Growth Factor Independence Research Articles

The Gfi1-SphK1 Axis Regulates the Growth and Survival of Myeloma Cells

In spite of major advances in treatment, multiple myeloma (MM) is currently an incurable hematologic malignancy due to emergence of drug-resistant clones. We previously reported that MM cells upregulate expression of the transcriptional repressor, Growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSC), which induces prolonged inhibition of osteoblast differentiation. We recently showed that Gfi1 levels are also increased in the majority of CD138+ cells from MM patients and cell lines ,and Gfi1 levels significantly correlated with Mcl-1 protein expression. Further, Gfi1 repressed MM cell death by inhibiting expression of apoptosis-inducing genes. Importantly, Gfi1 overexpression in MM cells enhanced MM cell growth and conferred resistance to proteasome-inhibitor- induced apoptosis. However, the mechanisms responsible for these effects of Gfi1are unknown.Sphingosine kinase 1 (SphK1) is overexpressed in many cancers including MM, and catalyzes the phosphorylation of sphingosine (SPH) to sphingosine-1-phosphate (S1P). S1P is a pleiotropic lipid mediator that regulates cell survival, migration, the recruitment of immune cells and angiogenesis, all of which contribute to cancer progression. Therefore, we hypothesized that adhesive interactions between MM cells and BMSC stimulate survival and growth of MM cells in part through the Gfi1-SphK1 axis by modulating their sphingolipid profile (Ceramide/SPH/S1P ratio).We found that SphK1 mRNA is highly expressed in CD138+ cells from MM patients and cell lines compared with normal donors. Further, Gfi1 protein expression correlated significantly with SphK1 protein level in CD138+ cells from MM patients and MM cell lines. Soluble factors (IL-6 and S1P) that are increased in the MM microenvironment, hypoxia (1% O2) and adhesive interactions of MM cells with BMSC further increased Gfi1 and SphK1 mRNA and protein levels in MM cells. Gfi1 Knockdown (KD) in MM cells induced a profound decrease of SphK1 mRNA and protein activity and inhibited MM cell growth and viability. In contrast, over-expression of Gfi1 in MM cells increased SphK1 levels that conferred a survival advantage to MM cells over empty vector-transduced control cells. Increased Gfi1 expression also resulted in increased intracellular S1P and decreased sphingosine levels, as measured by LC-MS/MS. Further, treatment of MM cells with a SphK1 specific inhibitor (SKI2), dose-dependently reduced MM cell viability at 24h, regardless of their p53 status. Interestingly, p53 null MM cells were more resistant to SK12, as compared with p53 replete cells. In p53 replete MM cells, SphK1 inhibition significantly reduced c-Myc protein expression, induced autophagy (as shown by increased LC3 II protein levels) and increased total ceramides levels. Moreover, BMSC protected p53 replete MM cells from the anti-survival effects of SKI2 in 3D cultures. These data suggest that Gfi1 regulates MM growth in part by enhancing the expression and activity of SphK1.Taken together, our results support that Gfi1 acts as a key regulator of MM growth and survival, at least partially through modulation of SphK1. Therefore, targeting lipid metabolism to modulate the levels of specific bioactive lipid components that can modify cancer cell fate may provide a new and attractive therapeutic approach for MM. DisclosuresRoodman:Amgen Denosumab: Membership on an entity's Board of Directors or advisory committees.

Haploinsufficiency of CALR Confers Hematopoietic Stem Cells (HSCs) with a Clonal Advantage over Wild-Type Cells, and, in Setting of Myeloproliferative Neoplasms, Compensates for the Functions of HSCs Impaired By the Calr Mutation

