NFE2 Research Articles

Abstract 1559: IKZF1 plays a crucial role in the spread of melanoma

Abstract Melanoma is one of the most fatal type of skin cancer. If melanoma has spread to distant sites, it is refractory to existing therapies with the 5-year survival rate declining to 17%. Metastasis remains as the main barrier to successful therapy, persisting as the main cause of cancer-related death. Thus, understanding metastasis mechanism is vital. First, we confirmed that IKZF1 gene was more increased in circulating tumor cells than primary tumor cells. Using GFP+ B16F10, we generated an orthotopic melanoma mouse model and isolated GFP+ tumor cells of primary footpad, blood, and lung. In single cell RNA sequencing, IKZF1, IRF8, NFE2 genes were more upregulated in circulating tumor cells than primary tumor cells. We also found these correlations in human melanoma cell line orthotopic mouse model and Braf/Pten mouse model. Next, we conduct intervention study to find causality in IKZF1 and metastasis. IKZF1 KO led to fewer CTCs and IKZF1 inhibitor, lenalidomide, reduced metastasis in mice. Collectively, we demonstrate that IKZF1 could contribute the formation of CTC and IKZF1 could be a target for metastasis treatment. Citation Format: Jongwook Oh, Dong Ki Lee, Soyeon Lee, Hyun Woo Park, Heon Yung Gee. IKZF1 plays a crucial role in the spread of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1559.

Transcriptional Changes in Patients with CLL Starting Venetoclax Therapy Identifies Inflammatory and Adaptive Stress Responses

Venetoclax is a selective BCL2 inhibitor that was recently approved for CLL treatment. Venetoclax inhibits the pro-survival protein BCL2 and induces deep and durable responses in a significant proportion of CLL patients. Herein, we aimed to study gene expression changes in leukemic cells from CLL patients during the ramp-up period of venetoclax treatment and to assess the transcriptional effect of escalating doses of BCL2 inhibition on tumor biology. We performed RNA sequencing on CD19+ selected tumor cells from 29 patients, ramped-up according to the standard Venetoclax schedule, with weekly increments from 20 mg to 400mg. Four samples were collected per patient, at baseline, and after completing one week on 50mg,100mg, and 400mg. Twelve (41%) patients switched from BTK inhibitor therapy to venetoclax due to disease progression on the BTKi. Those patients discontinued BTKi the day before they started Venetoclax ramp-up. 19 (59%) patients were either treatment-naïve or relapsed with at least months of treatment free-intervals before starting venetoclax. Circulating CD19+ cell counts, typically composed of >95% CLL cells, were enumerated using flow-cytometry. during the ramp-up period, revealed gradual reduction in CLL burden with escalating doses. In the off-BTKi group, median CLL cell counts started to decline on the 100mg dose and were reduced to 98% of baseline at the end of the ramp-up. In the other patients, reductions in median counts were already apparent after one week on 20mg and reached 99% at the end of the ramp-up. Principal component analysis (PCA) of transcriptome data grouped samples by patient, as we have commonly observed in such unsupervised analysis. After batch correcting the patient effect, PCA clearly separated on treatment timepoints from baseline (Figure). Interestingly, patients who recently stopped BTKi had distinct distribution on PCA plots in comparison to the other patients (Others). Indicating distinct transcriptomic changes in patients based on their recent treatment histories. To identify the impact of venetoclax on the CLL transcriptome, we carried out Gene set enrichment analysis using the Hallmark and C3 gene sets from the molecular signature database. Gene sets were identified using a false discovery rate (FDR) of < 0.05, and a normalized enrichment score (NES) of ≥ |1.8|. Gene sets were ranked by NES. In the off-BTKi patient group, 27 gene sets were identified as statistically significantly enriched at 100mg and 400 mg doses. In the “other” patients group, 15 gene sets were found significantly enriched at the same doses. Eight gene sets relating to inflammatory, interferon, and reactive oxygen species responses, cytokine signaling, and fatty acid metabolism. In the off-BTKi we prominently found enriched gene sets regulated by TP53 and related to DNA repair, cell cycle checkpoints, hypoxia and apoptosis. Consistent with an oxidative stress response we identified upregulation of NRF2, NFE2, TCF11, and BACH motif gene sets. The apparent more pronounced transcriptional response to venetoclax in the off-BTKi group prompted us to look more closely at changes in gene. At baseline, expression of BCL2L2 and NOXA was significantly lower in the off-BTKi patients in comparison to the “others” patient group. Surprisingly, on the 50mg dose expression of both the pro-apoptotic and anti-apoptotic BCL2 family members was significantly increased in the off-BTKi group in comparison to the others group; at the 100mg dose only pro-apoptotic genes remained significantly overexpressed in the off-BTKi group. In conclusion, venetoclax induced a distinct and prominent transcriptional response in CLL patients indicating broad impact on tumor biology, even at doses below 400mg. Intriguingly, CLL cells from patients who had progressed on BTKi therapy and switched to venetoclax showed a more extensive stress response that included activation of DNA damage and anti-oxidant pathways. It will be interesting to correlate the identified transcriptional changes with the depth and durability of the venetoclax response.

