Mutations In GFI1 Research Articles

P835: GENETIC BACKGROUND AND CLINICAL CHARACTERISTICS OF CONGENITAL NEUTROPENIAS IN ISRAEL

Background: Congenital neutropenias comprise a heterogenous group of disorders causing low neutrophil counts with a variable clinical presentation. Patients may suffer from severe infections and have a high risk of myeloid transformation. To date, over 30 genes are known to cause congenital neutropenias, with a diverse relative frequency in different ethnicities. Understanding the spectrum of genetic causes of neutropenia in ethnically diverse populations and the related clinical presentation may direct the molecular work-up and clinical care. Israel is enriched with multiple ethnicities and high consanguinity. Aims: To evaluate the clinical and genetic spectrum of patients with congenital neutropenias in Israel. Methods: All patients diagnosed with congenital neutropenia from the Israeli Inherited Bone Marrow Failure registry were included. Data were extracted from the registry and patients’ charts. Sanger sequencing was first performed for ELANE or G6PC3, followed by less commonly mutated genes. In recent years, patients with wild-type ELANE/G6PC3 were referred for targeted next generation sequencing, followed by whole exome sequencing. Results: The cohort encompasses 66 patients, from 50 families, including 7 with cyclic neutropenia. Fifty patients (76%) were genetically diagnosed; 22 (33% of the cohort) had mutations in ELANE, 9 (13.6%) had homozygous G6PC3 mutations, 7 (10.6%) SBDS mutations, 6 (9%) patients had SRP54 mutations, two each had heterozygous GFI1 and homozygous JAGN1 mutations and one each had TAZ and SLC37A1 mutations. All patients with G6PC3 and GFI1 mutations were of consanguineous Muslim Arab origin. Extra-hematopoietic manifestations were common among patients with mutations in G6PC3. Most patients (82%) presented with severe infections and this was also the most common cause of death. Eight patients (12%) developed myeloid transformation; in 6 of those a somatic molecular work-up was performed, and truncating mutations in the granulocyte-colony stimulating factor were detected in 4. Eighteen (27%) patients underwent hematopoietic stem cell transplantation, most often due to insufficient response to treatment with granulocyte-colony stimulating factor. Summary/Conclusion: The prevalence of genes causing congenital neutropenias in Israel is unique, as it includes a relatively high frequency of patients with mutations in G6PC3 and an absence of patients with HAX1 mutations, thus directing the genetic work-up and the clinical care. Similar to other registries, a molecular diagnosis was not achieved for 24% of the patients, suggesting yet unknown genetic causes.

Loss of murine Gfi1 causes neutropenia and induces osteoporosis depending on the pathogen load and systemic inflammation.

Gfi1 is a key molecule in hematopoietic lineage development and mutations in GFI1 cause severe congenital neutropenia (SCN). Neutropenia is associated with low bone mass, but the underlying mechanisms are poorly characterized. Using Gfi1 knock-out mice (Gfi1-ko/ko) as SCN model, we studied the relationship between neutropenia and bone mass upon different pathogen load conditions. Our analysis reveals that Gfi1-ko/ko mice kept under strict specific pathogen free (SPF) conditions demonstrate normal bone mass and survival. However, Gfi1-ko/ko mice with early (nonSPF) or late (SPF+nonSPF) pathogen exposure develop low bone mass. Gfi1-ko/ko mice demonstrate a striking rise of systemic inflammatory markers according to elevated pathogen exposure and reduced bone mass. Elevated inflammatory cytokines include for instance Il-1b, Il-6, and Tnf-alpha that regulate osteoclast development. We conclude that low bone mass, due to low neutrophil counts, is caused by the degree of systemic inflammation promoting osteoclastogenesis.

Abstract B19: Lysine specific demethylase-1 inhibition as a therapeutic strategy that leverages the requirement for growth factor independence-1 in Notch-driven T-ALL

Abstract The zinc-finger (ZnF) transcriptional repressor Growth Factor Independence (GFI) 1 is a master regulator of lineage allocation in hematopoietic, aerodigestive tract and central nervous system development. Transcriptional repression by GFI1 requires a SNAG domain at its N-terminus and a concatemer of six ZnFs at its C-terminus. The SNAG domain recruits Lysine Specific Demethylase (LSD) 1, a dominant effector of transcriptional repression by GFI1, while ZnFs 3-5 bind a consensus response element in the promoters of GFI1 target genes. A linker region separates the SNAG and ZnF domains in GFI1, and likely provides sites for protein—protein interactions and post-translational modifications that modulate GFI1 functions in cell fate determination. In hematopoiesis, GFI1 regulates self-renewal and maintenance within the hematopoietic stem cell (HSC) compartment, supports early events in lymphopoiesis and is required for maturation of granulocytic precursors. Mutations in GFI1 cause severe congenital neutropenia (SCN) type II, a pre-leukemic condition, and recent evidence shows that GFI1 maintains the malignant phenotype in lymphoid leukemias and lymphomas. These findings intimate that regulators and effectors of GFI1 may be attractive candidates for therapeutic development in hematologic malignancy and maturational failure syndromes. How GFI1 is regulated to control cell fates during hematopoiesis is unknown. We show that GFI1 contains a consensus SUMOylation motif, centered on lysine (K) 239 within its linker region that is required for GFI1 conjugation by SUMO/Ubiquitin-like (UBL) proteins. K239 is found within the binding site for PIAS3, an E3 SUMO ligase and known GFI1 binding protein. PIAS3 antagonizes GFI1-mediated transcriptional repression. Arginine substitution at K239 (GFI1-K239R) preserves transcriptional repression by GFI1 yet resists the inhibitory effects of PIAS3. Moreover, the GFI1-K239R derivative displays a prolonged half-life and preferentially segregates with chromatin in fractionated cells. These findings suggest that GFI1-mediated transcriptional repression is limited by SUMO/UBL protein conjugation and that its roles in cell fate determination may be altered by factors governing these modifications. Among these factors is the Notch1 intracellular domain (N1-ICD), which we show interacts with the same region in GFI1 that binds PIAS3 and competes with PIAS3 for GFI1 binding. Furthermore, N1-ICD abolishes GFI1 conjugation by SUMO/UBL proteins, prolongs GFI1 half-life and reverses the inhibitory effect of PIAS3 toward GFI1-mediated transcriptional repression. In so doing, N1-ICD supports the function of GFI1 as a transcriptional repressor. We also show that Gfi1 depletion causes profound apoptosis in T-ALL cells driven by N1-ICD, suggesting GFI1 inhibitors and/or small molecule inhibitors of its effectors may prove efficacious in T-ALL treatment. To address this hypothesis, we have developed reversible small molecule inhibitors of LSD1. These inhibitors display sub-micromolar IC50 toward T-ALL cell lines and primary patient isolates in the ex vivo setting, including those resistant to γ-secretase inhibitors. These findings suggest that the GFI1—LSD1 axis has significant influence over T-ALL viability and that LSD1 may be a novel therapeutic target in cancers driven by Notch activating mutations. Citation Format: Jason Singer, Daniel Andrade, Diana Bareyan, David McClellan, Helena Lucente, Matthew Velinder, Luke Maese, Mahesh Chandrasekharan, Emily Theisen, Fang Liu, Sunil Sharma, Michael Engel. Lysine specific demethylase-1 inhibition as a therapeutic strategy that leverages the requirement for growth factor independence-1 in Notch-driven T-ALL. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B19.