CALR exon9 frameshift mutations function as driver mutations in essential thrombocythemia (ET) and primary myelofibrosis patients with non-mutated JAK2 or MPL. The mutations augment signal transducer and activator of transcription activity in the presence of MPL, induce increased cell proliferation and growth factor independence in cell lines, and cause ET-like myeloproliferative neoplasms (MPN) in mice.In tumor cells bearing the CALR mutation, mutant CALR protein expression occurs while wild-type (WT) CALR expression is decreased by half. Although the biological activity of mutant CALR has been elucidated in detail, the significance of CALR haploinsufficiency is unclear. The purpose of this study was therefore to clarify the influence of CALR haploinsufficiency on hematopoiesis in normal and CALR-mutated MPN in mice.First, we analyzed the effect of CALR haploinsufficiency on hematopoiesis using CALR heterozygous knockout (CALR-hKO) mice (Tokuhiro et al. Sci Rep. 2015). Blood cell counts, liver and spleen weight, histology, cell fraction in bone marrow (BM) and spleen, and survival of CALR-hKO mice were all equivalent to that observed in WT mice. In the analysis of progenitor cells by fluorescence-activated cell sorting and colony formation assays, no difference was observed in the amount of progenitor cells and in colony replating capacity between WT mice and CALR-hKO mice. Interestingly, in competitive serial transplantation experiments using whole BM cells in primary and secondary transplanted mice, CALR-hKO cells showed higher levels of chimerism than WT cells.Next, the effect of CALR haploinsufficiency on hematopoiesis in mutant CALR-del52 overexpressing mice was analyzed. We compared three groups of mice, WT mice, CALR-del52 transgenic (TG) mice (Shide et al. Leukemia 2017) and CALR-del52 TG/CALR-hKO double-mutant (TG-hKO) mice. Both TG mice and TG-hKO mice developed ET-like MPN. Compared to WT mice, increases in megakaryocytes, platelets and hematopoietic progenitor cells (including HSCs) were observed in these mice. However, no differences were observed between TG mice and TG-hKO mice.Finally, 4000 LSK cells sorted from WT mice, TG mice, and TG-hKO mice (B6-Ly5.2) and B6-Ly5.1 competitor cells (1 × 106 WT BM cells) were mixed and injected into lethally irradiated B6-Ly5.1 recipient mice, and the percent chimerism of donor cells was followed for 1 year after transplantation. From 12 weeks after transplantation, the chimerism of TG cells was significantly lower than that of control WT cells, suggesting that the CALR mutation has a negative influence on clonal expansion of HSCs. The recipient mice transplanted with TG LSK cells showed very mild thrombocythemia. On the other hand, chimerism in TG-hKO cells was significantly higher than that in control WT cells up to 12 weeks after transplantation. Chimerism at 20 weeks after transplantation was equivalent to that in control WT cells, and the recipient mice transplanted with TG-hKO LSK cells showed severe thrombocythemia. These findings show that CALR haploinsufficiency compensates for HSCs functioning, which has been impaired by the CALR mutation, enhancing the ability of HSCs to develop ET.Mice experiments have showed that HSCs with the JAK2V617F mutation are fragile, and it is considered that mutations in DNMT3A or TET2 often occurred prior to mutations in JAK2 to compensate for the defect in self renewal capacity. Conversely, since the CALR mutation is not typically preceded by any mutations, it is considered that the CALR mutation may not require any preceding mutations. Our results showed that, like JAK2 V617F mutants, overexpression of the CALR-del52 mutant impairs HSC functioning. Furthermore, we found that CALR haploinsufficiency restores the functions of impaired HSCs to the same extent as that found in WT HSCs. When an HSC acquires the CALR mutation, “defect” and “recovery” states thus occur simultaneously in the cell. This finding may explain why mutant CALR clones expand without needing to undergo any preceding mutation. DisclosuresNo relevant conflicts of interest to declare.

Growth factor independence 1 expression in myeloma cells enhances their growth, survival, and osteoclastogenesis

