High Pretreatment DHEA Is Associated with Inferior Immunotherapy Response in Metastatic Non-Small Cell Lung Cancer.

Background: Sex difference in the immune response may influence patients' response to immune checkpoint inhibitors (ICIs). We conducted a prospective observation study to determine the correlation between pretreatment sex hormone levels and response to ICIs in metastatic non-small cell lung cancer (NSCLC). Method: Pretreatment plasma samples from 61 patients with newly diagnosed NSCLC prior to ICI therapy were collected. Six sex hormone levels [pyrazole triol, 17 ß-estradiol, 5-androstenediol, 3ß-androstenediol, dehydroepiandrosterone (DHEA), and S-equol] were measured using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). Overall survival (OS) and progression-free survival (PFS) were compared between the high- and low-level groups in the whole cohort. Result: Among the six sex hormones measured, DHEA levels were significantly higher among patients without clinical benefits in the discovery cohort; the remaining sex hormones did not differ significantly. In the whole cohort, median PFS was 22 months for patients with low DHEA levels vs. 3.8 months for those with high DHEA [hazard ratio, 14.23 (95% CI, 4.7-43); p < 0.001]. A significant association was also observed for OS [hazard ratio, 8.2 (95% CI, 2.89-23.35); p < 0.0001]. Conclusions: High pretreatment plasma DHEA levels were associated with poor clinical outcomes for patients with metastatic NSCLC treated with ICIs.

Modulating the polyamine/hypusine axis controls generation of CD8+ tissue-resident memory T cells.

Glutaminolysis is a hallmark of the activation and metabolic reprogramming of T cells. Isotopic tracer analyses of antigen-activated effector CD8+ T cells revealed that glutamine is the principal carbon source for the biosynthesis of polyamines putrescine, spermidine, and spermine. These metabolites play critical roles in activation-induced T cell proliferation, as well as for the production of hypusine, which is derived from spermidine and is covalently linked to the translation elongation factor eukaryotic translation initiation factor 5A (eIF5A). Here, we demonstrated that the glutamine/polyamine/hypusine axis controlled the expression of CD69, an important regulator of tissue-resident memory T cells (Trm). Inhibition of this circuit augmented the development of Trm cells ex vivo and in vivo in the BM, a well-established niche for Trm cells. Furthermore, blocking the polyamine/hypusine axis augmented CD69 expression as well as IFN-γ and TNF-α production in (a) human CD8+ T cells from peripheral blood and sarcoma tumor infiltrating lymphocytes and (b) human CD8+ CAR-T cells. Collectively, these findings support the notion that the polyamine-hypusine circuit can be exploited to modulate Trm cells for therapeutic benefit.

Associations of gut microbiome with endogenous estrogen levels in healthy postmenopausal women.

PURPOSE: The gut microbiome is a potentially important contributor to endogenous estrogen levels after menopause. In healthy postmenopausal women, we examined associations of fecal microbiome composition with levels of urinary estrogens, their metabolites, and relevant metabolic pathway ratios implicated in breast cancer risk. METHODS: Eligible postmenopausal women (n = 164) had a body mass index (BMI) ≤ 35 kg/m2 and no history of hormone use (previous 6 months) or cancer/metabolic disorders. Estrogens were quantified in spot urine samples with liquid chromatography-high resolution mass spectrometry (corrected for creatinine). Bacterial DNA was isolated from fecal samples and the V1-V2 hypervariable regions of 16S rRNA were sequenced on the Illumina MiSeq platform. We examined associations of gut microbiome's indices of within-sample (alpha) diversity (i.e., Shannon, Chao1, and Inverse Simpson), phylogenetic diversity, and the ratio of the two main phyla (Firmicutes and Bacteroidetes; F/B ratio) with individual estrogens and metabolic ratios, adjusted for age and BMI. RESULTS: In this sample of 164 healthy postmenopausal women, the mean age was 62.9 years (range 47.0-86.0). We found significant inverse associations of observed species with 4-pathway:total estrogens (p = 0.04) and 4-pathway:2-pathway (p = 0.01). Shannon index was positively associated with 2-catechols: methylated 2-catechols (p = 0.04). Chao1 was inversely associated with E1:total estrogens (p = 0.04), and 4-pathway:2-pathway (p = 0.02) and positively associated with 2-pathway:parent estrogens (p = 0.01). Phylogenetic diversity was inversely associated with 4-pathway:total estrogens (p = 0.02), 4-pathway:parent estrogens (p = 0.03), 4-pathway:2-pathway (p = 0.01), and 4-pathway:16-pathway (p = 0.03) and positively associated with 2-pathway:parent estrogens (p = 0.01). F/B ratio was not associated with any of the estrogen measures. CONCLUSION: Microbial diversity was associated with several estrogen metabolism ratios implicated in breast cancer risk. Further studies are warranted to confirm these findings in a larger and more representative sample of postmenopausal women, particularly with enrichment of minority participants.