Protein restriction impairs the response activation/responsivity of MAPK signaling pathway of hematopoietic stem cells

Protein restriction (PR) leads to bone marrow hypoplasia with changes in stromal cellularity components of the extracellular matrix in hematopoietic stem cells (HSCs). However, the underlying signaling mechanisms are poorly understood. We hypothesize that PR impairs the HSC mitogen-activated protein kinase (MAPK) signaling pathway response activation. Our aim is to evaluate the activation of MAPK and interleukin-3 (IL-3) proteins in HSC to explain PR-induced bone marrow hypoplasia, which causes altered proliferation and differentiation. C57BL/6 male mice were subjected to a low-protein diet (2% protein) or normoproteic (12% protein). PKC, PLCγ2, CaMKII, AKT, STAT3/5, ERK1/2, JNK, and p38d phosphorylation were evaluated by flow cytometry, and GATA1/2, PU.1, C/EBPα, NF-E2, and Ikz-3 genes (mRNAs) assessed by quantitative real-time-polymerase chain reaction. Pathway proteins, such as PLCγ2, JAK2, STAT3/5, PKC, and RAS do not respond to the IL-3 stimulus in PR, leading to lower activation of ERK1/2 and Ca2+ signaling pathways, consequently lowering the production of hematopoietic transcription factors. Colony forming units granulocyte-macrophage and colony forming units macrophage formation are impaired in PR even after being stimulated with IL-3. Long-term hematopoietic stem cells, short-term hematopoietic stem cells, granulocyte myeloid progenitor, and megakaryocyte-erythroid progenitor cells were significantly reduced in PR animals. This study shows for the first time that activation of MAPK pathway key proteins in HSCs is impaired in cases of PR. Several pathway proteins, such as PLCγ2, JAK2, STAT3, PKC, and RAS do not respond to IL-3 stimulation, leading to lower activation of extracellular signal-regulated protein kinase 1/2 and consequently lower production of hematopoietic transcription factors GATA1/2, PU.1, C/EBPa, NF-E2, and Ikz3. These changes result in a reduction in colony-forming units, proliferation, and differentiation, leading to hypocellularity.

Chronic Exposure to Cytoreductive Treatment Shapes Clonal Evolution in Myeloproliferative Neoplasms

