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Belatacept confers increased patient and graft survival in renal transplant recipients relative to calcineurin inhibitors, but is associated with an increased rate of acute rejection. Recent immunophenotypic studies comparing pre-transplant T cell phenotypes of patients who reject vs. those that remain stable on belatacept identified three potential “risky” memory T cell subsets that potentially underlie belatacept-resistant rejection: CD4(+) CD28(+) T(EM), CD8(+) CD28(null) and CD4(+) CD57(+) PD1(−) subsets. Here, we compared key phenotypic and functional aspects of these human memory T cell subsets, with the goal of identifying additional potential targets to modulate them. Results demonstrate that TIGIT, an increasingly well-appreciated immune checkpoint receptor, was expressed on all three risky memory T cell subsets in vitro and in vivo in the presence of belatacept. Co-culture of human memory CD4(+) and CD8(+) T cells with an agonistic anti-TIGIT mAb significantly increased apoptotic cell death of all three risky memory T cell subsets. Mechanistically, TIGIT-mediated apoptosis of risky memory T cells was dependent on FOXP3(+) Treg, suggesting that agonism of the TIGIT pathway increases FOXP3(+) Treg suppression of human memory T cell populations. Overall, these data suggest that TIGIT agonism could represent a new therapeutic target to inhibit belatacept-resistant rejection during transplantation.

Casitas B-lineage lymphoma (CBL) encodes an E3 ubiquitin ligase and signaling adaptor that regulates receptor and nonreceptor tyrosine kinases. Recurrent CBL mutations occur in myeloid neoplasms, including 10% to 20% of chronic myelomonocytic leukemia (CMML) cases, and selectively disrupt the protein’s E3 ubiquitin ligase activity. CBL mutations have been associated with poor prognosis, but the oncogenic mechanisms and therapeutic implications of CBL mutations remain incompletely understood. We combined functional assays and global mass spectrometry to define the phosphoproteome, CBL interactome, and mechanism of signaling activation in a panel of cell lines expressing an allelic series of CBL mutations. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced CBL phosphorylation, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) recruitment, and downstream phosphatidylinositol 3-kinase (PI3K)/AKT signaling in CBL-mutant cells. Signaling adaptor domains of CBL, including the tyrosine kinase–binding domain, proline-rich region, and C-terminal phosphotyrosine sites, were all required for the oncogenic function of CBL mutants. Genetic ablation or dasatinib-mediated inhibition of LYN reduced CBL phosphorylation, CBL-PIK3R1 interaction, and PI3K/AKT signaling. Furthermore, we demonstrated in vitro and in vivo antiproliferative efficacy of dasatinib in CBL-mutant cell lines and primary CMML. Overall, these mechanistic insights into the molecular function of CBL mutations provide rationale to explore the therapeutic potential of LYN inhibition in CBL-mutant myeloid malignancies.

One of the biggest barriers to achieving allograft tolerance is the presence of immunological memory within the recipient, which confers a faster, more robust immune response that is in most cases more resistant to pharmacologic immunosuppression. This review will identify the mechanisms by which alloreactive T cells arise within hosts prior to transplantation, and explore the properties of immunological memory that contribute to allograft rejection. In doing so we will also illuminate how targeting pathways that induce memory T cell exhaustion can promote allograft tolerance. Recent studies demonstrating the impact of the allograft microenvironment on memory cell survival and activation, as well as new therapeutic strategies that are being explored to mitigate memory driven allograft rejection, will also be reviewed.

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry-based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9-driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.

The cohesin complex plays an essential role in chromosome maintenance and transcriptional regulation. Recurrent somatic mutations in the cohesin complex are frequent genetic drivers in cancer, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Here, using genetic dependency screens of stromal antigen 2–mutant (STAG2-mutant) AML, we identified DNA damage repair and replication as genetic dependencies in cohesin-mutant cells. We demonstrated increased levels of DNA damage and sensitivity of cohesin-mutant cells to poly(ADP-ribose) polymerase (PARP) inhibition. We developed a mouse model of MDS in which Stag2 mutations arose as clonal secondary lesions in the background of clonal hematopoiesis driven by tet methylcytosine dioxygenase 2 (Tet2) mutations and demonstrated selective depletion of cohesin-mutant cells with PARP inhibition in vivo. Finally, we demonstrated a shift from STAG2- to STAG1-containing cohesin complexes in cohesin-mutant cells, which was associated with longer DNA loop extrusion, more intermixing of chromatin compartments, and increased interaction with PARP and replication protein A complex. Our findings inform the biology and therapeutic opportunities for cohesin-mutant malignancies., We developed models of cohesin-mutant myelodysplastic syndromes and acute myeloid leukemia and demonstrated a shift from STAG2- to STAG1-cohesin complexes, increased DNA damage, and sensitivity to PARP inhibition.

