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The genetics of relapsed pediatric acute myeloid leukemia (AML) has yet to be comprehensively defined. Here, we present the spectrum of genomic alterations in 136 relapsed pediatric AMLs. We identified recurrent exon 13 tandem duplications (TD) in upstream binding transcription factor (UBTF) in 9% of relapsed AML cases. UBTF-TD AMLs commonly have normal karyotype or trisomy 8 with cooccurring WT1 mutations or FLT3-ITD but not other known oncogenic fusions. These UBTF-TD events are stable during disease progression and are present in the founding clone. In addition, we observed that UBTF-TD AMLs account for approximately 4% of all de novo pediatric AMLs, are less common in adults, and are associated with poor outcomes and MRD positivity. Expression of UBTF-TD in primary hematopoietic cells is sufficient to enhance serial clonogenic activity and to drive a similar transcriptional program to UBTF-TD AMLs. Collectively, these clinical, genomic, and functional data establish UBTF-TD as a new recurrent mutation in AML.Significance:We defined the spectrum of mutations in relapsed pediatric AML and identified UBTF-TDs as a new recurrent genetic alteration. These duplications are more common in children and define a group of AMLs with intermediate-risk cytogenetic abnormalities, FLT3-ITD and WT1 alterations, and are associated with poor outcomes.See related commentary by Hasserjian and Nardi, p. 173.This article is highlighted in the In This Issue feature, p. 171.

Supplementary Data from Integrated Genomic Analysis Identifies UBTF Tandem Duplications as a Recurrent Lesion in Pediatric Acute Myeloid Leukemia

Supplementary Figure from Integrated Genomic Analysis Identifies UBTF Tandem Duplications as a Recurrent Lesion in Pediatric Acute Myeloid Leukemia

The genetics of relapsed pediatric acute myeloid leukemia (AML) has yet to be comprehensively defined. Here, we present the spectrum of genomic alterations in 136 relapsed pediatric AMLs. We identified recurrent exon 13 tandem duplications (TD) in upstream binding transcription factor (UBTF) in 9% of relapsed AML cases. UBTF-TD AMLs commonly have normal karyotype or trisomy 8 with cooccurring WT1 mutations or FLT3-ITD but not other known oncogenic fusions. These UBTF-TD events are stable during disease progression and are present in the founding clone. In addition, we observed that UBTF-TD AMLs account for approximately 4% of all de novo pediatric AMLs, are less common in adults, and are associated with poor outcomes and MRD positivity. Expression of UBTF-TD in primary hematopoietic cells is sufficient to enhance serial clonogenic activity and to drive a similar transcriptional program to UBTF-TD AMLs. Collectively, these clinical, genomic, and functional data establish UBTF-TD as a new recurrent mutation in AML. Significance: We defined the spectrum of mutations in relapsed pediatric AML and identified UBTF-TDs as a new recurrent genetic alteration. These duplications are more common in children and define a group of AMLs with intermediate-risk cytogenetic abnormalities, FLT3-ITD and WT1 alterations, and are associated with poor outcomes. See related commentary by Hasserjian and Nardi, p. 173. This article is highlighted in the In This Issue feature, p. 171.

