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•Cullin-5 regulates production of megakaryocyte-committed stem cells to prevent excess megakaryopoiesis.•Cullin-5-deficient megakaryopoiesis is largely independent of TPO and involves signaling via the beta-common and/or beta-IL-3 receptors.

•We identified 7 new genetic regions for factor VIII levels, 1 for von Willebrand factor levels, and 3 in a combined analysis.•Silencing B3GNT2and CD36reduced factor VIII release in vitro.Silencing B3GNT2, CD36, and PDIA3reduced von Willebrand factor release.

Germlinecoding and enhancer variants within the hematopoietic regulator GATA2create a bone marrow failure and leukemia predisposition (Soukup and Bresnick, 2020). We generated a mouse model of GATA2 deficiency syndrome by engineering mice with a single-nucleotide variant in an intronic enhancer (+9.5) from GATA2 deficiency patients combined with deletion of the enhancer from the second allele (compound heterozygous; CH). This system models patients harboring a germline pathogenic allele accompanied by epigenetic silencing of the normal allele. Unlike homozygous Gata2coding or regulatory deletions that are embryonic lethal, CH represents a unique system to analyze postnatal GATA2 function. We demonstrated that adult germline Gata2variation alters steady-state HSPCs, blocks HSPC expansion and differentiation upon chemotherapy, attenuates long-term repopulating activity following bone marrow transplantation, leads to bone marrow failure following chronic inflammation by the viral mimetic polyI:C, alters HSPC response to the bacterial cell wall component LPS and attenuates HSPC mobilization in response to granulocyte colony stimulating factor (G-CSF) (Soukup et al., 2019, Soukup et al., 2021).

A growing body of evidence from preclinical and human epidemiology studies of multiple cancer types indicate that physical activity can delay or avert the outgrowth of cancer, in a mechanistic process that may involve exercise-induced alterations to anti-cancer immunity. Many Chronic Lymphocytic leukaemia (CLL) patients present with asymptomatic, early-stage disease that is monitored until disease progression. Thus, exercise may be an effective way to manage disease burden and delay progression in treatment naïve CLL. The primary objective of this pilot study was to investigate the safety and feasibility of an exercise programme in people with treatment naïve CLL, and preliminarily explore the effects of exercise training on CLL counts, body composition, cardiorespiratory fitness, and immune cell phenotypes including T-cells.

Innate immune processes impact diverse hematopoietic stem and progenitor cell (HSPC) functions, yet how innate immune signaling networks are sensed by HSPC genomes and how HSPC genomic activity alters cell responsiveness to innate immune regulators are not fully understood. We demonstrated that E14.5 primary (-77 -/-) and immortalized (hi-77 -/-) GATA2-deficient fetal progenitors from Gata2-77 enhancer deletion mice upregulate Interferon-gamma (IFNγ) and Toll-like receptor (TLR) signaling components (Johnson et al. JEM2020) and are hypersensitive to IFNγ and TLR signaling (Tran et al. iScience2023). Combinatorial signaling involving both pathways regulates genes that are not regulated by individual pathways and amplifies responses beyond that induced by single pathways. Innate immune-activated genes harbor motifs for signal-dependent transcription factors, including the ETS factor PU.1, and GATA2 deficiency elevates PU.1 activity to upregulate B-lineage and myeloid genes (Jung et al. JCI2023). We hypothesize that PU.1 links innate immune signaling networks and HSPC genome control. In new multi-omic studies, we asked if PU.1 is required to sense exclusively IFNγ or TLR signaling, both pathways independently or combinatorially, or is not essential. We used hi-77 -/-progenitors lacking a PU.1 enhancer ( Spi1URE-/-) with ~2-fold lower PU.1 vs. control hi-77 -/-progenitors. RNA-seq was conducted with hi-77 -/-and hi-77 -/-; Spi1URE-/-progenitors, with or without IFNγ, TLR1/2 agonist Pam 3CSK 4, or both agents for 4 h. hi-77 -/-; Spi1URE-/-progenitors lost the responsiveness of 27 TLR-activated genes and retained the responsiveness of 6 TLR-activated genes. 80% of IFNγ-activated genes (115 of 144) retained IFNγ-responsiveness when PU.1 was reduced. In hi-77 -/-; Spi1URE-/-progenitors, 68.2% (148 of 217 genes) retained their responsiveness to combinatorial signaling. These studies revealed the disproportionate importance of PU.1 for TLR- vs. IFNγ-mediated transcriptional control and a PU.1 requirement for combinatorial signaling at the majority of loci.

Chronic Lymphocytic Leukaemia (CLL) is characterised by the proliferation and accumulation of clonal B cells (B-CLL cells) and is often treated with anti-CD20 monoclonal antibody immunotherapies, including rituximab. One of the mechanism-of-action of rituximab is antibody-dependent cellular cytotoxicity (ADCC) which occurs when natural killer (NK) cells detect rituximab bound to CD20 +target cells. Rituximab often fails to induce stringent disease eradication, due in part to the diffuse distribution of clonal cells across multiple lymphoid and non-lymphoid tissues where NK cell frequency can be low. It is well established that an individual bout of aerobic exercise induces a transient relocation of lymphocytes - including NK cells and B cells - into peripheral blood. We hypothesised that this exercise-induced lymphocytosis could be harnessed to enhance the efficacy of rituximab in CLL by relocating both target and effector cells together with rituximab into blood.

Heterozygous DDX41 germline mutations occur in familial myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) and acute erythroid leukemia (AEL). DDX41 encodes an RNA helicase that regulates RNA splicing, cGAS-Sting signaling in innate immunity, and maintains genome stability in erythropoiesis. We described the innovation of a facile genetic rescue system to compare RNA-regulatory activities of wild type and disease-associated DDX41 variants in engineered HoxB8-immortalized (hi) murine myeloid progenitor cells (hi-Ddx41+/+ and Ddx41+/- cells) (Kim et al., Leukemia 2023). We identified DDX41-regulated transcripts and membrane proteins as quantitative metrics of DDX41 activity that discriminate benign from pathogenic DDX41 alleles. As these transcripts and the cognate proteins were not known to be components of GATA2 genetic networks that govern hematopoietic stem/progenitor cell (HSPC) transitions, we hypothesized that DDX41 mechanisms in progenitors operate in parallel with or independent of GATA2 mechanisms. Considering the biological and pathological importance of DDX41 and many unanswered questions on how clinical variants impact DDX41 activity, we instituted studies to discover DDX41-regulated transcript isoforms. The datasets are being used to unveil how DDX41 controls cellular functions, how clinical variants derail mechanisms, and to establish interconnections or independence with GATA factor mechanisms.

