Growth factor independent 1 (Gfi1) controls myeloid differentiation by regulating gene expression and limits the activation of p53 by facilitating its de-methylation at Lysine 372. In human myeloid leukemia, low GFI1 levels correlate with an inferior prognosis. Here, we show that knockdown (KD) of Gfi1 in mice causes a fatal myeloproliferative disease (MPN) that could progress to leukemia after additional mutations. Both KO and KD mice accumulate myeloid cells that show signs of metabolic stress and high levels of reactive oxygen species. However, only KO cells have elevated levels of Lysine 372 methylated p53. This suggests that in contrast to absence of GFI1 , KD of GFI1 leads to the accumulation of myeloid cells because sufficient amount of GFI1 is present to impede p53-mediated cell death, leading to a fatal MPN. The combination of myeloid accumulation and the ability to counteract p53 activity under metabolic stress could explain the role of reduced GF1 expression in human myeloid leukemia.

Growth factor indepdendent 1 (GFI1) is a SNAG-domain, DNA binding transcriptional repressor which controls myeloid differentiation through molecular mechanisms and co-factors that still remain to be clearly identified. Here we show that GFI1 associates with the chromodomain helicase DNA binding protein 4 (CHD4) and other components of the Nucleosome remodeling and deacetylase (NuRD) complex. In granulo-monocytic precursors, GFI1, CHD4 or GFI1/CHD4 complexes occupy sites enriched for histone marks associated with active transcription suggesting that GFI1 recruits the NuRD complex to target genes regulated by active or bivalent promoters and enhancers. GFI1 and GFI1/CHD4 complexes occupy promoters that are either enriched for IRF1 or SPI1 consensus binding sites, respectively. During neutrophil differentiation, chromatin closure and depletion of H3K4me2 occurs at different degrees depending on whether GFI1, CHD4 or both are present, indicating that GFI1 is more efficient in depleting of H3K4me2 and -me1 marks when associated with CHD4. Our data suggest that GFI1/CHD4 complexes regulate histone modifications differentially to enable regulation of target genes affecting immune response, nucleosome organization or cellular metabolic processes and that both the target gene specificity and the activity of GFI1 during myeloid differentiation depends on the presence of chromatin remodeling complexes.

The X-linked gene DDX3X encodes an RNA helicase and is mutated at high frequencies in several types of human B-cell lymphoma. Females have two active DDX3X alleles and males carry a DDX3Y homolog on the Y chromosome. We show here that pan-hematopoietic, homozygous deletion of Ddx3x in female mice perturbs erythropoiesis causing early developmental arrest. However, both hemizygous male and heterozygous female embryos develop normally, suggesting that one Ddx3x allele is sufficient for fetal hematopoietic development in females and that the Ddx3y allele can compensate for the loss of Ddx3x in males. In adult mice, loss of DDX3X affects hematopoietic progenitors, early lymphoid development, marginal zone, and germinal center B-cells as well as lymphomagenesis driven by an Eμ-Myc or λ-Myc transgene in a sex-dependent manner. A B-cell-specific depletion of both Ddx3x alleles induces a delay in tumorigenesis in female mice while Ddx3x deletion in male mice does not impact tumorigenesis. However, male Eμ-Myc mice lacking Ddx3x on a pan-hematopoietic way remained for the majority lymphoma-free. The few tumors that appeared in these male mice showed upregulated expression of DDX3Y indicating a requirement of DDX3 activity for lymphomagenesis. Our data reveal sex-specific roles of DDX3X in erythro- and lymphopoiesis as well as in MYC-driven lymphomagenesis, which are important when considering inhibition of DDX3 as a treatment of Myc dependent B-cell lymphoma. Citation Format: Marion Lacroix, Hugues Beauchemin, Julie Ross, Jennifer Fraszczak, Peiman Shooshtarizadeh, Riyan Chen, Tarik Moroy. The X-linked gene for the helicase DDX3X is required for lymphoid differentiation and MYC-driven lymphomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB559.