Children with acute myeloid leukemia (AML) have a dismal prognosis due to a high relapse rate; however, the molecular basis leading to relapsed pediatric AML has not yet been fully characterized. To define the spectrum of alterations common at relapse, we performed integrated profiling of 136 relapsed pediatric AML cases with RNA sequencing (RNA-seq), whole-genome sequencing, and target-capture sequencing. In addition to well-characterized fusion oncoproteins, such as those involving KMT2A (n=36, 26.5%) or NUP98 (n=18, 13.2%), we also identified somatic mutations in UBTF (upstream binding transcription factor) in 12 of 136 cases (8.8%) of this relapsed cohort. Somatic alterations of the UBTF gene, which encodes a nucleolar protein that is a component of the RNA Pol I pre-initiation complex to ribosomal DNA promoters, have rarely been observed in AML. In our cohort, all alterations can be described as heterozygous in-frame exon 13 tandem duplications (UBTF-TD), either at the 3' end of exon 13 of UBTF or of the entire exon 13 (Fig. A). As we noticed limited detection in our pipeline as a result of complex secondary indels alongside the duplications, we established a soft-clipped read-based screening method to detect UBTF-TD more efficiently. Applying the screening to RNA-seq data of 417 additional pediatric AMLs from previous studies and our clinical service, we identified 15 additional UBTF-TDs, many of which have not been previously reported. At the amino acid level, UBTF-TDs caused amino acid insertions of variable sizes (15-181 amino acids), duplicating a portion of high mobility group domain 4 (HMG4), which includes short leucine-rich sequences. UBTF-TD AMLs commonly occurred in early adolescence (median age: 12.6, range: 2.4-19.6), and 19 of the total 27 cases had either normal karyotype (n=12) or trisomy 8 (n=7). UBTF-TD is mutually exclusive from other recurrent fusion oncoproteins, such as NUP98 and KMT2A rearrangements (Fig. B), but frequently occurred with FLT3-ITD (44.4%) or WT1 mutations (40.7%). The median variant allele fraction (VAF) of the UBTF-TD was 48.0% (range: 9.7-66.7%). In four cases with data at multiple disease time points, the identical UBTF-TDs were present at high allele fractions at all time points, suggesting that UBTF-TD is a clonal alteration. tSNE analysis of the transcriptome dataset showed that UBTF-TD AMLs share a similar expression pattern with NPM1 mutant and NUP98-NSD1 AML subtypes, including NKX2-3 and HOXB cluster genes (Fig. C) . Altogether, these findings suggest that UBTF-TD is a unique subtype of pediatric AML. To address the impact of UBTF-TD expression in primary hematopoietic cells, we introduced UBTF-TD and UBTF wildtype expression vectors into cord blood CD34+ cells via lentiviral transduction. UBTF-TD expression promotes colony-forming activity and cell growth, yielding cells with a persistent blast-like morphology (Fig. D). Further, transcriptional profiling of these cells demonstrated expression of HOXB genes and NKX2-3, similar to UBTF-TD AMLs in patients, indicating that UBTF-TD is sufficient to induce the leukemic phenotype. To investigate the prevalence of UBTF-TDs in larger de novo AML cohorts, we applied the above UBTF-TD screening method to the available de novo AML cohorts of TCGA (n=151, adult), BeatAML (n=220, pediatric and adult), and AAML1031 (n=1035, pediatric). We identified UBTF-TDs in 4.3% (45/1035) of the pediatric AAML1031 cohort, while the alteration is less common (0.9%: 3/329, p=0.002) in the adult AML cohorts (Fig. E). In the AAML1031 cohort, UBTF-TDs remain mutually exclusive with known molecular subtypes of AML and commonly occur with FLT3-ITD (66.7%) and WT1 (40.0%) mutations and either normal karyotype or trisomy 8. The presence of UBTF-TDs in the AAML1031 cohort is associated with a poor outcome (Fig. F, median overall survival, 2.3 years) and MRD positivity; multivariate analysis revealed that UBTF-TD and WT1 are independent risk factors for overall survival within FLT3-ITD+ AMLs. In conclusion, we demonstrate UBTF-TD defines a unique subtype of AMLs that previously lacked a clear oncogenic driver. While independent of subtype-defining oncogenic fusions, UBTF-TD AMLs are associated with FLT3-ITD and WT1 mutations, adolescent age, and poor outcomes. These alterations have been under-recognized by standard bioinformatic approaches yet will be critical for future risk-stratification of pediatric AML. Figure 1 Figure 1. Disclosures Iacobucci: Amgen: Honoraria; Mission Bio: Honoraria. Miller: Johnson & Johnson's Janssen: Current Employment. Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company.

Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving subunits of the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n=87) and adult (n=78) samples, including cases with RUNX1-RUNX1T1 (n=85) or CBFB-MYH11 (n=80) rearrangements, by whole-genome or whole-exome sequencing. In addition to previously reported somatic mutations in the Ras signaling pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a recurrent and previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated a remarkably different spectrum of cooperating mutations as RUNX1-RUNX1T1 cases harbored recurrent somatic mutations in DHX15 and ZBTB7A, as well as an enrichment of somatic mutations in epigenetic regulators, including ASXL2, and in components of the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscape of cooperating mutations between these related AML subtypes.

Janus kinases are essential for signal transduction by a variety of cytokine receptors and when inappropriately activated can cause hematopoietic disorders and oncogenesis. Consequently, it can be predicted that the interaction of the kinases with receptors and the events required for activation are highly controlled. In a screen to identify phosphorylation events regulating Jak2 activity in EpoR signaling, we identified a mutant (Jak2-Y613E) which has the property of being constitutively activated, as well as an inactivating mutation (Y766E). Although no evidence was obtained to indicate that either site is phosphorylated in signaling, the consequences of the Y613E mutation are similar to those observed with recently described activating mutations in Jak2 (Jak2-V617F and Jak2-L611S). However, unlike the V617F or L611S mutant, the Y613E mutant requires the presence of the receptor but not Epo stimulation for activation and downstream signaling. The properties of the Jak2-Y613E mutant suggest that under normal conditions, Jak2 that is not associated with a receptor is locked into an inactive state and receptor binding through the FERM domain relieves steric constraints, allowing the potential to be activated with receptor engagement.

