Progression from the Common Lymphoid Progenitor to B/Myeloid PreproB and ProB Precursors during B Lymphopoiesis Requires C/EBPα.

The C/EBPα transcription factor is required for myelopoiesis, with prior observations suggesting additional contributions to B lymphopoiesis. Cebpa expression is evident in common lymphoid progenitor (CLP) and preproB cells but is absent in proB and preB cells. We previously observed that marrow lacking the Cebpa +37 kb enhancer is impaired in producing B cells upon competitive transplantation. Additionally, a Cebpa enhancer/promoter-hCD4 transgene is expressed in B/myeloid CFU. Extending these findings, pan-hematopoietic murine Cebpa enhancer deletion using Mx1-Cre leads to expanded CLP, fewer preproB cells, markedly reduced proB and preB cells, and reduced mature B cells, without affecting T cell numbers. In contrast, enhancer deletion at the proB stage using Mb1-Cre does not impair B cell maturation. Further evaluation of CLP reveals that the Cebpa transgene is expressed almost exclusively in Flt3+ multipotent CLP versus B cell-restricted Flt3- CLP. In vitro, hCD4+ preproB cells produce both B and myeloid cells, whereas hCD4- preproB cells only produce B cells. Additionally, a subset of hCD4- preproB cells express high levels of RAG1-GFP, as seen also in proB cells. Global gene expression analysis indicates that hCD4+ preproB cells express proliferative pathways, whereas B cell development and signal transduction pathways predominate in hCD4- preproB cells. Consistent with these changes, Cebpa enhancer-deleted preproB cells downmodulate cell cycle pathways while upregulating B cell signaling pathways. Collectively, these findings indicate that C/EBPα is required for Flt3+ CLP maturation into preproB cells and then for proliferative Cebpaint B/myeloid preproB cells to progress to Cebpalo B cell-restricted preproB cells and finally to Cebpaneg proB cells.

Tankyrase inhibition promotes a stable human naïve pluripotent state with improved functionality.

The derivation and maintenance of human pluripotent stem cells (hPSCs) in stable naïve pluripotent states has a wide impact in human developmental biology. However, hPSCs are unstable in classical naïve mouse embryonic stem cell (ESC) WNT and MEK/ERK signal inhibition (2i) culture. We show that a broad repertoire of conventional hESC and transgene-independent human induced pluripotent stem cell (hiPSC) lines could be reverted to stable human preimplantation inner cell mass (ICM)-like naïve states with only WNT, MEK/ERK, and tankyrase inhibition (LIF-3i). LIF-3i-reverted hPSCs retained normal karyotypes and genomic imprints, and attained defining mouse ESC-like functional features, including high clonal self-renewal, independence from MEK/ERK signaling, dependence on JAK/STAT3 and BMP4 signaling, and naïve-specific transcriptional and epigenetic configurations. Tankyrase inhibition promoted a stable acquisition of a human preimplantation ICM-like ground state via modulation of WNT signaling, and was most efficacious in efficiently reprogrammed conventional hiPSCs. Importantly, naïve reversion of a broad repertoire of conventional hiPSCs reduced lineage-primed gene expression and significantly improved their multilineage differentiation capacities. Stable naïve hPSCs with reduced genetic variability and improved functional pluripotency will have great utility in regenerative medicine and human disease modeling.

ZNF143 protein is an important regulator of the myeloid transcription factor C/EBPα.

The transcription factor C/EBPα is essential for myeloid differentiation and is frequently dysregulated in acute myeloid leukemia. Although studied extensively, the precise regulation of its gene by upstream factors has remained largely elusive. Here, we investigated its transcriptional activation during myeloid differentiation. We identified an evolutionarily conserved octameric sequence, CCCAGCAG, ∼100 bases upstream of the CEBPA transcription start site, and demonstrated through mutational analysis that this sequence is crucial for C/EBPα expression. This sequence is present in the genes encoding C/EBPα in humans, rodents, chickens, and frogs and is also present in the promoters of other C/EBP family members. We identified that ZNF143, the human homolog of the Xenopus transcriptional activator STAF, specifically binds to this 8-bp sequence to activate C/EBPα expression in myeloid cells through a mechanism that is distinct from that observed in liver cells and adipocytes. Altogether, our data suggest that ZNF143 plays an important role in the expression of C/EBPα in myeloid cells.