Supplementary Data from The X-Linked Helicase DDX3X Is Required for Lymphoid Differentiation and MYC-Driven Lymphomagenesis

Gfi1b is a transcriptional repressor expressed in hematopoietic stem cells (HSCs) and megakaryocytes (MKs). Gfi1b deficiency leads to expansion of both cell types and abrogates the ability of MKs to respond to integrin. Here we show that Gfi1b forms complexes with β-catenin, its co-factors Pontin52, CHD8, TLE3 and CtBP1 and regulates Wnt/β-catenin-dependent gene expression. In reporter assays, Gfi1b can activate TCF-dependent transcription and Wnt3a treatment enhances this activation. This requires interaction between Gfi1b and LSD1 and suggests that a tripartite β-catenin/Gfi1b/LSD1 complex exists, which regulates Wnt/β-catenin target genes. Consistently, numerous canonical Wnt/β-catenin target genes, co-occupied by Gfi1b, β-catenin and LSD1, have their expression deregulated in Gfi1b-deficient cells. When Gfi1b-deficient cells are treated with Wnt3a, their normal cellularity is restored and Gfi1b-deficient MKs regained their ability to spread on integrin substrates. This indicates that Gfi1b controls both the cellularity and functional integrity of HSCs and MKs by regulating Wnt/β-catenin signaling pathway.

Here we demonstrate a mode of reciprocal regulation between GFI1 and p53 that controls the induction of apoptosis in T cells. We show that GFI1 prevents induction of p53 dependent apoptosis by recruiting LSD1 to p53, which leads to the demethylation of its C-terminal domain. This is accompanied by a decrease of the acetylation of lysine 117 within the core domain of the murine p53 protein, which is required for transcriptional induction of apoptosis. Our results support a model in which the effect of GFI1’s regulation of methylation at the c-terminus of p53 is ultimately mediated through control of acetylation at lysine 117 of p53. We propose that GFI1 acts prior to the occurrence of DNA damage to affect the post-translational modification state and limit the subsequent activation of p53. Once activated, p53 then transcriptionally activates GFI1, presumably in order to re-establish the homeostatic balance of p53 activity. These findings have implications for the activity level of p53 in various disease contexts where levels of GFI1 are either increased or decreased.

GFI1 is a transcriptional regulator expressed in lymphoid cells, and an "oncorequisite" factor required for development and maintenance of T-lymphoid leukemia. GFI1 deletion causes hypersensitivity to ionizing radiation, for which the molecular mechanism remains unknown. Here, we demonstrate that GFI1 is required in T cells for the regulation of key DNA damage signaling and repair proteins. Specifically, GFI1 interacts with the arginine methyltransferase PRMT1 and its substrates MRE11 and 53BP1. We demonstrate that GFI1 enables PRMT1 to bind and methylate MRE11 and 53BP1, which is necessary for their function in the DNA damage response. Thus, our results provide evidence that GFI1 can adopt non-transcriptional roles, mediating the post-translational modification of proteins involved in DNA repair. These findings have direct implications for treatment responses in tumors overexpressing GFI1 and suggest that GFI1's activity may be a therapeutic target in these malignancies.

Growth Factor Independent 1 (Gfi1) is a transcriptional repressor that regulates hematopoietic stem cells (HSC) and both early lymphoid and myeloid development. Gfi1 knockout (Gfi1 KO) mice have defects in myelopoiesis in particular lack of neutrophils and accumulation of monocytes and monocytic precursors in the bone marrow (BM). We report now that unlike Gfi1 KO mice, mice expressing a low level of Gfi1 (Gfi1 KD) develop spontaneously a fatal myeloproliferative disease (MPD) resembling leukemia when the mice are getting old, predisposing KD mice to myeloid leukemia after additional mutations. KD mice present a similar block of myeloid cell differentiation as the KO mice but KD BM cells have a better reconstitution capacity and survival due to a lower p53 activity. We found that a reduced expression of p53 in KO mice lead to the emergence of MPD with the same latency than observed in KD mice confirming the role of p53 in the disease severity. Thus it suggests that a knockout, but not a knockdown of Gfi1 initiates a p53 dependent cell death in myeloid cells and prevents the emergence of myeloid disease. In parallel, we also found that the metabolism especially pathways related to radical oxygen specie formation, was deregulated in sick KD BM cells suggesting a direct or indirect role of Gfi1 in metabolism regulation. This metabolism deregulation could increase the severity of the disease in the KD by supporting the tumorigenesis potential of the KD BM cells.