Rearrangement of immunoglobulin heavy-chain variable (V(H)) gene segments has been suggested to be regulated by interleukin 7 signaling in pro-B cells. However, the genetic evidence for this recombination pathway has been challenged. Furthermore, no molecular components that directly control V(H) gene rearrangement have been elucidated. Using mice deficient in the interleukin 7-activated transcription factor STAT5, we demonstrate here that STAT5 regulated germline transcription, histone acetylation and DNA recombination of distal V(H) gene segments. STAT5 associated with V(H) gene segments in vivo and was recruited as a coactivator with the transcription factor Oct-1. STAT5 did not affect the nuclear repositioning or compaction of the immunoglobulin heavy-chain locus. Therefore, STAT5 functions at a distinct step in regulating distal V(H) recombination in relation to the transcription factor Pax5 and histone methyltransferase Ezh2.

The Evi-1 gene was first identified as a site for viral integration in murine myeloid leukemia. Evi-1 is a zinc finger transcription factor that has been implicated in the development of myeloid neoplasia. In humans, disruption of the Evi-1 locus, by chromosomal rearrangements, is associated with myeloid leukemia and myelodyplastic syndromes. Here, we report the cloning and developmental pattern of expression of Xenopus Evi-1. xEvi-1 is expressed during oogenesis and during embryonic development. In situ hydridization reveals that xEvi-1 has a dynamic expression profile during early embryonic development. Expression of Evi-1 is detected by in situ hybridization in the pronephric tissue, the brain and in neural crest derivatives of the head and neck.

Suppressor of cytokine signaling 1 (SOCS1) is an obligate negative regulator of cytokine signaling and most importantly in vivo, signaling via the interferon-gamma (IFN-gamma) receptor. SOCS1, via its Src homology 2 domain, binds to phosphotyrosine residues in its targets, reducing the amplitude of signaling from cytokine receptors. SOCS1 is also implicated in blocking Toll-like receptor (TLR) signaling in macrophages activated by TLR agonists such as lipopolysaccharide (LPS), thus regulating multiple steps in the activation of innate immune responses. To rigorously test this, we isolated macrophages from Socs1-/- mice on multiple genetic backgrounds. We found no evidence that SOCS1 blocked TLR-activated pathways, endotoxin tolerance, or nitric oxide production. However, Socs1-/-;IFN-gamma-/- mice were extremely susceptible to LPS challenge, confirming previous findings. Because LPS induces IFN-beta production from macrophages, we tested whether SOCS1 regulates IFN-alpha/beta receptor signaling. We find that SOCS1 is required to inhibit IFN-alpha/beta receptor signaling in vitro. Furthermore, the absence of a single allele encoding TYK2, a JAK (Janus kinase) family member essential IFN-alpha/beta receptor signaling, rescued Socs1-/- mice from early lethality, even in the presence of IFN-gamma. We conclude that previous reports linking SOCS1 to TLR signaling are most likely due to effects on IFN-alpha/beta receptor signaling.

Puma encodes a BH3-only protein that is induced by the p53 tumor suppressor and other apoptotic stimuli. To assess its physiological role in apoptosis, we generated Puma knockout mice by gene targeting. Here we report that Puma is essential for hematopoietic cell death triggered by ionizing radiation (IR), deregulated c-Myc expression, and cytokine withdrawal. Puma is also required for IR-induced death throughout the developing nervous system and accounts for nearly all of the apoptotic activity attributed to p53 under these conditions. These findings establish Puma as a principal mediator of cell death in response to diverse apoptotic signals, implicating Puma as a likely tumor suppressor.

Suppressor of cytokine signaling (SOCS) proteins are feedback inhibitors of the Janus kinase (JAK) and signal transducer and activator of transcription (STAT) signaling pathway. SOCS3 is upregulated by several signals in macrophages and has been implicated as a regulator of various signaling pathways. Here we show that phosphorylation of STAT3 is prolonged in mouse Socs3-deficient macrophages after stimulation with interleukin-6 (IL-6) but not IL-10, indicating that SOCS3 specifically affects signaling mediated by IL-6 and gp130. IL-6 induces a wider transcriptional response in Socs3-deficient macrophages than in wild-type cells; this response is dominated by interferon (IFN)-regulated genes owing to an excess of STAT1 phosphorylation. Thus, SOCS3 functions to control the quality of the response to IL-6 and prevents the activation of an IFN-induced program of gene expression.