BackgroundIn spite of major advances in treatment, multiple myeloma (MM) is currently an incurable malignancy due to the emergence of drug-resistant clones. We previously showed that MM cells upregulate the transcriptional repressor, growth factor independence 1 (Gfi1), in bone marrow stromal cells (BMSCs) that induces prolonged inhibition of osteoblast differentiation. However, the role of Gfi1 in MM cells is unknown.MethodsHuman primary CD138+ and BMSC were purified from normal donors and MM patients’ bone marrow aspirates. Gfi1 knockdown and overexpressing cells were generated by lentiviral-mediated shRNA. Proliferation/apoptosis studies were done by flow cytometry, and protein levels were determined by Western blot and/or immunohistochemistry. An experimental MM mouse model was generated to investigate the effects of MM cells overexpressing Gfi1 on tumor burden and osteolysis in vivo.ResultsWe found that Gfi1 expression is increased in patient’s MM cells and MM cell lines and was further increased by co-culture with BMSC, IL-6, and sphingosine-1-phosphate. Modulation of Gfi1 in MM cells had major effects on their survival and growth. Knockdown of Gfi1 induced apoptosis in p53-wt, p53-mutant, and p53-deficient MM cells, while Gfi1 overexpression enhanced MM cell growth and protected MM cells from bortezomib-induced cell death. Gfi1 enhanced cell survival of p53-wt MM cells by binding to p53, thereby blocking binding to the promoters of the pro-apoptotic BAX and NOXA genes. Further, Gfi1-p53 binding could be blocked by HDAC inhibitors. Importantly, inoculation of MM cells overexpressing Gfi1 in mice induced increased bone destruction, increased osteoclast number and size, and enhanced tumor growth.ConclusionsThese results support that Gfi1 plays a key role in MM tumor growth, survival, and bone destruction and contributes to bortezomib resistance, suggesting that Gfi1 may be a novel therapeutic target for MM.

ETV6/FLT3 Fusion Is a Novel Client Protein of Hsp90.

FMS-like tyrosine kinase-3 fragments from exon 14 to the end without any mutations or deletions have been reported to fuse to ETV6 (TEL) in a few cases of myeloid/lymphoid neoplasms with eosinophilia carrying a translocation t(12;13)(p13;q12). This fusion protein confers constitutive activation on the FLT3 fragment and induces factor-independent growth in transfected Ba/F3 cells, indicating that it is an oncoprotein. However, the mechanism controlling the stability of this oncoprotein is unknown. In this study, we focus on finding factors controlling the stability of ETV6/FLT3. We have shown that the stability of ETV6/FLT3 is regulated by the Hsp90 chaperone. ETV6/FLT3 fusion protein forms a complex with Hsp90 by coimmunoprecipitation analyses using an Hsp90 antibody. The association between ETV6/FLT3 fusion protein and Hsp90 was impaired after treating ETV6/FLT3 transient transfection cos7 cells with 17-allylamino-17-demethoxygeldanamycin (17-AAG). 17-AAG induced a time- and dose-dependent downregulation of ectopically expressed ETV6/FLT3 protein in cos7 and HeLa-transfected cells. By using cycloheximide to block new protein translation, we found that 17-AAG accelerated the decay of ETV6/FLT3. Our findings could contribute to more understanding of the ETV6/FLT3 regulation through Hsp90 chaperone and open the way to finding effective treatment strategies for this rare disease.

L-Threonine improves intestinal mucin synthesis and immune function of intrauterine growth-retarded weanling piglets.

The aim of this study was to investigate the effects of dietary l-threonine supplementation on the growth performance, intestinal immune function, mucin synthesis, and goblet cell differentiation in weanling piglets with intrauterine growth retardation (IUGR). Eighteen litters of newborn piglets were selected at birth, with one normal birthweight (NBW) and two IUGR piglets in each litter. At weaning, the NBW piglet and one of the IUGR piglets were assigned to groups fed a basal diet (i.e., the NBW-CON and IUGR-CON groups). The other IUGR piglet was assigned to a group fed the basal diet supplemented with 2g l-threonine per kg of diet (i.e., IUGR-Thr group). Therefore, all piglets were distributed across three groups for a 3-wk feeding trial. Compared with NBW, IUGR decreased growth performance, increased ileal proinflammatory cytokine levels, and reduced ileal mucin 2 (Muc2) content and goblet cell density of weanling piglets. Supplementation of l-threonine increased the feed efficiency of the IUGR-Thr group compared with the IUGR-CON group. The l-threonine-supplemented diet attenuated ileal inflammatory responses of the IUGR-Thr piglets and increased production of Muc2 and secretory immunoglobulin A and density of goblet cells. In addition, L-threonine supplementation downregulated δ-like 1 and hes family bHLH transcription factor 1, whereas growth factor independence 1 and Kruppel-like factor 4 expression levels were upregulated. Dietary l-threonine supplementation attenuates inflammatory responses, facilitates Muc2 synthesis, and promotes goblet cell differentiation in the ileum of IUGR piglets.