Notch Alters Sumoylation To Govern GFI1 Protein Stability and Support Its Transcriptional Repression Function

The zinc-finger (ZF) transcriptional repressors, Growth factor independence (GFI)-1 and -1B are master regulators of lineage allocation in hematopoietic, aerodigestive tract and central nervous system development. GFI family members share near invariant SNAG and ZF domains at their N- and C-termini, respectively. Their SNAG domains recruit Lysine Specific Demethylase (LSD)-1, a dominant effector of transcriptional repression by GFI proteins, while ZFs 3-5 of both GFI1 and GFI1B recognize a common consensus element in target promoters. A non-homologous linker separates the SNAG and ZF domains. Despite near identical DNA binding and co-factor recruitment characteristics, GFI family proteins direct distinct hematopoietic outcomes. Granulopoiesis requires Gfi1, and Gfi1 mutations cause severe congenital neutropenia (SCN) type II. Gfi1 is also necessary for T-lymphopoiesis and maintenance of the malignant phenotype in lymphoid leukemias and lymphomas. In contrast, Gfi1b specifies erythroid and megakaryocytic fates and is often abundantly expressed in myeloid leukemias. These distinct outcomes cannot be explained by patterns of expression alone, as Gfi1 and Gfi1B transcripts partially overlap during hematopoiesis. These findings suggest a need to distinguish between GFI family proteins as they integrate developmental signals to coordinate alternate hematopoietic fates. How this occurs is not understood. We hypothesized that functional differences between GFI1 and GFI1B may arise from interactions and post-translational modifications occurring uniquely within their linker regions. We show the GFI1 linker contains a type-I SUMOylation consensus motif centered on K239 that is absolutely conserved among mammalian GFI1 proteins but absent from GFI1B. K239 is required for robust GFI1 SUMOylation, as arginine substitution (K239R) significantly impairs formation of high molecular weight, SUMO-conjugated GFI1 derivatives. Coincidentally, K239R substitution also significantly impairs LSD1 recruitment by the GFI1 SNAG domain. The SUMOylation site in the GFI1 linker is contained within the binding site for PIAS3, an E3 SUMO ligase. PIAS3 co-expression antagonizes transcriptional repression by GFI1, but not GFI1B. The GFI1-K239R derivative represses transcription comparable to wild type GFI1, but resists the inhibitory effects of PIAS3. Moreover, GFI1-K239R preferentially co-purifies with chromatin in fractionated cells, displaying a prolonged half-life relative to wild type GFI1. These data suggest that GFI1 SUMOylation occurs on chromatin, facilitates LSD1 recruitment and serves as a prelude to GFI1 degradation. We surmised that the Notch1 intracellular domain (N1-ICD), which requires GFI1 for both normal and malignant T-lymphopoiesis, might alter GFI1 SUMOylation to reinforce its functions as a transcriptional regulator. We show that N1-ICD interacts directly with the GFI1 linker via the same motif that binds PIAS3. N1-ICD and PIAS3 compete for GFI1 binding and display an oppositional relationship in GFI1-mediated transcriptional repression. N1-ICD reinforces GFI1 as a transcriptional repressor, blocks GFI1 conjugation by ubiquitin family proteins and prevents GFI1 degradation. Our findings suggest a post-transcriptional mechanism by which Notch signaling endorses GFI1 as a transcriptional regulator and intimate GFI1 binding partners like LSD1 as candidate biological response modifiers in Notch-driven malignancies. Disclosures:No relevant conflicts of interest to declare.

Deficiency Of JAGN1 Causes Severe Congenital Neutropenia Associated With Defective Secretory Pathway and Aberrant Myeloid Cell Homeostasis