Absence of host NF-κB p50 induces murine glioblastoma tumor regression, increases survival, and decreases T-cell induction of tumor-associated macrophage M2 polarization.

High-grade gliomas harbor abundant myeloid cells that suppress anti-tumor immunity and support tumor growth. Targeting transcription factors, such as NF-κB p50, that mediate suppressive myeloid M2 polarization may prove therapeutic. GL261-Luc glioblastoma cells were inoculated into wild-type and p50-/- mice, followed by analysis of tumor growth, survival, tumor myeloid cells, and T cells. The absence of host p50 slows tumor growth and enables regression in 30% of recipients, leading to prolonged survival. Tumors developing in p50-/- mice possess a greater concentration of tumor-infiltrating myeloid cells (TIMs) than those in wild-type mice. TIMs are predominantly F4/80hi macrophages which, along with tumor-associated microglia, express increased pro-inflammatory M1 and reduced immune-suppressive M2 markers. In p50-/- mice, total tumor CD4 T cells are threefold more abundant, whereas CD8 T-cell numbers are unchanged, and both produce increased IFNγ and Granzyme B. Naïve splenic p50-/- CD8 T cells manifest increased activation, whereas naïve p50-/- and WT CD4 T cells show similar Th1, Th2, and Th17 polarization. Antibody targeting CD4, but not CD8, fully obviates the p50-/- survival advantage. Combined CD4 and CD8 T-cell depletion reverses myeloid M2 polarization in wild-type hosts, without affecting myeloid M1 polarization in p50-/- hosts. Finally, gliomas grow similarly in p50(f/f) and p50(f/f);Lysozyme-Cre mice, the latter having reduced p50 specifically in myeloid cells and tumor microglia. Thus, high-grade glioma T cells play a key role in directing M2 polarization of tumor myeloid cells, and reducing NF-κB p50 in both tumor myeloid cells and T cells may contribute to glioma therapy.

Runx1 Phosphorylation by Src Increases Trans-activation via Augmented Stability, Reduced Histone Deacetylase (HDAC) Binding, and Increased DNA Affinity, and Activated Runx1 Favors Granulopoiesis.

Src phosphorylates Runx1 on one central and four C-terminal tyrosines. We find that activated Src synergizes with Runx1 to activate a Runx1 luciferase reporter. Mutation of the four Runx1 C-terminal tyrosines to aspartate or glutamate to mimic phosphorylation increases trans-activation of the reporter in 293T cells and allows induction of Cebpa or Pu.1 mRNAs in 32Dcl3 myeloid cells, whereas mutation of these residues to phenylalanine to prevent phosphorylation obviates these effects. Three mechanisms contribute to increased Runx1 activity upon tyrosine modification as follows: increased stability, reduced histone deacetylase (HDAC) interaction, and increased DNA binding. Mutation of the five modified Runx1 tyrosines to aspartate markedly reduced co-immunoprecipitation with HDAC1 and HDAC3, markedly increased stability in cycloheximide or in the presence of co-expressed Cdh1, an E3 ubiquitin ligase coactivator, with reduced ubiquitination, and allowed DNA-binding in gel shift assay similar to wild-type Runx1. In contrast, mutation of these residues to phenylalanine modestly increased HDAC interaction, modestly reduced stability, and markedly reduced DNA binding in gel shift assays and as assessed by chromatin immunoprecipitation with the -14-kb Pu.1 or +37-kb Cebpa enhancers after stable expression in 32Dcl3 cells. Affinity for CBFß, the Runx1 DNA-binding partner, was not affected by these tyrosine modifications, and in vitro translated CBFß markedly increased DNA affinity of both the translated phenylalanine and aspartate Runx1 variants. Finally, further supporting a positive role for Runx1 tyrosine phosphorylation during granulopoiesis, mutation of the five Src-modified residues to aspartate but not phenylalanine allows Runx1 to increase Cebpa and granulocyte colony formation by Runx1-deleted murine marrow.

RUNX1 and CBFß Mutations and Activities of Their Wild-Type Alleles in AML.

Mutations in RUNX1 and CBFB have long been recognized as important in hematological malignancies. Point mutations and deletions of RUNX1 are frequently found in myelodysplastic syndrome, myeloproliferative disease, and acute myeloid leukemia. Germline mutations in RUNX1 are associated with familial platelet disorder with predisposition to AML. In addition, as will be discussed in other chapters, both RUNX1 and CBFB are involved in recurrent chromosomal rearrangements in leukemia. More recently, roles for the non-mutated RUNX1 and CBFB genes have been identified in multiple leukemia subtypes. This chapter will discuss the roles of RUNX1 and CBFB, both in diseases caused by their mutations or deletions, as well as in the context of chromosomal rearrangements.