▪Introduction: Myeloproliferative neoplasms (MPN) are a heterogeneous group of chronic myeloid malignancies resulting from the combination of a driver mutated gene (JAK2, MPL or CALR) and a variety of acquired additional somatic mutations. Although next-generation sequencing (NGS) has identified high molecular risk mutations associated with adverse prognosis (Vannucchi et al., Leukemia, 2013 , Guglielmelli et al., Leukemia, 2014 ), the clonal evolution of these mutations remains poorly described. Chronic exposure to cytoreductive treatment, especially genotoxic drugs such as hydroxyurea (HU), could impact clonal evolution. A previous study suggested that Interferon-α (IFN) could limit the accumulation of cytogenetic abnormalities compared to HU (Mondello et al., Blood, 2018). The objective of our study was to describe the long-term evolution of the mutational landscape in the era of NGS in a large cohort of MPN patients.Methods: A total of 1538 consecutive patients were diagnosed with MPN according to WHO criteria and followed in our hospital between January 2011 and January 2021. This study included 1039 of them in whom a NGS molecular analysis targeting 36 myeloid genes with a sensitivity of 1% was performed at diagnosis and/or during follow-up. Patients with only one NGS (n=588), AML/MDS transformation at either the first (n=3) or the second NGS (n=2) were excluded from the analysis. Serial NGS data obtained in chronic MPN phase were thus analyzed for 446 patients. Clinical and biological characteristics at time of diagnosis and follow-up were collected from medical charts and electronic medical records. Mutation rates per year were calculated for each gene as the difference in the number of mutations between first and last NGS divided by the time interval (in years) between both NGS.Results : Median age at MPN diagnosis in our whole cohort was 51 years [IQR 41-60]. Our cohort included 167 (37%), 205 (46%) and 64 (14%) patients with Polycythemia Vera (PV), Essential thrombocythemia (ET) and primary myelofibrosis (MF) respectively. 279 patients (63%) had at least one additional mutation at first NGS, and respectively 27 (6%) and 104 (23%) patients had TP53 and high molecular risk mutations. Median interval between MPN diagnosis and the first NGS was 6.5 years [IQR 1.7-13] while median time between the first and the last NGS was 2.5 years [IQR 1.6-4, range 0.3-14.3]. Overall, 178 patients (39.9%) acquired an additional mutation at last NGS evaluation, most frequently involving TET2, DNMT3A, ASXL1, TP53 and NFE2 genes . To study the impact of chronic MPN therapy on clonal evolution, we focused on patients who electively received HU (n=112) or IFN (n=92) as a monotherapy, or did not receive any cytoreductive treatment (n=119) between the first and the last NGS. The remaining patients received ruxolitinib (n=44), anagrelide (n=10), vercyte (n=7) or polytherapy (n=62). At last follow-up, 74 patients receiving IFN (80.4%) and 65 (58%) treated with HU had a complete hematological response. When combining all additional mutations, the global mutation rate per year did not significantly differ between treatment groups. When analyzing individual genes, TP53 mutation rate was higher in patients treated with HU compared to the patients receiving IFN (p=0.014) or not treated (p=0.008) (Figure). MDS/AML evolution occurred in 4 patients (3.6%) treated with HU, 2 (1.7%) without cytoreductive therapy versus none of the 92 patients treated with IFN (ns). In the whole cohort, MDS/AML evolution was significantly increased in patients harboring TP53 mutations (p= 0.004). In contrast, DNMT3A mutation rate was significantly increased in patients receiving IFN compared to patients treated with HU (p=0.045) (Figure). The latest result is in line with previous observations showing that loss of DNMT3A could confer resistance to IFN in a JAK2-V617F mouse model (Stetka et al., Blood, 2020).Conclusion: Our results highlight the impact of chronic cytoreductive therapy on clonal evolution shaping in MPN. IFN limits the emergence of TP53 mutated clones compared to HU, thus potentially reducing the risk of leukemogenesis. Emergence of DNMT3A mutated clones under IFN therapy requires further exploration and could potentially play a role in therapeutic resistance. This study on a large clinically and biologically annotated cohort illustrates how serial NGS analysis may guide therapeutic options for MPN patients. [Display omitted] DisclosuresRaffoux: PFIZER: Consultancy; ASTELLAS: Consultancy; ABBVIE: Consultancy; CELGENE/BMS: Consultancy. Kiladjian: AbbVie: Membership on an entity's Board of Directors or advisory committees; AOP Orphan: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Incyte Corporation: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Taiho Oncology, Inc.: Research Funding; PharmaEssentia: Other: Personal fees. Benajiba: Pfizer: Research Funding; Gilead: Research Funding.