Growth Factor Independent 1 (Gfi1) is a transcriptional repressor that regulates hematopoietic stem cells and both early lymphoid and myeloid development. Gfi1 knockout (Gfi1-KO) mice have defects in lympho- and myelopoiesis and in particular lack neutrophils and accumulate monocytes and monocytic precursors in bone marrow and blood, without developing a spontaneous fatal myeloid malignancy. We have recently shown that AML patients with low GFI1 expression levels have a worse prognosis than patients with high GFI1 expression. Also, leukemic cells with low GFI1 expression show a gene signature associated with Leukemia Stem Cells (LSC). To study this further, we have generated a humanized mouse strain with reduced GFI1 expression (GFI1-knockdown mice, or GFI1-KD). Using these mice, we have shown that AML development induced by onco-fusion proteins such as MLL-AF9 or NUP98-HOXD13 is accelerated in mice with low human GFI1 expression. We report now that Gfi1-KD mice present the block of myeloid cell differentiation as the Gfi1-KO mice but with an even higher accumulation of monocytes in bone marrow, spleen and blood. Interestingly, unlike Gfi1-KO mice, Gfi1-KD animals spontaneously develop a fatal myelo-proliferative disease resembling leukemia, without the need to introduce any additional oncogenic mutations. In the absence of p53, both Gfi1-KO and GFI1–KD mice developed the same fatal myelo-proliferative disease now with almost the same latency. Thus suggests that a knockout, but not a knockdown of Gfi1 initiates a p53 dependent cell death in myeloid cells and prevents the emergence of a myeloid disease by a similar mechanism that we previously described for Gfi1 in T-cells. Indeed, GFI1-KD cells show much lower level of p53 K372 methylation and activation of p53 target genes and RNA-Seq analyses reveal enrichment of a p53 gene expression signature in KO BM cells versus sick KD BM cells. Our data suggest that Gfi1 plays a critical role in regulating both the differentiation and survival of myeloid cells to prevent the emergence of myelo-proliferative diseases and leukemia.

A regulatory circuit that controls myeloid versus B lymphoid cell fate in hematopoietic progenitors has been proposed, in which a network of the transcription factors Egr1/2, Nab, Gfi1 and PU.1 forms the core element. Here we show that a direct link between Gfi1, the transcription factor E2A and its inhibitor Id1 is a critical element of this regulatory circuit. Our data suggest that a certain threshold of Gfi1 is required to gauge E2A activity by adjusting levels of Id1 in multipotent progenitors, which are the first bipotential myeloid/lymphoid-restricted progeny of hematopoietic stem cells. If Gfi1 levels are high, Id1 is repressed enabling E2A to activate a specific set of B lineage genes by binding to regulatory elements for example the IL7 receptor gene. If Gfi1 levels fall below a threshold, Id1 expression increases and renders E2A unable to function, which prevents hematopoietic progenitors from engaging along the B lymphoid lineage.

Growth factor independent 1b (Gfi1b) is a SNAG domain transcription factor that regulates gene expression by recruiting histone-modifying enzymes such as deacetylases (HDAC), de-methylases (LSD1) or methyl-transferases (G9a) to target gene promoters. Gfi1b regulates hematopoietic stem cell cellularity and differentiation along the megakaryocyte-erythrocyte lineage. Ablation of Gfi1b in mice causes an expansion of hematopoietic stem- and -precursor cells and megakaryocytes in bone marrow and peripheral blood. Using mass spectrometry and immune precipitation analysis, we found that Gfi1b interacts with beta-catenin and several inhibitory members of the canonical Wnt/beta-catenin pathway such as APC, CHD8, CtBP, Pontin 52 and the Groucho proteins TLE1 and TLE3. Similar to LSD1, CtBP interacts with the SNAG domain of Gfi1b, but Pontin52, CHD8 and TLE3 bind to a newly identified N-terminal domain in Gfi1b that is conserved in several species. Interestingly, Gfi1b can induce TCF-beta-catenin dependent transcription in a TOP/FOP reporter assay, which could be even enhanced by Wnt treatment. Since this activity of Gfi1b was abrogated when the SNAG domain was deleted or mutated, it is likely that LSD1 or CtBP mediate this new function of Gfi1b. Indeed, immune complexes collected with anti beta-catenin antibodies contained LSD1 only when Gfi1b was present, suggesting that Gfi1b recruits LSD1 to beta-catenin. Comparison of gene expression profiles between wt and Gfi1b deficient cells revealed deregulated expression of genes belonging to the canonical Wnt pathway in the absence of Gfi1b. Comparison of Ch-IP seq datasets demonstrated that a significant portion of beta-catenin target genes is also co-occupied by Gfi1b and LSD1. These genes are down-regulated in the absence of Gfi1b, which suggests that the Gfi1b/LSD1 complex can act as an activator for beta-catenin target genes