The discovery of the GATA binding protein (GATA factor) transcription factor family revolutionized hematology. Studies of GATA proteins have yielded vital contributions to our understanding of how hematopoietic stem and progenitor cells develop from precursors, how progenitors generate red blood cells, how hemoglobin synthesis is regulated, and the molecular underpinnings of nonmalignant and malignant hematologic disorders. This thrilling journey began with mechanistic studies on a β-globin enhancer- and promoter-binding factor, GATA-1, the founding member of the GATA family. This work ushered in the cloning of related proteins, GATA-2-6, with distinct and/or overlapping expression patterns. Herein, we discuss how the hematopoietic GATA factors (GATA-1-3) function via a battery of mechanistic permutations, which can be GATA factor subtype, cell type, and locus specific. Understanding this intriguing protein family requires consideration of how the mechanistic permutations are amalgamated into circuits to orchestrate processes of interest to the hematologist and more broadly.

Somatic mutations in TP53 and NRAS are associated with transformation of human chronic myeloid diseases to acute myeloid leukemia (AML). Here, we report that concurrent RAS pathway and TP53 mutations are identified in a subset of AML patients and confer an inferior overall survival. To further investigate the genetic interaction between p53 loss and endogenous NrasG12D/+ in AML, we generated conditional NrasG12D/+p53−/− mice. Consistent with the clinical data, recipient mice transplanted with NrasG12D/+p53−/− bone marrow cells rapidly develop a highly penetrant AML. We find that p53−/− cooperates with NrasG12D/+ to promote increased quiescence in megakaryocyte-erythroid progenitors (MEPs). NrasG12D/+p53−/− MEPs are transformed to self-renewing AML-initiating cells and are capable of inducing AML in serially transplanted recipients. RNA sequencing analysis revealed that transformed MEPs gain a partial hematopoietic stem cell signature and largely retain an MEP signature. Their distinct transcriptomes suggests a potential regulation by p53 loss. In addition, we show that during AML development, transformed MEPs acquire overexpression of oncogenic Nras, leading to hyperactivation of ERK1/2 signaling. Our results demonstrate that p53−/− synergizes with enhanced oncogenic Nras signaling to transform MEPs and drive AML development. This model may serve as a platform to test candidate therapeutics in this aggressive subset of AML.

Somatic mutations in TP53and NRASare associated with transformation of human chronic myeloid diseases to acute myeloid leukemia (AML). Here, we report that concurrent RASpathway and TP53mutations are identified in a subset of AML patients and confer an inferior overall survival. To further investigate the genetic interaction between p53loss and endogenous NrasG12D/+in AML, we generated conditional NrasG12D/+p53−/−mice. Consistent with the clinical data, recipient mice transplanted with NrasG12D/+p53−/−bone marrow cells rapidly develop a highly penetrant AML. We find that p53−/−cooperates with NrasG12D/+to promote increased quiescence in megakaryocyte-erythroid progenitors (MEPs). NrasG12D/+p53−/−MEPs are transformed to self-renewing AML-initiating cells and are capable of inducing AML in serially transplanted recipients. RNA sequencing analysis revealed that transformed MEPs gain a partial hematopoietic stem cell signature and largely retain an MEP signature. Their distinct transcriptomes suggests a potential regulation by p53loss. In addition, we show that during AML development, transformed MEPs acquire overexpression of oncogenic Nras, leading to hyperactivation of ERK1/2 signaling. Our results demonstrate that p53−/−synergizes with enhanced oncogenic Nras signaling to transform MEPs and drive AML development. This model may serve as a platform to test candidate therapeutics in this aggressive subset of AML.

Several approaches for controlling hematopoietic stem and progenitor cell expansion, lineage commitment, and maturation have been investigated for improving clinical interventions. We report here that amino acid substitutions in a thrombopoietin receptor (Mpl)--containing cell growth switch (CGS) extending receptor stability improve the expansion capacity of human cord blood CD34+ cells in the absence of exogenous cytokines. Activation of this CGS with a chemical inducer of dimerization (CID) expands total cells 99-fold, erythrocytes 70-fold, megakaryocytes 0.5-fold, and CD34+ stem/progenitor cells 4.4-fold by 21 days of culture. Analysis of cells in these expanded populations identified a CID-dependent bipotent erythrocyte-megakaryocyte precursor (PEM) population, and a CID-independent macrophage population. The CD235a+/CD41a+ PEM population constitutes up to 13% of the expansion cultures, can differentiate into erythrocytes or megakaryocytes, exhibits very little expansion capacity, and exists at very low levels in unexpanded cord blood. The CD206+ macrophage population constitutes up to 15% of the expansion cultures, exhibits high-expansion capacity, and is physically associated with differentiating erythroblasts. Taken together, these studies describe a fundamental enhancement of the CGS expansion platform, identify a novel precursor population in the erythroid/megakaryocytic differentiation pathway of humans, and implicate an erythropoietin-independent, macrophage-associated pathway supporting terminal erythropoiesis in this expansion system.

Complex genetic networks control hematopoietic stem cell differentiation into progenitors that give rise to billions of erythrocytes daily. Previously, we described a role for the master regulator of erythropoiesis, GATA-1, in inducing genes encoding components of the autophagy machinery. In this context, the Forkhead transcription factor, Foxo3, amplified GATA-1–mediated transcriptional activation. To determine the scope of the GATA-1/Foxo3 cooperativity, and to develop functional insights, we analyzed the GATA-1/Foxo3-dependent transcriptome in erythroid cells. GATA-1/Foxo3 repressed expression of Exosc8, a pivotal component of the exosome complex, which mediates RNA surveillance and epigenetic regulation. Strikingly, downregulating Exosc8, or additional exosome complex components, in primary erythroid precursor cells induced erythroid cell maturation. Our results demonstrate a new mode of controlling erythropoiesis in which multiple components of the exosome complex are endogenous suppressors of the erythroid developmental program.

