[This corrects the article DOI: 10.1371/journal.pone.0344471.].

Supplementary Figures S1-S2 and Supplementary Tables S1-S3.

Background: The inflammaging modulator, S100A9, mediates immunosuppression and plays a key role in the pathogenesis of low-risk myelodysplastic syndrome (MDS). Specifically, S100A9’s feedforward activation in the bone marrow (BM) induces pyroptotic cell death of hematopoietic stem and progenitor cells (HSPC) and the activation and accumulation of myeloid-derived suppressor cells (MDSC). This process creates a suppressive microenvironment including the secretion of transforming growth factor β (TGFβ) that signals through the TGFβ receptor 1 (TGFBR1) to induce inhibitory processes that contribute to this phenotype. We hypothesize that targeting with an investigational TGFBR1 inhibitor, TP-6379, reduces immune suppression and restores hematopoiesis in MDS. Methods: Primary low risk MDS BM mononuclear cells (BMMNC) were obtained from the Moffitt Total Cancer Care Protocol and cultured in vitro with TP-6379. Healthy BMMNC were purchased, treated with or without recombinant S100A9 and TP-6379. Results: Treatment of primary MDS BMMNC (n=15) and S100A9-treated healthy BMMNC (n=5) with TP-6379 for 48 hours was observed to significantly improve proliferation of hematopoietic progenitor cells as measured by colony forming capacity. Total colonies, and BFU-E specific colonies, showed significant increase. This expansion of progenitors with treatment was validated by flow cytometric analysis of Lineage−HLA-DR−CD34+ HSPC and showed a reduction in genomic instability, measured by γH2AX. In addition, TP-6379 was observed to reduce the numbers of suppressive mediators; MDSC, T regs and senescent lymphocytes and increase the number of cytotoxic cells, including NK cells. Our analysis revealed that BMMNC with spliceosomal mutations, including SF3B1, have elevated sensitivity to TP-6379 treatment. Therefore, we tested wild type and SF3B1 K700E CRISPR knock-in K562 cells in the presence of TP-6379 for 48 hours. K562 is a well-established cell line model for study of MDS. The SF3B1 mutant expressing cells have significantly reduced BFU-E colony forming capacity, compared to wild type cells, concordant with higher pyroptosis activation and consistent with the BMMNC results. Hence, we measured phospho-SMAD activity, as a direct measure of TGFBR1 inhibition. SF3B1 mutant expressing cells were shown to be highly sensitive to TP-6379, with a significant loss of SMAD activation. Expression analysis of primary MDS, S100A9-treated healthy BMMNC cells and K562 SF3B1 mutant cells revealed elevated expression of SERPINE1 which was rescued by TP-6379 treatment. Conclusions: TP-6379 restores healthy hematopoiesis in low-risk MDS, which may translate into a strong therapeutic option in a disease with few clinical options. Importantly, our work also suggests a specific role for spliceosome dysfunction in bone marrow failure and a novel role for SERPINE1 in MDS and S100A9-induced suppression. Citation Format: Erika A. Eksioglu, Gabriela M. Wright, Jason M. Foulks, Matthew Lalonde, Steven L. Warner, Kenneth L. Wright. Improvement of hematopoiesis in primary low risk MDS with the TGFBR1 investigational inhibitor TP-6379. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5150.

Advances in the understanding of the tumor microenvironment have led to devel-opment of immunotherapeutic strategies, such as chimeric antigen receptor T cells (CAR-Ts). However, despite success in blood malignancies, CAR-T therapies in solid tumors have been hampered by their restricted infiltration. Here, we used our understanding of early cytotoxic lymphocyte infiltration of human lymphocytes in solid tumors in vivo to investigate the receptors in normal, adjacent, and tumor tissues of primary non-small-cell lung cancer specimens. We found that CX3CL1-CX3CR1 reduction restricts cytotoxic cells from the solid-tumor bed, contributing to tumor escape. Based on this, we designed a CAR-T construct using the well-established natural killer group 2, member D (NKG2D) CAR-T expression together with overexpression of CX3CR1 to promote their infiltration. These CAR-Ts infiltrate tumors at higher rates than control-activated T cells or IL-15-overexpressing NKG2D CAR-Ts. This construct also had similar functionality in a liver-cancer model, demonstrating potential efficacy in other solid malignancies.

