No abstract

Somatic mutations in CREBBP occur frequently in B-cell lymphoma. Here, we show that loss of CREBBP facilitates development of germinal center derived lymphomas in mice. In both human and murine lymphomas CREBBP loss of function resulted in focal depletion of enhancer H3K27 acetylation and aberrant transcriptional silencing of genes that regulate B-cell signaling and immune responses including class II MHC. Mechanistically, CREBBP regulated enhancers are counter-regulated by the BCL6 transcriptional repressor in a complex with SMRT and HDAC3, which we find bind extensively to MHC class II loci. HDAC3 loss of function rescued repression of these enhancers and corresponding genes including MHC class II, and more profoundly suppress CREBPP mutant lymphomas in vitro and in vivo. Hence CREBBP loss of function contributes to lymphomagenesis by enabling unopposed suppression of enhancers by BCL6/SMRT/HDAC3 complexes, suggesting HDAC3 targeted therapy as a precision approach for CREBBP mutant lymphomas.

The Rho family of GTPases represents a class of Ras-related signaling molecules often deregulated in cancer. Rho GTPases switch from a GDP-bound, inactive state to a GTP-bound, active state in response to extracellular stimuli such as mitogens and extracellular matrix. In addition, Rho GTPase signaling can be altered in response to cell intrinsic factors such as changes in oncogenic or tumor suppressor signaling. In their active form, these proteins bind to a number of effector molecules, activating signaling cascades which regulate a variety of cellular processes including cytoskeletal reorganization, cell cycle progression, cell polarity and transcription. Here, we focus on one Rho family member, Cdc42, which is overexpressed in a number of human cancers. Consistent with a role in the promotion of tumorigenesis, activating mutations in Cdc42 and guanine nucleotide exchange factors are transforming, while inhibition of Cdc42 activity can impinge on cellular transformation following the activation of oncoproteins or loss of tumor suppressor function. Furthermore, Cdc42 activity has been implicated in the invasive phenotype which characterizes tumor metastasis, further suggesting that Cdc42 may be a useful target for therapeutic intervention. However, several recent studies in mice have unveiled a putative tumor suppressor function of Cdc42 in several tissue types which may involve cell polarity maintenance, suggesting that the role of Cdc42 in cancer development is complex and may be cell type specific.

SUMMARY Following myocardial infarction (MI), myeloid cells derived from the hematopoietic system drive a sharp increase in systemic leukocyte levels that correlate closely with mortality. The origin of these myeloid cells, and the response of hematopoietic stem and progenitor cells (HSPCs) to MI, however, is unclear. Here, we identify a CCR2+CD150+CD48− LSK hematopoietic subset as the most upstream contributor to emergency myelopoiesis after ischemic organ injury. CCR2+ HSPC have fourfold higher proliferation rates than CCR2−CD150+CD48− LSK cells, display a myeloid differentiation bias, and dominate the migratory HSPC population. We further demonstrate the myeloid translocation gene 16 (Mtg16) regulates CCR2+ HSPC emergence. Mtg16−/− mice have decreased levels of systemic monocytes and infarct-associated macrophages and display compromised tissue healing and post-MI heart failure. Together, these data provide insights into regulation of emergency hematopoiesis after ischemic injury, and identify potential therapeutic targets to modulate leukocyte output after MI.

RhoA, the founding member of mammalian Rho GTPase family, is thought to be essential for actomyosin regulation. To date, the physiologic function of RhoA in mammalian cell regulation has yet to be determined genetically. Here we have created RhoA conditional knock-out mice. Mouse embryonic fibroblasts deleted of RhoA showed no significant change in actin stress fiber or focal adhesion complex formation in response to serum or LPA, nor any detectable change in Rho-kinase signaling activity. RhoA is a founding member of the mammalian Rho GTPase family and has been implicated in a variety of roles in cell regulation, mostly by the dominant negative mutant expression approach or applying C3 bacterial toxin that covalently modifies the effector domain of RhoA and other related Rho GTPases (1-3). Among the widely accepted cell functions, RhoA is considered essential for actin stress fiber formation and actomyosin contractility, focal adhesion complex and adherens junction complex formation, gene transcription, cell cycle progression, and survival (1, 4, 5). Concomitant knock-out or knockdown of RhoB and RhoC in theVertebrates have two closely related family members, RhoB and RhoC, that share significant sequence homology with RhoA (6). RhoB is localized primarily on endosome membranes, whereas RhoC, like RhoA, is cytosolic (7,8). Additionally, RhoB and RhoC appear to show different, and sometimes opposite, cell functions (8, 9). Neither RhoB nor RhoC knockout mice display detectable developmental defects, and no clear phenotypes were reported from primary cells derived from these gene-targeted animals (10 -12).In the present studies, we present genetic data to unambiguously demonstrate an essential role of RhoA in regulating cell cytokinesis and a dispensable, redundant role with RhoB and RhoC in regulating the actomyosin and adhesion machineries in primary mouse embryonic fibroblasts (MEFs). 2 EXPERIMENTAL PROCEDURESMice-Mice harboring conditional RhoA alleles, in which exon 3 is flanked by loxP sites (RhoA f/f ), were generated as depicted in supplemental Fig. S1.Mouse Embryonic Fibroblast Isolation and Cell CulturePrimary MEFs were isolated from mouse embryos at embryonic day 12.5 as described previously (13). See supplemental Experimental Procedures for additional details. RESULTS AND DISCUSSIONRhoA Is Dispensable for Actomyosin Signaling-Our current knowledge of the cellular functions of RhoA is based primarily on numerous studies performed in fibroblast or epithelial cells by nonspecific mutant overexpression or toxin treatment (13)(14)(15)(16)(17)(18). To investigate the physiologic role of RhoA in cell regulation, we generated a conditional knock-out mouse model in which exon 3 of the RhoA gene, which encodes the guanine nucleotide binding sequences, was sandwiched between loxP sequences to allow Cre recombinase-mediated gene targeting (supplemental Fig. S1). Primary MEFs were derived from homozygous embryos, and deletion of RhoA gene was achieved by infection of the cells with adenovirus transiently expressin...