A review of the literature for intra-arterial chemotherapy used to treat retinoblastoma.

Retinoblastoma is a malignancy of the retina that usually presents before the age of 5 years. Sporadic retinoblastoma is most often unilateral and with no hereditary influence, whereas familial retinoblastoma presents unilaterally or bilaterally in conjunction with genetic inheritance. Several treatments have been attempted with the goals of saving the child's life, salvaging the eye, and preserving vision. Alternative methods including external beam radiation, systemic chemotherapy and focal therapies have been shown to be effective but carry a risk of enucleation and other complications proportional to the severity of the tumor. Selective intra-arterial chemotherapy for retinoblastoma began in 1988 in Japan and has emerged in the last 7 years in the United States as a feasible, effective and minimally invasive treatment option. We review the retinoblastoma treatment literature focusing on intra-arterial chemotherapy.

Runx1 deletion or dominant inhibition reduces Cebpa transcription via conserved promoter and distal enhancer sites to favor monopoiesis over granulopoiesis.

Deletion of Runx1 in adult mice produces a myeloproliferative phenotype. We now find that Runx1 gene deletion increases marrow monocyte while reducing granulocyte progenitors and that exogenous RUNX1 rescues granulopoiesis. Deletion of Runx1 reduces Cebpa mRNA in lineage-negative marrow cells and in granulocyte-monocyte progenitors or common myeloid progenitors. Pu.1 mRNA is also decreased, but to a lesser extent. We also transduced marrow with dominant-inhibitory RUNX1a. As with Runx1 gene deletion, RUNX1a expands lineage-Sca-1+c-kit+ and myeloid cells, increased monocyte CFUs relative to granulocyte CFUs, and reduced Cebpa mRNA. Runx1 binds a conserved site in the Cebpa promoter and binds 4 sites in a conserved 450-bp region located at +37 kb; mutation of the enhancer sites reduces activity 6-fold in 32Dcl3 myeloid cells. Endogenous Runx1 binds the promoter and putative +37 kb enhancer as assessed by ChIP, and RUNX1-ER rapidly induces Cebpa mRNA in these cells, even in cycloheximide, consistent with direct gene regulation. The +37 kb region contains strong H3K4me1 histone modification and p300-binding, as often seen with enhancers. Finally, exogenous C/EBPα increases granulocyte relative to monocyte progenitors in Runx1-deleted marrow cells. Diminished CEBPA transcription and consequent impairment of myeloid differentiation may contribute to leukemic transformation in acute myeloid leukemia cases associated with decreased RUNX1 activity.

SHP2 tyrosine phosphatase stimulates CEBPA gene expression to mediate cytokine-dependent granulopoiesis.

G-CSF signals contribute to granulocyte lineage specification. We previously found that G-CSF induces SHP2 tyrosine phosphorylation and that chemical inhibition of SHP1/SHP2 reduces CFU-G and prevents G-CSF but not M-CSF activation of ERK. We now find that SHP2 shRNA knockdown in the 32Dcl3 granulocytic line reduces ERK activation, diminishes CEBPA protein and RNA expression and promoter histone acetylation, and inhibits granulopoiesis. Exogenous, shRNA-resistant SHP2 rescues these effects of SHP2 knockdown, exogenous C/EBPα rescues granulocytic markers, and exogenous RUNX1 rescues C/EBPα. 32Dcl3 lines with knockdown of ERK1 and ERK2 retain normal levels of C/EBPα and differentiate normally in G-CSF despite also having reduced proliferation. SHP2 knockdown reduces CEBPA levels in lineage-negative murine marrow cells cultured in TPO, Flt3 ligand, and SCF, without affecting the rate of cell expansion. On transfer to IL-3, IL-6, and SCF to induce myelopoiesis, levels of granulocytic RNAs are reduced and monocyte-specific RNAs are increased by SHP2 knockdown, and there is a reduction in the percentage of CFU-G that form in methylcellulose and of granulocytes that develop in liquid culture. In summary, SHP2 is required for induction of C/EBPα expression and granulopoiesis in response to G-CSF or other cytokines independent of SHP2-mediated ERK activation.