Myelodysplastic Syndromes (MDSs) are bone marrow (BM) failure malignancies characterized by constitutive innate immune activation, including NLRP3 inflammasome driven pyroptotic cell death. We recently reported that the danger-associated molecular pattern (DAMP) oxidized mitochondrial DNA (ox-mtDNA) is diagnostically increased in MDS plasma although the functional consequences remain poorly defined. We hypothesized that ox-mtDNA is released into the cytosol, upon NLRP3 inflammasome pyroptotic lysis, where it propagates and further enhances the inflammatory cell death feed-forward loop onto healthy tissues. This activation can be mediated via ox-mtDNA engagement of Toll-like receptor 9 (TLR9), an endosomal DNA sensing pattern recognition receptor known to prime and activate the inflammasome propagating the IFN-induced inflammatory response in neighboring healthy hematopoietic stem and progenitor cells (HSPCs), which presents a potentially targetable axis for the reduction in inflammasome activation in MDS. We found that extracellular ox-mtDNA activates the TLR9-MyD88-inflammasome pathway, demonstrated by increased lysosome formation, IRF7 translocation, and interferon-stimulated gene (ISG) production. Extracellular ox-mtDNA also induces TLR9 redistribution in MDS HSPCs to the cell surface. The effects on NLRP3 inflammasome activation were validated by blocking TLR9 activation via chemical inhibition and CRISPR knockout, demonstrating that TLR9 was necessary for ox-mtDNA-mediated inflammasome activation. Conversely, lentiviral overexpression of TLR9 sensitized cells to ox-mtDNA. Lastly, inhibiting TLR9 restored hematopoietic colony formation in MDS BM. We conclude that MDS HSPCs are primed for inflammasome activation via ox-mtDNA released by pyroptotic cells. Blocking the TLR9/ox-mtDNA axis may prove to be a novel therapeutic strategy for MDS.

Immunotherapy has improved survival for many cancer patients, especially for immunogenic malignancies such as lung cancer. However, even in lung cancer, the response rate for anti-PD1 therapy (for example) does not exceed 20%. Thus, it is critical to identify mechanisms that block the response to immune checkpoint therapies. STK11 (also known as LKB1) is encoded by the serine threonine kinase 11 gene (STK11). Patients harboring tumors with STK11 mutations have reduced infiltrates of cytotoxic T-cells and clinical studies have shown that they respond poorly to anti-PD1 or anti-PDL-1 therapies regardless of PDL-1 status, which otherwise predicts benefit. Herein, we have used gene expression data from a cohort of 442 lung adenocarcinoma patients to identify CX3CL1 (fractalkine) as a gene silenced in STK11 mutant tumors with potential to be a key direct modulator of the immune system relevant to STK11 loss. To further explore this hypothesis, we have edited the STK11 gene in A549 cells back to wild type (STK11 corrected). As predicted, restoration of STK11 function resulted in modest expression of CX3CL1 as measured by Western blotting. Unexpectedly, exposure of STK11 corrected cells to human immune cells isolated from the blood of healthy donors resulted in a 5 to 10-fold increase in CX3CL1 suggesting interactive signaling between tumor and immune cells. Using transwell assays and STK11 corrected A549 cells we find that restoration of functional STK11 increases immune cell migration, adhesion and invasion in vitro. Finally, cytotoxicity assays demonstrate that STK11 corrected A549 cells are 3 to 5-fold more sensitive to immune cell killing. Taken together these results suggest the hypothesis that CX3CL1 loss mediates immune evasion in STK11 mutant lung adenocarcinomas. Future work will further explore the mechanisms underlying this pathway in preclinical models. Citation Format: Eria Eksioglu, Gabriela M. Wright, Trent R. Percy, Kenneth L. Wright, W. Douglas Cress. Loss of CX3CL1 expression mediates immune evasion in STK11 mutated lung adenocarcinomas. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4454.