▪Analysis of patients with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling differentiation, maintenance, and decay of neutrophil granulocytes. Mutations in ELANE, GFI1, HAX1, G6PC3, WAS, and VPS45 are known to cause SCN.We here describe a new monogenetic SCN variant with biallelic mutations in the gene encoding Jagunal homolog 1 (JAGN1). We studied an index family from Northern Africa with a total of 5 children suffering from SCN. An Affymetrix SNP array-based genetic linkage analysis was performed and identified a single interval of perfect segregation with highly significant multi-marker LOD scores of at least 4.5spanning approximately 1.5Mbp from 9.52Mb to 11.04Mb on chromosome 3 of NCBI's human genome build 36.3. This interval contained a total of 30 genes, including JAGN1 which encodes an ER-resident protein. Sanger sequencing revealed a homozygous mutation c.3G>A in exon 1 of the JAGN1 gene; this mutation leads to disruption of the defined start of translation.Systematic analysis of a cohort of 90 SCN patients identified 9 distinct homozygous mutations in the gene encoding Jagunal homolog 1 (JAGN1) in 14 SCN patients, thus accounting for approximately 10% of SCN patients. The clinical phenotype was variable and included failure to thrive, developmental delay and bone skeletal abnormalities. The only consistent finding in all JAGN1-deficient patients was SCN and partial or complete refractoriness to therapy with rh-GCSF.JAGN1 is the human ortholog of a gene originally identified in Drosophila melanogaster. Jagunal-deficient oocytes are characterized by defective ER reorganization and aberrant membrane trafficking during vitellogenesis.We found that JAGN1-mutant human granulocytes showed aberrantly enlarged ER structures and paucity of secretory vesicles. We hypothesized that that ER aberrations may be associated with defective N-glycosylation of multiple proteins in neutrophil granulocytes and found that JAGN1-mutant neutrophil granulocytes exhibited anomalous N-glycomic profiles characterized by a marked reduction in fucosylation of all their multi-antennary glycans. JAGN1-deficient neutrophil granulocytes showed increased apoptosis in response to TNFa and staurosporine, likely accounting for the lack of mature neutrophils in these patients. Additional studies in JAGN1-knockdown cells indicate that JAGN1 participates in the secretory pathway and is required for granulocyte-colony stimulating factor receptor-mediated signaling. Global proteomic analysis of the JAGN1-interactome identified a limited number of interaction partners including members of the Coat Protein I (COPI) complex (COPA, COPB2, and COPG2) which suggest a role for JAGN1 in vesicular trafficking from Golgi to ER. Taken together, JAGN1 emerges as a hitherto unrecognized factor necessary in differentiation and survival of neutrophil granulocytes and a novel gene implicated in SCN. Disclosures:No relevant conflicts of interest to declare.

Abstract LB-357: Notch and PIAS3 differentially regulate sumoylation of GFI1 to modulate transcriptional repression.

Abstract The zinc-finger (ZF) transcriptional repressor, Growth factor independence (GFI)-1, is a master regulator of lineage allocation in hematopoietic, aerodigestive tract and central nervous system development. Gfi1 is necessary for T-lymphopoiesis following Notch activation, while Notch-driven T-cell leukemia/lymphoma requires Gfi1 to maintain a malignant phenotype. Granulopoiesis also requires Gfi1, and Gfi1 mutations cause severe congenital neutropenia. GFI1 shares near invariant SNAG and ZF domains with its paralog, GFI1B, at their N- and C-termini, respectively. These conserved regions confer comparable DNA binding and transcriptional repression abilities to both proteins, yet Gfi1b specifies erythroid and megakaryocytic fates and is often abundantly expressed in myeloid leukemias. A non-homologous linker separates SNAG and ZF domains in GFI proteins. We hypothesized Notch dependence on GFI1 in normal and malignant T-lymphopoiesis arises from interactions and post-translational modifications occurring uniquely within its linker region. We show the GFI1 linker contains a type-I sumoylation consensus motif centered on K239 that is absolutely conserved among mammalian GFI1 proteins but absent from GFI1B. GFI1 is sumoylated on K239, and arginine substitution (K239R) abolishes modification by ubiquitin family proteins. We confirm GFI1 binds the E3 sumo ligase, PIAS3, and extend this observation to mammalian cells. The binding interface includes the PIAS3 RING domain and a subdomain within the GFI1 linker that contains the sumoylation consensus motif. Transcriptional repression by GFI1 is impaired by PIAS3 co-expression. GFI1-K239R resists this effect of PIAS3, and its half-life is prolonged 3-fold over wild type GFI1. These data suggested GFI1 sumoylation and turnover limit its functions in transcriptional control, and that responses dependent upon GFI1 might alter sumoylation to reinforce GFI1 functions. To explore this notion, we leveraged the dependence of Notch on GFI1. We show that Notch intracellular domain (NICD) expression blocks GFI1 sumoylation and reverses the inhibitory effect of PIAS3 on GFI1-mediated transcriptional repression. Moreover, NICD binds the GFI1 linker and competes with PIAS3 for GFI1 binding. These results define an oppositional relationship between NICD and PIAS3 in GFI1 sumoylation and transcription, and intimate a post-translational mechanism by which Notch signaling supports GFI1 functions in normal and malignant T-lymphopoiesis. Citation Format: Daniel Andrade, Jason Singer, Diana Bareyan, David McClellan, Helena Lucente, Mahesh Chandrasekharan, Michael E. Engel. Notch and PIAS3 differentially regulate sumoylation of GFI1 to modulate transcriptional repression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-357. doi:10.1158/1538-7445.AM2013-LB-357

Abstract 3089: Regulation of GFI proteins by Notch intracellular domains

Abstract Growth factor independence (GFI)-1 and its homolog, GFI1B, are transcriptional regulators that play critical roles during hematopoiesis. GFI family proteins are characterized by near-invariant SNAG and zinc finger domains that direct transcriptional repression and DNA-binding, respectively. A linker region with limited homology and unknown function separates the conserved SNAG and zinc-finger regions. GFI proteins display distinct expression patterns within the myelo-erythroid compartment and make similarly distinct contributions to cell fate specification. GFI1 expression is necessary for hematopoietic stem cell maintenance and is required for granulocytic differentiation beyond the promyelocyte stage. Enforced expression of GFI1 promotes IL-2-independent growth of hematopoietic progenitors and inhibits apoptosis. Mutations in GFI1 lead to a severe congenital neutropenia syndrome that carries an increased risk of myeloid leukemia development. GFI1B expression predominates in erythroid and megakaryocytic precursors and is required for terminal differentiation along both lineages. Moreover, elevated GFI1B expression occurs preferentially in erythroid and megakaryoblastic leukemias, and contributes to the malignant phenotype by supporting growth and preventing apoptosis. Given their complementary roles as growth and survival factors, yet distinct contributions to lineage allocation in hematopoiesis, it is essential to define how GFI family proteins are integrated into the signaling machinery governing cell fate specification. We show that both GFI1 and GFI1B interact with intracellular domains of Notch receptors. Enforced expression of the Notch1 intracellular domain (N1-ICD) impairs GFI-mediated transcriptional repression. Using deletion mutagenesis, we have mapped the regions required for the N1-ICD–GFI1 interaction. Both the ankyrin repeat and transactivation domains of N1-ICD contribute binding surfaces for GFI1. In reciprocal mapping experiments, we show that N1-ICD binding to GFI1 requires a 128-amino acid motif in the divergent linker region near the zinc finger repeat elements. This region contains a high probability consensus sumoylation element that is absolutely conserved in mammalian species but not present in GFI1B. These data suggest that Notch activation may directly impact transcriptional targets regulated by GFI family proteins and expand the potential sphere of influence for Notch in hematopoietic development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3089. doi:1538-7445.AM2012-3089