C/EBPα and C/EBPα oncoproteins regulate nfkb1 and displace histone deacetylases from NF-κB p50 homodimers to induce NF-κB target genes.

Mutated CEBPA defines a subgroup of acute myeloid leukemia (AML). We have previously shown that C/EBPα or its AML mutants synergize with NF-κB p50 to activate antiapoptotic genes, including BCL2 and FLIP. Furthermore, p50 binds and activates the CEBPA gene in myeloid cells. We now report that C/EBPα or C/EBPα leucine zipper AML mutants bind in vivo to the nfkb1 (p50) promoter and induce its expression even in the presence of cycloheximide. Induction of p50 by C/EBPα depends on 2 conserved κB sites in the nfkb1 promoter. C/EBPα did not induce p65 expression. Thus, C/EBPα and p50 reciprocally regulate each other's expression, establishing a positive feedback relationship. Although p50 homodimers inhibit transcription, C/EBPα and p50 synergistically activate antiapoptotic genes. ChIP analysis showed that C/EBPα diminishes the occupation of histone deacetylase 1 (HDAC1) or HDAC3 on the endogenous FLIP promoter but not in mice lacking p50. Coimmunoprecipitation confirmed that C/EBPα, its AML variants, or C/EBPß disrupt interaction between p50 and HDACs dependent on the C/EBP basic region. These findings suggest that C/EBPs displace HDACs from p50 homodimers bound to antiapoptotic genes, contributing to NF-κB dysregulation in leukemia, and that the C/EBPα:p50 complex is a potential therapeutic target.

Identification of a specific intronic PEAR1 gene variant associated with greater platelet aggregability and protein expression.

Genetic variation is thought to contribute to variability in platelet function; however, the specific variants and mechanisms that contribute to altered platelet function are poorly defined. With the use of a combination of fine mapping and sequencing of the platelet endothelial aggregation receptor 1 (PEAR1) gene we identified a common variant (rs12041331) in intron 1 that accounts for ≤ 15% of total phenotypic variation in platelet function. Association findings were robust in 1241 persons of European ancestry (P = 2.22 × 10⁻8) and were replicated down to the variant and nucleotide level in 835 persons of African ancestry (P = 2.31 × 10⁻²7) and in an independent sample of 2755 persons of European descent (P = 1.64 × 10⁻5). Sequencing confirmed that variation at rs12041331 accounted most strongly (P = 2.07 × 10⁻6) for the relation between the PEAR1 gene and platelet function phenotype. A dose-response relation between the number of G alleles at rs12041331 and expression of PEAR1 protein in human platelets was confirmed by Western blotting and ELISA. Similarly, the G allele was associated with greater protein expression in a luciferase reporter assay. These experiments identify the precise genetic variant in PEAR1 associated with altered platelet function and provide a plausible biologic mechanism to explain the association between variation in the PEAR1 gene and platelet function phenotype.

Phosphorylation of RUNX1 by cyclin-dependent kinase reduces direct interaction with HDAC1 and HDAC3.

RUNX1 regulates formation of the definitive hematopoietic stem cell and its subsequent lineage maturation, and mutations of RUNX1 contribute to leukemic transformation. Phosphorylation of Ser-48, Ser-303, and Ser-424 by cyclin-dependent kinases (cdks) increases RUNX1 trans-activation activity without perturbing p300 interaction. We now find that endogenous RUNX1 interacts with endogenous HDAC1 or HDAC3. Mutation of the three RUNX1 serines to aspartic acid reduces co-immunoprecipitation with HDAC1 or HDAC3 when expressed in 293T cells; mutation of these three serines to alanine increases HDAC interaction, and mutation of each serine individually to aspartic acid also reduces these interactions. GST-RUNX1 isolated from bacterial extracts bound in vitro translated HDAC1 or HDAC3, and these interactions were weakened by mutation of Ser-48, Ser-303, and Ser-424 to aspartic acid. The ability of RUNX1 phosphorylation and not only serine to aspartic acid conversion to reduce HDAC1 binding was demonstrated using wild-type GST-RUNX1 phosphorylated in vitro using cdk1/cyclinB and by exposure of 293T cells transduced with RUNX1 and HDAC1 to roscovitine, a cdk inhibitor. Finally, RUNX1 or RUNX1(tripleD), in which Ser-48, Ser-303, and Ser-424 are mutated to aspartic acid, stimulated proliferation of transduced, lineage-negative murine marrow progenitors more potently than did RUNX1(tripleA), in which these serines are mutated to alanine, suggesting that stimulation of RUNX1 trans-activation by cdk-mediated reduction in HDAC interaction increases marrow progenitor cell proliferation.