An obstacle to cancer immunotherapy has been that the affinity of T-cell receptors (TCRs) for antigens expressed in tumors is generally low. We initiated clinical testing of engineered T cells expressing an affinity-enhanced TCR against HLA-A*01–restricted MAGE-A3. Open-label protocols to test the TCRs for patients with myeloma and melanoma were initiated. The first two treated patients developed cardiogenic shock and died within a few days of T-cell infusion, events not predicted by preclinical studies of the high-affinity TCRs. Gross findings at autopsy revealed severe myocardial damage, and histopathological analysis revealed T-cell infiltration. No MAGE-A3 expression was detected in heart autopsy tissues. Robust proliferation of the engineered T cells in vivo was documented in both patients. A beating cardiomyocyte culture generated from induced pluripotent stem cells triggered T-cell killing, which was due to recognition of an unrelated peptide derived from the striated muscle-specific protein titin. These patients demonstrate that TCR-engineered T cells can have serious and not readily predictable off-target and organ-specific toxicities and highlight the need for improved methods to define the specificity of engineered TCRs.

Previous reports of GATA2mutations have focused on the coding region of the gene or full gene deletions. We recently identified 2 patients with novel insertion/deletion mutations predicted to result in mRNA nonsense-mediated decay, suggesting haploinsufficiency as the mechanism of GATA2 deficient disease. We therefore screened patients without identified exonic lesions for mutations within conserved noncoding and intronic regions. We discovered 1 patient with an intronic deletion mutation, 4 patients with point mutations within a conserved intronic element, and 3 patients with reduced or absent transcription from 1 allele. All mutations affected GATA2transcription. Full-length cDNA analysis provided evidence for decreased expression of the mutant alleles. The intronic deletion and point mutations considerably reduced the enhancer activity of the intron 5 enhancer. Analysis of 512 immune system genes revealed similar expression profiles in all clinically affected patients and reduced GATA2transcript levels. These mutations strongly support the haploinsufficient nature of GATA2 deficiency and identify transcriptional mechanisms and targets that lead to MonoMAC syndrome.

Master transcriptional regulators of development often function through dispersed cis elements at endogenous target genes. While cis-elements are routinely studied in transfection and transgenic reporter assays, it is challenging to ascertain how they function in vivo. To address this problem in the context of the locus encoding the critical hematopoietic transcription factor Gata2, we engineered mice lacking a cluster of GATA motifs 2.8 kb upstream of the Gata2 transcriptional start site. We demonstrate that the −2.8 kb site confers maximal Gata2 expression in hematopoietic stem cells and specific hematopoietic progenitors. By contrast to our previous demonstration that a palindromic GATA motif at the neighboring −1.8 kb site maintains Gata2 repression in terminally differentiating erythroid cells, the −2.8 kb site was not required to initiate or maintain repression. These analyses reveal qualitatively distinct functions of 2 GATA motif-containing regions in vivo.

In megakaryocytes, the maturation process and oxidative stress response appear to be closely related. It has been suggested that increased oxygen tension and reactive oxygen species (ROS) promote megakaryopoiesis and that the expression of stress-responsive genes responsible for ROS elimination declines during megakaryocytic maturation. NF-E2 p45 is an essential regulator of megakaryopoiesis, whereas Nrf2 is a key activator of stress-responsive genes. Because p45 and Nrf2 have similar DNA-binding specificities, we hypothesized that p45 competes with Nrf2 to repress stress-responsive genes and achieves favorable intracellular conditions to allow ROS to be efficiently used as signaling molecules. We conducted comprehensive gene expression profiling with wild-type and p45-null megakaryocytes and examined the functional relationship between p45 and Nrf2. We found that 2 characteristic gene clusters are defined within p45 target genes: platelet genes and cytoprotective genes. The former are unique targets activated by p45, whereas the latter are common targets of p45 and Nrf2. Further analysis suggested that, as a less efficacious activator, p45 maintains moderate expression of cytoprotective genes through competing with Nrf2 and promotes ROS accumulation. Increased ROS enhanced platelet gene expression. These results suggest that p45 dominates over Nrf2 to enhance megakaryocytic maturation by promoting ROS accumulation.

Genome-wide analyses of the relationship between H3 K79 dimethylation and transcription have revealed contradictory results. To clarify this relationship at a single locus, we analyzed expression and H3 K79 modification levels of wild-type (WT) and transcriptionally impaired β-globin mutant genes during erythroid differentiation. Analysis of fractionated erythroid cells derived from WT/Δ locus control region (LCR) heterozygous mice reveals no significant H3 K79 dimethylation of the β-globin gene on either allele prior to activation of transcription. Upon transcriptional activation, H3 K79 di-methylation is observed along both WT and ΔLCR alleles, and both alleles are located in proximity to H3 K79 dimethylation nuclear foci. However, H3 K79 di-methylation is significantly increased along the ΔLCR allele compared with the WT allele. In addition, analysis of a partial LCR deletion mutant reveals that H3 K79 dimethylation is inversely correlated with β-globin gene expression levels. Thus, while our results support a link between H3 K79 dimethylation and gene expression, high levels of this mark are not essential for high level β-globin gene transcription. We propose that H3 K79 dimethylation is destabilized on a highly transcribed template.

Genome-wide analyses of the relationship between H3 K79 dimethylation and transcription have revealed contradictory results. To clarify this relationship at a single locus, we analyzed expression and H3 K79 modification levels of wild-type (WT) and transcriptionally impaired β-globin mutant genes during erythroid differentiation. Analysis of fractionated erythroid cells derived from WT/Δ locus control region (LCR) heterozygous mice reveals no significant H3 K79 dimethylation of the β-globin gene on either allele prior to activation of transcription. Upon transcriptional activation, H3 K79 di-methylation is observed along both WT and ΔLCR alleles, and both alleles are located in proximity to H3 K79 dimethylation nuclear foci. However, H3 K79 di-methylation is significantly increased along the ΔLCR allele compared with the WT allele. In addition, analysis of a partial LCR deletion mutant reveals that H3 K79 dimethylation is inversely correlated with β-globin gene expression levels. Thus, while our results support a link between H3 K79 dimethylation and gene expression, high levels of this mark are not essential for high level β-globin gene transcription. We propose that H3 K79 dimethylation is destabilized on a highly transcribed template.

