression and that this relationship is necessary for leukemic progression.

Insights into pulmonary phosphate homeostasis and osteoclastogenesis emerge from the study of pulmonary alveolar microlithiasis.

Pulmonary alveolar microlithiasis 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 pulmonary alveolar microlithiasis 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 and other proteins suggested a role for osteoclast-like cells in the host response to 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 activation in a receptor activator of nuclear factor-κB ligand 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.

Label-Free Quantification (LFQ) of Fecal Proteins for Potential Pregnancy Detection in Polar Bears.

Reliable pregnancy diagnostics would be beneficial for monitoring polar bear (Ursus maritimus) populations both in situ and ex situ, but currently there is no method of non-invasive pregnancy detection in this species. Recent reports in several carnivore species described the identification of fecal proteins that may serve as pregnancy biomarkers; however, repeatability has been limited. The objective of the current analysis was to utilize an unbiased, antibody-free, label-free method for the identification and quantification of fecal proteins to determine if differences associated with pregnancy are detectable in polar bears. Protein was extracted from fecal samples (n = 48) obtained from parturient (n = 6) and non-parturient (n = 6) profiles each at four timepoints: pre-breeding season, embryonic diapause, early placental pregnancy, and mid-placental pregnancy. Protein was prepared and analyzed on the Thermo Orbitrap Eclipse nanoLC-MS/MS system. A total of 312 proteins was identified and quantified; however, coefficients of variation (CV) were high for both abundance ratio variability (384.8 ± 61.0% SEM) and within group variability (86.8 ± 1.5%). Results of this study suggest that the inconsistencies in specific protein concentrations revealed previously by antibody-based assays may not be due to that methodology's limitations, but rather, are reflective of true variation that exists among samples.

Tyrosine nitration of a humanized anti-cocaine mAb differentially affects ligand binding of cocaine and its metabolites.

Tetranitromethane was used to selectively modify tyrosine residues of a humanized anti-cocaine mAb (h2E2), under development for the treatment of cocaine use disorders. The effect of mild tyrosine nitration on the affinity of cocaine and two high affinity cocaine metabolites, cocaethylene and benzoylecgonine, was assessed using differential scanning fluorimetry to measure ligand affinities via ligand-induced thermal stabilization of the mAb antigen binding region. Nitrated tyrosine residues were identified by mass spectral analysis of thermolysin peptides. One objective was to understand the binding affinity differences observed for these three ligands, which are not explained by the published crystal structure of the h2E2 mAb Fab fragment co-crystalized with benzoylecgonine, since the carboxylic acid of benzoylecgonine that is esterified to form cocaine and cocaethylene is not in contact with the mAb. Importantly, the binding affinity of the cocaine metabolite benzoylecgonine was not decreased by mild nitration, whereas the binding affinities of cocaine and cocaethylene were decreased about two-fold. These ligands differ only in the substituent attached to the carboxylate moiety of the compound, with benzoylecgonine having an unesterified carboxylate, and cocaine and cocaethylene having methyl and ethyl esters, respectively, at this position. The results are consistent with nitration of light chain tyrosine residue 34, resulting in a less favorable interaction with cocaine and cocaethylene carboxylate esters, while not affecting binding of benzoylecgonine. Thus, light chain Tyr34 residue may have molecular interactions with cocaine and cocaethylene not present for benzoylecgonine, leading to the observed affinity differences for these three ligands.

Blocking UBE2N abrogates oncogenic immune signaling in acute myeloid leukemia.

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.

Cutting Edge: l-Arginine Transfer from Antigen-Presenting Cells Sustains CD4+ T Cell Viability and Proliferation.

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.

Oxidation of specific tryptophan residues inhibits high-affinity binding of cocaine and its metabolites to a humanized anticocaine mAb.

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.

TRAF6 functions as a tumor suppressor in myeloid malignancies by directly targeting MYC oncogenic activity.

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.

FBXO11 is a candidate tumor suppressor in the leukemic transformation of myelodysplastic syndrome.

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.

Publisher Correction: Recognition of RNA N6-methyladenosine by IGF2BP proteins enhances mRNA stability and translation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Phosphoproteomic analysis identifies phospho-Threonine-17 site of phospholamban important in low molecular weight isoform of fibroblast growth factor 2-induced protection against post-ischemic cardiac dysfunction.

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 Selected 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 < 0.05), and this functional improvement was significantly abrogated in hearts expressing LMW FGF2 and Thr17Ala PLB (p < 0.05). CONCLUSION: The findings indicate that LMW FGF2 modulates intracellular calcium handling/cycling via regulatory changes in SR proteins essential for recovery from I/R injury, and thereby protects the heart from post-ischemic cardiac dysfunction.

Biotherapy of Brain Tumors with Phosphatidylserine-Targeted Radioiodinated SapC-DOPS Nanovesicles.

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-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.

Mouse models of neutropenia reveal progenitor-stage-specific defects.

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.

SWATH-Proteomics of Ibrutinib's Action in Myeloid Leukemia Initiating Mutated G-CSFR Signaling.

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 show the efficacy of ibrutinib (a Bruton's tyrosine kinase inhibitor) in mutated G-CSFR leukemia models but do not address broader signaling mechanisms. EXPERIMENTAL DESIGN: A label-free quantitative proteomics workflow to evaluate the cellular effects of ibrutinib treatment is established. This includes three 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 shows about 1000 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 is very robust with coefficients of variation for quantitation at 13.0-20.4% resulting in dramatic patterns of protein differences among the groups. CONCLUSIONS AND CLINICAL RELEVANCE: This robust dataset 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.

Ablation of miR-144 increases vimentin expression and atherosclerotic plaque formation.

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.

TIFAB Regulates USP15-Mediated p53 Signaling during Stressed and Malignant Hematopoiesis.

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.

Phospho serine and threonine analysis of normal and mutated granulocyte colony stimulating factor receptors.

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.