Clinical and Genetic Characteristics of Patients with Severe Congenital Neutropenia in Japan,

Abstract 3213▪▪This icon denotes a clinically relevant abstractSevere congenital neutropenia (SCN) includes a variety of hematologic disorders characterized by severe neutropenia, with absolute neutrophil counts (ANC) below 0.5 × 109/L, and associated with severe systemic bacterial infections from early infancy. Mutations in ELANE, HAX1, G6PC3, WAS and GFI1 have been so far identified in patients with SCN. The Severe Chronic Neutropenia International Registry (SCNIR) has collected data to monitor the clinical course, treatments, and disease outcomes for SCN patients. In this study, we analyzed the clinical and genetic characteristics of patients with SCN based on the Japanese cohort study collecting the data on chronic neutropenia using a standardized case report form. This study is approved by the ethics committees of the Hiroshima University School of Medicine. Forty-six patients with SCN in Japan were enrolled in this study. Mean present age of patients was 13.6 years old ranged from 1 to 39. The Mean age at diagnosis was 4.6 months ranged from 0 to 24 months. Approximately 90% of patients were diagnosed before 12 months. Twenty-three patients were female. As initial clinical presentation, subcutaneous abscess and cutaneous cellulitis were dominated (37%), followed by unknown fever (17%), stomatitis (13%) and lymphadenitis (13%). On the other hand, infections which patients had experienced after diagnosis were bacterial pneumonia (50%), cutaneous infections (50%) and oral infections such as stomatitis (48%) and gingivitis (48 %). Total 8 patients (17%) including 4 patients with mutations in HAX1 suffered from psychomotor retardation that was higher than that in general Japanese population (about 1%), suggesting that psychomotor retardation may be one of considerable complications in patients with SCN. Thirty-three out of 46 patients with SCN were preformed gene analysis and 29 patients (approximately 88 %) were identified mutations; 25 patients had heterozygous mutations in ELANE, 4 patients had homozygous mutations in HAX1. The proportion of mutations in ELANE (76%) was higher and that in HAX1 (12%) was relatively lower in Japan compared with those in Western counties. Thirty-six patients (78%) were treated with G-CSF, including regular use in 26 patients (56%) and on demand use during infections in 10 patients (22%). In 26 patients treated with regular G-CSF therapy, the mean cumulative duration was 6.6 years, ranged from 0.4 to 19. Two patients showed no response to G-CSF therapy. Four patients among total 46 patients developed MDS/AML and 3 patients were alive after receiving hematopoietic stem cell transplantation (HSCT). Total 12 out of 46 patients (26%) with SCN were underwent HSCT before malignant transformations due to recurrent infections and/or the long-term use of G-CSF. Reduced intensity conditionings (RIC) were used in part of patients. All patients were alive after HSCT. In Japan, high proportion of patients was treated with HSCT using RIC before malignant transformations. The accumulation of cases is necessary to establish suitable conditioning regimen and appropriate indication of HSCT in patients with SCN. Disclosures:No relevant conflicts of interest to declare.

A Microrna Feed-Forward Loop Amplifies GFI1N382S Transcriptional Reprogramming In Severe Congenital Neutropenia

AbstractAbstract 2239The Growth factor independent -1 (Gfi1) transcriptional repressor regulates both hematopoietic stem cell self renewal and myeloid differentiation. Humans with severe congenital neutropenia (SCN) display mutations in GFI1 that generate dominant negative acting proteins. Moreover, GFI1-mutant SCN patients and Gfi1-/- mice display a unique accumulation of myeloid progenitors. Recently we showed that Gfi1 regulation of HoxA9, Pbx1 and Meis1 underlies these phenomena, in that the Gfi1-Hox transcriptional circuit controls the accumulation of myeloid progenitors in vivo. We have also shown that Gfi1 regulates miR-21 during myelopoiesis, and that miR-21 is deregulated by Gfi1N382S expression. Our new data link these concepts by demonstrating that forced expression of miR-21 in bone marrow cells results in the accumulation of myeloid progenitors in transplant recipients. Moreover, miR-21 directly targets the Ski oncoprotein, and Ski-/- bone marrow cells show an accumulation of myeloid progenitors. Thus, Gfi1-/-, miR-21 overexpressing-, and Ski-/- myeloid progenitors accumulate in the marrow. Strikingly, Ski is dramatically reduced in miR-21 overexpressing Lin- bone marrow cells. Nearly undetectable Ski expression in Gfi1-/- bone marrow cells can be completely rescued by antagonizing miR-21 activity. Since Ski is a corepressor and Gfi1 is a transcriptional repressor, we next tested whether the two proteins physically interact. Indeed, endogenous Ski and Gfi1 can be coimmunoprecipitated. Synthetic Ski and Gfi1 proteins reveal that the interaction is mediated through Ski carboxy-terminal and Gfi1 zinc-finger domains. Chromatin immunoprecipitation reveals Ski and Gfi1 co-occupy several Gfi1 target genes (including HoxA9), which are derepressed upon Gfi1 or Ski knockdown. However, while Gfi1 binds and regulates the miR-21 gene, Ski is not bound to the miR-21 gene, and Ski knockdown has no effect upon miR-21 levels. Thus, the data point to a novel feed-forward transcriptional circuit. Gfi1N382S deregulation of miR-21 amplifies the dominant-negative effect of Gfi1N382S through miR-21 targeting of Ski, leading to further derepression of Gfi1-Ski target genes. Disclosures:No relevant conflicts of interest to declare.