Activation of PI3K is among the earliest signaling events observed in T cells after conjugate formation with antigen-presenting cells (APCs). The relevant PI3K catalytic isoform and relative contribution of the TcR and CD28 to PI3K activity at the immune synapse have not been determined unequivocally. Using a quantitative imaging-based assay, we show that the PI3K activity at the T cell–APC contact area is dependent on the p110δ, but not the p110γ, isoform of PI3K. CD28 enhanced PIP3 production at the T-cell synapse independently of its YMNM PI3K-recruitment motif that instead was required for efficient PKCθ recruitment. CD28 could partially compensate for the lack of p110δ activity during T-cell activation, which indicates that CD28 and p110δ act in parallel and complementary pathways to activate T cells. Consistent with this, CD28 and p110δ double-deficient mice were severely immune compromised. We therefore suggest that combined pharmaceutic targeting of p110δ activity and CD28 costimulation has potent therapeutic potential.

The GATA-1–interacting protein Friend Of GATA-1 (FOG-1) is essential for the proper transcriptional activation and repression of numerous GATA-1 target genes. Although FOG-1–independent activation by GATA-1 has been described, all known examples of GATA-1–mediated repression are FOG-1 dependent. In the GATA-1–null G1E cell line, estrogen receptor ligand binding domain (ER) chimeras of either wild-type GATA-1 or a FOG-1–binding defective mutant of GATA-1 repressed several genes similarly upon activation with β-estradiol. Repression also occurred in a FOG-1–null cell line expressing ER–GATA-1 and during ex vivo erythropoiesis. At the Lyl1and Rgs18loci, we found highly restricted occupancy by GATA-1 and GATA-2, indicating that these genes are direct targets of GATA factor regulation. The identification of genes repressed by GATA-1 independent of FOG-1 defines a novel mode of GATA-1–mediated transcriptional regulation.

No relevant conflicts of interest to declare.

The transcription factor GATA2 is essential for the generation and function of hematopoietic stem and progenitor cells (HSPCs), erythroid precursors, and endothelial cells. A conserved intronic GATA2enhancer, 9.8 kb downstream of the transcriptional start site (+9.5 in the mouse), is mutated in patients with GATA2 deficiency syndrome. Patient mutations within this region include a c.1017+512del28 deletion, removing E-box and GATA motifs, c.1017+532T>A that disrupts the E-box, and, most frequently, C>T in a 3’ Ets motif (c.1017+572C>T) (Soukup and Bresnick, 2020). Homozygous mutation of the Ets motif in mice allows normal developmental and steady-state hematopoiesis but impairs hematopoietic regeneration (Soukup et al., 2019).

Mouse gene–targeting studies have documented a central role of the p110δ isoform of phosphoinositide 3-kinase (PI3K) in B-cell development and function. A defect in B-cell antigen receptor (BCR) signaling is key to this B-cell phenotype. Here we further characterize this signaling defect and report that a p110δ-selective small molecule inhibitor mirrors the effect of genetic inactivation of p110δ in BCR signaling. p110δ activity is indispensable for BCR-induced DNA synthesis and phosphorylation of Akt/protein kinase B (PKB), forkhead transcription factor/forkhead box O3a (FOXO3a), and p70 S6 kinase (p70 S6K), with modest effects on the phosphorylation of glycogen synthase kinase 3 α/β (GSK3α/β) and extracellular signal-regulated kinase (Erk). The PI3K-dependent component of intracellular calcium mobilization also completely relies on p110δ catalytic activity. Resting B cells with inactive p110δ fail to enter the cell cycle, correlating with an incapacity to up-regulate the expression of cyclins D2, A, and E, and to phosphorylate the retinoblastoma protein (Rb). p110δ is also critical for interleukin 4 (IL-4)–induced phosphorylation of Akt/PKB and FOXO3a, and protection from apoptosis. Taken together, these data show that defects observed in p110δ mutant mice are not merely a consequence of altered B-cell differentiation, and emphasize the potential utility of p110δ as a drug target in autoimmune diseases in which B cells play a crucial role.

Previous studies demonstrated that DNase I hypersensitive site -40 (HS-40) of the α-globin locus is capable of greatly enhancing expression of a hybrid β/γ-globin transcriptional unit in plasmid-transfected murine erythroleukemia (MEL) cells. However, as reported here, this same γ-globin gene expression cassette was only transcribed at trace amounts in erythroid cells of transgenic mice. This lack of expression was not directly attributable to the β/γ-globin transcriptional unit, since this same unit linked to a composite β-globin locus control region was expressed at high levels in transgenic mice. This lack of expression was also not directly attributable to chromosomal position effects, since addition of chromatin insulators failed to increase the frequency of expression. DNase I hypersensitivity and chromatin immunoprecipitation assays demonstrated that the lack of expression was correlated with a closed chromatin structure. We hypothesize that transgenes undergo dynamic changes in chromatin conformation following chromosomal integration and that the discrepant results reported here can be attributed to the relatively high level of chromatin remodeling that occurs in the transgenic mouse model, coupled with the relative inability of the HS-40 element to maintain an open chromatin state under such conditions. (Blood. 2005;105: 3346-3352)

Rheumatoid arthritis (RA) is a chronic, inflammatory disease of the synovium of uncertain pathogenesis. A number of phenotypic and functional T-cell defects have been described in RA, including abnormal clonal expansions and suppressed proliferative responses, which suggest a defect in T-cell differentiation. Here, we show that RA patients possess fewer naive CD4+T cells than healthy controls. Furthermore, a smaller proportion of these cells contains a T-cell receptor excision circle (TREC). Patients with RA also have unusual populations of T cells. These include immature cells characterized as CD45RBbrightCD45RA+CD62L−by flow cytometry and a large population that coexpresses CD45RA and CD45RO. These cells are hyperresponsive to mitogen and TCR stimulation when compared to naive cells. Additionally, an unusual putative central memory subset expressing CD62L, but not CD45RA, appears in RA patients at the expense of more typical cells. Levels of C-reactive protein correlate inversely with the TREC content of naive T cells and positively with the sizes of naive and immature atypical T-cell subsets. These data suggest that inflammation drives proliferation of naive T cells in RA and encourages their differentiation into atypical, hyperresponsive progeny. TREC content of individual naive and atypical T-cell subsets suggests an ontogeny consistent with this hypothesis. These studies provide further evidence of a T-cell differentiation defect in RA, which could explain some of the well-characterized immunologic features of the disease.