3120 - Dominant-Negative GFI1B Mutations are Causal in Rare Inherited Platelet Disorders and Cause Defects in Stress Thrombopoiesis

Inherited platelet disorders represent a heterogeneous group of rare diseases often characterized by lifelong bleeding diatheses. Specific therapies are lacking because the affected molecular processes are poorly understood. Previously, heterozygous nonsense mutations in Growth Factor Independence 1b (GFI1B) in families with autosomal-dominant bleeding disorders were identified. This disease is characterized by a plethora of megakaryocyte (MK) and platelet abnormalities, including macro thrombocytopenia, dysmorphic megakaryocytes, paucity of platelet /-granules, CD34 expression on MKs and platelets, increased MK numbers and their dislocation in the marrow. GFI1B is a transcription factor that contains an evolutionary conserved N-terminal SNAG domain and six C-terminal zinc fingers of which zinc finger three to five are crucial for DNA binding. Through the SNAG domain GFI1B recruits chromatin-modifying enzymes to promoters of target genes to regulate gene expression. Many mutations in the GFI1B gene found in inherited platelet disorders are located in zinc finger five, resulting in a DNA binding defective protein. To study the consequences of these mutations in vivo, we used Crispr/Cas technology and established three independent mouse lines that carry one Gfi1b allele in which the 5th and 6th zinc fingers are truncated or eliminated. All three lines of GFI1B DN mice had a mild but significant reduction of platelet counts resembling the situation found in patients. Moreover, GFI1B DN mice showed an expansion MKs and Mk precursors as well as hematopoietic stem cells. To test whether the DN mutations affect platelet recovery, we injected GFI1B DN mice and controls with an anti GP-IB antibody, which leads to an almost complete depletion of platelets. Clearly, GFI1B DN mice showed a delayed recovery of platelet numbers compared to controls. In addition, in many cases the platelets of DN mice were larger and appeared less granular after recovery than their counterparts in wt mice. Our data provide evidence for a causal relationship between inherited mutations in GFI1B in patients with bleeding disorders and reveal a new role for GFI1B in stress thrombopoiesis.

LSD1 inhibitors disrupt the GFI1 transcription repressor complex

ABSTRACTPharmacologic inhibition of KDM1A (Lysine Demethylase 1A) induces differentiation in certain subtypes of acute myeloid leukemia. Our recent studies reveal this is dependent upon drug-induced disruption of the GFI1 (Growth Factor Independent 1) transcription repressor complex, leading to activation of enhancers distributed close to genes controlling monocytic lineage differentiation.