Dose-escalation trial of combination dabrafenib, trametinib, and AT13387 in patients with BRAF-mutant solid tumors.

BACKGROUND: Combination BRAF and MEK inhibitor therapy is an active regimen in patients who have BRAF V600E-mutated tumors; however, the clinical efficacy of this therapy is limited by resistance. Preclinically, the addition of heat shock protein 90 (HSP90) inhibition improves the efficacy of BRAF inhibitor therapy in both BRAF inhibitor-sensitive and BRAF inhibitor-resistant mutant cell lines. METHODS: Cancer Therapy Evaluation Program study 9557 (ClinicalTrials.gov identifier NCT02097225) is a phase 1 study that was designed to assess the safety and efficacy of the small-molecule HSP90 inhibitor, AT13387, in combination with dabrafenib and trametinib in BRAF V600E/K-mutant solid tumors. Correlative analyses evaluated the expression of HSP90 client proteins and chaperones. RESULTS: Twenty-two patients with metastatic, BRAF V600E-mutant solid tumors were enrolled using a 3 + 3 design at four dose levels, and 21 patients were evaluable for efficacy assessment. The most common tumor type was colorectal cancer (N = 12). Dose-limiting toxicities occurred in one patient at dose level 3 and in one patient at dose level 4; specifically, myelosuppression and fatigue, respectively. The maximum tolerated dose was oral dabafenib 150 mg twice daily, oral trametinib 2 mg once daily, and intravenous AT13387 260 mg/m2 on days 1, 8, and 15. The best response was a partial response in two patients and stable disease in eight patients, with an overall response rate of 9.5% (90% exact confidence interval [CI], 2%-27%), a disease control rate of 47.6% (90% CI, 29%-67%), and a median overall survival of 5.1 months (90% CI, 3.4-7.6 months). There were no consistent proteomic changes associated with response or resistance, although responders did have reductions in BRAF expression, and epidermal growth factor receptor downregulation using HSP90 inhibition was observed in one patient who had colorectal cancer. CONCLUSIONS: HSP90 inhibition combined with BRAF/MEK inhibition was safe and produced evidence of modest disease control in a heavily pretreated population. Additional translational work may identify tumor types and resistance mechanisms that are most sensitive to this approach.

Fucosylation of HLA-DRB1 regulates CD4+ T cell-mediated anti-melanoma immunity and enhances immunotherapy efficacy.

Immunotherapy efficacy is limited in melanoma, and combinations of immunotherapies with other modalities have yielded limited improvements but also adverse events requiring cessation of treatment. In addition to ineffective patient stratification, efficacy is impaired by paucity of intratumoral immune cells (itICs); thus, effective strategies to safely increase itICs are needed. We report that dietary administration of L-fucose induces fucosylation and cell surface enrichment of the major histocompatibility complex (MHC)-II protein HLA-DRB1 in melanoma cells, triggering CD4+ T cell-mediated increases in itICs and anti-tumor immunity, enhancing immune checkpoint blockade responses. Melanoma fucosylation and fucosylated HLA-DRB1 associate with intratumoral T cell abundance and anti-programmed cell death protein 1 (PD1) responder status in patient melanoma specimens, suggesting the potential use of melanoma fucosylation as a strategy for stratifying patients for immunotherapies. Our findings demonstrate that fucosylation is a key mediator of anti-tumor immunity and, importantly, suggest that L-fucose is a powerful agent for safely increasing itICs and immunotherapy efficacy in melanoma.