We have previously described the development of oncoretrovirus vectors for human γ-globin using a truncated β-globin promoter, modified γ-globin cassette, and α-globin enhancer. However, one of these vectors is genetically unstable, and both vectors exhibit variable expression patterns in cultured cells, common characteristics of oncoretrovirus vectors for globin genes. To address these problems, we identified and removed the vector sequences responsible for genetic instability and flanked the resultant vector with the chicken β-globin HS4 chromatin insulator to protect expression from chromosomal position effects. After determining that flanking with the cHS4 element allowed higher, more uniform levels of γ-globin expression in MEL cell lines, we tested these vectors using a mouse bone marrow transduction and transplantation model. When present, the γ-globin cassettes from the uninsulated vectors were expressed in only 2% to 5% of red blood cells (RBCs) long term, indicating they are highly sensitive to epigenetic silencing. In contrast, when present the γ-globin cassette from the insulated vector was expressed in 49% ± 20% of RBCs long term. RNase protection analysis indicated that the insulated γ-globin cassette was expressed at 23% ± 16% per copy of mouse α-globin in transduced RBCs. These results demonstrate that flanking a globin vector with the cHS4 insulator increases the likelihood of expression nearly 10-fold, which in turn allows for γ-globin expression approaching the therapeutic range for sickle cell anemia and β thalassemia.

Current chemotherapeutic and butyrate therapeutics that induce fetal hemoglobin expression generally also suppress erythropoiesis, limiting the production of cells containing fetal hemoglobin (F cells). Recently, selected short-chain fatty acid derivatives (SCFADs) were identified that induce endogenous γ-globin expression in K562 cells and human burst-forming units–erythroid and that increase proliferation of human erythroid progenitors and a multilineage interleukin-3–dependent hematopoietic cell line. In this report, γ-globin inducibility by these SCFADs was further demonstrated in mice transgenic for the locus control region and the entire β-globin gene locus in a yeast artificial chromosome and in 2 globin promoter-reporter assays. Conditioned media experiments strongly suggest that their proliferative activity is a direct effect of the test compounds. Investigation of potential mechanisms of action of these SCFADs demonstrates that these compounds induce prolonged expression of the growth-promoting genes c-myb andc-myc. Both butyrate and specific growth-stimulatory SCFADs induced prolonged signal transducer and activator of transcription (STAT)-5 phosphorylation and activation, and c-cisexpression, persisting for more than 120 minutes, whereas with IL-3 alone phosphorylation disappeared within minutes. In contrast to butyrate treatment, the growth-stimulating SCFADs did not result in bulk histone H4 hyperacetylation or induction of p21Waf/Cip, which mediates the suppression of cellular growth by butyrate. These findings suggest that the absence of bulk histone hyperacetylation and p21 induction, but prolonged induction of cis, myb, myc, and STAT-5 activation, contribute to the cellular proliferation induced by selected SCFADs.

The human ß-globin locus control region (LCR) confers high-level, tissue-specific expression to the ß-globin genes. Tandem Maf recognition elements (MAREs) within the hypersensitive site 2 (HS2) subregion of the LCR are important for the strong enhancer activity of the LCR. Multiple proteins are capable of interacting with these sites in vitro, including the erythroid cell- and megakaryocyte-specific transcription factor, NF-E2. The importance of NF-E2 for ß-globin gene expression is evident in murine erythroleukemia cells lacking the p45 subunit of NF-E2. These CB3 cells have a severe defect in ?- and ß-globin gene transcription, which can be restored by expression of NF-E2. However, mice nullizygous for p45 express nearly normal levels of ß-globin. Thus, either a redundant factor(s) exists in mice that can functionally replace NF-E2, or NF-E2 does not function through the LCR to regulate ß-globin gene expression. To address this issue, we asked whether NF-E2 binds directly to the tandem MAREs of HS2 in intact cells. Using a chromatin immunoprecipitation assay, we provide evidence for NF-E2 binding directly and specifically to HS2 in living erythroleukemia cells and in mouse fetal liver. The specific immunoisolation of HS2 sequences was dependent on the presence of p45 and on intact MAREs within HS2. These results support a direct role for NF-E2 in the regulation of ß-globin gene expression through activation of the LCR.

Genomic analyses in clinical and experimental contexts have accelerated discoveries of human genetic variants. While elucidating the consequences of conspicuously loss-of-function variants is highly tractable, decoding the impact of missense or non-coding variants is considerably more challenging. Previously, we described a germline variant in GATA2in a patient with GATA2-deficiency syndrome, which inserts nine amino acids between the two zinc fingers (9aa-Ins), one of which mediates sequence-specific DNA binding (Cavalcante de Andrade Silva M. et al., Leukemia, 2021). Unlike other GATA2 coding region and enhancer variants identified (Bresnick E.H. et al., Blood Adv., 2020), it was unclear whether the altered zinc finger spacing would be inhibitory, stimulatory or of no consequence. The 9aa-Ins variant was defective in activating several target genes (Hdc, Ear2and Tpsb2) in a genetic complementation assay with Gata2-77 enhancer-mutant (-77 -/-) primary hematopoietic progenitor cells. As only several target genes were tested, we used RNA-seq to conduct a genome-wide comparison of the capacity of GATA2 and 9aa-Ins to activate and repress transcription. To elucidate mechanisms, we considered the following models: 1) 9aa-Ins fails to regulate all genes normally controlled by GATA2; 2) 9aa-Ins fails to repress all genes normally controlled by GATA2; 3) 9aa-Ins fails to activate genes normally controlled by GATA2; 4) 9aa-Ins ectopically regulates genes not controlled by GATA2. Using a genetic complementation approach with -77 -/-cells that were immortalized by the Hoxb8 transcription factor (hi-77 -/-) (Wang G.G. et al., Nat. Methods, 2006; Johnson K.D. et al., JEM, 2020), we compared GATA2 and 9aa-Ins activities when expressed at a comparable level. This analysis revealed 2,138 GATA2-regulated, 525 GATA2 and 9aa-Ins-regulated, and 414 ectopically-regulated genes (at least two-fold change, adjusted P-value <0.05). A similar number of genes were GATA2-activated (1,061) and repressed (1,077). Only 144 out of the 1,061 (14%) were 9aa-Ins-activated and 381 out of 1,077 (35%) were 9aa-Ins-repressed, illustrating the severe consequences of this mutation and a greater impact on activation versus repression. Statistical analysis with a range of P-values constraints (0.01 to 0.1) verified that activation by 9aa-Ins was more significantly impaired than repression (86% were no longer activated, and 65% were no longer repressed, P = 5.4 x 10 -6). Gene ontology analysis revealed that the 9aa insertion impaired GATA2-mediated activation of genes related to GPCR signaling and GATA2-mediated repression of genes related to innate immune machinery. The ectopically-regulated genes did not conform to a particular mechanism or pathway.