Myc and E2f1 promote cell cycle progression, but overexpression of either can trigger p53-dependent apoptosis. Mice expressing an Eμ- Myc transgene in B lymphocytes develop lymphomas, the majority of which sustain mutations of either the Arf or p53 tumor suppressors. Eμ- Myc transgenic mice lacking one or both E2f1 alleles exhibited a slower onset of lymphoma development associated with increased expression of the cyclin-dependent kinase inhibitor p27 Kip1 and a reduced S phase fraction in precancerous B cells. In contrast, Myc-induced apoptosis and the frequency of Arf and p53 mutations in lymphomas were unaffected by E2f1 loss. Therefore, Myc does not require E2f1 to induce Arf, p53, or apoptosis in B cells, but depends upon E2f1 to accelerate cell cycle progression and downregulate p27 Kip1 .

Defining signals that can support the self-renewal of multipotential hemopoietic progenitor cells (MHPCs) is pertinent to understanding leukemogenesis and may be relevant to developing stem cell-based therapies. Here we define a set of signals, JAK2 plus either c-kit or flt-3, which together can support extensive MHPC self-renewal. Phenotypically and functionally distinct populations of MHPCs were obtained, depending on which receptor tyrosine kinase, c-kit or flt-3, was activated. Self-renewal was abrogated in the absence of STAT5a/b, and in the presence of inhibitors targeting either the mitogen-activated protein kinase or phosphatidylinositol 3' kinase pathways. These findings suggest that a simple two-component signal can drive MHPC self-renewal.

Bcl-2 family proteins are key regulators of apoptosis and function as cell death antagonists (e.g., Bcl-2, Bcl-XL, and Mcl-1) or agonists (e.g., Bax, Bad, and Bak). Here we report that among the Bcl-2 family of proteins tested (Bcl-2, Bcl-XL, Mcl-1, Bax, Bad, and Bak), Bcl-XLwas unique in that its protein levels were tightly regulated by hemopoietins in both immortal and primary myeloid progenitors. Investigating signaling pathways utilized by cytokine receptors established that the regulation of Bcl-XLprotein levels is mediated by the Jak kinase pathway and is independent of other signaling effectors including STATs, PI-3′ kinase, and Ras. Moreover, we provide the first direct evidence thatBcl-Xis altered in cancer, becausebcl-Xexpression was activated selectively by retroviral insertions in murine myeloid and T-cell hemopoietic malignancies. Tumors harboringbcl-Xinsertions had alteredbcl-XRNAs, expressed elevated levels of Bcl-XLprotein, and lacked the requirements for cytokines normally essential for cell survival. Finally, overexpression of Bcl-XLeffectively protected IL-3-dependent myeloid cells from apoptosis following removal of trophic factors. Therefore, Bcl-XLfunctions as a key cytokine regulated anti-apoptotic protein in myelopoiesis and contributes to leukemia cell survival.

Two genes have been implicated in leukemias of patients with abnormalities of chromosome 3, band q26: EVI1, which can be activated over long distances by chromosomal rearrangements involving 3q26, and EAP, a ribosomal gene that fuses with AML1 in a therapy-related myelodysplasia patient with a t(3;21)(q26.2;q22). AML1 was identified by its involvement in the t(8;21)(q22;q22) of acute myeloid leukemia. Here we report the consistent identification of fusion transcripts between AML1 and EAP or between AML1 and previously unidentified sequences that we named MDS1 (MDS-associated sequences) in the leukemic cells of four patients with therapy-related myelodysplasia/acute myeloid leukemia and in one patient with chronic myelogenous leukemia in blast crisis, all of whom had a t(3;21). In addition, we have identified a third chimeric transcript, AML1/EVI1, in one of the therapy-related acute myeloid leukemia patients. Pulsed-field gel electrophoresis established the order of the genes as EAP, the most telomeric, and EVI1, the most centromeric, gene. The results indicate that translocations could involve multiple genes and affect gene expression over long distances.