Branched-chain keto acids promote an immune-suppressive and neurodegenerative microenvironment in leptomeningeal disease.

Leptomeningeal disease (LMD) occurs when tumors seed into the leptomeningeal space and cerebrospinal fluid (CSF), leading to severe neurological deterioration and poor survival outcomes. We utilized comprehensive multi-omics analyses of CSF from patients with lymphoma LMD to demonstrate an immunosuppressive cellular microenvironment and identified dysregulations in proteins and lipids indicating neurodegenerative processes. Strikingly, we found a significant accumulation of toxic branched-chain keto acids (BCKA) in the CSF of patients with LMD. The BCKA accumulation was found to be a pan-cancer occurrence, evident in lymphoma, breast cancer, and melanoma LMD patients. Functionally, BCKA disrupted the viability and function of endogenous T lymphocytes, chimeric antigen receptor (CAR) T cells, neurons, and meningeal cells. Treatment of LMD mice with BCKA-reducing sodium phenylbutyrate significantly improved neurological function, survival outcomes, and efficacy of anti-CD19 CAR T cell therapy. This is the first report of BCKA accumulation in LMD and provides preclinical evidence that targeting these toxic metabolites improves outcomes.

Inhibition of Epidermal Growth Factor Receptor Signaling by Antisense Oligonucleotides as a Novel Approach to Epidermal Growth Factor Receptor Inhibition.

We report a novel method to inhibit epidermal growth factor receptor (EGFR) signaling using custom morpholino antisense oligonucleotides (ASOs) to drive expression of dominant negative mRNA isoforms of EGFR by ASO-induced exon skipping within the transmembrane (16) or tyrosine kinase domains (18 and 21). In vivo ASO formulations induced >95% exon skipping in several models of nonsmall cell lung cancer (NSCLC) and were comparable in efficacy to erlotinib in reducing colony formation, cell viability, and migration in EGFR mutant NSCLC (PC9). However, unlike erlotinib, ASOs maintained their efficacy in both erlotinib-resistant subclones (PC9-GR) and wild-type overexpressing EGFR models (H292), in which erlotinib had no significant effect. The most dramatic ASO-induced phenotype resulted from targeting the EGFR kinase domain directly, which resulted in maximal inhibition of phosphorylation of EGFR, Akt, and Erk in both PC9 and PC9GR cells. Phosphoproteomic mass spectrometry confirmed highly congruent impacts of exon 16-, 18-, and 21-directed ASOs compared with erlotinib on PC9 genome-wide cell signaling. Furthermore, EGFR-directed ASOs had no impact in EGFR-independent NSCLC models, confirming an EGFR-specific therapeutic mechanism. Further exploration of synergy of ASOs with existing tyrosine kinase inhibitors may offer novel clinical models to improve EGFR-targeted therapies for both mutant and wild-type NSCLC patients.

Macrophages use apoptotic cell-derived methionine and DNMT3A during efferocytosis to promote tissue resolution.