Human genetic variants are classified based on potential pathogenicity to guide clinical decisions. However, mechanistic uncertainties often preclude definitive categorization. Germline coding and enhancer variants within the hematopoietic regulator GATA2create a bone marrow failure and leukemia predisposition (Soukup and Bresnick, 2020). The conserved murine enhancer (+9.5) promotes hematopoietic stem cell (HSC) genesis, and a single-nucleotide human variant in an Ets motif attenuates chemotherapy- and transplantation-induced hematopoietic regeneration in a cell-autonomous manner (Soukup et al., 2019). Since an epigenetically-silenced normal allele can exacerbate phenotypes of a pathogenic heterozygous variant, we engineered a bone marrow failure model harboring the Ets motif variant and a severe E-box:GATA enhancer mutation on the second allele (CH) that renders the enhancer chromatin inaccessible, to determine how perturbations of the hematopoietic system trigger pathogenesis.

Cell type-specific transcription factors governing hematopoietic stem and progenitor cell transitions establish networks containing hundreds of genes and proteins. Network complexity renders it challenging to discover essential versus modulatory or redundant components. This scenario is exemplified by GATA2 mechanisms that control hematopoiesis during embryogenesis. Loss of a far upstream Gata2enhancer (-77) disrupts the GATA2-dependent genetic network governing hematopoietic progenitor cell differentiation (Johnson KD. et al., Sci. Adv., 2015). The aberrant network includes the transcription factor Interferon Regulatory Factor-8 and a host of innate immune regulators, including Toll-like receptors (TLRs) (Johnson KD. et al., J. Exp. Med., 2020). Mutant embryonic progenitors lose the capacity to balance production of diverse hematopoietic progeny and generate excessive monocytic progeny. As IRF8 is vitally important for monocytic and dendritic cell differentiation (Yanez A. and Goodridge H., Curr. Opin. Hematol., 2016), we asked whether IRF8 is essential, contributory, or inconsequential. Using a double-mutant genetic rescue in vivosystem, we demonstrated that reducing Irf8, in the context of the -77 mutant allele, reversed granulocytic deficiencies and the excessive accumulation of dendritic cell-committed progenitors. In -77 -/-E14.5 fetal livers, monocyte progenitors (MPs) increased 2.3-fold (P = 0.006), granulocyte progenitors (GPs) decreased 2.2-fold (P = 0.003) and common dendritic cell progenitors (CDPs) increased 10.2-fold (P = 0.021) relative to wildtype littermates. Ablating Irf8in -77 mutants (-77 -/-; Irf8-/-) restored MPs and CDPs to wildtype levels and reversed the GP deficiency; further increasing GPs 4.2-fold relative to wildtype (P = 0.0009). Despite many dysregulated components that control vital transcriptional, signaling and immune processes, the aberrant elevation of a single transcription factor deconstructed the embryonic hematopoiesis program. We analyzed the mechanistic and biological implications of IRF8 dysregulation concomitant with ectopic upregulation of other innate immune genes (including Toll-like receptors (TLRs) in GATA2-deficient embryonic progenitors. In principle, such genes might function upstream, downstream, or in parallel with IRF8. Based on TLR upregulation and TLR roles in progenitor mechanisms (Nagai Y. et al., Immunity, 2006; Schuettpelz L. et al., Leukemia, 2014; Caiado F. et al., J. Exp. Med., 2021), we tested whether GATA2 deficiency in embryonic progenitors impacts cellular responsiveness to TLR ligands. Wild type and -77 enhancer-mutant progenitors were treated with increasing concentrations of the TLR1/2 ligand Pam 3CSK 4. The mutant progenitors were hypersensitive to Pam 3CSK 4, which resulted in supra-physiological induction of Tnfexpression (2.8-fold at 34 nM, P = 0.004; 3.2-fold at 68 nM, P = 0.0003). Quantitative analyses indicated that hypersensitivity reflected increased Pam 3CSK 4efficacy, but not potency. GATA2 re-expression in the mutant progenitors attenuated the elevated IRF8 expression and TLR signaling, normalizing Tnfand Ccl3expression to a level comparable to that of wild type progenitors. In GATA2-rescued mutant progenitors, Tnfand Ccl3expression decreased 3.9-fold (P = 0.005) and 2.5-fold (P = 0.047), respectively. Thus, GATA2 suppresses TLR signaling in embryonic progenitors. Ongoing studies are elucidating the mechanistic interconnections between IRF8- and TLR-dependent inflammatory networks in GATA2 deficiency during embryonic and adult hematopoiesis in cell populations and single cells, relationships between murine and human mechanisms, and the impact of targeted interventions that modulate these mechanisms.

Introduction:

Progress toward gene therapy of β-chain hemoglobinopathies has been limited in part by poor expression of globin genes in virus vectors. To derive an optimal expression cassette, we systematically analyzed the sequence requirements and relative strengths of theAγ- and β-globin promoters, the activities of various erythroid-specific enhancers, and the importance of flanking and intronic sequences. Expression was analyzed by RNase protection after stable plasmid transfection of the murine erythroleukemia cell line, MEL585. Promoter truncation studies showed that theAγ-globin promoter could be deleted to −159 without affecting expression, while deleting the β-globin promoter to −127 actually increased expression compared with longer fragments. Expression from the optimal β-globin gene promoter was consistently higher than that from the optimal Aγ-globin promoter, regardless of the enhancer used. Enhancers tested included a 2.5-kb composite of the β-globin locus control region (termed a μLCR), a combination of the HS2 and HS3 core elements of the LCR, and the HS-40 core element of the α-globin locus. All three enhancers increased expression from the β-globin gene to roughly the same extent, while the HS-40 element was notably less effective with theAγ-globin gene. However, the HS-40 element was able to efficiently enhance expression of a Aγ-globin gene linked to the β-globin promoter. Inclusion of extended 3′ sequences from either the β-globin or the Aγ-globin genes had no significant effect on expression. A 714-bp internal deletion ofAγ-globin intron 2 unexpectedly increased expression more than twofold. With the combination of a −127 β-globin promoter, anAγ-globin gene with the internal deletion of intron 2, and a single copy of the HS-40 enhancer, γ-globin expression averaged 166% of murine α-globin mRNA per copy in six pools and 105% in nine clones. When placed in a retrovirus vector, this cassette was also expressed at high levels in MEL585 cells (averaging 75% of murine α-globin mRNA per copy) without reducing virus titers. However, recombined provirus or aberrant splicing was observed in 5 of 12 clones, indicating a significant degree of genetic instability. Taken together, these data demonstrate the development of an optimal expression cassette for γ-globin capable of efficient expression in a retrovirus vector and form the basis for further refinement of vectors containing this cassette.