M-CSF elevates c-Fos and phospho-C/EBPalpha(S21) via ERK whereas G-CSF stimulates SHP2 phosphorylation in marrow progenitors to contribute to myeloid lineage specification.

The role of hematopoietic cytokines in lineage commitment remains uncertain. To gain insight into the contribution of cytokine signaling to myeloid lineage specification, we compared granulocyte colony-stimulating factor (G-CSF) and macrophage colony-stimulating factor (M-CSF) signaling in Ba/F3 cells expressing both the G-CSF and M-CSF receptors and in lineage-negative murine marrow cells. G-CSF and M-CSF serve as prototypes for additional cytokines that also influence immature myeloid cells. G-CSF specifically activated signal transducer and activator of transcription 3 and induced Src homology region 2 domain-containing phosphatase 2 (SHP2) phosphorylation, whereas M-CSF preferentially activated phospholipase Cgamma2, and thereby extracellular signal-regulated kinase (ERK), to stabilize c-Fos and stimulate CCAAT/enhancer-binding protein (C/EBP)alpha(S21) phosphorylation. In contrast, activation of Jun kinase or c-Jun was similar in response to either cytokine. Inhibition of ERK prevented induction of c-Fos by M-CSF and reduced C/EBPalpha phosphorylation and formation of colony-forming unit-monocytes. SHP2 inhibition reduced ERK activation in G-CSF, but not M-CSF, and reduced colony-forming unit-granulocytes, underscoring divergent pathways to ERK activation. Phorbol ester mimicked the effect of M-CSF, activating ERK independent of SHP2. In summary, M-CSF activates ERK more potently than G-CSF, and thereby induces higher levels of c-Fos and phospho-C/EBPalpha(S21), which may directly interact to favor monopoiesis, whereas G-CSF activates signal transducer and activator of transcription 3 and SHP2, potentially shifting the balance to granulopoiesis via gene induction by C/EBPalpha homodimers and via effects of SHP2 on regulators besides ERK.

Twist-2 controls myeloid lineage development and function.

Basic helix-loop-helix (bHLH) transcription factors play critical roles in lymphoid and erythroid development; however, little is known about their role in myeloid lineage development. In this study, we identify the bHLH transcription factor Twist-2 as a key negative regulator of myeloid lineage development, as manifested by marked increases in mature myeloid populations of macrophages, neutrophils, and basophils in Twist-2-deficient mice. Mechanistic studies demonstrate that Twist-2 inhibits the proliferation as well as differentiation of granulocyte macrophage progenitors (GMP) by interacting with and inhibiting the transcription factors Runx1 and C/EBPalpha. Moreover, Twist-2 was found to have a contrasting effect on cytokine production: inhibiting the production of proinflammatory cytokines such as interleukin-12 (IL-12) and interferon-gamma (IFNgamma) while promoting the regulatory cytokine IL-10 by myeloid cells. The data from further analyses suggest that Twist-2 activates the transcription factor c-Maf, leading to IL-10 expression. In addition, Twist-2 was found to be essential for endotoxin tolerance. Thus, this study reveals the critical role of Twist-2 in regulating the development of myeloid lineages, as well as the function and inflammatory responses of mature myeloid cells.

NF-kappa B p50 regulates C/EBP alpha expression and inflammatory cytokine-induced neutrophil production.

NF-kappaB is a key transcriptional inducer of the inflammatory response in mature myeloid cells, and also stimulates cell survival, but its role in immature myeloid cell development has not been well characterized. C/EBPalpha is required for the development of monocytic and granulocytic myeloid cells from early progenitors, and NF-kappaB and C/EBPbeta cooperatively induce several inflammatory mediators. Having found that C/EBPalpha binds NF-kappaB p50 preferentially compared with NF-kappaB p65, we have now investigated myelopoiesis in nfkb1(-/-) mice lacking NF-kappaB p50. Absence of p50 leads to a significant reduction in the number of granulocytic progenitors, CFU-granulocyte, obtained with G-CSF or GM-CSF in vitro and reduces neutrophil production in vivo in response to G-CSF, with preservation of monopoiesis in vitro in response to cytokines or LPS. To gain insight into the mechanism underlying reduced granulopoiesis in the absence of NF-kappaB p50, we assessed the expression of several myeloid regulatory proteins in lineage-negative, immature myeloid cells. Although PU.1, C/EBPbeta, and STAT3 levels were unchanged, C/EBPalpha protein and RNA levels were reduced approximately 3-fold in the absence of NF-kappaB p50. In addition, NF-kappaB p50 and C/EBPalpha bound the endogenous C/EBPalpha promoter in a chromatin immunoprecipitation assay, and NF-kappaB p50 trans activated the C/EBPalpha promoter, alone or in cooperation with C/EBPalpha. Despite reduction of C/EBPalpha, G-CSFR and M-CSFR levels were maintained in total marrow and in lineage-negative cells. Together, these data indicate that acute inflammation not only activates mature myeloid cells, but also stimulates neutrophil production via NF-kappaB p50 induction of C/EBPalpha transcription.