Disruption of p53/Puma-mediated apoptosis protects against lethality due to DNA damage. Here we demonstrate the unexpected requirement of the pro-apoptotic p53-target gene Puma to mount a successful innate immune response to bacterial sepsis. Puma−/− mice rapidly died when challenged with bacteria. While the immune response in Puma−/− mice was unchanged in cell migration, phagocytosis and bacterial killing, sites of infection accumulated large abscesses and sepsis was progressive. Blocking p53/Puma-induced apoptosis during infection caused resistance to ROS-induced cell death in the CD49d+ neutrophil subpopulation, resulting in insufficient immune resolution. This study identifies a biological role for p53/Puma apoptosis in optimizing neutrophil lifespan so as to ensure the proper clearance of bacteria and exposes a counter-balance between the innate immune response to infection and survival from DNA damage., Author Summary Protection against fulminant, lethal bacterial sepsis requires a robust and rapid response from the neutrophil subclass of white blood cells. However, prolonged survival of these activated cells leads to organ damage. The cellular processes determining the optimal life span of neutrophils are not understood. The p53/Puma pathway drives programmed cell death by initiating apoptosis. This response is beneficial in preventing cellular transformation by eliminating abnormal cells that have sustained DNA damage. Here we demonstrate an opposing effect of the pathway in the context of a successful immune response to bacterial sepsis. Puma−/− mice rapidly died when challenged with bacteria and sites of infection accumulated large abscesses. Blocking p53/Puma-induced apoptosis during infection prolonged the life of the CD49d+ neutrophil subpopulation, resulting in insufficient immune resolution. This study expands the biological role of p53/Puma-induced apoptosis to the proper clearance of bacteria by neutrophils and exposes a counter-balance between the innate immune response to infection and survival from DNA damage.

Retroviral activation of Evi-1 gene expression is one of the most common transforming events in murine myeloid leukemias. To evaluate the role of the EVI1 gene in human acute myelogenous leukemia (AML), leukemic blasts or cell lines from 116 patients were examined. In eight patients the EVI1 gene was expressed and all but one had cytogenetically detectable translocations of chromosome 3q26 where the EVI1 gene has been localized. To identify breakpoints, a restriction map that spans 1700 kilobases (kb) of the EVI1 locus was developed by pulsed-field gel electrophoresis. In one case, t(3;3)(q21;q26), a rearrangement was localized to 170-330 kb 5' of the gene. In a second case, t(3;3)(q21;q26), there was a rearrangement 13 kb 5' of the gene. This rearrangement was cloned and shown to be due to the fusion of sequences from 3q21-22 with the EVI1 locus. In the third case, ins(3)-(q21q25q27), there was a rearrangement that mapped 150 kb downstream from the 5' end of the gene.

Expression of the Evi-1 gene is frequently activated in murine myeloid leukemias by retroviral insertions immediately 5' or 90 kb 5' of the gene. The Evi-1 gene product is a nuclear, DNA-binding zinc finger protein of 145 kDa. On the basis of the properties of the myeloid cell lines in which the Evi-1 gene is activated, it has been hypothesized that its expression blocks normal differentiation. To explore this proposed role, we have constructed a retrovirus vector containing the gene and examined its effects on an interleukin-3-dependent myeloid cell line that differentiates in response to granulocyte colony-stimulating factor (G-CSF). Expression of the Evi-1 gene in these cells did not alter the normal growth factor requirements of the cells. However, expression of the Evi-1 gene blocked the ability of the cells to express myeloperoxidase and to terminally differentiate to granulocytes in response to G-CSF. This effect was not due to altered expression of the G-CSF receptor or to changes in the initial responses of the cells to G-CSF. These results support the hypothesis that the inappropriate expression of the Evi-1 gene in myeloid cells interferes with the ability of the cells to terminally differentiate.

Janus kinase 2 (Jak2) has a pivotal role in erythropoietin (Epo) signaling pathway, including erythrocyte differentiation and Stat5 activation. In the course of screening for critical phosphorylation of tyrosine residues in Jak2, we identified tyrosine 913 (Y(913)) as a novel and functional phosphorylation site, which negatively regulates Jak2. Phosphorylation at Y(913) rapidly occurred and was sustained for at least 120 min after Epo stimulation, in contrast to the transient phosphorylation of Y(1007/1008) in the activation loop of Jak2. Interestingly, phosphorylation defective mutation of Y(913) (Y(913)F) results in a significant enhancement of Epo-induced Jak2 activation, whereas phosphorylation mimic mutation of Y(913) (Y(913)E) completely abrogated its activation. Furthermore, Jak2 deficient fetal liver cells expressing Y(913)F mutant generated many mature erythroid BFU-E and CFU-E colonies, while Y(913)E mutant failed to reconstitute Jak2 deficiency. We also demonstrate, in Jak1, phosphorylation of Y(939), a corresponding tyrosine residue with Y(913), negatively regulated Jak1 signaling pathway. Accordingly, our results suggest that this tyrosine phosphorylation in JH1 domain may be involved in common negative regulation mechanism for Jak family.