Histone deacetylase 3 (HDAC3) contributes to the regulation of gene expression, chromatin structure, and genomic stability. Because HDAC3 associates with oncoproteins that drive leukemia and lymphoma, we engineered a conditional deletion allele in mice to explore the physiological roles of Hdac3 in hematopoiesis. We used the Vav-Cre transgenic allele to trigger recombination, which yielded a dramatic loss of lymphoid cells, hypocellular bone marrow, and mild anemia. Phenotypic and functional analysis suggested that Hdac3 was required for the formation of the earliest lymphoid progenitor cells in the marrow, but that the marrow contained 3-5 times more multipotent progenitor cells. Hdac3 -/-stem cells were severely compromised in competitive bone marrow transplantation. In vitro, Hdac3 -/-stem and progenitor cells failed to proliferate, and most cells remained undifferentiated. Moreover, one-third of the Hdac3 -/-stem and progenitor cells were in S phase 2 hours after BrdU labeling in vivo, suggesting that these cells were impaired in transit through the S phase. DNA fiber-labeling experiments indicated that Hdac3 was required for efficient DNA replication in hematopoietic stem and progenitor cells. Thus, Hdac3 is required for the passage of hematopoietic stem/progenitor cells through the S phase, for stem cell functions, and for lymphopoiesis.

Given the fundamental roles of histone deacetylases (HDACs) in the regulation of DNA repair, replication, transcription and chromatin structure, it is fitting that therapies targeting HDAC activities are now being explored as anti-cancer agents. In fact, two histone deacetylase inhibitors (HDIs), SAHA and Depsipeptide, are FDA approved for single-agent treatment of refractory cutaneous T cell lymphoma (CTCL). An important target of these HDIs, histone deacetylase 3 (HDAC3), regulates processes such as DNA repair, metabolism, and tumorigenesis through the regulation of chromatin structure and gene expression. Here we show that HDAC3 inhibition using a first in class selective inhibitor, RGFP966, resulted in decreased cell growth in CTCL cell lines due to increased apoptosis that was associated with DNA damage and impaired S phase progression. Through isolation of proteins on nascent DNA (iPOND), we found that HDAC3 was associated with chromatin and is present at and around DNA replication forks. DNA fiber labeling analysis showed that inhibition of HDAC3 resulted in a significant reduction in DNA replication fork velocity within the first hour of drug treatment. These results suggest that selective inhibition of HDAC3 could be useful in treatment of CTCL by disrupting DNA replication of the rapidly cycling tumor cells, ultimately leading to cell death.

Summary Bromodomain and extra-terminal domain (BET) family inhibitors offer an approach to treating hematological malignancies. We used precision nuclear run-on transcription sequencing (PRO-seq) to create high-resolution maps of active RNA polymerases across the genome in t(8;21) acute myeloid leukemia (AML) which are exceptionally sensitive to BET inhibitors. PRO-seq identified over 1400 genes showing impaired release of promoter-proximal paused RNA polymerases, including the stem cell factor receptor tyrosine kinase KIT that is mutated in t(8;21) AML. PRO-seq also identified an enhancer 3′ to KIT. Chromosome conformation capture confirmed contacts between this enhancer and the KIT promoter, while CRISPRi-mediated repression of this enhancer impaired cell growth. PRO-seq also identified microRNAs, including MIR29C and MIR29B2, that target the anti-apoptotic factor MCL1 and were repressed by BET inhibitors. MCL1 protein was up-regulated, and inhibition of BET proteins sensitized t(8:21)-containing cells to MCL1 inhibition, suggesting a potential mechanism of resistance to BET inhibitor-induced cell death.