Efferocytosis, the clearance of apoptotic cells (ACs) by macrophages, is critical for tissue resolution, with defects driving many diseases. Mechanisms of efferocytosis-mediated resolution are incompletely understood. Here, we show that AC-derived methionine regulates resolution through epigenetic repression of the extracellular signal-regulated kinase 1/2 (ERK1/2) phosphatase Dusp4. We focus on two key efferocytosis-induced pro-resolving mediators, prostaglandin E2 (PGE2) and transforming growth factor beta 1 (TGF-ß1), and show that efferocytosis induces prostaglandin-endoperoxide synthase 2/cyclooxygenase 2 (Ptgs2/COX2), leading to PGE2 synthesis and PGE2-mediated induction of TGF-ß1. ERK1/2 phosphorylation/activation by AC-activated CD36 is necessary for Ptgs2 induction, but this is insufficient owing to an ERK-DUSP4 negative feedback pathway that lowers phospho-ERK. However, subsequent AC engulfment and phagolysosomal degradation lead to Dusp4 repression, enabling enhanced p-ERK and induction of the Ptgs2-PGE2-TGF-ß1 pathway. Mechanistically, AC-derived methionine is converted to S-adenosylmethionine, which is used by DNA methyltransferase-3A (DNMT3A) to methylate Dusp4. Bone-marrow DNMT3A deletion in mice blocks COX2/PGE2, TGF-ß1, and resolution in sterile peritonitis, apoptosis-induced thymus injury and atherosclerosis. Knowledge of how macrophages use AC-cargo and epigenetics to induce resolution provides mechanistic insight and therapeutic options for diseases driven by impaired resolution.

Associations of established breast cancer risk factors with urinary estrogens in postmenopausal women.

PURPOSE: Circulating estrogens are an established risk factor for postmenopausal breast cancer (BCa). We describe the distribution of urinary estrogens, their metabolites, and relevant metabolic pathway ratios among healthy postmenopausal women and examine associations of several known BCa factors with these estrogen measures. METHODS: Eligible postmenopausal women (n = 167) had no history of hormone use (previous 6 months) and cancer/metabolic disorders and had a body mass index (BMI) ≤ 35 kg/m2. Estrogens were quantified in spot urine samples with liquid chromatography-high-resolution mass spectrometry and corrected for creatinine. We assessed overall distributions of estrogens and associations of age, BMI, race/ethnicity, parity/age at first birth, age at menarche, alcohol, and smoking with log-transformed estrogen measures using multivariate regression. RESULTS: BMI was positively associated with estrone (ß per unit = 0.04, 95% Confidence Interval [CI] 0.00; 0.07), combined parent estrogens (ß = 0.04, 95% CI 0.01; 0.07), and E2:total estrogens (ß = 0.04, 95% CI 0.02; 0.06), and inversely associated with 4-MeOE1 (ß = - 0.17, 95% CI - 0.33; - 0.02), E3:parent estrogens (ß = - 0.04, 95% CI - 0.07; - 0.00), and 16-pathway:parent (ß = - 0.04, 95% CI - 0.07; - 0.01). Being African American vs. white was associated with higher levels of 4-MeOE1 (ß = 3.41, 95% CI 0.74; 6.08), 17-epiE3 (ß = 1.19, 95% CI 0.07; 2.31), 2-pathway:parent (ß = 0.54, 95% CI 0.04; 1.04), and lower levels of E2:total estrogens (ß = - 0.48, 95% CI - 0.83; - 0.13). Having < 7 alcohol drinks/week vs. none was associated with higher levels of 16-ketoE2 (ß = 1.32, 95% CI 0.36; 2.27), 16-epiE3 (ß = 1.02, 95% CI 0.24; 1.79), and 17-epiE3 (ß = 0.55, 95% CI 0.02; 1.08). Smoking was positively associated with E3:parent (ß = 0.29, 95% CI 0.01; 0.57), 16-pathway:parent (ß = 0.25, 95% CI 0.01; 0.49), and inversely associated with estradiol (ß = - 0.52, 95% CI - 0.93; - 0.10). As compared to nulliparous, parous women with age at first birth ≥ 25 years had lower levels of estrone, combined parent estrogens, 2-OHE1, and 2-OHE2. CONCLUSION: Our findings suggest that BMI, race/ethnicity, and some reproductive and lifestyle factors may contribute to postmenopausal BCa through their effects on circulating estrogens.