Gfi1 is a transcriptional regulator expressed in B and T cells as well as lymphoid precursors and is required for the development and maintenance of T-cell lymphoid leukemia. Since previous work from our laboratory had shown that mice with a genetic deletion of Gfi1 were more sensitive to ionizing radiation (IR), we hypothesized that Gfi1 played either a transcriptional role or a direct role in the response of cells to DNA damage. Using mouse and cell culture models of genetic knock-out (KO) and knock-down (KD) as well as overexpression of Gfi1, we demonstrate that there is a dose-dependant relation between the level of Gfi1 expression and the apoptotic response of cells, their ability to repair DNA damage induced by IR and the signalling that follows the induction of damage. This suggested a direct implication of Gfi1 in the DDR and in DNA repair. Transcriptome analysis using RNA-Sequencing and confirmed by independent experiments showed that GFI1 activates the expression of key genes involved in the DNA damage response including ATR, ATM, CHEK1, CHEK2, 53BP1. Furthermore, Mass Spec analysis and co-immune precipitation experiments show that Gfi1 interacts with members of the MRN complex as well as the ATM kinase and that this interaction is strengthened following IR exposure. This indicates both a role for Gfi1 in the transcriptional activation of genes involved in the DNA damage response and a direct role in the response through interaction with proteins such as the MRN complex. It would be conceivable that, prior to damage, Gfi1’s transcriptional activity maintains adequate levels of a set of DDR Proteins with which it then interacts following DNA damage to regulate their activity. Further work on this project aims to improve our understanding of the role of Gfi1 in the context of the response of lymphoid tumor cells to radiotherapy and chemotherapy and to suggest methods by which such tumors may be rendered more sensitive to treatments.

Most patients with acute lymphoblastic leukemia (ALL) fail current treatments highlighting the need for better therapies. Because oncogenic signaling activates a p53-dependent DNA damage response and apoptosis, leukemic cells must devise appropriate countermeasures. We show here that growth factor independence 1 (Gfi1) can serve such a function because Gfi1 ablation exacerbates p53 responses and lowers the threshold for p53-induced cell death. Specifically, Gfi1 restricts p53 activity and expression of proapoptotic p53 targets such as Bax, Noxa (Pmaip1), and Puma (Bbc3). Subsequently, Gfi1 ablation cures mice from leukemia and limits the expansion of primary human T-ALL xenografts in mice. This suggests that targeting Gfi1 could improve the prognosis of patients with T-ALL or other lymphoid leukemias.

The coding single nucleotide polymorphism GFI136N in the human gene growth factor independence 1 (GFI1) is present in 3%-7% of whites and increases the risk for acute myeloid leukemia (AML) by 60%. We show here that GFI136N, in contrast to GFI136S, lacks the ability to bind to the Gfi1 target gene that encodes the leukemia-associated transcription factor Hoxa9 and fails to initiate histone modifications that regulate HoxA9 expression. Consistent with this, AML patients heterozygous for the GFI136N variant show increased HOXA9 expression compared with normal controls. Using ChipSeq, we demonstrate that GFI136N specific epigenetic changes are also present in other genes involved in the development of AML. Moreover, granulomonocytic progenitors, a bone marrow subset from which AML can arise in humans and mice, show a proliferative expansion in the presence of the GFI136N variant. In addition, granulomonocytic progenitors carrying the GFI136N variant allele have altered gene expression patterns and differ in their ability to grow after transplantation. Finally, GFI136N can accelerate a K-RAS driven fatal myeloproliferative disease in mice. Our data suggest that the presence of a GFI136N variant allele induces a preleukemic state in myeloid precursors by deregulating the expression of Hoxa9 and other AML-related genes.