CBL encodes an E3 ubiquitin ligase and signaling adaptor that acts downstream of cytokine receptors. Recurrent CBL mutations occur in myeloid malignancies, but the mechanism by which these mutations drive oncogenesis remains incompletely understood. Here we performed a series of studies to define the phosphoproteome, CBL interactome and molecular mechanisms of signaling activation in cells expressing an allelic series of CBL mutants. Our analyses revealed that increased LYN activation and interaction with mutant CBL are key drivers of enhanced PIK3R1 recruitment and downstream PI3K/AKT signaling in CBL-mutant cells. Furthermore, we demonstrated in vitro and in vivo efficacy of LYN inhibition by dasatinib in CBL-mutant cell lines and primary chronic myelomonocytic leukemia cells. Overall, our data provide rationale for exploring the therapeutic potential of LYN inhibition in patients with CBL-mutated myeloid malignancies.Statement of SignificanceWe investigated the oncogenic mechanisms of myeloid malignancy-associated CBL mutations by mass spectrometry-based proteomics and interactomics. Our findings indicate that increased LYN kinase activity in CBL-mutant cells stimulates PI3K/AKT signaling, revealing opportunities for the use of targeted inhibitors in CBL-mutated myeloid malignancies.

Summary Previous work has demonstrated that Th17 memory cells but not Th1 cells are resistant to CD28/CTLA-4 blockade with CTLA-4 Ig, leading us to investigate the individual roles of the CD28 and CTLA-4 cosignaling pathways on Th1 versus Th17 cells. We found that selective CD28 blockade with a domain antibody (dAb) inhibited Th1 cells but surprisingly augmented Th17 responses. CD28 agonism resulted in a profound increase in CTLA-4 expression in Th17 cells as compared with Th1 cells. Consistent with these findings, inhibition of the CD28 signaling protein AKT revealed that CTLA-4 expression on Th17 cells was more significantly reduced by AKT inhibition relative to CTLA-4 expression on Th17 cells. Finally, we found that FOXO1 and FOXO3 overexpression restrained high expression of CTLA-4 on Th17 cells but not Th1 cells. This study demonstrates that the heterogeneity of the CD4+ T cell compartment has implications for the immunomodulation of pathologic T cell responses., Graphical Abstract, Highlights • CD28 blockade resulted in augmentation of human Th17 cells relative to Th1 cells • Th17 polarized mice exhibited graft rejection in the presence of CD28 blockade • A significant portion of Th17 cell CTLA-4 expression was induced by CD28 ligation • Overexpression of FOXO1 or FOXO3 inhibited Th17 cell CTLA-4 expression, Molecular Mechanism of Behavior; Immunology; Immune Response

Tumor epitopes – peptides that are presented on surface-bound MHC I proteins - provide targets for cancer immunotherapy and have been identified extensively in the annotated protein-coding regions of the genome. Motivated by the recent discovery of translated novel unannotated open reading frames (nuORFs) using ribosome profiling (Ribo-seq), we hypothesized that cancer-associated processes could generate nuORFs that can serve as a new source of tumor antigens that harbor somatic mutations or show tumor-specific expression. To identify cancer-specific nuORFs, we generated Ribo-seq profiles for 29 malignant and healthy samples, developed a sensitive analytic approach for hierarchical ORF prediction, and constructed a high-confidence database of translated nuORFs across tissues. Peptides from 3,555 unique translated nuORFs were presented on MHC I, based on analysis of an extensive dataset of MHC I-bound peptides detected by mass spectrometry, with >20-fold more nuORF peptides detected in the MHC I immunopeptidomes compared to whole proteomes. We further detected somatic mutations in nuORFs of cancer samples and identified nuORFs with tumor-specific translation in melanoma, chronic lymphocytic leukemia and glioblastoma. NuORFs thus expand the pool of MHC I-presented, tumor-specific peptides, targetable by immunotherapies.