Adoptive transfer of immature myeloid cells lacking NF-κB p50 (p50-IMC) impedes the growth of MHC-matched high-risk neuroblastoma.

High-risk neuroblastomas harbor abundant myeloid cells that suppress antitumor immunity and support tumor growth. Macrophages lacking the inhibitory NF-κB p50 subunit adopt a pro-inflammatory phenotype. We now report that murine 9464D neuroblastoma cells, which express high levels of exogenous MYCN, grow slower in syngeneic p50(f/f);Lys-Cre mice that lack p50 in macrophages and neutrophils, compared with p50(f/f) littermates. Tumors in p50(f/f);Lys-Cre mice possess increased numbers of total and activated CD4+ and CD8+ T cells, and depletion of both of these T-cell populations accelerates tumor growth. Anti-PD-1 T-cell checkpoint blockade, or DNA methyltransferase and histone deacetylase inhibition, further slows tumor growth. In addition, adoptive transfer of immature myeloid cells lacking NF-κB p50 (p50-IMC), generated either from the bone marrow of p50-/- mice or via nucleofection of a p50 sgRNA:Cas9 complex into wild-type hematopoietic progenitors, also slowed growth of MHC-matched 9464D tumors but not of MHC-mismatched Neuro2A tumors. These findings further validate the utility of targeting myeloid NF-κB p50 as a strategy for cancer therapy and demonstrate activity of p50-IMC generated by gene editing of syngeneic marrow cells, a cell product relevant to clinical translation.

C/EBPα induces Ebf1 gene expression in common lymphoid progenitors.

C/EBPα is required for formation of granulocyte-monocyte progenitors (GMP) and also participates in B lymphopoiesis. The common lymphoid progenitor (CLP) and preproB populations but not proB cells express Cebpa, and pan-hematopoietic deletion of the +37 kb Cebpa enhancer using Mx1-Cre leads not only to reduced GMP but also to 2-fold reduced marrow preproB and >15-fold reduced proB and preB cells. We now show that IL7Rα-Cre-mediated deletion of the +37 kb Cebpa enhancer, which occurs in 89% of Ly6D+ and 65% of upstream Ly6D- CLP, leads to a 2-fold reduction of both preproB and proB cells, and a 3-fold reduction in preB cells, with no impact on GMP numbers. These data support a direct role for C/EBPα during B lineage development, with reduced enhancer deletion in Ly6D- CLP mediated by IL7Rα-Cre diminishing the effect on B lymphopoiesis compared to that seen with Mx1-Cre. Amongst mRNAs encoding key transcriptional regulators that initiate B lymphoid specification (PU.1, E2A, IKAROS, EBF1, FOXO1, and BACH2), only Ebf1 levels are altered in CLP upon Mx1-Cre-mediated Cebpa enhancer deletion, with Ebf1 reduced ~40-fold in Flt3+Sca-1intc-kitintIL7Rα+ CLP. In addition, Cebpa and Ebf1 RNAs were 4- and 14-fold higher in hCD4+ versus hCD4- CLP from Cebpa-hCD4 transgenic mice. Histone modification ChIP-Seq data for CLP indicate the presence of active, intronic Ebf1 enhancers located 270 and 280 kb upstream of the transcription start sites. We identified a cis element in this region that strongly binds C/EBPα using the electrophoretic mobility shift assay. Mutation of this C/EBPα-binding site in an Ebf1 enhancer-TK-luciferase reporter leads to a 4-fold reduction in C/EBPα-mediated trans-activation. These findings support a model of B lymphopoiesis in which induction of Ebf1 by C/EBPα in a subset of CLP contributes to initiation of B lymphopoiesis.