Abstract 223 A coding variant form of GFI1 (GFI136N) increases the risk to develop AML by 60% and is present in about 10–15 % of all Caucasian AML patients. To determine the underlying molecular mechanism and potentially develop new therapeutic approaches, we generated “knockin” mouse strains wherein the endogenous murine Gfi1 gene was replaced either by the human GFI1 variant ( GFI136N , the form predisposing to AML) or by the more common form of GFI1 ( GFI136S ). In most hematopoietic compartments no difference was observable between GFI136N and GFI136S expressing mice; however, there was a 3–5 fold increase in the number of granulocytic monocytic progenitors (GMPs) and common myeloid progenitors (CMPs) in Gfi136N expressing (either homozygous or heterozygous) mice compared to wild-type or Gfi136S expressing mice(p≤0.01). Interestingly, both human and murine AML leukemic cells are thought to originate from GMPs and CMPs. To assess functional differences, we seeded GMPs from GFI136N or GFI136S knockin mice on methylcelluose or transplanted them into into syngenic animals. We found that GFI136N expressing GMPs proliferate faster and have an increased self-renewal capacity both in-vitro and in-vivo compared to GMPs carrying Gfi136S alleles. A gene expression array analysis showed that GFI136N GMPs have a stem cell-like gene signature with elevated levels of Hoxa9 expression and a deregulation of a number of oncogenes involved in the development of human AML such as Trib2, Tet2 or Idh2. It is of particular interest that Hoxa9, a known GFI1 target gene, was up-regulated 3–4 fold in GFI136N GMPs compared to in GFI136S GMPs (p≤0.01). It is known that high levels of Hoxa9 accelerate AML development in mice and are associated with a poor prognosis in AML patients. GFI1 is a transcriptional repressor and exerts its function by recruiting different histone modifying enzymes, in particular LSD1, which de-methylates histone 3 (H3) at lysine 4 (K4), or histone deacetylases (HDACs), which remove acetyl groups from H3K9 residues and G9a, which initiates dimethylation of H3K9. Both H3K4 methylation and H3K9 acetylation correlate with actived gene expression, whereas H3K9dimethyl correlates with repession. Chromatin-immuno-precipitation (ChIP) of Gfi1-bound chromatin from Lin−Sca1−c-Kit+ cells, which contains the GMP population, showed that GFI136N binds to a lesser degree to the Hoxa9 locus than GFI136S. This diminished binding of Gfi136N correlated with an increased H3K4 dimethylation and H3K9 acetylation as well as diminished H3K9 dimethylation across the Hoxa9 locus in GFI136N cells. It is likely that these epigenetic changes lead to the increased Hoxa9 expression observed in GFI136N GMPs. A more exhaustive ChIP-Seq analysis with antibodies recognizing H3K4dimethyl in Lin−Sca1−c-Kit+ cells from Gfi136N or Gfi136S mice showed significant epigenetic alterations throughout the Hoxa9 locus genome and at other GFI1 target genes. It is conceivable that these epigenetic alterations explain, at least in part, the changed gene expression signatures in GFI136N GMPs. To investigate the role of GFI136N in myeloid leukemogenesis, we induced the expression of a mutated form of KRAS (K12D) in both GFI136N and GFI136S mice. All mice developed a deadly myelo-proliferative disorder, but animals carrying the GFI136N allele succumbed to the disease within a significantly shorter latency period (17 against 31 days, p≤0.01) than GFI136S mice. We also transduced GFI136N and GFI136S GMPs with retroviral vectors directing the expression of either the AML1-Eto9a or the MLL-AF9 onco-fusion proteins typically found in human AML. We observed that GFI136N GMPs expressing MLL-AF9 or AML1-Eto9a generated 5–10 fold more colonies (p≤0.01) on methylcellulose and exhibited a higher replating efficiency than the respective GFI136S GMPs. Finally, AML blast cells from GFI136N heterozygous patients expressed higher levels of HOXA9 compared to AML blasts from GFI136S homozygous patients, suggesting that our mouse model reflects the disease predisposition in human patients. Our knockin mice are, to our knowledge, the first animal model for a human genetic variation that predisposes to leukemia. Based on the findings with this model, we propose that the human GFI136N variant predisposes to AML by inducing epigenetic changes affecting the expression of important regulators with oncogenic potential such as Hoxa9. Disclosures: No relevant conflicts of interest to declare.