Fucosylated Proteome Profiling Identifies a Fucosylated, Non-Ribosomal, Stress-Responsive Species of Ribosomal Protein S3.

Alterations in genes encoding for proteins that control fucosylation are known to play causative roles in several developmental disorders, such as Dowling-Degos disease 2 and congenital disorder of glycosylation type IIc (CDGIIc). Recent studies have provided evidence that changes in fucosylation can contribute to the development and progression of several different types of cancers. It is therefore important to gain a detailed understanding of how fucosylation is altered in disease states so that interventions may be developed for therapeutic purposes. In this report, we find that fucosylation occurs on many intracellular proteins. This is an interesting finding, as the fucosylation machinery is restricted to the secretory pathway and is thought to predominately affect cell-membrane-bound and secreted proteins. We find that Ribosomal protein S3 (RPS3) is fucosylated in normal tissues and in cancer cells, and that the extent of its fucosylation appears to respond to stress, including MAPK inhibitors, suggesting a new role in posttranslational protein function. Our data identify a new ribosome-independent species of fucosylated RPS3 that interacts with proteins involved in posttranscriptional regulation of RNA, such as Heterogeneous nuclear ribonucleoprotein U (HNRNPU), as well as with a predominance of non-coding RNAs. These data highlight a novel role for RPS3, which, given previously reported oncogenic roles for RPS3, might represent functions that are perturbed in pathologies such as cancer. Together, our findings suggest a previously unrecognized role for fucosylation in directly influencing intracellular protein functions.

Cell Type-specific Adaptive Signaling Responses to KRASG12C Inhibition.

PURPOSE: Covalent inhibitors of KRASG12C specifically target tumors driven by this form of mutant KRAS, yet early studies show that bypass signaling drives adaptive resistance. Although several combination strategies have been shown to improve efficacy of KRASG12C inhibitors (KRASi), underlying mechanisms and predictive strategies for patient enrichment are less clear. EXPERIMENTAL DESIGN: We performed mass spectrometry-based phosphoproteomics analysis in KRASG12C cell lines after short-term treatment with ARS-1620. To understand signaling diversity and cell type-specific markers, we compared proteome and phosphoproteomes of KRASG12C cells. Gene expression patterns of KRASG12C cell lines and lung tumor tissues were examined. RESULTS: Our analysis suggests cell type-specific perturbation to ERBB2/3 signaling compensates for repressed ERK and AKT signaling following ARS-1620 treatment in epithelial cell type, and this subtype was also more responsive to coinhibition of SHP2 and SOS1. Conversely, both high basal and feedback activation of FGFR or AXL signaling were identified in mesenchymal cells. Inhibition of FGFR signaling suppressed feedback activation of ERK and mTOR, while AXL inhibition suppressed PI3K pathway. In both cell lines and human lung cancer tissues with KRASG12C, we observed high basal ERBB2/3 associated with epithelial gene signatures, while higher basal FGFR1 and AXL were observed in cells/tumors with mesenchymal gene signatures. CONCLUSIONS: Our phosphoproteomic study identified cell type-adaptive responses to KRASi. Markers and targets associated with ERBB2/3 signaling in epithelial subtype and with FGFR1/AXL signaling in mesenchymal subtype should be considered in patient enrichment schemes with KRASi.

Plasma complement activation mechanisms differ in ornate (Terrapene ornata ornata) and eastern box turtles (Terrapene carolina carolina).

Eastern (Terrapene carolina carolina) and ornate (Terrapene ornata ornata) box turtles have robust plasma antibacterial activity, however, the mechanism behind this activity is unknown. We used sheep red blood cell (SRBC) hemolysis assays, mannan-affinity chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) to explore the mechanisms of complement activity in box turtles. Plasma from both species demonstrated volume, time, and temperature-dependent SRBC hemolysis, with significantly greater hemolytic activity in ornate box turtle plasma. Hemolytic activity was highly attenuated following treatment with heat, EDTA, and salicylaldoxime in both species, but was unchanged after treatment with methylamine and ammonium hydroxide. Two abundant mannan-binding proteins (presumed C-type lectins) were identified in eastern box turtle plasma using SDS-PAGE and MALDI-TOF, but ornate box turtles did not express either protein. Eastern box turtles appear to rely on the lectin pathway of complement activation while ornate box turtles utilize the alternative pathway. This study provides further evidence that mechanisms underlying immune function are not always conserved between closely related species. This finding may have important implications for explaining species differences in susceptibility to emerging threats such as disease, toxicants, and climate change.