Insights into the function of Hax1 are provided by a study showing that Hax1 facilitates the processing of the mitochondrial protease HtrA2 by the mitochondrial rhomboid protease Parl. It is the protease activity of HtrA2 that is essential to suppress apoptosis in both lymphocytes and striatal neurons. Cytokines affect a variety of cellular functions, including regulation of cell numbers by suppression of programmed cell death1. Suppression of apoptosis requires receptor signalling through the activation of Janus kinases and the subsequent regulation of members of the B-cell lymphoma 2 (Bcl-2) family. Here we demonstrate that a Bcl-2-family-related protein, Hax1, is required to suppress apoptosis in lymphocytes and neurons. Suppression requires the interaction of Hax1 with the mitochondrial proteases Parl (presenilin-associated, rhomboid-like) and HtrA2 (high-temperature-regulated A2, also known as Omi). These interactions allow Hax1 to present HtrA2 to Parl, and thereby facilitates the processing of HtrA2 to the active protease localized in the mitochondrial intermembrane space. In mouse lymphocytes, the presence of processed HtrA2 prevents the accumulation of mitochondrial-outer-membrane-associated activated Bax, an event that initiates apoptosis. Together, the results identify a previously unknown sequence of interactions involving a Bcl-2-family-related protein and mitochondrial proteases in the ability to resist the induction of apoptosis when cytokines are limiting.

Mast cells are critical effectors of allergic disease, and are now implicated in immune responses observed in arthritis, multiple sclerosis, and heart disease. Because of their role in inflammation, understanding how mast cells develop is of clinical importance. In this study we determined the effects of IFN-γ on mast cell survival. Using in vitro culture of bone marrow cells in IL-3 plus stem cell factor, we found that the addition of IFN-γ induced apoptosis, as exhibited by the presence of subdiploid DNA and caspase activation. IFN-γ-mediated apoptosis was Stat1-dependent, and was accompanied by loss of mitochondrial membrane potential. Apoptosis was reduced in cultures of bone marrow cells derived from p53- or Bax-deficient mice, as well as H2K-Bcl-2 transgenic mice. IFN-γ hyperresponsiveness has been shown to result in inflammatory disease and death in mice lacking the regulatory protein suppressor of cytokine signaling (SOCS)-1. Bone marrow cells from SOCS-1 knockout (KO) mice failed to give rise to viable mast cells after culture in IL-3 plus stem cell factor, with profound apoptosis occurring as the cultures matured. However, bone marrow cells lacking both SOCS-1 and IFN-γ survived normally. This in vitro defect in mast cell development was recapitulated in vivo. SOCS-1 KO mice demonstrated a 67% decrease in peritoneal mast cell numbers relative to wild-type mice, a deficiency that was reversed in SOCS-1/IFN-γ KO mice. These data demonstrate the potent regulatory effects of IFN-γ on mast cell survival and show that this cytokine can elicit mast cell death in vitro and in vivo.

The tyrosine kinase, Janus kinase-2 (Jak2), plays a pivotal role in signal transduction through a variety of cytokine receptors, including the receptor for erythropoietin (Epo). Although the physiological relevance of Jak2 has been definitively established, less is known about its regulation. In studies assessing the roles of sites of tyrosine phosphorylation, we identified Y(119) in the FERM (band 4.1, Ezrin, radixin and moesin) domain as a phosphorylation site. In these studies, we demonstrate that the phosphorylation of Y(119) in response to Epo downregulates Jak2 kinase activity. Using a phosphorylation mimic mutation (Y(119)E), downregulation is shown to involve dissociation of Jak2 from the receptor complex. Conversely, a Y(119)F mutant is more stably associated with the receptor complex. Thus, in cytokine responses, ligand binding induces activation of receptor associated Jak2, autophosphorylation of Y(119) in the FERM domain and the subsequent dissociation of the activated Jak2 from the receptor and degradation. This regulation occurs with the receptors for Epo, thrombopoietin and growth hormone but not with the receptor for interferon-gamma.