We have reported previously that CD33hi myeloid-derived suppressor cells (MDSCs) play a direct role in the pathogenesis of myelodysplastic syndromes (MDSs) and that their sustained activation contributes to hematopoietic and immune impairment, including modulation of PD1/PDL1. MDSCs can also limit the clinical activity of immune checkpoint inhibition in solid malignancies. We hypothesized that depletion of MDSCs may ameliorate resistance to checkpoint inhibitors and, hence, targeted them with AMV564 combined with anti-PD1 in MDS bone marrow (BM) mononuclear cells (MNCs) enhanced activation of cytotoxic T cells. AMV564 was active in vivo in a leukemia xenograft model when co-administered with healthy donor peripheral blood MNCs (PBMCs). Our findings provide a strong rationale for clinical investigation of AMV564 as a single agent or in combination with an anti-PD1 antibody and in particular for treatment of cancers resistant to checkpoint inhibitors.

Inflammatory molecules and their receptors serve as regulatory cues driving the proliferation of hematopoietic stem and progenitors (HSPC) in myelodysplastic syndromes (MDS). Recent studies indicate that inflammaging (inflammation in aging) in the bone marrow (BM) microenvironment contributes to hematopoietic impairment and MDS clone expansion. However, attempts to develop therapeutics targeting these processes have been hampered by potential off target effects due to the lack of identification of selective biomarkers to specifically target the MDS malignant clone. Therefore, a better understanding of the mechanisms of MDS inflammaging processes for selection and target of malignant clones remains a key goal. Several groups, including ours, have found that innate immune receptors are over expressed in MDS BM HSPC, including Toll-like receptors (TLRs). Overexpression of these receptors was associated with secretion of inflammatory cytokines and mediators including S100A9. S100A9, a Damage-Associated Molecular Pattern (DAMPs) molecule, causes pyroptosis of HSPC and induces genomic instability leading to MDS clonal expansion in an autocrine amplification loop. However, most of the TLRs and associated factors are hard to target due to their wide expression on healthy immune cells. We found that TLR9, usually an intracellular receptor, is upregulated and translocated to the surface of MDS progenitors, which thereby presents an opportunity for disease-specific targeting through its natural ligand, CpG oligonucleotides. TLR9 is one of the main pathogen recognition receptors expressed intracellularly in innate immune cells and recognizes bacterial nucleic acid moieties. Its gene expression has been previously found to be upregulated in MDS and now our work demonstrates a functional upregulation and translocation in low-risk disease. Flow cytometric sorting of low risk MDS BM cells with labeled CpG separated cells based on mutational burden suggesting that surface TLR9 is specific to malignant clones. Furthermore, pyroptotic release of nucleic acid DAMPs, such as RNA:DNA hybrids and our recently published biomarker oxidized mitochondrial DNA, is linked to the translocation of TLR9 from intracellular endosomes to the surface of these cells. This overexpression was validated in our unique inflammaging animal model, the S100A9 transgenic (Tg) mice, that phenocopies human MDS. Moreover, treatment of human BM mononuclear cells (MNC) with recombinant human S100A9 induced surface expression of TLR9, through the accumulation of S100A9-induced RNA:DNA hybrids and oxidized mitochondrial DNA. Therefore, this paradoxical surface expression of TLR9 in MDS BM HSPCs provides an opportunity to selectively target malignant cells through the targeted delivery of cytotoxins using our newly developed CpG-linked payload deliver strategy. CpG has been used safely in phase I and Phase II clinical trials where it has been shown to be well tolerated, although not effective as a stand-alone therapy. Therefore, we linked CpG to dendrimer particles (CpG-Den) providing a cationic lipid surface structure capable of disrupting cells only after internalization through TLR9. The CpG-Den linkage ratio can be manipulated to modify cytotoxicity strength and the particle size avoids leukocyte engulfment. Using both primary MDS BM MNC and BM MNC from S100A9Tg mice we demonstrate that treatment in vitro and in vivo with CpG-Den selectively targets surface TLR9+ HSPC, increasing healthy hematopoiesis in colony forming assays, and BM expression of HSPC, as well ameliorating anemia in vivo in the S100A9Tg mice. Use of this therapy also led to a decrease in suppressive myeloid cells likely aiding in the reduction of the inflammaging cycle. Localized targeting was confirmed in vivo by labeling CpG-Den with IR800 revealing specific accumulation in the mice BM. Our data supports TLR9-targeting potential in a proof-of-concept strategy to selectively target primary MDS malignant cells to restore and enhance hematopoiesis while avoiding toxicities.