Tumor-associated epitopes presented on MHC-I that can activate the immune system against cancer cells are typically identified from annotated protein-coding regions of the genome, but whether peptides originating from novel or unannotated open reading frames (nuORFs) can contribute to antitumor immune responses remains unclear. Here we show that peptides originating from nuORFs detected by ribosome profiling of malignant and healthy samples can be displayed on MHC-I of cancer cells, acting as additional sources of cancer antigens. We constructed a high-confidence database of translated nuORFs across tissues (nuORFdb) and used it to detect 3,555 translated nuORFs from MHC-I immunopeptidome mass spectrometry analysis, including peptides that result from somatic mutations in nuORFs of cancer samples as well as tumor-specific nuORFs translated in melanoma, chronic lymphocytic leukemia and glioblastoma. NuORFs are an unexplored pool of MHC-I-presented, tumor-specific peptides with potential as immunotherapy targets. New tumor epitopes are discovered by ribosome profiling and immunopeptidome mass spectrometry.

Identification of human leukocyte antigen (HLA)-bound peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS) is poised to provide a deep understanding of rules underlying antigen presentation. However, a key obstacle is the ambiguity that arises from the co-expression of multiple HLA alleles. Here, we have implemented a scalable mono-allelic strategy for profiling the HLA peptidome. By using cell lines expressing a single HLA allele, optimizing immunopurifications, and developing an application-specific spectral search algorithm, we identified thousands of peptides bound to 16 different HLA class I alleles. These data enabled the discovery of subdominant binding motifs and an integrative analysis quantifying the contribution of factors critical to epitope presentation, such as protein cleavage and gene expression. We trained neural-network prediction algorithms with our large dataset (>24,000 peptides) and outperformed algorithms trained on data-sets of peptides with measured affinities. We thus demonstrate a strategy for systematically learning the rules of endogenous antigen presentation.

Therapies currently in preclinical development for prion disease seek to lower prion protein (PrP) expression in the brain. Trials of such therapies are likely to rely on quantification of PrP in cerebrospinal fluid (CSF) as a pharmacodynamic biomarker and possibly as a trial endpoint. Studies using PrP ELISA kits have reproducibly shown that CSF PrP is lowered in the symptomatic phase of disease, a potential confounder for reading out the effect of PrP-lowering drugs in symptomatic patients. To date it has been unclear whether the reduced abundance of PrP in CSF results from its incorporation into plaques, retention in intracellular compartments, downregulation as a function of the disease process, or other factors. Because misfolding or proteolytic cleavage could potentially render PrP invisible to ELISA even if its concentration were constant or increasing in disease, we sought to establish an orthogonal method for CSF PrP quantification. We developed a targeted mass spectrometry method based on multiple reaction monitoring (MRM) of nine PrP tryptic peptides quantified relative to known concentrations of isotopically labeled standards. Analytical validation experiments showed process replicate coefficients of variation below 15%, good dilution linearity and recovery, and suitable performance for both CSF and brain homogenate and across humans as well as preclinical species of interest. InN=55 CSF samples from individuals referred to prion surveillance centers with rapidly progressive dementia, all six human PrP peptides, spanning the N- and C-terminal domains of PrP, were uniformly reduced in prion disease cases compared to individuals with non-prion diagnoses. This confirms the findings from ELISA studies, demonstrating that lowered CSF PrP concentration in prion disease is a genuine result of the disease process and not merely an artifact of ELISA-based measurement. We provide a targeted mass spectrometry-based method suitable for preclinical and clinical quantification of CSF PrP as a tool for drug development.

Prediction of HLA epitopes is important for the development of cancer immunotherapies and vaccines. However, current prediction algorithms have limited predictive power, in part because they were not trained on high-quality epitope datasets covering a broad range of HLA alleles. To enable prediction of endogenous HLA class I-associated peptides across a large fraction of the human population, we used mass spectrometry to profile >185,000 peptides eluted from 95 HLA-A, -B, -C and -G mono-allelic cell lines. We identified canonical peptide motifs per HLA allele, unique and shared binding submotifs across alleles and distinct motifs associated with different peptide lengths. By integrating these data with transcript abundance and peptide processing, we developed HLAthena, providing allele-and-length-specific and pan-allele-pan-length prediction models for endogenous peptide presentation. These models predicted endogenous HLA class I-associated ligands with 1.5-fold improvement in positive predictive value compared with existing tools and correctly identified >75% of HLA-bound peptides that were observed experimentally in 11 patient-derived tumor cell lines.