Suppressor of cytokine signaling 3 (SOCS3) binds cytokine receptors and thereby suppresses cytokine signaling. Deletion of SOCS3 causes an embryonic lethality that is rescued by a tetraploid rescue approach, demonstrating an essential role in placental development and a non-essential role in embryo development. Rescued SOCS3-deficient mice show a perinatal lethality with cardiac hypertrophy. SOCS3-deficient placentas have reduced spongiotrophoblasts and increased trophoblast secondary giant cells. Enforced expression of SOCS3 in a trophoblast stem cell line (Rcho-1) suppresses giant cell differentiation. Conversely, SOCS3-deficient trophoblast stem cells differentiate more readily to giant cells in culture, demonstrating that SOCS3 negatively regulates trophoblast giant cell differentiation. Leukemia inhibitory factor (LIF) promotes giant cell differentiation in vitro, and LIF receptor (LIFR) deficiency results in loss of giant cell differentiation in vivo. Finally, LIFR deficiency rescues the SOCS3-deficient placental defect and embryonic lethality. The results establish SOCS3 as an essential regulator of LIFR signaling in trophoblast differentiation.

The identification of common virus integration sites (cVIS) in retrovirally induced tumors in mice provides a powerful strategy to isolate novel transforming genes. Applying virus LTR-specific inverse-PCR and RT-PCR combined with automated sequencing on CasBr-M Murine Leukemia Virus (MuLV) induced myeloid leukemias, 126 virus integration sites were cloned. Using locus- and LTR-specific primers, a nested-PCR/Southern-blotting procedure was developed on genomic DNA from a large panel of MuLV-induced leukemias, to analyse whether a particular virus insertion represented a cVIS. In fact 39 out of 41 integrations analysed this way appeared to represent a common virus integration. We recognized six previously cloned cVISs, i.e. Evi1, Hoxa7, c-Myb, Cb2/Evi11, Evi12, and His1 and 33 novel common insertions, designated Cas-Br Virus Integration Site (Casvis). Among this group we found integrations in or near genes encoding nuclear proteins, e.g. Dnmt-2, Nm23-M2, Ctbp1 or Erg, within receptor genes, e.g. Cb2 or mrc1, novel putative signaling or transporter genes, the ringfinger-protein gene Mid1 and a panel of genes encoding novel proteins with unknown function. The finding that 39 out of 41 integrations analysed represented a cVIS, suggests that the majority of the other virus insertions that were not yet analysed by the PCR/Southern-blotting method are located in a cVIS as well and may therefore also harbor novel disease genes.

Many receptors activate phospholipase Cgamma1 or -gamma2. To assess the role of PLCgamma2, we derived enzyme-deficient mice. The mice are viable but have decreased mature B cells, a block in pro-B cell differentiation, and B1 B cell deficiency. IgM receptor-induced Ca2+ flux and proliferation to B cell mitogens are absent. IgM, IgG2a, and IgG3 levels are reduced, and T cell-independent antibody production is absent. The similarity to Btk- or Blnk-deficient mice demonstrates that PLCgamma2 is downstream in Btk/Blnk signaling. FcRgamma signaling is also defective, resulting in a loss of collagen-induced platelet aggregation, mast cell FcepsilonR function, and NK cell FcgammaRIII and 2B4 function. The results define a signal transduction pathway broadly utilized by immunoglobulin superfamily receptors.

PEG-rmMGDF (60 μg/kg) given to mice immediately after lethal myelosuppression (Carboplatin 80 mg/kg + 750R Cs-137), prevents death and promotes multilineage hematopoietic reconstitution (Pestina et al ., Exp Hematol 27, Suppl 1: 100, 1999). Our data from study of p53-deficient mice suggests that this myeloprotection results in part by preventing p53-dependent apoptosis of c-Mpl-expressing multipotential hematopoietic cells. To define molecules involved in the antiapoptotic signaling pathways induced by c-Mpl ligation, we performed in vivo studies using mice with specific gene deletions. Jak-2 tyrosine kinase is rapidly activated as a result of c-Mpl ligation, but its role in antiapoptotic signaling has not been examined in vivo . Because Jak2 −/− is an embryonic lethal due to failed development of hematopoiesis, we examined whether adult Jak2 +/− mice were protected from lethal myelosuppression by PEG-rmMGDF. The survival rate of Jak2 +/ − mice (68%) was lower than that of wild-type (WT) mice (91%), indicating that Jak2 activation plays a role in the myeloprotective signaling pathway in vivo. We next investigated the importance of the Jak2 substrates Stat5a and b in this pathway using mice with combined deficiency of these Stat proteins. Intact Stat5ab −/− mice had normal steady-state platelet counts and similar elevations in platelet numbers in response to PEG-rmMGDF to those of WT mice, suggesting that neither Stat5 protein is required during megakaryocytopoieses. However, Stat5 −/− mice exposed to the myelosuppressive regimen were severely compromised in their ability to be protected by PEG-rmMGDF (19% survival vs. 100% for Stat5ab +/− and 90% for WT mice). These findings provide in vivo evidence that Jak2 and Stat5 play important roles in the myeloprotective signaling pathways initiated by c-Mpl ligation.