The clinical utility of histone/protein deacetylase (HDAC) inhibitors in combinatorial regimens with proteasome inhibitors for patients with relapsed and refractory multiple myeloma (MM) is often limited by excessive toxicity due to HDAC inhibitor promiscuity with multiple HDACs. Therefore, more selective inhibition minimizing off-target toxicity may increase the clinical effectiveness of HDAC inhibitors. We demonstrated that plasma cell development and survival are dependent upon HDAC11, suggesting this enzyme is a promising therapeutic target in MM. Mice lacking HDAC11 exhibited markedly decreased plasma cell numbers. Accordingly, in vitro plasma cell differentiation was arrested in B cells lacking functional HDAC11. Mechanistically, we showed that HDAC11 is involved in the deacetylation of IRF4 at lysine103. Further, targeting HDAC11 led to IRF4 hyperacetylation, resulting in impaired IRF4 nuclear localization and target promoter binding. Importantly, transient HDAC11 knockdown or treatment with elevenostat, an HDAC11-selective inhibitor, induced cell death in MM cell lines. Elevenostat produced similar anti-MM activity in vivo, improving survival among mice inoculated with 5TGM1 MM cells. Elevenostat demonstrated nanomolar ex vivo activity in 34 MM patient specimens and synergistic activity when combined with bortezomib. Collectively, our data indicated that HDAC11 regulates an essential pathway in plasma cell biology establishing its potential as an emerging theraputic vulnerability in MM.

Dendritic cells (DCs) are professional antigen presenting cells with a great capacity for cross-presentation of exogenous antigens from which robust anti-tumor immune responses ensue. However, this function is not always available and requires DCs to first be primed to induce their maturation. In particular, in the field of DC vaccine design, currently available methodologies have been limited in eliciting a sustained anti-tumor immune response. Mechanistically, part of the maturation response is influenced by the presence of stimulatory receptors relying on ITAM-containing activating adaptor molecules like DAP12, that modulates their function. We hypothesize that activating DAP12 in DC could force their maturation and enhance their potential anti-tumor activity for therapeutic intervention. For this purpose, we developed constitutively active DAP12 mutants that can promote activation of monocyte-derived DC. Here we demonstrate its ability to induce the maturation and activation of monocyte-derived DCs which enhances migration, and T cell stimulation in vitro using primary human cells. Moreover, constitutively active DAP12 stimulates a strong immune response in a murine melanoma model leading to a reduction of tumor burden. This provides proof-of-concept for investigating the pre-activation of antigen presenting cells to enhance the effectiveness of anti-tumor immunotherapies.

Mantle Cell Lymphoma (MCL) is a non-Hodgkin lymphoma with a median survival rate of five years. Standard treatment with high-dose chemotherapy plus rituximab (anti-CD20 antibody) has extended overall survival although, the disease remains incurable. Histone deacetylases (HDAC) are a family of enzymes that regulate multiple proteins and cellular pathways through post-translational modification. Broad spectrum HDAC inhibitors have shown some therapeutic promise, inducing cell cycle inhibition and apoptosis in leukemia and non-Hodgkin’s lymphoma. However, the therapeutic effects of these broad-spectrum HDAC inhibitors can detrimentally dampen Natural Killer (NK) cell cytotoxicity, reduce NK viability, and downregulate activation receptors important for NK mediated anti-tumor responses. Impairment of NK function in MCL patients during therapy potentially limits therapeutic activity of rituximab. Thus, there is an unmet need to decipher specific roles of individual HDACs in order to preserve and/or enhance NK function, while, directly impairing MCL viability. We investigated the impact of HDAC8 in MCL cell lines. Inhibition or genetic loss of HDAC8 caused MCL cells to undergo apoptosis. In contrast, exposure of primary human NK cells to an HDAC8 inhibitor does not alter viability, receptor expression, or antibody dependent cellular cytotoxicity (ADCC). However, an increase in effector cytokine interferon-gamma (IFNγ) producing NK cells was observed in response to HDAC8 inhibition. Taken together these data suggest that selective HDAC8 inhibitors may simultaneously preserve NK functional activity, while impairing MCL tumor growth, establishing a rationale for future clinical evaluation.