Correspondence: Cyclic neutropenia associated with T cell immunity to granulocyte proteases and a double de novo mutation in GFI1, a transcriptional regulator of ELANE

correspondence: Cyclic neutropenia associated with T cell immunity to granulocyte proteases and a double <i>de novo</i> mutation in <i>GFI1</i>, a transcriptional regulator of <i>ELANE</i>

Abstract 1188: Cell-of-origin determines genetic selection and defines T-cell acute lymphocytic leukemia sub-types in mouse models

Abstract The cell-of-origin has yet to be identified for most forms of cancer. However, such knowledge would provide insight into the earliest events that initially distinguish a tumor cell from its normal cell-of-origin. Understanding and identifying these events are critical, as these early genetic changes may create targets that can be exploited therapeutically. Unfortunately, the tools to address this question have only recently become available. We have recently completed a forward genetic screen to examine how altering the cell-of-origin affects genetic selection in cancer. To accomplish this we made use of a Cre-inducible Sleeping Beauty (SB) system to initiate transposon mutagenesis in hematopoietic stem cells (HSCs), thymic progenitor cells (TP) or late stage CD4+/CD8+ thymocytes (DP). Each of these approaches produced predominantly T-cell lymphomas with different characteristics. Analysis of transposon-induced mutations in each of the three lymphoma models revealed clear differences in their genetic profiles. For instance, the HSC model was defined by frequent mutations in Notch1 (∼58%). By contrast, lymphomas in the DP model did not have Notch1 mutations, but instead had frequent mutations in Myc, Gfi1, Whsc1 and Akt2. Interestingly, tumors that were initiated in thymic progenitor cells had a genetic profile similar to that seen in the HSC or the DP model. Recently, a sub-type of T-cell acute lymphoblastic leukemia (T-ALL), called early thymic progenitor ALL (ETP-ALL) was described in which the tumor cells resembled ETPs in both surface marker and mRNA expression. In addition, ETP-ALL patients have a much worse prognosis than typical T-ALL patients. We compared gene expression signatures from a subset of HSC and DP lymphomas to that of ETP-ALL and typical T-ALL. Interestingly, tumors from the DP model have a similar gene expression to human ETP-ALL, while HSC tumors show a greater resemblence to typical T-ALL. Our data indicate that ETP-ALL may not arise from an early thymic progenitor, but from a differentiated T-cell that has undergone anaplastic transformation. The implications of this study are that cell-of-origin cannot be inferred from analysis of protein or gene expression patterns in tumors. Our findings also suggest that differentiation state of the cell-of-origin plays a critical role in determining what types of mutations will be selected during transformation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1188.

Congenital neutropenia

Congenital neutropenia comprises a variety of genetically heterogeneous phenotypic traits. Molecular elucidation of the underlying genetic defects has yielded important insights into the physiology of neutrophil differentiation and function. Non-syndromic variants of congenital neutropenia are caused by mutations in ELA2, HAX1, GFI1, or WAS. Syndromic variants of congenital neutropenia may be due to mutations in genes controlling glucose metabolism (SLC37A4, G6PC3) or lysosomal function (LYST, RAB27A, ROBLD3/p14, AP3B1, VPS13B). Furthermore, defects in genes encoding ribosomal proteins (SBDS, RMRP) and mitochondrial proteins (AK2, TAZ) are associated with congenital neutropenia syndromes. Despite remarkable progress in the field, many patients with congenital neutropenia cannot yet definitively be classified by genetic terms. This review addresses diagnostic and therapeutic aspects of congenital neutropenia and covers recent molecular and pathophysiological insights of selected congenital neutropenia syndromes.

Gfi1 as a Regulator of Hematopoietic Differentiation.

Abstract Growth Factor Independent-1 (Gfi1) is a transcriptional repressor originally identified as a target of Moloney leukemia virus insertion mutagenesis. Gfi1 potently collaborates with Myc and Pim oncogenes to mediate T cell transformation. Retroviral insertions which activate Gfi1 are among the most frequent events in MoMLV-induced T cell, but not myeloid, leukemias. Orthologous proteins in Drosophila (Senseless) and C.elegans (Pag-3) are key regulators of developmental decisions. Gfi1−/− mice display lymphopenia, neutropenia, and abnormally proliferative HSC and progenitors which eventually lead to bone marrow failure. Humans with bone marrow failure syndromes, such as Severe Congenital Neutropenia or Non-Immune Chronic Idiopathic Neutropenia of Adults, display mutations in GFI1, which produce dominant negative acting GFI1 proteins. The SCN-associated GFI1N382S mutant proteins derepress a subset of GFI1 target genes, including CSF1 and CSF1R, to block granulopoeisis. Normally, Gfi1 antagonizes monopoiesis mediated by Pu.1, Egr1, Egr2, and Nab2. Thus, Gfi1 loss of function may impair granulopoiesis by failing to successfully repress monopoeitic differentiation. GFI1 dysfunction may also underlie Specific Granule Deficiency. GFI1 is able to synergize with C/EBP transcription factors to activate genes, such as neutrophil collagenase. Gfi1 may, therefore, enhance granulopoietic differentiation through C/EBP factors. Though great progress has been made in understanding Gfi1 biological and biochemical functions, we are only beginning to understand the role of Gfi1 in integrating hematopoietic transcriptional programming. We find that Gfi1 signaling controls both the differentiation and transformation of myeloid progenitors through an evolutionarily conserved transcriptional and post-transcriptional network.

Incidence of SCN-Assoicated Gene Mutations in Severe Congenital Neutropenia Patients in North America

Severe congenital neutropenia is a genetically heterogeneous syndrome associated with mutations in several different genes including ELA2, HAX1, GFI1, WAS, and CSF3R. The goal of this study was to define the mutation frequency of these genes in the North American SCN patient population. We also sequenced SBDS, since mutations of SBDS have been associated with congenital and acquired neutropenia. A total of 159 patients were identified in the North American Severe Chronic Neutropenia International Registry (SCNIR) for whom informed consent and genomic DNA samples adequate for sequencing were available. To accommodate our semi-automated high-throughput sequencing pipeline, 94 samples were chosen for sequencing. Since ELA2 sequencing had already been performed in most cases, preference was given to those samples without known ELA2 mutation. Among the samples, 73 were from patients with SCN, 4 with cyclic neutropenia, 10 with idiopathic neutropenia, 2 with Shwachman-Diamond Syndrome (SDS), and 3 with Barth syndrome. Two samples were excluded because of poor sequence quality. Singleton cases with validated mutations of GFI1 (N382S) and WAS (L270P) were observed. The N382S GFI1 mutation was associated with striking monocytosis. A novel nonsense mutation of GFI1 (R412X) was detected in one additional case. As expected, compound heterozygous mutations of SBDS were present in the two cases of SDS. In addition, heterozygous mutations of SBDS (84Cfs3X and Q94X) were observed in two cases of SCN. Typical truncation mutations of CSF3R were detected in 4 cases, all developing MDS or AML. Surprisingly, no mutations of HAX1 were detected. Considering only patients with a diagnosis of SCN who were from North America (125 of the total 159 cases), the incidence of ELA2 mutations was 68%. Eleven novel ELA2 mutations were identified. In 28.8% of cases, no mutation of any gene were detected. Based on these data, we recommend that ELA2 genotyping be performed in all patients with suspected SCN. In the North American population mutations in HAX1, GFI1, SBDS, and WAS are rare and routine genotyping is not indicated. Finally, the data suggest that there are yet undiscovered genetic causes of SCN.