SOCS3 (CIS3/JAB2) is an SH2-containing protein that binds to the activation loop of Janus kinases, inhibiting kinase activity, and thereby suppressing cytokine signaling. During embryonic development, SOCS3 is highly expressed in erythroid lineage cells and is Epo independent. Transgene-mediated expression blocks fetal erythropoiesis, resulting in embryonic lethality. SOCS3 deletion results in an embryonic lethality at 12–16 days associated with marked erythrocytosis. Moreover, the in vitro proliferative capacity of progenitors is greatly increased. SOCS3-deficient fetal liver stem cells can reconstitute hematopoiesis in lethally irradiated adults, indicating that its absence does not disturb bone marrow erythropoiesis. Reconstitution of lymphoid lineages in JAK3-deficient mice also occurs normally. The results demonstrate that SOCS3 is critical in negatively regulating fetal liver hematopoiesis.

A variety of cytokines activate receptor-associated members of the Janus family of protein tyrosine kinases (Jaks). To assess the role of Jak2, we have derived Jak2-deficient mice. The mutation causes an embryonic lethality due to the absence of definitive erythropoiesis. Fetal liver myeloid progenitors, although present based on the expression of lineage specific markers, fail to respond to erythropoietin, thrombopoietin, interleukin-3 (IL-3), or granulocyte/macrophage colony-stimulating factor. In contrast, the response to granulocyte specific colony-stimulating factor is unaffected. Jak2-deficient fibroblasts failed to respond to interferon gamma (IFNgamma), although the responses to IFNalpha/beta and IL-6 were unaffected. Lastly, reconstitution experiments demonstrate that Jak2 is not required for the generation of lymphoid progenitors, their amplification, or functional differentiation. Therefore, Jak2 plays a critical, nonredundant role in the function of a specific group of cytokines receptors.

SOCS1 is an SH2-containing protein that is primarily expressed in thymocytes in a cytokine- and T cell receptor–independent manner. SOCS1 deletion causes perinatal lethality with death by 2–3 weeks. During this period thymic changes include a loss of cellularity and a switch from predominantly CD4 + CD8 + to single positive cells. Peripheral T cells express activation antigens and proliferate to IL-2 in the absence of anti-CD3. In addition, IFNγ is present in the serum. Reconstitution of the lymphoid lineage of JAK3-deficient mice with SOCS1-deficient stem cells recapitulates the lethality and T cell alterations. Introducing a RAG2 or IFNγ deficiency eliminates lethality. The results demonstrate that lymphocytes are critical to SOCS1-associated perinatal lethality and implicate SOCS1 in lymphocyte differentiation or regulation.

The EVI1 gene encodes a zinc-finger, DNA-binding protein originally described as the transforming gene associated with a common ecotropic viral insertion site in myeloid leukemias. Previous studies demonstrated EVI1 expression in human leukemias in cases with 3q26 translocations, but not in normal blood or bone marrow. These studies also suggested an association between EVI1 expression and chromosome 7 deletion (del). Because of this association, we examined expression of EVI1 using RNA polymerase chain reaction (PCR) in patients with myelodysplastic syndromes (MDS) and acute leukemia with and without 3q26 translocations. EVI1 RNA was expressed in 29% of 34 (95% confidence interval, 20% to 50%) patients with the MDS subtypes refractory anemia (RA), refractory anemia with excess blasts (RAEB), or refractory anemia with excess blasts in transformation (RAEB-T). The vast majority of these cases occurred in patients with RAEB and RAEB-T. EVI1 expression was not detected in patients with chronic myelomonocytic leukemia (CMML), normal bone marrow or cord blood, or a variety of other hematologic malignancies. EVI1 RNA was detected in three of 18 patients with acute myelogenous leukemia (AML) and in two of four patients with acute promyelocytic leukemia (APL). Karyotypes showed that only one AML patient had karyotype 3q26 abnormalities, indicating that EVI1 expression is associated with cases that do not have structural abnormalities involving chromosome 3q26. These studies document for the first time the abnormal expression of EVI1 RNA by patients with MDS, and suggest an important role for EVI1 in the pathogenesis or progression of some myeloid malignancies.