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.

The Janus kinase 2 (JAK2)-driven myeloproliferative neoplasms (MPNs) are associated with clonal myelopoiesis, elevated risk of death due to thrombotic complications, and transformation to acute myeloid leukemia (AML). JAK2 inhibitors improve the quality of life for MPN patients, but these approved therapeutics do not readily reduce the natural course of disease or antagonize the neoplastic clone. An understanding of the molecular and cellular changes requisite for MPN development and progression are needed to develop improved therapies. Recently, murine MPN models were demonstrated to exhibit metabolic vulnerabilities due to a high dependence on glucose. Neoplastic hematopoietic progenitor cells in these mice express elevated levels of glycolytic enzymes and exhibit enhanced levels of glycolysis and oxidative phosphorylation, and the disease phenotype of these MPN model mice is antagonized by glycolytic inhibition. While all MPN-driving mutations lead to aberrant JAK2 activation, these mutations often co-exist with mutations in genes that encode epigenetic regulators, including loss of function mutations known to enhance MPN progression. In this perspective we discuss how altered activity of epigenetic regulators (e.g., methylation and acetylation) in MPN-driving stem and progenitor cells may alter cellular metabolism and contribute to the MPN phenotype and progression of disease. Specific metabolic changes associated with epigenetic deregulation may identify patient populations that exhibit specific metabolic vulnerabilities that are absent in normal hematopoietic cells, and thus provide a potential basis for the development of more effective personalized therapeutic approaches.

With the exponential growth of bandwidth demands on present data centers, we are researching the feasibility of deploying cryogenic DRAM based memory subsystems for the next generation of higher bandwidth, more efficient, lower cost and smaller footprint data centers of the future. As part of our early research, we are designing DRAM based subsystems that operate at 77K and are submerged in liquid nitrogen. To effectively test the memory subsystem at cryogenic temperatures, creative test methods must be employed. This paper illustrates the test setups, procedures and methodologies that were used to successfully test DRAM based subsystems at cryogenic temperatures