Progress toward gene therapy of ß-chain hemoglobinopathies has been limited in part by poor expression of globin genes in virus vectors. To derive an optimal expression cassette, we systematically analyzed the sequence requirements and relative strengths of theA?- and ß-globin promoters, the activities of various erythroid-specific enhancers, and the importance of flanking and intronic sequences. Expression was analyzed by RNase protection after stable plasmid transfection of the murine erythroleukemia cell line, MEL585. Promoter truncation studies showed that theA?-globin promoter could be deleted to -159 without affecting expression, while deleting the ß-globin promoter to -127 actually increased expression compared with longer fragments. Expression from the optimal ß-globin gene promoter was consistently higher than that from the optimal A?-globin promoter, regardless of the enhancer used. Enhancers tested included a 2.5-kb composite of the ß-globin locus control region (termed a µLCR), a combination of the HS2 and HS3 core elements of the LCR, and the HS-40 core element of the ?-globin locus. All three enhancers increased expression from the ß-globin gene to roughly the same extent, while the HS-40 element was notably less effective with theA?-globin gene. However, the HS-40 element was able to efficiently enhance expression of a A?-globin gene linked to the ß-globin promoter. Inclusion of extended 3' sequences from either the ß-globin or the A?-globin genes had no significant effect on expression. A 714-bp internal deletion ofA?-globin intron 2 unexpectedly increased expression more than twofold. With the combination of a -127 ß-globin promoter, anA?-globin gene with the internal deletion of intron 2, and a single copy of the HS-40 enhancer, ?-globin expression averaged 166% of murine ?-globin mRNA per copy in six pools and 105% in nine clones. When placed in a retrovirus vector, this cassette was also expressed at high levels in MEL585 cells (averaging 75% of murine ?-globin mRNA per copy) without reducing virus titers. However, recombined provirus or aberrant splicing was observed in 5 of 12 clones, indicating a significant degree of genetic instability. Taken together, these data demonstrate the development of an optimal expression cassette for ?-globin capable of efficient expression in a retrovirus vector and form the basis for further refinement of vectors containing this cassette.

Human basophils possess receptors for interleukin-2 (IL-2) and IL-4. The effect of 3 days of intravenous administration of IL-2 and/or IL-4 on basophil histamine release was examined in three groups of patients receiving IL-2, IL-4, or the combination of agents as part of a protocol to treat malignant melanoma or renal cell carcinoma. Because all patients received ranitidine for control of side effects, a control group of patients receiving ranitidine for Zollinger-Ellison's syndrome was also studied. IL-4 significantly inhibited IgE-mediated histamine release, while there was a trend for enhancement of IgE-mediated histamine release by IL-2. Administration of the combination of IL-2 and IL-4 did not alter IgE-mediated basophil histamine release. Both IL- 2 and IL-4, alone and in combination, enhanced basophil histamine release induced by histamine releasing factors in human nasal washings. The effect of IL-2 alone was significantly greater than that of IL-4 alone or the combination of IL-2 plus IL-4. Taken together, the data suggest that when coadministered, IL-4 may inhibit the effects of IL-2 on basophils. Neither cytokine exerted any effect on basophil histamine release induced by the calcium ionophore A23187, nor did ranitidine cause any effects on histamine release induced by any of the stimulants. Thus, human basophil reactivity can be affected by IL-2 and by IL-4. The role that these two cytokines play in basophil function in vivo is likely to be complex.

The differentiation status of Sternberg-Reed (SR) cells is still not well defined, primarily because of their scarcity in tumor biopsies of Hodgkin's disease (HD). In this study we have determined the genomic differentiation status of SR cells by quantitation of recombinase activating gene (RAG) expression. RAG genes are selectively transcribed in immature lymphoid cells. In B cells they are silent after genomic rearrangement has occurred, whereas in T cells they are downregulated during positive selection of double-positive thymocytes into single- positive cells. RNA from tumor biopsies either with numerous (11 cases) or a with few SR cells (16 cases) was assessed by a sensitive reverse transcriptase polymerase chain reaction (RT-PCR) and the results compared with established positive and negative controls. In all except two cases levels of RAG expression were within the range of those determined in negative controls. In both positive cases and in the positive control RAG mRNA was further quantitated by competitive PCR. In cases with abundant SR cells RAG expression was still below that observed in 10(-2) dilutions of positive controls. These results suggest that SR cells are derived from lymphoid cells, more differentiated than the pre-B or common thymocyte stage, which have already undergone genomic rearrangement. They show the value of assessing RAG expression by RT-PCR in the characterization of lymphoid malignancies.

The Epstein-Barr virus (EBV) has been increasingly detected in Hodgkin's disease (HD), but its role in pathogenesis remains uncertain. We analyzed 20 specimens of HD known to contain EBV DNA by a sensitive reverse transcriptase polymerase chain reaction (RT-PCR). The cases were assessed for the presence of RNA transcripts of the BNLF1 gene (coding for the viral latent membrane protein [LMP]) and the late replicative gene BLLF1 (coding for the principle envelope glycoprotein [gp220/350]). LMP RNA transcripts were found in 9 of 20 (45%) cases, mostly those containing many copies of viral DNA and of mixed cellularity (MC) histological subtype. Only one LMP RNA-positive case was also positive for RNA transcripts of the active replication gene BLLF1. Our results show that viral burden in HD is not primarily related to active viral replication, but is associated with LMP gene expression.