A typo in the 'Reviewer information' section of this Letter was corrected online.

The human genome contains thousands of long non-coding RNAs1, but specific biological functions and biochemical mechanisms have been discovered for only about a dozen2-7. A specific long non-coding RNA-non-coding RNA activated by DNA damage (NORAD)-has recently been shown to be required for maintaining genomic stability8, but its molecular mechanism is unknown. Here we combine RNA antisense purification and quantitative mass spectrometry to identify proteins that directly interact with NORAD in living cells. We show that NORAD interacts with proteins involved in DNA replication and repair in steady-state cells and localizes to the nucleus upon stimulation with replication stress or DNA damage. In particular, NORAD interacts with RBMX, a component of the DNA-damage response, and contains the strongest RBMX-binding site in the transcriptome. We demonstrate that NORAD controls the ability of RBMX to assemble a ribonucleoprotein complex-which we term NORAD-activated ribonucleoprotein complex 1 (NARC1)-that contains the known suppressors of genomic instability topoisomerase I (TOP1), ALYREF and the PRPF19-CDC5L complex. Cells depleted for NORAD or RBMX display an increased frequency of chromosome segregation defects, reduced replication-fork velocity and altered cell-cycle progression-which represent phenotypes that are mechanistically linked to TOP1 and PRPF19-CDC5L function. Expression of NORAD in trans can rescue defects caused by NORAD depletion, but rescue is significantly impaired when the RBMX-binding site in NORAD is deleted. Our results demonstrate that the interaction between NORAD and RBMX is important for NORAD function, and that NORAD is required for the assembly of the previously unknown topoisomerase complex NARC1, which contributes to maintaining genomic stability. In addition, we uncover a previously unknown function for long non-coding RNAs in modulating the ability of an RNA-binding protein to assemble a higher-order ribonucleoprotein complex.

Primordial germ cells (PGCs) form during early embryogenesis with a supply of maternal mRNAs that contain shorter poly(A) tails. How translation of maternal mRNAs is regulated during PGC development remains elusive. Here we describe a small-molecule screen with zebrafish embryos that identified primordazine, a compound that selectively ablates PGCs. Primordazine's effect on PGCs arises from translation repression through primordazine-response elements in the 3' UTRs. Systematic dissection of primordazine's mechanism of action revealed that translation of mRNAs during early embryogenesis occurs by two distinct pathways, depending on the length of their poly(A) tails. In addition to poly(A)-tail-dependent translation (PAT), early embryos perform poly(A)-tail-independent noncanonical translation (PAINT) via deadenylated 3' UTRs. Primordazine inhibits PAINT without inhibiting PAT, an effect that was also observed in quiescent, but not proliferating, mammalian cells. These studies reveal that PAINT is an alternative form of translation in the early embryo and is indispensable for PGC maintenance.