T-Cell Autoimmunity May Contribute to Neutropenia in a Patient with Cyclic Neutropenia (CN) and Double De-Novo Mutations in Gfi-1.

CN is a rare autosomal dominant disease characterized by predictable cycling of patients' neutrophils from normal levels to a nadir of ≤100 cells / μL. While heterozygous mutations in the neutrophil elastase gene (ELA2) occur in the majority of patients and altered subcellular trafficking of neutrophil elastase (NE) has also been demonstrated, it remains unknown how these defects lead to the CN phenotype. Similarly, mutations in the gene for the beta subunit of adapter protein 3 (AP3), which binds NE and shuttles it to endosomal vesicles, are present in severely neutropenic patients with Hermansky-Pudlak syndrome (type 2). Interestingly, separate single base-pair mutations in the zinc finger domains of Gfi-1, a gene that maintains the long-term potential of hematopoeitic stem cells and that negatively regulates ELA2 expression, were reported in two different patients with severe congenital neutropenia (SCN). Decreased immunity against recall antigens, and an increase in the proportion of T-cells bearing a mature memory phenotype have also been described in patients with Gfi-1 mutations. As yet, there is no unifying hypothesis linking these various abnormalities. We hypothesized that aberrantly expressed NE, resulting from mutations in ELA2 or associated regulatory genes, could predispose autologous T-cells to target myeloid precursors that aberrantly express NE. Such a reversal of immune tolerance could contribute to neutropenia in CN and SCN patients. We report in a 25 year-old man with CN from birth the simultaneous occurrence of heterozygous mutations in Gfi-1 (N382S and K403R), which were previously identified in separate patients with SCN. Neither mutation was found in this patient's parents however, and sequencing of mRNA encoding PRTN3, ELA2 and AP3 showed no mutations. Because Gfi-1 serves as a transcriptional repressor of ELA2, we hypothesized that NE may be overexpressed and / or mis-trafficked in neutrophils. To address this hypothesis, we analyzed granulocytes by FACS staining and confocal microscopy. NE was disproportionately expressed in the plasma membrane of 75% of the patient's granulocytes compared to only 3% of granulocytes from healthy donors (HD). We have previously demonstrated that cytotoxic T-lymphocytes (CTLs) that recognize proteinase 3 (P3) and NE-derived peptides, such as the HLA-A2 restricted peptide PR1 (VLQELNVTV), can kill leukemia but not normal myeloid cells due to aberrant expression of P3 and NE in the leukemia and that these PR1 specific CTLs (PR1-CTL) are not detected in healthy donors. Because this patient expressed the HLA-A2 allele, we used fluorescently labeled PR1/HLA-A2 tetramers to study whether PR1-CTL were present. Strikingly, 0.5% to 1% of total CD8+ cells from the patient were specific for PR1, as measured over 2 years, whereas no PR1-CTL were present in control HD or the patient's mother, who is also HLA-A2+. The PR1-CTLs co-expressed CCR7 and CD45, consistent with a naïve phenotype, and cytokine secretion after crosslinking the T-cell receptors was similar to controls. These data suggest that aberrant NE expression can lead to a reversal of immune tolerance and a corresponding increase in PR1-CTL. The mechanism of neutrophil cycling in CN patients is not known; however, an autoimmune response against peptides derived from aberrantly expressed granule proteins in myeloid precursors could lead to a CTL-mediated feedback loop that would also be consistent with the chalone hypothesis of feedback regulation already proposed for this disease.

Mosaic Tetraploidy and Transient Mutations in the GFI1 Gene in a Patient with Severe Chronic Neutropenia.

Severe congenital neutropenia (SCN), also known as Kostmann disease, is a rare disorder characterized by peripheral blood agranulocytosis and maturation arrest of neutrophils within the bone marrow at the promyelocytic stage. Patients usually present with an absolute neutrophil count below 0.2 x 109/L and severe infections, with the typical onset of symptoms during infancy. Most patients respond to treatment with granulocyte colony-stimulating factor, but retain an increased risk for death from sepsis and for development of myelodysplastic syndrome and acute myeloid leukemia (MDS/AML). Several reports have described karyotype abnormalities associated with this syndrome, most typically clonal loss of chromosome 7 or abnormalities of chromosome 21 associated with transformation to MDS/AML. The etiology of these abnormalities and their role in progression to MDS/AML remains unclear. Here we present a case of a 15 year-old boy with SCN, lymphopenia, and persistent tetraploid mosaicism in the bone marrow and in subpopulations of peripheral blood leukocytes. His phenotype, including growth and development, was otherwise normal. The patient's father, who had mild neutropenia and a history of monoclonal gammopathy, also showed a tetraploid subpopulation in his bone marrow. Flow cytometric analysis of the DNA content of CD45+ nucleated cells in the patient's peripheral blood showed that 20% of CD13+ granulocytes, and 15% of CD14+ monocytes were tetraploid. The lifelong persistence of the mosaicism and the similar finding in the patient's father suggest that it represents a congenital propensity towards acquiring tetraploidy. However, cytogenetic evaluation of the patient's skin fibroblasts detected no tetraploidy, indicating failure of the putative tetraploid embryonic cells to contribute to that lineage. The fact that the patient's father also had a population of tetraploid cells in his bone marrow and a history of neutropenia suggests a common underlying genetic cause for both conditions in this kindred.Mutational analysis revealed that the ELA2 gene sequence was normal, but the GFI1 gene exhibited transient, simultaneous appearance of two novel mutations: a missense substitution in exon 1 and a single base change in the promoter region, at a putative binding site for the myeloid-specific transcription factor MZF1. The GFI1 mutations were repeatedly identified in a DNA sample obtained in 2003, but not in 2001, 2002, or 2006 samples. Sequencing of DNA obtained from buccal swabs of the patient and his parents also revealed no GFI1 mutation. The transient appearance may represent the emergence and subsequent loss of a clone of hematopoietic stem or progenitor cells with GFI1 mutations. This hypothesis is supported by a recent report demonstrating that GFI1 mutation results in premature apoptosis of myeloid cells, similar to apoptosis-inducing mutations in the neutrophil elastase gene ELA2. The fact that GFI1 mutations were identified in a DNA sample from one year, but not from others, raises the possibility that GFI1 mutations reported in other neutropenic patients may also be transient. We speculate that an underlying genetic defect, inherited in an autosomal dominant pattern, leads to both disordered mitosis and leukopenia in this kindred.

