Salicylate and acetylsalicylic acid are potent and widely used anti-inflammatory drugs. They are thought to exert their therapeutic effects through multiple mechanisms, including the inhibition of cyclo-oxygenases, modulation of NF-κB activity, and direct activation of AMPK. However, the full spectrum of their activities is incompletely understood. Here we show that salicylate specifically inhibits CBP and p300 lysine acetyltransferase activity in vitro by direct competition with acetyl-Coenzyme A at the catalytic site. We used a chemical structure-similarity search to identify another anti-inflammatory drug, diflunisal, that inhibits p300 more potently than salicylate. At concentrations attainable in human plasma after oral administration, both salicylate and diflunisal blocked the acetylation of lysine residues on histone and non-histone proteins in cells. Finally, we found that diflunisal suppressed the growth of p300-dependent leukemia cell lines expressing AML1-ETO fusion protein in vitro and in vivo. These results highlight a novel epigenetic regulatory mechanism of action for salicylate and derivative drugs.

To mount an effective immune response against infectious pathogens or tissue injury, hematopoietic stem cells (HSCs) increase their proliferation and production of myeloid cells, including macrophages, which destroy the pathogens and repair the damaged tissue. Proper resolution of inflammation is essential to restore hematopoietic homeostasis, as unrestrained inflammation can result in life-threatening pathologies such as sepsis, autoimmune disorders and cancer. The molecular mechanisms that control the resolution of inflammation, and how these contribute to disease phenotypes, are poorly understood. BAF (SWI/SNF) complexes are ATPase dependent chromatin-remodeling complexes that play fundamental roles in transcription. BAF complexes use the energy of ATP to modulate the accessibility of transcription factors to DNA and thus, orchestrate the gene expression programs that control proliferation and cellular identity. Genes encoding for BAF subunits are frequently mutated in cancer and developmental disorders. In hematopoietic malignancies, loss-of-function mutations and low expression of specific BAF subunits have been reported in patients with anemia and bone marrow failure. Work from our lab previously demonstrated that the hematopoietic-specific BAF complex subunit Dpf2 cooperates with the transcription factor Runx1 to regulate myeloid differentiation. Based on these studies, we generated a hematopoietic-specific Dpf2 knock-out mouse model and found that mice lacking Dpf2 develop pancytopenia, anemia and an uncontrolled inflammatory response that results in early death. Dpf2-/- peripheral blood samples showed dysplastic features including increased number of polychromatophilic blood cells and Howell-Jolly bodies in erythrocytes. Histopathological analyses revealed the presence of fibrosis and prominent infiltration of histiocytes in multiple organs, including lungs, liver and spleen. Detailed chemical profiling of plasma showed increased levels of multiple pro-inflammatory cytokines, indicative of systemic inflammation. Flow cytometry analyses and Mass cytometry profiling further revealed an expansion of myeloid lineages, specifically monocytes and macrophages, concomitant with severe defects in lymphoid and erythroid differentiation. We also found that Dpf2-/-HSCs had increased serial re-plating capacity and a marked myeloid differentiation bias. To identify the transcription factor networks underlying these phenotypes, we performed RNAseq and ATACseq on control and Dpf2-/- HSCs. Gene Set Enrichment Analyses indicated that Dpf2-/- HSCs have extensive gene expression alterations in immune signaling and interferon response pathways, as well as leukocyte and erythroid differentiation. We also found that Dpf2 loss results in pronounced changes in expression and genomic accessibility of specific transcription factors that control macrophage differentiation and proliferation. Together, our mechanistic studies support a model whereby the absence of Dpf2 results in misregulation of the transcription factor networks that establish macrophage cell identity, leading to a marked increase in macrophage infiltrations and shortened survival of the mice. Treatment of the Dpf2-/-mice with clodronate-containing liposomes, which deplete macrophages from bone marrow and spleen, prolonged survival of the mice. Our work uncovers a novel role of Dpf2 in restraining inflammatory responses by controlling macrophage proliferation and function. Moreover, we propose that, in addition to their tumor suppressive roles in cancer, BAF complexes may have a central role in the prevention of immunopathologies. Disclosures Kadoch: Foghorn Therapeutics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Scientific founder, fiduciary board of directors member, scientific advisory board member, shareholder, and consultant for Foghorn Therapeutics (Cambridge, MA). . Vega:NCI: Research Funding.

During emergency hematopoiesis, hematopoietic stem cells (HSCs) rapidly proliferate to produce myeloid and lymphoid effector cells, a response that is critical against infection or tissue injury. If unresolved, this process leads to sustained inflammation, which can cause life-threatening diseases and cancer. Here, we identify a role of double PHD fingers 2 (DPF2) in modulating inflammation. DPF2 is a defining subunit of the hematopoiesis-specific BAF (SWI/SNF) chromatin-remodeling complex, and it is mutated in multiple cancers and neurological disorders. We uncovered that hematopoiesis-specific Dpf2-KO mice developed leukopenia, severe anemia, and lethal systemic inflammation characterized by histiocytic and fibrotic tissue infiltration resembling a clinical hyperinflammatory state. Dpf2 loss impaired the polarization of macrophages responsible for tissue repair, induced the unrestrained activation of Th cells, and generated an emergency-like state of HSC hyperproliferation and myeloid cell-biased differentiation. Mechanistically, Dpf2 deficiency resulted in the loss of the BAF catalytic subunit BRG1 from nuclear factor erythroid 2-like 2-controlled (NRF2-controlled) enhancers, impairing the antioxidant and antiinflammatory transcriptional response needed to modulate inflammation. Finally, pharmacological reactivation of NRF2 suppressed the inflammation-mediated phenotypes and lethality of Dpf2Δ/Δ mice. Our work establishes an essential role of the DPF2-BAF complex in licensing NRF2-dependent gene expression in HSCs and immune effector cells to prevent chronic inflammation.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.