LC-HRMS of derivatized urinary estrogens and estrogen metabolites in postmenopausal women.

In order to undertake an epidemiologic study relating levels of parent estrogens (estrone and estradiol) and estrogen metabolites (EMs) to other breast cancer risk factors, we have optimized methods for EM quantification with ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). A two-step approach was adopted; the first step comprised method development and evaluation of the method performance. The second step consisted of applying this method to quantify estrogens in postmenopausal women and determine if the observed patterns are consistent with the existing literature and prior knowledge of estrogen metabolism. First, 1-methylimidazole-2-sulfonyl chloride (MIS) was used to derivatize endogenous estrogens and estrogen metabolites in urine from study participants. Since C18 reversed phase columns have not been able to separate all the structurally related EMs, we used a C18-pentafluorophenyl (PFP) column. The parent estrogens and EMs were baseline resolved with distinct retention times on this C18-PFP column using a 30 min gradient. This method was used to quantify the parent estrogens and 13 EMs in urine samples collected in an initial pilot study involving males as well as pre- and peri-menopausal females to assess a range of EM levels in urine samples and enable comparison to the previous literature for assay evaluation. Detection limits ranged from 1 - 20 pg/mL depending on the EM. We evaluated matrix effects and interference as well as the intra- and inter-batch reproducibility including hydrolysis, extraction, derivatization and LC-MS analysis using charcoal-stripped human urine as a matrix. Methods were then applied to the measurement of estrogens in urine samples from 169 postmenopausal women enrolled in an epidemiological study to examine relationships between breast cancer risk, the intestinal microbiome, and urinary EMs. The results from our cohort are comparable to previous reports on urinary EMs in postmenopausal women and enabled thorough evaluation of the method.

Cereblon harnesses Myc-dependent bioenergetics and activity of CD8+ T lymphocytes.

Immunomodulatory drugs, such as thalidomide and related compounds, potentiate T-cell effector functions. Cereblon (CRBN), a substrate receptor of the DDB1-cullin-RING E3 ubiquitin ligase complex, is the only molecular target for this drug class, where drug-induced, ubiquitin-dependent degradation of known "neosubstrates," such as IKAROS, AIOLOS, and CK1α, accounts for their biological activity. Far less clear is whether these CRBN E3 ligase-modulating compounds disrupt the endogenous functions of CRBN. We report that CRBN functions in a feedback loop that harnesses antigen-specific CD8+ T-cell effector responses. Specifically, Crbn deficiency in murine CD8+ T cells augments their central metabolism manifested as elevated bioenergetics, with supraphysiological levels of polyamines, secondary to enhanced glucose and amino acid transport, and with increased expression of metabolic enzymes, including the polyamine biosynthetic enzyme ornithine decarboxylase. Treatment with CRBN-modulating compounds similarly augments central metabolism of human CD8+ T cells. Notably, the metabolic control of CD8+ T cells by modulating compounds or Crbn deficiency is linked to increased and sustained expression of the master metabolic regulator MYC. Finally, Crbn-deficient T cells have augmented antigen-specific cytolytic activity vs melanoma tumor cells, ex vivo and in vivo, and drive accelerated and highly aggressive graft-versus-host disease. Therefore, CRBN functions to harness the activation of CD8+ T cells, and this phenotype can be exploited by treatment with drugs.

Macrophage Metabolism of Apoptotic Cell-Derived Arginine Promotes Continual Efferocytosis and Resolution of Injury.