LIMITING THE TOXICITY OF CHEMOTHERAPY BY ENHANCING REGENERATION OF INTESTINAL STEM CELLS

IntroductionChemotherapy is the backbone treatment for most malignancies. Typical cytotoxic chemotherapy drugs, such as doxorubicin, a topoisomerase II inhibitor, also harm normal cells that divide rapidly, such as the gastrointestinal lining, and cause morbidity and mortality that limits medical treatment. There is a need to reduce chemotherapy toxicity and thus provide a therapeutic benefit and improve overall quality of life of cancer patients. Intestinal stem cells are selectively killed by chemotherapy drugs, which trigger a poorly characterized activation of quiescent stem cells and/or progenitors for crypt regeneration. Growth Factor‐Independent 1 (GFI1) is a zinc finger transcriptional repressor implicated in the differentiation of secretory precursors into goblet and Paneth cells in the intestinal epithelium. Here, we hypothesize that following injury, increasing reversion of Gfi1+ secretory cells into stem cells will improve intestinal epithelium regeneration and mitigate chemo‐induced injury.MethodsIn our injury model, mice were treated with 20 mg/kg Doxorubicin through intraperitoneal injection leading to Lgr5+ stem cell injury and crypt loss. Gfi1 reporter mice (Gfi1cre; ROSA26floxSTOP‐YFP) mice and intestinal organoids were used to investigate intestinal stem cell reversion and repair mechanism following injury.ResultsUnder homeostatic conditions, Gfi1‐lineal cells were secretory Paneth and Goblet cells, which were not part of the stem cell pool. After injury, we found that Gfi1+ secretory cells can re‐enter the cell cycle and give rise to all cell lineages of the intestinal epithelium, indicating that Gfi1‐lineal cells revert to become active stem cells and repopulate the stem cell pool following tissue injury. The extent of reversion was generally correlated with the severity of injury as assessed by dysmorphic crypts. To identify potential boosters of intestinal regeneration, we generated three‐dimensional organoid cultures from Gfi1 reporter mice which provide a convenient and physiologically relevant model to identify key pathways regulating stem cell injury and regeneration caused by chemotherapy drugs. Our results demonstrated that PI3kinase/AKT activation using PTEN inhibitor improved cell survival, and elevated WNT signaling using high R‐spondin treatment increased efficiency of Gfi1+ cell reversion upon injury. High R‐spondin treatment and PTEN inhibition combined to enhance both survival and reversion upon injury. Additionally, we used AKT inhibitor to suppress AKT activity. AKT inhibition prevented survival of organoids after injury despite R‐spondin or PTEN inhibitor co‐treatment, indicating that AKT is epistatic to WNT signaling, and activation of AKT after injury is critical for intestinal epithelium regeneration.ConclusionsThese findings indicate that Gfi1+ secretory cells display plasticity and can reacquire stemness upon tissue damage. Moreover, PI3kinase/AKT and WNT are key regulators of cell survival and stem cell reversion after severe tissue injury. Our studies in intestinal stem cells will improve our current understanding injury‐induced regeneration and identify potential therapeutic strategies to mitigate the effects of chemo‐induced normal tissue injury and improve cancer chemotherapy.Support or Funding InformationNIH R01 DK092306U01 DK103117This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PIK3CA C2 Domain Deletions Hyperactivate Phosphoinositide 3-kinase (PI3K), Generate Oncogene Dependence, and Are Exquisitely Sensitive to PI3Kα Inhibitors.

Purpose: We describe herein a novel P447_L455 deletion in the C2 domain of PIK3CA in a patient with an ER+ breast cancer with an excellent response to the PI3Kα inhibitor alpelisib. Although PIK3CA deletions are relatively rare, a significant portion of deletions cluster within amino acids 446-460 of the C2 domain, suggesting these residues are critical for p110α function.Experimental Design: A computational structural model of PIK3CAdelP447-L455 in complex with the p85 regulatory subunit and MCF10A cells expressing PIK3CAdelP447-L455 and PIK3CAH450_P458del were used to understand the phenotype of C2 domain deletions.Results: Computational modeling revealed specific favorable inter-residue contacts that would be lost as a result of the deletion, predicting a significant decrease in binding energy. Coimmunoprecipitation experiments showed reduced binding of the C2 deletion mutants with p85 compared with wild-type p110α. The MCF10A cells expressing PIK3CA C2 deletions exhibited growth factor-independent growth, an invasive phenotype, and higher phosphorylation of AKT, ERK, and S6 compared with parental MCF10A cells. All these changes were ablated by alpelisib treatment.Conclusions: C2 domain deletions in PIK3CA generate PI3K dependence and should be considered biomarkers of sensitivity to PI3K inhibitors. Clin Cancer Res; 24(6); 1426-35. ©2017 AACR.

Overexpression of PIK3CA in head and neck squamous cell carcinoma is associated with poor outcome and activation of the YAP pathway

ObjectivesPhosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) is commonly altered in many human tumors, leading to the activation of p110α enzymatic activity that stimulates growth factor-independent cell growth. PIK3CA alterations such as mutation, gene amplification and overexpression are common in head and neck squamous cell carcinoma (HNSCC) and. We aim to explore how these alterations and clinical outcome are associated, as well as the molecular mechanisms involved. Material and methodsMutation and copy-number variation in PIK3CA, and whole-genome expression profiles, were analyzed in primary HNSCC tumors from The Cancer Genome Atlas (TCGA) cohort (n = 243). The results were validated in an independent cohort form the University Hospital of A Coruña (UHAC, n = 62). Expression of the PIK3CA gene protein product (PI3K p110α) and nuclear YAP were assessed in tissue microarrays in a cohort from the University Hospital 12 de Octubre (UH12O, n = 91). ResultsOnly high expression of the PIK3CA gene was associated with poor clinical outcome. The study of gene expression, transcription factor and protein signatures suggested that the activation of the Hippo-YAP pathway, involved in organ size, stem cell maintenance and tumorigenesis, could underlie tumor progression in PI3KCA overexpressing tumors. Tissue arrays showed that PI3K p110α levels correlated with YAP nuclear localization in HNSCC tumors. ConclusionsHigh expression of PIK3CA in HNSCC primary tumors identifies patients at high risk for recurrence. In these tumors, progression could rely on the Hippo-YAP pathway instead of the canonical Akt/mTOR pathway. This observation could have important implications in the therapeutic options for patients.

Hhex induces promyelocyte self-renewal and cooperates with growth factor independence to cause myeloid leukemia in mice

AbstractThe hematopoietically expressed homeobox (Hhex) transcription factor is overexpressed in human myeloid leukemias. Conditional knockout models of murine acute myeloid leukemia indicate that Hhex maintains leukemia stem cell self-renewal by enabling Polycomb-mediated epigenetic repression of the Cdkn2a tumor suppressor locus, encoding p16Ink4a and p19Arf. However, whether Hhex overexpression also affects hematopoietic differentiation is unknown. To study this, we retrovirally overexpressed Hhex in hematopoietic progenitors. This enabled serial replating of myeloid progenitors, leading to the rapid establishment of interleukin-3 (IL-3)–dependent promyelocytic cell lines. Use of a Hhex-ERT2 fusion protein demonstrated that continuous nuclear Hhex is required for transformation, and structure function analysis demonstrated a requirement of the DNA-binding and N-terminal–repressive domains of Hhex for promyelocytic transformation. This included the N-terminal promyelocytic leukemia protein (Pml) interaction domain, although deletion of Pml failed to prevent Hhex-induced promyelocyte transformation, implying other critical partners. Furthermore, deletion of p16Ink4a or p19Arf did not promote promyelocyte transformation, indicating that repression of distinct Hhex target genes is required for this process. Indeed, transcriptome analysis showed that Hhex overexpression resulted in repression of several myeloid developmental genes. To test the potential for Hhex overexpression to contribute to leukemic transformation, Hhex-transformed promyelocyte lines were rendered growth factor–independent using a constitutively active IL-3 receptor common β subunit (βcV449E). The resultant cell lines resulted in a rapid promyelocytic leukemia in vivo. Thus, Hhex overexpression can contribute to myeloid leukemia via multiple mechanisms including differentiation blockade and enabling epigenetic repression of the Cdkn2a locus.

JAK1 somatic mutation in myeloproliferative neoplasm

For most triple-negative MPNs a molecular diagnosis is not available because access to clinical next-generation sequencing (NGS) is limited or expensive. We employed a custom designed NGS panel to identify potential driver mutations in a case of triple-negative MPN. Review of the blood morphology and bone marrow histology of our patient (78-year-old woman presenting with leucocytosis, left shift and thrombocytopenia) was undertaken. A provisional diagnosis of MPN-U was made. Testing for BCR-ABL, JAK2, CALR and MPL mutations was negative. A comprehensive NGS panel was therefore employed to search for a responsible driver mutation. Two single nucleotide variants (SNVs) were detected in >200 kb of sequenced DNA, including a heterozygous SNV in JAK1 leading to a predicted missense mutation, V658I. The V658 amino acid in JAK1 is equivalent to V617 in JAK2, and transduction of JAK1-V658I leads to factor independent cell growth of BaF3 cells, so it is likely pathogenic. To our knowledge, JAK1 mutations have not been described in MPNs. We suggest consideration of JAK1 mutations in cases of MPN-U, aCML or CMML which do not have a molecular diagnosis. This is of clinical value since the disease is likely to respond to JAK inhibitors which have affinity for JAK1, such as ruxolitinib.