Introduction: Acetylated histone and non-histone proteins are pharmacologic targets for both solid and hematological cancers including myeloproliferative neoplasms (MPNs), a group of clonal hematological malignancies driven by aberrant JAK2/STAT signaling. MPNs are characterized by epigenetic alterations, including aberrant acetylation, which makes this disease particularly interesting for targeting with HDAC inhibitors. Four classes of histone deacetylases (Class I-IV HDACs) regulate gene transcription and modulate cellular processes that drive the initiation and progression of cancer. Pan-HDAC and class I-selective HDAC inhibitors have gained traction in clinical settings, yet we reasoned that specific targeting of the 18 distinct HDAC proteins may establish roles for select HDACs as therapeutic vulnerabilities in MPNs. Methods: To explore the roles of individual HDACs in MPN, we first conducted an inhibitor screen of compounds having distinct HDAC selectivity based on electrophoretic mobility shift assays with full-length human HDAC proteins expressed in baculovirus and unique peptide substrates. Ultra-specific HDAC6 compounds were initially targeted for analysis based on its previously defined role in HSP90-mediated JAK2 stabilization and translation. Survival of MPN cell line models, MPN patient samples, leukemia cell lines, and MPN disease progression in mice transplanted with Hdac6-/-, and Hdac11-/- hematopoietic stem cells (HSCs) transduced with the MPLW515L oncogene, as well as Tg-Hdac11-eGfp mice were used to show the role of HDAC6 and HDAC11 in oncogene-driven and homeostatic hematopoiesis. As further proof of specificity, HDAC6 and HDAC11 were genetically ablated in MPN model cell lines using either RNA interference or inducible shRNA. For HDAC11 substrate identification, a combination of RNA-seq, acetylated proteome (SILAC), global metabolomics (LC-MS), Seahorse metabolic assays (Agilent Technologies), enzymatic assays, and acetylation-specific immunoblotting and mutation profiling were performed (Fig. 1). Results: Despite the established interplay between HDAC6, HSP90 and JAK2, neither a highly selective HDAC6 inhibitor, HDAC6 silencing, nor the Hdac6 deficiency suppressed MPN pathogenesis, although there were clear effects on the acetylation of α-tubulin, a well characterized HDAC6-selective substrate. Intriguingly, both inhibition of HDAC11 activity with highly-specific HDAC11 inhibitors and silencing HDAC11 using an inducible validated shRNA, identified HDAC11 as a therapeutic vulnerability for multiple human MPN cell lines. The Tg-Hdac11-eGFP reporter mice showed that HDAC11 is expressed in several hematopoietic cell types, including myeloid cells, erythroblasts, and megakaryocytes. Thus, Hdac11-/- and Hdac11+/+MPLWT bone marrow were examined for steady-state hematopoiesis and transplantation chimerism. These studies demonstrated that HDAC11 does not contribute to homeostatic or transplantated bone marrow reconstitution. However, in the oncogenic MPL model, recipient mice transplanted withoncogenic MPLW515L-expressing Hdac11-deficient HSCs displayed markedly impaired cytokine-independent colony-formation, had less fibrosis, and displayed improved survival in primary and secondary MPN hematopoietic stem cell transplantation; thus HDAC11 contributes to MPN pathogenesis (Fig. 1). Studies in additional leukemia cell lines, including THP-1, HL-60, and mantle lymphoma cell lines, but not in Ramos or K562 cells, established that HDAC11 contributes to oncogene-driven events in other cell types. Mechanistically, RNA-seq, SILAC proteomics, and metabolic profiling revealed that HDAC11 controls aerobic glycolysis by deacetylating Lys343 of the glycolytic enzyme enolase-1 (ENO1), functionally inactivating ENO1. Finally, the effects of targeting HDAC11 on metabolism were augmented by blocking compensatory pathways of oxidative phosphorylation that are induced via JAK2V617Fand MPLW515L oncogenic signaling. Conclusions: Our comprehensive screens of HDAC inhibitors, coupled with our biological, in vivo and molecular studies, indicate that HDAC11 is an attractive and potent target for disabling MPN metabolism and pathogenesis. These finding support the rationale for further development of clinical HDAC11 inhibitors for the treatment of metabolically-active cancers such as MPNs. Disclosures Pinilla Ibarz: Teva: Consultancy; TG Therapeutics: Consultancy; Sanofi: Speakers Bureau; Bayer: Speakers Bureau; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Abbvie: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau. Reuther:Incyte Corporation: Research Funding. Levine:Loxo: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Research Funding; Lilly: Honoraria; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Isoplexis: Membership on an entity's Board of Directors or advisory committees; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Gilead: Consultancy; Celgene: Consultancy, Research Funding; Qiagen: Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Amgen: Honoraria. Verma:BMS: Research Funding; Janssen: Research Funding; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria. Epling-Burnette:Incyte Corporation: Research Funding; Celgene Corporation: Patents & Royalties, Research Funding; Forma Therapeutics: Research Funding.