NF-κB p50-deficient immature myeloid cell (p50-IMC) adoptive transfer slows the growth of murine prostate and pancreatic ductal carcinoma.

BACKGROUND: Macrophages and dendritic cells lacking the transcription factor nuclear factor kappa B p50 are skewed toward a proinflammatory phenotype, with increased cytokine expression and enhanced T cell activation; additionally, murine melanoma, fibrosarcoma, colon carcinoma, and glioblastoma grow slower in p50-/- mice. We therefore evaluated the efficacy of p50-negative immature myeloid cells (p50-IMCs) adoptively transferred into tumor-bearing hosts. Immature cells were used to maximize tumor localization, and pretreatment with 5-fluorouracil (5FU) was examined due to its potential to impair marrow production of myeloid cells, to target tumor myeloid cells and to release tumor neoantigens. METHODS: Wild-type (WT)-IMC or p50-IMC were generated by culturing lineage-negative marrow cells from WT or p50-/- mice in media containing thrombopoietin, stem cell factor and Flt3 ligand for 6 days followed by monocyte colony-stimulating factor for 1 day on ultralow attachment plates. Mice inoculated with Hi-Myc prostate cancer (PCa) cells or K-RasG12D pancreatic ductal carcinoma (PDC)-luciferase cells received 5FU followed 5 days later by three doses of 107 immature myeloid cells (IMC) every 3-4 days. RESULTS: PCa cells grew slower in p50-/- mice, and absence of host p50 prolonged the survival of mice inoculated orthotopically with PDC cells. IMC from Cytomegalovirus (CMV)-luciferase mice localized to tumor, nodes, spleen, marrow, and lung. 5FU followed by p50-IMC slowed PCa and PDC tumor growth, ~3-fold on average, in contrast to 5FU followed by WT-IMC, 5FU alone or p50-IMC alone. Slowed tumor growth was evident for 93% of PCa but only 53% of PDC tumors; we therefore focused on PCa for additional IMC analyses. In PCa, p50-IMC matured into F4/80+ macrophages, as well as CD11b+F4/80-CD11c+ conventional dendritic cells (cDCs). In both tumor and draining lymph nodes, p50-IMC generated more macrophages and cDCs than WT-IMC. Activated tumor CD8+ T cells were increased fivefold by p50-IMC compared with WT-IMC, and antibody-mediated CD8+ T cell depletion obviated slower tumor growth induced by 5FU followed by p50-IMC. CONCLUSIONS: 5FU followed by p50-IMC slows the growth of murine prostate and pancreatic carcinoma and depends on CD8+ T cell activation. Deletion of p50 in patient-derived marrow CD34+ cells and subsequent production of IMC for adoptive transfer may contribute to the therapy of these and additional cancers.

BRAF V600E-mutated metastatic pediatric Wilms tumor with complete response to targeted RAF/MEK inhibition.

Wilms tumor (WT) is the most common renal malignancy of childhood and accounts for 6% of all childhood malignancies. With current therapies, the 5-yr overall survival (OS) for children with unilateral favorable histology WT is greater than 85%. The prognosis is worse, however, for the roughly 15% of patients who relapse, with only 50%-80% OS reported in those with recurrence. Herein, we describe the extended and detailed clinical course of a rare case of a child with recurrent, pulmonary metastatic, favorable histology WT harboring a BRAF V600E mutation. The BRAF V600E mutation, commonly found in melanoma and other cancers, and previously undescribed in WT, has recently been reported by our group in a subset of epithelial-predominant WT. This patient, who was included in that series, presented with unilateral, stage 1, favorable histology WT and was treated with standard chemotherapy. Following the completion of therapy, the patient relapsed with pulmonary metastatic disease, that then again recurred despite an initial response to salvage chemotherapy and radiation. Next-generation sequencing (NGS) on the metastatic pulmonary nodule revealed a BRAF V600E mutation. After weighing the therapeutic options, a novel approach with dual BRAF/MEK inhibitor combination therapy was initiated. Complete radiographic response was observed following 4 months of therapy with dabrafenib and trametinib. At 12 months following the start of BRAF/MEK combination treatment, the patient continues with a complete response and has experienced minimal treatment-related side effects. This represents the first case, to our knowledge, of effective treatment with BRAF/MEK molecularly targeted therapy in a pediatric Wilms tumor patient.