Introduction: Cancer vaccine therapies rely on accurate personalized selection of immunizing peptides in order to potentiate tumor-specific immune responses against neoepitopes derived from somatic mutations. Given the unique accumulation of mutations in each tumor as well as the patient’s particular complement of HLA class I alleles, the ability to accurately predict which epitopes will be presented by tumor cells is a fundamental prerequisite for successful vaccine design. By utilizing a mono-allelic mass spectrometry (MS) strategy for profiling the endogenous HLA class I peptidome, we recently showed that prediction of endogenous presentation can be drastically improved when model training integrates peptide sequence along with intracellular signals such as likelihood of proteasomal processing and peptide abundance. Yet the limited set of mono-allelic data did not allow for deep comparative analysis across HLA- A, B, and C alleles, which can better inform pan-allele predictor design. Moreover, the significant variability in per-allele model performance remains unexplained. Methods: We recently developed a scalable mono-allelic MS technique to profile naturally presented peptides on HLA molecules, whereby the HLA class I deficient B721.221 cell line is transfected with HLA expression vectors coding for a single allele of interest and eluted HLA peptides are analyzed by LC-MS/MS. In addition, endogenously presented antigens on primary tumor-derived cell lines from 4 melanoma patients were also identified via MS. To extract knowledge from this unique dataset, we implemented computational tools to summarize, visualize, and compare the characteristics of HLA- A, B, C, and G alleles and developed a novel approach to define allele similarity that takes into account the collection of sub-motifs per allele. We trained neural network prediction models, validated their performance on internal and external datasets, and analyzed the variability in performance across alleles. Results: To date, we have generated binding data for 92 HLA- A, B, C and G alleles, identifying more than 190,000 peptides and covering the most frequent alleles in the population. Extensive mono-allelic profiling revealed that some alleles present non-9-mer peptides with high frequency. The availability of large number of non-9-mer peptides allowed us to build length-specific models that often performed better than the corresponding non-length-specific models currently used. We observe that HLA- A and B alleles present more peptides of length 10 and 11 than C alleles, while C alleles have a higher propensity for 8-mers. Correlation-based analysis of binding motifs revealed that HLA-A and B motifs are more specific whereas C motifs are less stringent and thus share more overlapping binders. Since binding data are available only for a fraction of all known alleles, pan-allele models implicitly embed allele similarity to predict for uncharacterized alleles based on the sequence of the binding pocket. By clustering allele-specific peptides into sub-motifs, we propose a novel explicit approach to delineate allele similarity at finer granularity that can improve pan-allele model design. We show that our allele-specific models are better at discriminating tumor-presented epitopes than state of the art predictors and investigate the relationship between false discovery rate and natural abundance of anchor residues to better understand differences in model accuracy amongst alleles. Finally, deconvolution of tumor-presented peptides demonstrated that ~10% of peptides are presented on HLA-C, which has been historically understudied. Conclusions: We have vastly expanded the collection of endogenous HLA-specific peptides deriving biologic insights into the principles of epitope presentations and valuable considerations for prediction model design and epitope selection for tumor vaccines. Citation Format: Siranush Sarkizova, Susan Klaeger, Derin B. Keskin, Karl Clauser, Hasmik Keshishian, Christina R. Hartigan, Nir Hacohen, Steven A. Carr, Catherine J. Wu. Broad analysis and more accurate predictions of HLA class I epitope binding in 92 common HLA alleles profiled by mono-allelic mass spectrometry [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B042.