Continual efferocytic clearance of apoptotic cells (ACs) by macrophages prevents necrosis and promotes injury resolution. How continual efferocytosis is promoted is not clear. Here, we show that the process is optimized by linking the metabolism of engulfed cargo from initial efferocytic events to subsequent rounds. We found that continual efferocytosis is enhanced by the metabolism of AC-derived arginine and ornithine to putrescine by macrophage arginase 1 (Arg1) and ornithine decarboxylase (ODC). Putrescine augments HuR-mediated stabilization of the mRNA encoding the GTP-exchange factor Dbl, which activates actin-regulating Rac1 to facilitate subsequent rounds of AC internalization. Inhibition of any step along this pathway after first-AC uptake suppresses second-AC internalization, whereas putrescine addition rescues this defect. Mice lacking myeloid Arg1 or ODC have defects in efferocytosis in vivo and in atherosclerosis regression, while treatment with putrescine promotes atherosclerosis resolution. Thus, macrophage metabolism of AC-derived metabolites allows for optimal continual efferocytosis and resolution of injury.

HDAC11 deficiency disrupts oncogene-induced hematopoiesis in myeloproliferative neoplasms.

Protein acetylation is an important contributor to cancer initiation. Histone deacetylase 6 (HDAC6) controls JAK2 translation and protein stability and has been implicated in JAK2-driven diseases best exemplified by myeloproliferative neoplasms (MPNs). By using novel classes of highly selective HDAC inhibitors and genetically deficient mouse models, we discovered that HDAC11 rather than HDAC6 is necessary for the proliferation and survival of oncogenic JAK2-driven MPN cells and patient samples. Notably, HDAC11 is variably expressed in primitive stem cells and is expressed largely upon lineage commitment. Although Hdac11is dispensable for normal homeostatic hematopoietic stem and progenitor cell differentiation based on chimeric bone marrow reconstitution, Hdac11 deficiency significantly reduced the abnormal megakaryocyte population, improved splenic architecture, reduced fibrosis, and increased survival in the MPLW515L-MPN mouse model during primary and secondary transplantation. Therefore, inhibitors of HDAC11 are an attractive therapy for treating patients with MPN. Although JAK2 inhibitor therapy provides substantial clinical benefit in MPN patients, the identification of alternative therapeutic targets is needed to reverse MPN pathogenesis and control malignant hematopoiesis. This study establishes HDAC11 as a unique type of target molecule that has therapeutic potential in MPN.

Correction: Different mechanisms of serum complement activation in the plasma of common (Chelydra serpentina) and alligator (Macrochelys temminckii) snapping turtles.

[This corrects the article DOI: 10.1371/journal.pone.0217626.].

An immunoproteomic approach to characterize the CAR interactome and signalosome.

Adoptive transfer of T cells that express a chimeric antigen receptor (CAR) is an approved immunotherapy that may be curative for some hematological cancers. To better understand the therapeutic mechanism of action, we systematically analyzed CAR signaling in human primary T cells by mass spectrometry. When we compared the interactomes and the signaling pathways activated by distinct CAR-T cells that shared the same antigen-binding domain but differed in their intracellular domains and their in vivo antitumor efficacy, we found that only second-generation CARs induced the expression of a constitutively phosphorylated form of CD3ζ that resembled the endogenous species. This phenomenon was independent of the choice of costimulatory domains, or the hinge/transmembrane region. Rather, it was dependent on the size of the intracellular domains. Moreover, the second-generation design was also associated with stronger phosphorylation of downstream secondary messengers, as evidenced by global phosphoproteome analysis. These results suggest that second-generation CARs can activate additional sources of CD3ζ signaling, and this may contribute to more intense signaling and superior antitumor efficacy that they display compared to third-generation CARs. Moreover, our results provide a deeper understanding of how CARs interact physically and/or functionally with endogenous T cell molecules, which will inform the development of novel optimized immune receptors.