Genomic Landscape of RUNX1-Familial Platelet Disorder with Myeloid Malignancies Reveals Rising Clonal Hematopoiesis

Familial platelet disorder with associated myeloid malignancies (FPDMM) is a rare autosomal dominant disease caused by germline RUNX1 mutations. FPDMM patients have defective megakaryocytic development, low platelet counts, prolonged bleeding times, and a life-long risk (20-50%) of developing hematological malignancies. FPDMM is a rare genetic disease in need of comprehensive clinical and genomic studies.In early 2019 we launched a longitudinal natural history study of patients with FPDMM at the NIH Clinical Center and by May 2021 we have enrolled 98 patients and 100 family controls from 55 unrelated families. Genomic data have been generated from 56 patients in 24 families, including whole exome sequencing (WES), RNA-seq, and single-nucleotide polymorphism (SNP) array. We have identified 21 different germline RUNX1 variants among these 24 families, which include lost-of-function mutations throughout the RUNX1 gene, but pathogenic/likely pathogenic missense mutations are mostly clustered in the runt-homology domain (RHD). As an important form of RUNX1 germline mutations, five splice site variants located between exon 4-5 and exon 5-6 were identified in 6 families, which led to the productions of novel transcript forms that are predicted to generate truncated RUNX1 proteins. Large deletions affecting the RUNX1 gene are also common, ranging from 50 Kb to 1.5Mb, which were detected in 8 of the 55 enrolled families. Besides RUNX1, copy number variation (CNV) analysis from both SNP array and WES showed limited CNV events in non-malignant FPDMM patients. In addition, fusion gene analysis did not detect any in-frame fusion gene in these patients, indicating a relatively stable chromosome status in FPDMM patients.Somatic mutation landscape shows that the overall mutation burden in non-malignant FPDMM patients is lower than AML or other cancer types. However, in 13 of the 44 non-malignant patients (30%), somatic mutations were detected in at least one of the reported clonal hematopoiesis of indeterminate potential (CHIP) genes, significantly higher than the general population (4.3%). Moreover, 85% of our patients who carried CHIP mutations are under 65 years of age; in the general population, only 10% of people above 65 years of age and 1% of people under 50 were reported to carry CHIP mutations. Among mutated genes related to clonal hematopoiesis, BCOR is the most frequently mutated gene (5/44) in our FPDMM cohort, which is not a common CHIP gene among the general population. Mutations in known CHIP genes including SF3B1, TET2, and DNMT3A were also found in more than one patient. In addition, sequencing of 5 patients who already developed myeloid malignancies detected somatic mutations in BCOR, TET2, NRAS, KRAS, CTCF, KMT2D, PHF6, and SUZ12. Besides reported CHIP genes or leukemia driver genes, 3 unrelated patients carried somatic mutations in the NFE2 gene, which is essential for regulating erythroid and megakaryocytic maturation and differentiation. Two of the NFE2 mutations are nonsense mutations, and the other is a missense mutation in the important functional domain. NFE2 somatic mutations may play important roles in developing malignancy because 2 of the 3 patients already developed myeloid malignancies. For multiple patients in our cohort, we have sequenced their DNA on multiple timepoints. We have observed patients with expanding clones carrying FKBP8, BCOR or FOXP1 mutations. We have also observed a patient with relatively stable clone(s) with somatic BCOR, DNMT3A, and RUNX1T1, who have been sampled over more than four years. We will follow these somatic mutations through sequencing longitudinally and correlate the findings with clinical observations to see if the dynamic changes of CHIP clones harboring the mutations give rise to MDS or leukemia.In summary, the genomic analysis of our new natural history study demonstrated diverse types of germline RUNX1 mutations and high frequency of somatic mutations related to clonal hematopoiesis in FPDMM patients. These findings indicate that monitoring the dynamic changes of these CHIP mutations prospectively will benefit patients' clinical management and help us understand possible mechanisms for the progression from FPDMM to myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Involvement of a Transcription factor, Nfe2, in Breast Cancer Metastasis to Bone.