The possibility of inducing transplantation tolerance by somatic gene transfer is under investigation in our miniature swine model. As a crucial step in this project, we have used a retroviral vector engineered to express both a drug-resistance gene (Neo) and a swine class II DRB cDNA to transduce porcine bone marrow (BM) cells. Analysis of cultured swine fibroblasts exposed to high-titer viral supernatants demonstrated that drug resistance had been conferred and that transferred vector sequences were transcribed appropriately. Similar transduction studies with swine BM demonstrated the transfer of drug resistance to as high as 14% of colony-forming unit-granulocyte- macrophage (CFU-GM). Using polymerase chain reaction (PCR) of cDNA, vector-derived allogeneic DRB transcripts were detected in colonies derived from primitive CFU-Mix and high proliferative potential-colony- forming cell (HPP-CFC), as well as in drug-resistant GM colonies grown from transduced bone marrow (BM) maintained in long-term BM cultures (LTBMCs) for up to 5 weeks. These results indicate that a significant proportion of both colony-forming progenitors and LTBMC-initiating cells were transduced with the DRB-recombinant retroviral vector and that both vector-derived genes were expressed in the differentiated progeny of these cells.

Two infants with absent radii are described. Both had hemorrhagic manifestations which occurred within 24 hours of birth, and from which they eventually died. The most important hematologic feature was absence of megakaryocytes in the bone marrow. Five cases showing a similar picture have been recorded, four as amegakaryocytic thrombocytopenia, and one as congenital leukemia. It is suggested that these cases are related on the one hand to Fanconi’s anemia, and on the other to congenital leukemia.

This report describes 41 dogs that received 1200-1600 r of whole body irradiation followed by infusions of homologous marrow. The clinical and hematologic events in these animals are described. Five dogs given Methotrexate following irradiation lived beyond four months and appeared to have survived the period of secondary syndromes.

In rats whose intestinal or thoracic duct lymph was drained externally for several days, lymphopenia occurred. Large numbers of cells were collected in the lymph each day, as many, apparently, from the intestinal lymph alone as from the thoracic duct. Hepatic lymph contributed relatively few cells. Augmentation of lymph flow decreased the concentration of cells in the lymph but did not affect the total number of cells collected each day. Fasting for several days likewise did not decrease the first day’s output. With each day’s lymph flow, however, the daily output of cells spontaneously decreased. The decrease was not prevented by the intravenous injection of fresh lymph or of fresh rat plasma in large amounts. In view of this unexplained effect, one must be cautious in interpreting the results of experiments on the lymph lymphocyte.

The master regulator of hematopoiesis GATA2 controls generation and function of hematopoietic stem and progenitor cells, and heterozygous GATA2mutations create a predisposition to develop immunodeficiency, myelodysplasia, and acute myeloid leukemia (Spinner et al. Blood, 2014; Dickinson et al. Blood, 2014; Churpek and Bresnick J. Clin. Invest. 2019). Although mechanisms that trigger the transition of a non-pathogenic GATA2mutation into overt pathology are enigmatic, a paradigm has arisen in which GATA2mutations are considered to be loss-of-function. We developed a genetic rescue assay to quantify the function of wild type GATA2 and GATA2 disease mutants when expressed at near-physiological levels in primary progenitor cells and demonstrated that GATA2 disease mutations abrogate certain biological and molecular activities, while enabling others (Katsumura et al., 2018, PNAS). We isolated lineage-negative (Lin-) or Lin-Kit+cells from fetal liver of mice with a homozygous mutation of the Gata2-77 enhancer, which downregulates Gata2expression by ~80%. The mutant progenitor cells are largely defective in erythroid, megakaryocytic and granulocytic differentiation and exhibit a predominant monocytic differentiation fate (Johnson et al., 2015, Science Adv.). We compared GATA2 and GATA2 disease mutant activities in the rescue system using a colony formation assay. GATA2, R307W mutant (in N-finger) and T354M mutant (in DNA-binding C-finger) rescued myeloid colony formation and promoted granulocyte proliferation. Surprisingly, R307W and T354M induced more CFU-GM than GATA2. GATA2 and R307W, but not T354M, rescued BFU-E. These data indicated that GATA2 disease mutations were not strictly inhibitory, and in certain contexts, mutant activities exceeded that of GATA2.

Epperla: Pharmacyclics: Honoraria; Verastem Oncology: Speakers Bureau. Costa:Abbvie: Consultancy; Karyopharm: Consultancy; Fujimoto Pharmaceutical Corporation Japan: Other: Advisor; Sanofi: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding. Cashen:Novartis: Other: Speaker's Bureau; Seattle Genetics: Other: Speaker's Bureau; Celgene: Other: Speaker's Bureau. Hamadani:Otsuka: Research Funding; Takeda: Research Funding; Pharmacyclics: Consultancy; ADC Therapeutics: Consultancy, Research Funding; Merck: Research Funding; Sanofi Genzyme: Research Funding, Speakers Bureau; Celgene: Consultancy; Janssen: Consultancy; Medimmune: Consultancy, Research Funding. Barta:Mundipharma: Honoraria; Celgene: Research Funding; Takeda: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Honoraria, Research Funding; Bayer: Consultancy, Research Funding; Mundipharma: Honoraria; Merck: Research Funding; Celgene: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Reddy:Celgene: Consultancy; BMS: Consultancy, Research Funding; Genentech: Research Funding; Abbvie: Consultancy; KITE Pharma: Consultancy. Karmali:Gilead/Kite; Juno/Celgene: Consultancy, Speakers Bureau; Takeda, BMS: Other: Research Funding to Institution; Astrazeneca: Speakers Bureau. Bello:Celgene: Speakers Bureau. Chavez:Novartis: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Svoboda:AstraZeneca: Consultancy; Celgene: Research Funding; Incyte: Research Funding; Pharmacyclics: Consultancy, Research Funding; Kyowa: Consultancy; Merck: Research Funding; BMS: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding. Glenn:BMS: Research Funding; Merck: Research Funding; Genentech: Research Funding. Cohen:Lymphoma Research Foundation: Research Funding; Seattle Genetics, Inc.: Consultancy, Research Funding; ASH: Research Funding; Takeda Pharmaceuticals North America, Inc.: Research Funding; Gilead/Kite: Consultancy; Bristol-Meyers Squibb Company: Research Funding; Genentech, Inc.: Consultancy, Research Funding; Janssen Pharmaceuticals: Consultancy; LAM Therapeutics: Research Funding; UNUM: Research Funding; Hutchison: Research Funding; Astra Zeneca: Research Funding.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.