Recurrent somatic mutations in core components and modulators of the cohesin ring - a multimeric protein complex that forms a ring structure around DNA and provides spatial genome organization - have been identified across multiple cancer types, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), where they are associated with poor overall survival. Cohesin proteins are involved in sister chromatid cohesion, chromatin organization into loops, transcriptional activation, and DNA damage repair. The mechanisms underlying clonal expansion of these driver mutations are unknown and no therapies have selective efficacy in cohesin-mutant cancers. We sought to determine the effects of mutations in the most frequently mutated cohesin subunit, STAG2, on cohesin complex composition using immunoprecipitation followed by quantitative mass spectrometry (IP-MS), genetic dependencies of STAG2-mutant cells by genome-wide CRISPR screening, and mutant cohesin association with chromatin using chromatin immunoprecipitation followed by sequencing (ChIP-Seq). Our goal was to understand how these mutations contribute to cellular transformation and to identify possible therapeutic targets. Applying IP-MS in AML cell lines engineered with different STAG2 mutations, we identified and validated a switch from STAG2- to its paralog STAG1-containing cohesin complexes. In addition, we observed changes in the interaction of the mutant cohesin complex with proteins involved in DNA repair and replication, including PARP1, and RNA-mediated interaction with RNA splicing machinery, including SF3B family members. We next hypothesized that these cohesin-dependent alterations could lead to shifts in genetic dependencies. Using genome-scale CRISPR-Cas9 screens, we identified preferential dependency of STAG2-mutant cells on STAG1, consistent with our proteomics studies. We also found a striking concordance between additional cellular processes highlighted by IP-MS experiments and observed increased dependency of STAG2-mutant cells on DNA damage repair and mRNA processing. Therefore, STAG2 mutations lead to changes in cohesin complex structure and alter interactions with proteins involved in DNA damage, replication, and RNA modification, which become genetic dependencies in this context. Prompted by this concordance, we evaluated DNA replication, DNA damage and splicing in cohesin-mutant cells. We observed a 4-fold increase in replication fork stalling in STAG2-mutant cells, which was associated with accumulation of double strand DNA breaks and activation of the ATR and ATM DNA damage checkpoints. STAG2-mutant cells demonstrated ~100-fold increased sensitivity to the PARP inhibitor talazoparib, which was consistent across models of other cohesin-mutant subunits. In addition, cohesin-mutant cells showed aberrant splicing and increased sensitivity to treatment with SF3B1 inhibitors E7107 and H3B-8800. In aggregate, genetic or pharmacologic perturbation of DNA damage repair or splicing created a synthetic vulnerability for cohesin-mutant cells in vitro and in vivo. Finally, we explored how STAG1-containing complexes alter cohesin-mediated genome compartmentalization in cohesin-mutant cells. Using ChIP-Seq, we observed that STAG2 loss leads to a global decrease in cohesin binding to chromatin, including at sites of insulated neighborhood boundaries, with subsequent gene expression changes. Loss of cohesin binding was associated with increased enhancer activity and super-enhancer expansion in STAG2-mutant cells. In addition, we identified changes in the co-localization of the mutant cohesin complex with super-enhancer enriched factors, DNA damage repair and splicing machinery. These findings are consistent with a model in which wild type and mutant cohesin complexes, defined by their unique composition and patterns of chromatin binding and architecture, have differential abilities to maintain chromatin organization as it relates to spatial organization of super-enhancers, coactivators and transcription factors, as well as DNA damage repair and splicing machinery. Perturbation of any of these components, which have been recently proposed to form phase-separated nuclear bodies, creates vulnerabilities that may be exploited therapeutically with existing drugs in patients with cohesin-mutated malignancies. Disclosures Abraham: Syros Pharmaceuticals: Equity Ownership. Seiler:H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. D'Andrea:Intellia Therapeutics: Consultancy; Cedilla Therpeutics: Consultancy, Equity Ownership; EMD-Serono: Consultancy, Research Funding; Sierra: Consultancy, Research Funding; Ideaya: Consultancy, Equity Ownership; Lilly: Consultancy, Research Funding; Formation Biologics: Consultancy. Young:Omega Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Syros Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Camp4 Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Both sickle cell disease and β-thalassemia are major sources of morbidity and mortality worldwide. Continued production of the β-like γ-globin genes that form fetal hemoglobin after infancy has been shown to ameliorate the severity of these disorders. As a result, there has been considerable interest in understanding the underlying regulation of the physiologic fetal-to-adult hemoglobin switch in humans to be able to better manipulate this process for therapeutic purposes. To date, only a single factor, BCL11A, has been identified as being involved in the developmental regulation of human hemoglobin switching. BCL11A is a direct transcriptional repressor of the γ-globin genes. Moreover, BCL11A is expressed in a developmental stage-specific manner to regulate human hemoglobin switching. However, despite extensive studies, the mechanisms that act upstream to regulate BCL11A expression and thereby control hemoglobin switching have remained elusive. To gain further insights, we have directly explored the developmental regulation of BCL11A expression at various stages of human erythropoiesis. We find that BCL11A is regulated at the level of mRNA translation during development. While BCL11A mRNA is comparably expressed in a similar manner at all developmental stages in erythroid cells, robust protein expression only occurs in adult erythroid cells. Importantly, we demonstrate that at the earlier stages of development, the observed reduction in protein expression is attributable to decreased synthesis and not increased degradation of BCL11A through direct assessment of both protein synthesis and degradation rates in primary erythroid cells from various stages of human development. Interestingly, while BCL11A protein is not well synthesized at these earlier stages of development, we find that its mRNA curiously continues to be associated with ribosomes in a comparable manner between newborn and adult erythroid precursors using polysome fractionation of stage-matched erythroid cells. Through use of an unbiased proteomic analysis approach involving RNA affinity purification of the 18S ribosomal RNA in erythroid cells, we demonstrate that the RNA-binding protein LIN28B, which is developmentally expressed in a reciprocal pattern to BCL11A, directly interacts with ribosomes. We additionally show that the observed suppression of BCL11A protein translation is mediated by LIN28B. Using a newly developed RNA isolation and crosslinking immunoprecipitation approach coupled to massively parallel sequencing we mapped the direct interaction of LIN28B with BCL11A mRNA at nucleotide resolution. Through a number of functional assays in both newborn and adult erythroid cells, we additionally demonstrate that this alteration of BCL11A translation is entirely independent of the role of LIN28B in the biogenesis of let-7 microRNAs. Finally, we show that BCL11A is the major functional target in LIN28B-mediated fetal hemoglobin induction through functional complementation experiments in primary human erythroid cells. Our results reveal a previously unappreciated regulatory mechanism underlying human hemoglobin switching. Moreover, our findings highlight opportunities for developing improved treatments for sickle cell disease and β-thalassemia by understanding the upstream regulation of human hemoglobin switching. Disclosures No relevant conflicts of interest to declare.

Type 2 diabetes (T2D) affects Latinos at twice the rate seen in populations of European descent. We recently identified a risk haplotype spanning SLC16A11 that explains ∼20% of the increased T2D prevalence in Mexico. Here, through genetic fine-mapping, we define a set of tightly linked variants likely to contain the causal allele(s). We show that variants on the T2D-associated haplotype have two distinct effects: (1) decreasing SLC16A11 expression in liver and (2) disrupting a key interaction with basigin, thereby reducing cell-surface localization. Both independent mechanisms reduce SLC16A11 function and suggest SLC16A11 is the causal gene at this locus. To gain insight into how SLC16A11 disruption impacts T2D risk, we demonstrate that SLC16A11 is a proton-coupled monocarboxylate transporter and that genetic perturbation of SLC16A11 induces changes in fatty acid and lipid metabolism that are associated with increased T2D risk. Our findings suggest that increasing SLC16A11 function could be therapeutically beneficial for T2D. Video Abstract [Figure presented] Keywords: type 2 diabetes (T2D); genetics; disease mechanism; SLC16A11; MCT11; solute carrier (SLC); monocarboxylates; fatty acid metabolism; lipid metabolism; precision medicine

Impaired erythropoiesis in the deletion 5q (del(5q)) subtype of myelodysplastic syndrome (MDS) has been linked to heterozygous deletion of RPS14, which encodes the ribosomal protein small subunit 14. We generated mice with conditional inactivation of Rps14 and demonstrated an erythroid differentiation defect that is dependent on the tumor suppressor protein p53 (encoded by Trp53 in mice) and is characterized by apoptosis at the transition from polychromatic to orthochromatic erythroblasts. This defect resulted in age-dependent progressive anemia, megakaryocyte dysplasia and loss of hematopoietic stem cell (HSC) quiescence. As assessed by quantitative proteomics, mutant erythroblasts expressed higher levels of proteins involved in innate immune signaling, notably the heterodimeric S100 calcium-binding proteins S100a8 and S100a9. S100a8--whose expression was increased in mutant erythroblasts, monocytes and macrophages--is functionally involved in the erythroid defect caused by the Rps14 deletion, as addition of recombinant S100a8 was sufficient to induce a differentiation defect in wild-type erythroid cells, and genetic inactivation of S100a8 expression rescued the erythroid differentiation defect of Rps14-haploinsufficient HSCs. Our data link Rps14 haploinsufficiency in del(5q) MDS to activation of the innate immune system and induction of S100A8-S100A9 expression, leading to a p53-dependent erythroid differentiation defect.