Abstract 560 More than 50% of patients diagnosed with B or T-cell leukemia and lymphoma will fail current treatment protocols. This highlights the urgent need for new and improved therapies. Since the transcription factor Growth factor independent-1 (Gfi1) plays an important role in lymphoid differentiation, we explored whether it might be a suitable target for therapy. Using mouse models in which T-cell leukemia can be induced by transgenic expression of mutated forms of Notch1 , by injection of the carcinogen N-Ethyl–nitrosourea (ENU) or infection with a Murine Moloney Leukemia Virus, we found that Gfi1 knockout mice had a significantly lower incidence and a longer latency period of T-ALL. To verify whether targeting Gfi1 would be a novel approach to treat B- or T-cell lymphoma, we used Mx1Cre Gfi1 fl/fl mice. In these mice, injection of polyinosinic-polycytidylic acid (pIpC) activates the Mx1 promoter driven Cre expression, which ultimately leads to the deletion of the floxed Gfi1 alleles. As controls, we used Gfi1 fl/fl mice, which lack the Cre recombinase and thus still express Gfi1 after pIpC injection. To elicit a T- or B-cell lymphoma, we used ENU injection combined with expression of a mutated Notch1 transgene for T-ALL , or transgenic over-expression of c-Myc for B-cell leukemia ( Eμ-Myc ). Using in-vivo ultrasound supported imaging, we observed complete regression of tumour masses when Gfi1 was eliminated in Mx1Cre Gfi1 fl/fl mice, curing the mice of the either the T or B-cell malignancies. Strikingly, this effect was observed in the absence of any other treatment regimen. To explore the mechanisms underlying this phenomenon, we explanted tumor samples from mice, in which Gfi1 expression was either maintained or deleted and performed gene expression arrays. A comparative analysis of the array data demonstrated that loss of Gfi1 affects pathways of key importance for leukemia such as metabolism, cell cycle progression, basal transcription and apoptosis but also the response to DNA damage. It has been shown previously for non hematological malignancies that oncogenic transformation in conjuction with dysregulated cell cycle induces DNA strand breaks (DSBs), which leads to an increased p53 dependent apoptotic response in tumours compared to non-transformed cells. This forces the tumor cells to counteract this effect – for instance by selection for the loss of p53. Consistent with this concept, we noted that leukemic cells from our tumor models displayed a greater amount of DSBs and also higher rates of spontaneous apoptosis than normal cells. Interestingly, the number of apoptotic cells was further increased in those tumors where Gfi1 had been deleted. We hypothesized that Gfi1 protects leukemia cells against DSB induced apoptosis. To test this hypothesis, we irradiated in Gfi1 +/+ and Gfi1 −/− thymocytes, which induces DSB in thymocytes. In line with our hypothesis we found that Gfi1 −/− thymocytes showed increased rates of apoptosis compared to irradiated Gfi1 +/+ thymocytes and that loss of Gfi1 led to an increased induction of pro-apoptotic genes such as Bax , Noxa and Puma after irradiation. Since Bax, Noxa and Puma are all p53 target genes, we investigated a possible link between Gfi1 and p53 and found that (I) Gfi1 binds to p53, (II) that Gfi1 inhibits the transcription of p53 target genes and (III) that Gfi1 occupies p53 target gene at the same sites as p53. In summary, Gfi1 antagonizes p53 function and loss of Gfi1 sensitizes cells to p53-mediated apoptosis. Next, we used different human T-ALL cell lines and treated these cells either with sh-RNA lentivirus or morpholinos to abrogate GFI1 expression. In all cases, down-regulation of GFI1 expression led to increased apoptosis and impeded growth of human leukemia cells. Finally, we transplanted leukemic cells of T-ALL patients into NOD-Scid, IL2R null (NSG) mice, waited for the leukemic cells to engraft, and then injected GFI1 specific- or control morpholinos. While mice treated with control morpholino died of leukemia, the animals treated with GFI1-specific morpholinos survived showing a significant reduction of human leukemic cells in the blood, bone marrow and spleen, even with samples from patients who did not respond to first line therapy. Since morpholinos have received approval for use in humans, our data suggest that targeting GFI1 in human T-ALL patients may be a promising therapeutic target and a feasible way to complement current T-ALL treatment regimens. Disclosures: No relevant conflicts of interest to declare.