Gfi1-N382S Mutants from Human Severe Congenital Neutropenia Patients Function through a Transcriptional Dominant Negative Mechanism.

The Growth factor independence 1 (Gfi1) zinc finger transcriptional repressor is a T cell leukemia oncoprotein that also plays a crucial role in granulopoiesis in both mice and humans. A single point mutation in the amino terminal SNAG repressor domain (P2A) is able to ablate both Gfi1 transcriptional repression activity and linked oncogenic activity in T lymphoctyes. Mice deleted for Gfi1 are lymphopenic, but also lack mature neutrophils. Gfi1−/− mice display a profound block to myeloid differentiation and abnormal promyelocytes accumulate in the blood. Humans with Severe Congenital Neutropenia (SCN) with heterozygous mutations in Gfi1 have similar abnormal promyelocytes. We introduced the SCN patient Gfi1N382S DNA-binding-deficient mutation into murine Gfi1 and overexpressed it in primary murine bone marrow cells. While expression of the wild type Gfi1 resulted almost exclusively in mature granulocyte differentiation, forced expression of the N382S mutant resulted almost exclusively in monocytic differentiation. Flow cytometric analysis revealed a population of N382S-expressing cells with markers of both monocytes and neutrophils resembling the atypical Gfi1−/− promyelocytes. To determine if mutation of the N382 residue is uniquely able to block Gfi1 function, we constructed a virtual model of Gfi1 zinc fingers 3, 4 and 5 interacting with DNA. The model revealed several possible protein-DNA interactions. In order to validate the model we mutated those residues to alanine and performed EMSA with in vitro transcribed/translated proteins. The same alanine substitution mutants were expressed in primary murine bone marrow and tested for their ability to control myelopoiesis. Lack of DNA binding in EMSA tightly correlated with impaired granulopoesis in our in vitro model, suggesting the necessity of intact DNA binding for proper Gfi1 function. These data suggested that the non-DNA binding mutants were able to inhibit repression by wild type endogenous Gfi1, perhaps through the sequestration of limiting corepressor proteins. The Gfi1P2A mutant is unable to repress transcription. We therefore tested the effect of Gfi1P2A expression on myelopoiesis and found that it blocked granulopoiesis equivalently to Gfi1N382S. To rigorously determine whether the titration of limiting corepressors was the cause of N382S neutropenia, we constructed a compound mutant containing both N382S and P2A and expressed it in primary murine bone marrow cells. Expression of Gfi1P2A-N382S had little effect on myelopoiesis. We conclude that SCN patients with heterozygous Gfi1 mutations have blocked granulopoiesis because the non-DNA binding mutant protein competes with the wild type allele for titratable associated cofactors.

Neutropenia-Associated Mutations in PRDM5, a Novel Epigenetic Regulator of Hematopoiesis.

The growth factor independent 1 (Gfi1) oncoprotein is an intrinsic regulator of hematopoiesis, and its mutations are associated with congenital neutropenia. We performed a yeast two-hybrid screen with Gfi1, in order to begin dissecting the gene network through which it acts and also toward identifying other genes potentially responsible for cases of hereditary neutropenia lacking mutations in Gfi1 or ELA2 (encoding neutrophil elastase, the most common cause of hereditary neutropenia). From a limited number of proteins found to interact with Gfi1 in the yeast two-hybrid screen, we evaluated PRDM5, a previously uncharacterized zinc finger protein belonging to the PR domain (PRDI-BF1 and RIZ homology) -containing family of tumor suppressor genes. We screened for mutations in the gene encoding PRDM5 among 230 unrelated patients with otherwise genetically unexplained congenital neutropenia and discovered two with heterozygous amino acid sequence substitutions that were not detected in twice the number of controls. Consequently, we systematically characterized PRDM5, and found that it functions as a novel epigenetic regulator of hematopoiesis: First, PRDM5 is a sequence-specific DNA-binding transcription factor as revealed by random sequence selection. Second, by using chromatin immunoprecipitation (ChIP)-based cloning along with “ChIP-on-chip” assays, we identified approximately two hundred protein-coding genes (including hematopoietic transcription factors) and thirty-four microRNA genes as putative PRDM5 targets, a subset of which we confirmed by additionally performing gel retardation and transfection reporter assays. Significantly, one of the targets of PRDM5—just as is true of Gfi1—is ELA2. Third, depletion of PRDM5 by siRNA in both HeLa and HEK293 cells changed the cell cycle program, and, as revealed by microarray and real-time RT-PCR assays, PRDM5 knockdown in HEK293 cells altered the expression of ninety of the protein-coding targets and eight of the microRNA targets. Fourth, transcriptional profiling revealed that the expression of about five thousand genes, including many involved in the regulation of cell proliferation and differentiation, changed more than five-fold following PRDM5 knockdown in HEK293 cells. Fifth, we found that PRDM5 regulates transcription by associating with the histone deacetylase HDAC1 and the histone methyltransferase G9a and recruits them to its target genes. Treatment of cells with the histone deacetylase inhibitors trichostatin A (TSA) or 4-phenylbutyric acid (PBA) deregulates PRDM5 target genes. Finally, the two variants observed in patients with congenital neutropenia alter the DNA-binding activity of PRDM5 and interfere with its function as a transcriptional repressor in reporter assays, suggesting that, just as with Gfi1, rare mutations in a global regulator of hematopoiesis may contribute to the genetic mechanisms causing neutropenia.