Immunosuppressant drugs (ISDs) are commonly prescribed to solid organ transplant patients. Their narrow therapeutic index and potential for toxicity necessitates careful monitoring of blood concentrations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods are increasingly used for ISD measurement. However, there remain many challenges with this methodology, particularly regarding interassay variability. The Thermo Scientific Prelude is an online extraction/liquid chromatography platform that uses turbulent flow technology coupled with MS/MS. A multicenter evaluation of the Prelude for the measurement of cyclosporine A, tacrolimus, and sirolimus is described. ISDs were measured at each site using standardized protocols. Sample preparation liquid chromatography-MS/MS was performed using the Prelude coupled to a TSQ Vantage. Chromatography was achieved with a Cyclone-P TurboFlow/Accucore C8 column combination using a multisolvent loading and eluting pump system. Mass spectrometry acquisitions were performed in selective reaction monitoring mode and data processed using TraceFinder (version 3.1). Multisite mean imprecision for cyclosporine A ranged from 8.8% (54 mcg/L) to 9.8% (450 mcg/L); for tacrolimus, 4.7% (15.5 mcg/L) to 12.6% (2.5 mcg/L); for sirolimus, 7.4% (19.9 mcg/L) to 16.5% (2.6 mcg/L). Approximately 110 specimens were used for method comparison. For cyclosporine A, mean bias against the multisite mean ranged from -18% to 1%; for tacrolimus, values ranged from -7% to 4%; for sirolimus, values ranged from -4% to 2%. Comparisons of multisite mean Prelude results with routine ISD method results was also performed for cyclosporine A (slope = 0.7878, intercept = 24.16, r = 0.98), tacrolimus (slope = 0.9391, intercept = 0.1017, r = 98), and sirolimus (slope = 0.9618, intercept = 0.1483, r = 0.97). The Prelude ISD method offers acceptable and comparable multisite performance. This study has also highlighted the importance of adopting standardized protocols and LC-MS/MS methods for better comparability between ISD assays.

Cancer mutations yield neo-antigens, which are instrumental to immune-mediated recognition and control of cancer. Vaccine-based therapies targeting neo-antigens will require accurate prediction of which mutations yield peptides presented on polymorphic HLA class I. While in vitro methods have produced increasingly accurate predictors of peptide:MHC binding, there remains a need to define rules for endogenous antigen presentation. Here, we use rapid, high-resolution liquid chromatography mass spectrometry (LC-MS/MS) to identify >24,000 peptides associated with 16 HLA alleles in B cell lines that each express a single HLA allele. The elution of peptides from single HLA alleles allowed us to develop improved rules for endogenous peptide presentation based on the physicochemical properties of binding peptides, patterns of peptide cleavage and abundance of cognate transcripts. Finally, we trained models that integrated MS-derived peptide data and gene expression and demonstrate improved prediction of endogenous peptide presentation in independent datasets. Our strategy thus improves the performance of current predictive algorithms and provides a rapid and scalable method to generate rules for the massive and diverse set of human HLA alleles. Citation Format: Michael S. Rooney, Jennifer G. Abelin, Derin B. Keskin, Siranush Sarkizova, Christina Hartigan, Wandi Zhang, John Sidney, Jonathan Stevens, William J. Lane, Guang L. Zhang, Karl R. Clauser, Nir Hacohen, Steven A. Carr, Catherine J. Wu. High-throughput profiling of HLA allele-specific peptides by MS for improved epitope prediction [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B089.

High cancer death rates indicate the need for new anti-cancer therapeutic agents. Approaches to discover new cancer drugs include target-based drug discovery and phenotypic screening. Here, we identify phosphodiesterase 3A modulators as cell-selective cancer cytotoxic compounds by phenotypic compound library screening and target deconvolution by predictive chemogenomics. We found that sensitivity to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, across 766 cancer cell lines correlates with expression of the phosphodiesterase 3A gene, PDE3A. Like DNMDP, a subset of known PDE3A inhibitors kill selected cancer cells while others do not. Furthermore, PDE3A depletion leads to DNMDP resistance. We demonstrate that DNMDP binding to PDE3A promotes an interaction between PDE3A and Schlafen 12 (SLFN12), suggesting a neomorphic activity. Co-expression of SLFN12 with PDE3A correlates with DNMDP sensitivity, while depletion of SLFN12 results in decreased sensitivity to DNMDP. Our results implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery. Citation Format: Lucian de Waal, Timothy A. Lewis, Matthew G. Rees, Aviad Tsherniak, Xiaoyun Wu, Peter S. Choi, Lara Gechijian, Christina Hartigan, Patrick W. Faloon, Mark J. Hickey, Nicola Tolliday, Steven A. Carr, Paul A. Clemons, Benito Munoz, Bridget K. Wagner, Alykhan F. Shamji, Angela N. Koehler, Monica Schenone, Alex B. Burgin, Stuart L. Schreiber, Heidi Greulich, Matthew Meyerson. Identification of selective cancer cytotoxic modulators of phosphodiesterase 3a by predictive chemogenomics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C136.