Label-free quantitative mass spectrometry analysis of differential protein expression in the developing cochlear sensory epithelium.

BACKGROUND: The sensory epithelium of the inner ear converts the mechanical energy of sound to electro-chemical energy recognized by the central nervous system. This process is mediated by receptor cells known as hair cells that express proteins in a timely fashion with the onset of hearing. METHODS: The proteomes of 3, 14, and 30 day-old mice cochlear sensory epithelia were revealed, using label-free quantitative mass spectrometry (LTQ-Orbitrap). Statistical analysis using a one-way ANOVA followed by Bonferroni's post-hoc test was used to show significant differences in protein expression. Ingenuity Pathway Analysis was used to observe networks of differentially expressed proteins, their biological processes, and associated diseases, while Cytoscape software was used to determine putative interactions with select biomarker proteins. These candidate biomarkers were further verified using Western blotting, while coimmunoprecipitation was used to verify putative partners determined using bioinformatics. RESULTS: We show that a comparison across all three proteomes shows that there are 447 differentially expressed proteins, with 387 differentially expressed between postnatal day 3 and 30. Ingenuity Pathway Analysis revealed ~ 62% of postnatal day 3 downregulated proteins are involved in neurological diseases. Several proteins are expressed exclusively on P3, including Parvin α, Drebrin1 (Drb1), Secreted protein acidic and cysteine rich (SPARC), Transmembrane emp24 domain-containing protein 10 (Tmed10). Coimmunoprecipitations showed that Parvin and SPARC interact with integrin-linked protein kinase and the large conductance calcium-activated potassium channel, respectively. CONCLUSIONS: Quantitative mass spectrometry revealed the identification of numerous differentially regulated proteins over three days of postnatal development. These data provide insights into functional pathways regulating normal sensory and supporting cell development in the cochlea that include potential biomarkers. Interacting partners of two of these markers suggest the importance of these complexes in regulating cellular structure and synapse development.

Combined BRAF and HSP90 Inhibition in Patients with Unresectable BRAF V600E-Mutant Melanoma.

Purpose: BRAF inhibitors are clinically active in patients with advanced BRAFV600-mutant melanoma, although acquired resistance remains common. Preclinical studies demonstrated that resistance could be overcome using concurrent treatment with the HSP90 inhibitor XL888.Patients and Methods: Vemurafenib (960 mg p.o. b.i.d.) combined with escalating doses of XL888 (30, 45, 90, or 135 mg p.o. twice weekly) was investigated in 21 patients with advanced BRAFV600-mutant melanoma. Primary endpoints were safety and determination of a maximum tolerated dose. Correlative proteomic studies were performed to confirm HSP inhibitor activity.Results: Objective responses were observed in 15 of 20 evaluable patients [75%; 95% confidence interval (CI), 51%-91%], with 3 complete and 12 partial responses. Median progression-free survival and overall survival were 9.2 months (95% CI, 3.8-not reached) and 34.6 months (6.2-not reached), respectively. The most common grade 3/4 toxicities were skin toxicities, such as rash (n = 4, 19%) and cutaneous squamous cell carcinomas (n = 3, 14%), along with diarrhea (n = 3, 14%). Pharmacodynamic analysis of patients' peripheral blood mononuclear cells (PBMC) showed increased day 8 HSP70 expression compared with baseline in the three cohorts with XL888 doses ≥45 mg. Diverse effects of vemurafenib-XL888 upon intratumoral HSP client protein expression were noted, with the expression of multiple proteins (including ERBB3 and BAD) modulated on therapy.Conclusions: XL888 in combination with vemurafenib has clinical activity in patients with advanced BRAFV600-mutant melanoma, with a tolerable side-effect profile. HSP90 inhibitors warrant further evaluation in combination with current standard-of-care BRAF plus MEK inhibitors in BRAFV600-mutant melanoma. Clin Cancer Res; 24(22); 5516-24. ©2018 AACR See related commentary by Sullivan, p. 5496.