We previously reported that S100A9 promotes ineffective hematopoiesis and the development of MDS in a feed forward age-dependent fashion. Nonetheless, the precise mechanism by which S100A9 may foster DNA damage in MDS remains unclear. We recently showed that S100A9 directs overexpression of the fat-mass and obesity-associated gene (FTO) encoding an m6A RNA demethylase, which leads to nuclear exclusion of SRSF2. Removal of SRSF2 from its functional domain in the nucleosome leads to stalling of RNA polymerase II and formation of the nucleic acid R-loops, comprising DNA:RNA hybrids with the associated non-template single-stranded DNA. S100A9/FTO axis activation leads to SRSF2 deregulation through suppression of its main nuclear transport protein RanBP2, thereby stalling transcription machinery with resulting accumulation of nuclear R-loops and cytosolic/extracellular RNA:DNA hybrids. Persistent R-loops induce DNA damage while also compromising DNA repair. Here we identify an S100A9/FTO-regulated pathway responsible for induction of genomic instability through the accumulation of cytoplasmic RNA:DNA hybrids and modification of the spliceosomal patterns of aged S100A9Tg mice matching MDS hematopoietic stem and progenitor cells (HSPC). We first investigated which components of the S100A9/FTO axis are critical to hematopoiesis and those that are important for both the development of RNA:DNA hybrids and γH2AX activation. We analyzed the contribution of RanBP2 and the effects of elimination of R-loop formation via overexpression of RNAse H1, an enzyme that removes stalled R-loops in the nucleus by degrading DNA-hybridized RNA, thereby reducing the accumulation of cytoplasmic RNA:DNA hybrids. CRISPR knock-down of RanBP2 showed that the protein is critical for accumulation of yH2AX defined by double stranded breaks (DSB). Importantly, overexpression of RNAse H1 degraded R-loops and restored colony-forming capacity, indicating that RNA:DNA hybrids induced by the S100A9/FTO have profound effects on hematopoietic potential. However, while the FTO exclusion of SRSF2 from the nucleus explains the accumulation of γH2AX, it should potentially impact global RNA splicing. To investigate this, we performed a comparative RNAseq analysis on WT and S100A9Tg mice (young and old) to understand both changes induced through the normal aging process as well as those compounded by S100A9. We found that genes linked to splicing, RNA development, nucleotide excision repair and genomic instability and ribosome function were downregulated in aged S100A9Tg mice. Further analysis comparing splicing patterns of S100A9Tg and WT mice with human MDS BM HSPC led to ~200 common genes that were analyzed further. These genes showed that there are splicing changes enriched in spliceosomal assembly and mRNA splice selection site genes. We also found that the enriched genes affect the nucleolus and ribosome formation matching what is seen phenotypically with MDS. Dysregulation of these pathways are highly consistent with our observations of the pathways affected by the S100A9/FTO-induced inflammaging process, validating our hypothesis of S100A9 as a common initiator of dysfunction that can give rise to MDS. Importantly, our data demonstrates the potential for spliceosomal dysfunction regardless of the presence of spliceosomal mutations in MDS. We are currently in the process of performing both DRIPseq and m6A-seq of primary human MDS specimens and S100A9Tg mice to further assess the role of the S100A9/FTO pathway in the selection of sites for RNA/DNA hybrid formation and rise of genomic dysfunction that gives rise to MDS. We conclude that S100A9/FTO-induced nuclear exclusion of SRSF2 aids in the formation of RNA:DNA-hybrids that lead to genomic instability and the disruption of normal spliceosomal patterns in both human HSPC and the S100A9Tg MDS murine model, representing a previously uncharacterized mechanism contributing to MDS pathogenesis. Our studies provide evidence that targeting this cascade offers significant potential for development of novel, biologically rational therapeutics for MDS. Disclosures List: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract Therapeutic molecules targeting the activity of histone deacetylases (HDACs) are currently under investigation for the treatment of several malignancies. There are currently eighteen human HDACs and while histone deacetylation is associated with transcriptional repression, acetylated lysine targets are functionally diverse and include cytoplasmic, nuclear, and mitochondrial proteins. Here, we used a new class of novel small molecule inhibitors that are highly selective for HDAC11 to identify its role in the regulation of non-histone proteins. Stable isotope labeling with amino acids in cell culture (SILAC) followed by mass spectrometry in the presence of HDAC11 selective inhibitors identified proteins with acetylation and/or expression changes after treatment and were compared to known HDAC substrates to establish a unique set of putative HDAC11 target proteins. Metabolic processes were highly enriched in this data set. Specifically, acetylated enolase 1 (ENO1, 2-phospho-D-glycerate hydrolase) which catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate (PEP) in the glycolytic pathway was highly altered after HDAC11 inhibition. Using acetylated lysine specific immunoprecipitation, we validated the hyperacetylated state of ENO1 upon HDAC11 inhibition. Functional assays confirmed that the HDAC11 inhibition lowered ENO1-mediated PEP production, and reduced proliferation and viability of hematopoietic and solid tumor cells. Similar observations were obtained in HDAC11 knock down cell lines confirming that HDAC11 is a required molecule in the regulation of ENO1-mediated metabolic regulation. The proteomics data also mapped three distinct target lysine residues of HDAC11 in ENO1 and each of these residues were substituted to either an acetylated or an un-acetylated lysine mimic to test their function in ENO1 activity and stability. We confirmed that K335 is the major target site of HDAC11 and its substitution to the acetylated mimic (glutamine) causes loss of enolase activity. Concomitantly, using proton nuclear magnetic resonance spectroscopy we identified some glycolytic intermediates upstream of ENO1 to be increased and downstream intermediates quantitatively reduced after HDAC11 inhibition suggesting that glycolysis is functionally suppressed. Glycolytic pathway disruption was associated with a compensatory increase in oxygen consumption and ATP production through oxidative phosphorylation in these oncogene transformed tumor cells, but not in their non-transformed counterparts. Suppression of fatty acid oxidation by inhibiting carnitine palmitoyltransferase 1 (CPT-1) or blocking glutamine utilization by inhibiting glutaminase (GLS1) in combination with HDAC11 inhibition resulted in a cooperative reduction in cellular ATP levels further supporting a direct role of HDAC11 in regulating glycolysis in tumor cells. For the first time, this study mechanistically and functionally defines a cytoplasmic non-histone protein regulated by HDAC11. Citation Format: Vasundhara Sharma, Agni Christodoulidou, Lanzhu Yue, Aileen Y. Alontaga, William E. Goodheart, Rebecca Hesterberg, Xiaozhang Zheng, Matthew W. Martin, Jennifer Y. Lee, Pearlie K. Burnette, Kenneth L. Wright. HDAC11 regulates lysine acetylation of enolase 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-249.