Neutropenia-Associated Mutations of PFAAP5, a Novel Protein Mediating Transcriptional Repressor Interaction between Gfi1 and Neutrophil Elastase.

Mutations in ELA2, encoding the protease neutrophil elastase, cause cyclic neutropenia and are also the most common cause of the Kostmann syndrome of congenital neutropenia. Mutations in the transcriptional repressor and intrinsic regulator of hematopoiesis, Gfi1, are another rare cause of congenital neutropenia. ELA2 is a target of Gfi1; consequently, mutation of Gfi1 leads to over-expression of neutrophil elastase. Defects in a predicted feedback circuit offer a possible explanation for the periodic oscillations of cyclic neutropenia, but how neutrophil elastase and Gfi1 regulate hematopoiesis with respect to each other, and what additional factors may be involved, has remained undefined.To identify other components of a potential hematopoietic feedback circuit, we performed yeast two-hybrid screens with Gfi1 and neutrophil elastase using bone marrow cDNA libraries. We found 25 proteins that interacted with Gfi1. Only two proteins, however, interacted with the isoform of neutrophil elastase containing an intact carboxyl terminal extension (deletion of which is the most frequently occurring type of mutation in congenital neutropenia). Remarkably, one of the two, the previously uncharacterized protein PFAAP5, was among the 25 found to also interact with Gfi1. PFAAP5 may therefore play a unique role in mediating interactions between neutrophil elastase and Gfi1.We further studied PFAAP5. Immunofluorescent localization with an epitope-tagged construct transfected into cultured cells demonstrates its localization in the nucleus. To determine if PFAAP5 influences Gfi1's activities as a transcription factor in the presence or absence of neutrophil elastase, we performed transient transfection assays with a Gfi1 responsive reporter and PFAAP5 siRNA knockdown in NIH3T3 cells, which express PFAAP5, but not Gfi1 or neutrophil elastase, endogenously. Not surprisingly, transfected neutrophil elastase demonstrated no effect as a transcriptional repressor by itself. Unexpectedly, however, transcriptional repression by transfected Gfi1 increased with co-expression of neutrophil elastase. Knockdown of PFAAP5 had no effect on transfected Gfi1 repressor activity alone, but knockdown of PFAAP5 blocked the ability of co-expressed neutrophil elastase to increase Gfi1 repressor activity. Therefore, neutrophil elastase can increase Gfi1 repressor function, but only in the presence of PFAAP5.Finally, we evaluated PFAAP5 as a candidate gene in cases of congenital neutropenia in which ELA2 or Gfi1 mutations are absent. One homozygous and two different heterozygous coding sequence substitutions were detected in four of 230 unrelated patients, yet absent among double the number of controls. The mutations prevent PFAAP5 from interacting with either neutrophil elastase or Gfi1 in the yeast two-hybrid assay. Thus, mutations of neutrophil elastase, Gfi1, or PFAAP5 may each similarly disrupt a common circuit in which Gfi1 represses ELA2 and neutrophil elastase feeds back to cooperate with Gfi1 in regulating its own transcription.

A 17q25.3 Duplication Defines a New Dosage-Sensitive Congenital Neutropenia Locus and Implicates SOCS3 as a Candidate Gene for Cases Unexplained by ELA2 Mutation.

We have investigated the etiology of congenital neutropenia in a girl with de novo duplication of chromosome 17q25.3. She presented during the first year of life with neutropenia, episodic hypothermia, failure to thrive, and other congenital abnormalities. Peripheral blood karyotype demonstrated 46,XX,add(17)(q25.3), and molecular cytogenetic studies confirmed interstitial duplication of chr17-derived material. We excluded ELA2 mutation (the most common cause of hereditary neutropenia) and reasoned that the neutropenia and other medical problems most likely were the result of the chromosomal abnormality. In the duplicated 17q25.3 region, SOCS3 emerged as a promising candidate gene responsible for neutropenia, because SOCS3 is a well-characterized negative regulator of G-CSF-receptor signaling and, in murine conditional knockout models, acts as a physiologic negative regulator of granulopoiesis. As we had previously demonstrated that mutations of the Gfi1 transcriptional repressor are a rare cause of human neutropenia, we searched for potential Gfi1 binding sites in the SOCS3 promoter and noted a total of five, and chromatin immunoprecipitation analysis validated occupancy of the SOCS3 promoter by Gfi1 in Jurkat, HL-60, and U937 cells. Thus, several lines of evidence suggested SOCS3 as a plausible neutropenia gene. We confirmed that SOCS3 is indeed duplicated in this patient by both FISH and quantitative genomic PCR. In an effort to identify other patients with SOCS3-related neutropenia, we sequenced both exons of SOCS3 in a total of 66 patients with severe congenital neutropenia (SCN) or cyclic neutropenia (CN) and 94 patients with chronic idiopathic neutropenia of adults (CINA) in whom ELA2 mutations were absent. No coding mutations were detected, but we did identify several variants in the SOCS3 promoter and 5′- and 3′- UTRs, including a single base pair substitution (+1779AtoG) in the 3′-UTR in a SCN patient and deletion of a single G (-1318delG) occurring in a six-G tract immediately adjacent to a predicted STAT binding site in a highly conserved region of the SOCS3 promoter (found in heterozygous and homozygous form in several patients with CN and SCN). The 3′-UTR alteration was absent in 270 control chromosomes and is located within a predicted binding site for miR-449 that is well-conserved across mammalian species. In sum, we have identified a patient with congenital neutropenia and a de novo duplication of 17q25.3 defining a new candidate locus for congenital neutropenia, and the evaluation to date suggests that SOCS3 is a promising dosage sensitive candidate gene in this interval that additionally could be a key target in neutropenia associated with Gfi1 mutations.