Modification of histones is critically involved in regulating chromatin structure and gene expression. The zinc finger protein Gfi1 silences transcription by recruiting a complex of histone modifying enzymes such as LSD-1/CoRest and HDAC-1 to target gene promoters. Here we present evidence that Gfi1 forms a complex with the p150 subunit of the histone chaperone chromatin assembly factor-1 (Caf-1). Gfi1 and p150 interact at endogenous expression levels and co-localize in distinct sub-nuclear structures. We show that p150 enhances Gfi1-mediated transcriptional repression and that it occupies Gfi1 target gene promoters in transfected cells and primary murine T cells only in the presence of Gfi1. Finally, size exclusion chromatography shows a fraction of p150 to coelute with Gfi1, LSD-1 and HDAC-1 and thus provides evidence that p150 is part of the Gfi1 repression complex. Since p150 binds directly to histones H3 and H4, our findings suggest that p150 may link the DNA-bound Gfi1 repressor complex to histones enabling modifications required for transcriptional silencing.

The adaptors SAP, EAT-2 and ERT are specific to cells of the immune system and belong to the SAP family. All three are expressed in natural killer (NK) cells. Here we examined the global function of the SAP family using mice lacking SAP, EAT-2 and ERT. These adaptors acted together in a mechanism that was essential for the elimination of hematopoietic but not nonhematopoietic cells by NK cells. This function was mediated by many receptors of the SLAM family on NK cells that were engaged by ligands found solely on hematopoietic cells. In the absence of SAP-related adaptors, SLAM receptors lost their activating function and became inhibitory receptors that repressed other activating receptors, such as NKG2D. Hence, the SAP family is essential for the elimination of unwanted hematopoietic cells by NK cells.

SAP is an intracellular adaptor molecule composed almost exclusively of an SH2 domain. It is mutated in patients with X-linked lymphoproliferative disease, a human immunodeficiency. Several immune abnormalities were also identified in SAP-deficient mice. By way of its SH2 domain, SAP interacts with tyrosine-based motifs in the cytoplasmic domain of SLAM family receptors. SAP promotes SLAM family receptor-induced protein tyrosine phosphorylation, due to its capacity to recruit the Src-related kinase FynT. This unusual property relies on the existence of a second binding surface in the SAP SH2 domain, centered on arginine 78 of SAP, that binds directly to the FynT SH3 domain. Herein, we wanted to further understand the mechanisms controlling the interaction between SLAM-SAP and FynT. Our experiments showed that, unlike conventional associations mediated by SH3 domains, the interaction of the FynT SH3 domain with SLAM-SAP was strictly inducible. It was absolutely dependent on engagement of SLAM by extracellular ligands. We obtained evidence that this inducibility was not due to increased binding of SLAM to SAP following SLAM engagement. Furthermore, it could occur independently of any appreciable SLAM-dependent biochemical signal. In fact, our data indicated that the induced association of the FynT SH3 domain with SLAM-SAP was triggered by a change in the conformation of SLAM-associated SAP caused by SLAM engagement. Together, these data elucidate further the events initiating SLAM-SAP signaling in immune cells. Moreover, they identify a strictly inducible interaction mediated by an SH3 domain.