Rapidly evolving workloads and exploding data volumes place great pressure on data-center compute, IO, and memory performance, and especially on memory capacity. Increasing memory capacity requires a commensurate reduction in memory cost per bit. DRAM technology scaling has been steadily delivering affordable capacity increases, but DRAM scaling is rapidly reaching physical limits. Other technologies such as Flash, enhanced Flash, Phase Change Memory, and Spin Torque Transfer Magnetic RAM hold promise for creating high capacity memories at lower cost per bit. However, these technologies have attributes that require careful management. We propose a hybrid DIMM architecture that uses a hardware-managed DRAM in front of enhanced Flash, which has much lower read latencies than conventional Flash. We explore the design space of such SCM devices in the context of different technology parameters, evaluating performance and endurance for data-center workloads. Our hybrid memory architecture is commercially realizable and can use standard DIMM form factors, giving it a low barrier to market entry. We find that for workloads like media streaming, enhanced Flash can be combined with DRAM to enable 88% of the performance of a DRAM-only system of the same capacity at 23% of the cost, even when factoring in replacement costs due to wear-out. The bottom line is that cost per performance is a factor of 3.8 better than DRAM.

The data presented here are related to the research article entitled "Selective expression of the transcription elongation factor ELL3 in B cells prior to ELL2 drives proliferation and survival" (Alexander et al., 2017) [1]. The cited research article characterizes Eleven-nineteen Lysine-rich Leukemia 3 (ELL3) expression in the B cell compartment and functional dependence in B lymphoma cell lines. This data report describes the mRNA expression pattern in a panel of cell lines representing the B cell compartment, supplementing the protein expression data presented in the associated research report. In addition, a reanalysis is presented of publicly available mRNA expression data from primary murine B cells to reveal dynamic regulation of the ELL family members post LPS stimulation (Barwick et al., 2016) [2]. The effect of ELL3 depletion on cell morphology, latent Epstein Barr Virus (EBV) lytic replication and differentiation markers in a Burkitt's lymphoma (BL) cell line cells are presented.