Gfi1b: a key player in the genesis and maintenance of acute myeloid leukemia and myelodysplastic syndrome.

Differentiation of hematopoietic stem cells is regulated by a concert of different transcription factors. Disturbed transcription factor function can be the basis of (pre)malignancies such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Growth factor independence 1b (Gfi1b) is a repressing transcription factor regulating quiescence of hematopoietic stem cells and differentiation of erythrocytes and platelets. Here, we show that low expression of Gfi1b in blast cells is associated with an inferior prognosis of MDS and AML patients. Using different models of human MDS or AML, we demonstrate that AML development was accelerated with heterozygous loss of Gfi1b, and latency was further decreased when Gfi1b was conditionally deleted. Loss of Gfi1b significantly increased the number of leukemic stem cells with upregulation of genes involved in leukemia development. On a molecular level, we found that loss of Gfi1b led to epigenetic changes, increased levels of reactive oxygen species, as well as alteration in the p38/Akt/FoXO pathways. These results demonstrate that Gfi1b functions as an oncosuppressor in MDS and AML development.

Enforced GFI1 expression impedes human and murine leukemic cell growth

The differentiation of haematopoietic cells is regulated by a plethora of so-called transcription factors (TFs). Mutations in genes encoding TFs or graded reduction in their expression levels can induce the development of various malignant diseases such as acute myeloid leukaemia (AML). Growth Factor Independence 1 (GFI1) is a transcriptional repressor with key roles in haematopoiesis, including regulating self-renewal of haematopoietic stem cells (HSCs) as well as myeloid and lymphoid differentiation. Analysis of AML patients and different AML mouse models with reduced GFI1 gene expression levels revealed a direct link between low GFI1 protein level and accelerated AML development and inferior prognosis. Here, we report that upregulated expression of GFI1 in several widely used leukemic cell lines inhibits their growth and decreases the ability to generate colonies in vitro. Similarly, elevated expression of GFI1 impedes the in vitro expansion of murine pre-leukemic cells. Using a humanized AML model, we demonstrate that upregulation of GFI1 expression leads to myeloid differentiation morphologically and immunophenotypically, increased level of apoptosis and reduction in number of cKit+ cells. These results suggest that increasing GFI1 level in leukemic cells with low GFI1 expression level could be a therapeutic approach.

Mechanisms of bone destruction in multiple myeloma.

Osteolytic bone disease is a frequent complication of multiple myeloma, resulting in skeletal complications that are a significant cause of morbidity and mortality. It is the result of an increased activity of osteoclasts, which is not followed by reactive bone formation by osteoblasts. Recent studies have revealed novel molecules and pathways that are implicated in osteoclast activation and osteoblast inhibition. Among them, the most important include the receptor activator of nuclear factor-kappa B ligand/osteoprotegerin pathway, the macrophage inflammatory proteins and the activin-A that play a crucial role in osteoclast stimulation in myeloma, while the wingless-type (Wnt) signalling inhibitors (sclerostin and dickkopf-1) along with the growth factor independence-1 are considered the most important factors for the osteoblast dysfunction of myeloma patients. Finally, the role of osteocytes, which is the key cell for normal bone remodelling, has also revealed during the last years through their interaction with myeloma cells that leads to their apoptosis and the release of RANKL and sclerostin maintaining bone loss in these patients. This review focuses on the latest available data for the mechanisms of bone destruction in multiple myeloma.

Prognostic significance of high GFI1 expression in AML of normal karyotype and its association with a FLT3-ITD signature

Growth Factor Independence 1 (GFI1) is a transcriptional repressor that plays a critical role during both myeloid and lymphoid haematopoietic lineage commitment. Several studies have demonstrated the involvement of GFI1 in haematological malignancies and have suggested that low expression of GFI1 is a negative indicator of disease progression for both myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML). In this study, we have stratified AML patients into those defined as having a normal karyotype (CN-AML). Unlike the overall pattern in AML, those patients with CN-AML have a poorer survival rate when GFI1 expression is high. In this group, high GFI1 expression is paralleled by higher FLT3 expression, and, even when the FLT3 gene is not mutated, exhibit a FLT3-ITD signature of gene expression. Knock-down of GFI1 expression in the human AML Fujioka cell line led to a decrease in the level of FLT3 RNA and protein and to the down regulation of FLT3-ITD signature genes, thus linking two major prognostic indicators for AML.

Single methyl groups can act as toggle switches to specify transmembrane Protein-protein interactions.

Transmembrane domains (TMDs) engage in protein-protein interactions that regulate many cellular processes, but the rules governing the specificity of these interactions are poorly understood. To discover these principles, we analyzed 26-residue model transmembrane proteins consisting exclusively of leucine and isoleucine (called LIL traptamers) that specifically activate the erythropoietin receptor (EPOR) in mouse cells to confer growth factor independence. We discovered that the placement of a single side chain methyl group at specific positions in a traptamer determined whether it associated productively with the TMD of the human EPOR, the mouse EPOR, or both receptors. Association of the traptamers with the EPOR induced EPOR oligomerization in an orientation that stimulated receptor activity. These results highlight the high intrinsic specificity of TMD interactions, demonstrate that a single methyl group can dictate specificity, and define the minimal chemical difference that can modulate the specificity of TMD interactions and the activity of transmembrane proteins.

Growth factor independence 1 (Gfi1) regulates the AML supporting function of mesenchymal stromal cells

Growth factor independence 1 (Gfi1) regulates the AML supporting function of mesenchymal stromal cells

GFI1 functions in transcriptional control and cell fate determination require SNAG domain methylation to recruit LSD1.

Proper hematopoietic cell fate decisions require coordinated functions of transcription factors, their associated co-regulators, and histone modifying enzymes. Growth factor independence 1 (GFI1) is a zinc finger transcriptional repressor and master regulator of normal and malignant hematopoiesis. While several GFI1-interacting proteins have been described, how GFI1 leverages these relationships to carry out transcriptional repression remains unclear. Here we describe a functional axis involving GFI1, SMYD2 and LSD1 that is a critical contributor to GFI1-mediated transcriptional repression. SMYD2 methylates lysine-8 (K8) within a -8KSKK11- motif embedded in the GFI1 SNAG domain. Methylation-defective GFI1 SNAG domain lacks repressor function due to failure of LSD1 recruitment and persistence of promoter H3K4 di-methyl marks. Methylation-defective GFI1 also fails to complement GFI1 depletion phenotypes in developing zebrafish and lacks pro-growth and survival functions in lymphoid leukemia cells. Our data show a discrete methylation event in the GFI1 SNAG domain that facilitates recruitment of LSD1 to enable transcriptional repression and coordinate control of hematopoietic cell fate in both normal and malignant settings.

The HMGCR inhibitor Fluvastatin induces apoptosis and autophagy in primary and neoplastic mast cells

Abstract Mast cells are best known for their role in inflammatory and allergic disease. Mast cell leukemia is a rare subtype of acute myelogenous leukemia, typically expressing the c-Kit mutation, D816V, that yields factor-independent growth. Understanding and treating mast cell hyperplasia and leukemia could be beneficial to both inflammatory and malignant diseases. Recently the family of statin drugs, widely employed as HMG CoA Reductase (HMGCR) inhibitors to lower serum cholesterol, have also been found to be immunosuppressive. However, statin effects on mast cell survival are not known. We report that Fluvastatin induces apoptosis in mouse bone marrow-derived mast cells (BMMC) and P815 mastocytoma cells in a dose- and time-dependent manner. Apoptosis was evidenced by DNA fragmentation and caspase activation. Importantly, the mastocytoma line P815, which expresses mutant c-Kit, was readily killed by Fluvastatin, with an IC50 of 5–10μM. P815 cell death was marked by c-Kit upregulation preceding mitochondrial membrane instability and Caspase-9 activation. Chemical blockade of autophagy reduced Fluvastatin-mediated apoptosis in primary mast cells, but augmented apoptosis in transformed mast cells. These data suggest that the HMGCR pathway may be targeted to suppress mast cell survival, and that its combination with autophagy inhibitors may be useful for treating mast cell neoplasias.