Simple SummaryBreast cancer patients are frequently complicated by bone metastasis, which deteriorates their life expectancy. Bone metastasis is treated with the drugs targeting osteoclast activation, which is mostly observed at the metastasis sites. However, these drugs cannot directly inhibit cancer cell growth and therefore, a novel therapeutic strategy is required to impede cancer cell proliferation at bone metastasis sites. Here, we proved that a transcription factor, NFE2, was expressed selectively in breast cancer cells at bone metastasis sites and contributed crucially to their enhanced proliferation therein, by activating Wnt pathway. Thus, NFE2 can be a novel molecular target to treat breast cancer bone metastasis.Patients with triple negative breast cancer (TNBC) is frequently complicated by bone metastasis, which deteriorates the life expectancy of this patient cohort. In order to develop a novel type of therapy for bone metastasis, we established 4T1.3 clone with a high capacity to metastasize to bone after orthotopic injection, from a murine TNBC cell line, 4T1.0. To elucidate the molecular mechanism underlying a high growth ability of 4T1.3 in a bone cavity, we searched for a novel candidate molecule with a focus on a transcription factor whose expression was selectively enhanced in a bone cavity. Comprehensive gene expression analysis detected enhanced Nfe2 mRNA expression in 4T1.3 grown in a bone cavity, compared with in vitro culture conditions. Moreover, Nfe2 gene transduction into 4T1.0 cells enhanced their capability to form intraosseous tumors. Moreover, Nfe2 shRNA treatment reduced tumor formation arising from intraosseous injection of 4T1.3 clone as well as another mouse TNBC-derived TS/A.3 clone with an augmented intraosseous tumor formation ability. Furthermore, NFE2 expression was associated with in vitro growth advantages of these TNBC cell lines under hypoxic condition, which mimics the bone microenvironment, as well as Wnt pathway activation. These observations suggest that NFE2 can potentially contribute to breast cancer cell survival in the bone microenvironment.

Shared roles for Scl and Lyl1 in murine platelet production and function

AbstractThe stem cell leukemia (Scl or Tal1) protein forms part of a multimeric transcription factor complex required for normal megakaryopoiesis. However, unlike other members of this complex such as Gata1, Fli1, and Runx1, mutations of Scl have not been observed as a cause of inherited thrombocytopenia. We postulated that functional redundancy with its closely related family member, lymphoblastic leukemia 1 (Lyl1) might explain this observation. To determine whether Lyl1 can substitute for Scl in megakaryopoiesis, we examined the platelet phenotype of mice lacking 1 or both factors in megakaryocytes. Conditional Scl knockout (KO) mice crossed with transgenic mice expressing Cre recombinase under the control of the mouse platelet factor 4 (Pf4) promoter generated megakaryocytes with markedly reduced but not absent Scl. These Pf4Sclc-KO mice had mild thrombocytopenia and subtle defects in platelet aggregation. However, Pf4Sclc-KO mice generated on an Lyl1-null background (double knockout [DKO] mice) had severe macrothrombocytopenia, abnormal megakaryocyte morphology, defective pro-platelet formation, and markedly impaired platelet aggregation. DKO megakaryocytes, but not single-knockout megakaryocytes, had reduced expression of Gata1, Fli1, Nfe2, and many other genes that cause inherited thrombocytopenia. These gene expression changes were significantly associated with shared Scl and Lyl1 E-box binding sites that were also enriched for Gata1, Ets, and Runx1 motifs. Thus, Scl and Lyl1 share functional roles in platelet production by regulating expression of partner proteins including Gata1. We propose that this functional redundancy provides one explanation for the absence of Scl and Lyl1 mutations in inherited thrombocytopenia.

The Effect of Mesenchymal Stem Cell-Derived Microvesicles on Erythroid Differentiation of Umbilical Cord Blood-Derived CD34+ Cells.

Purpose: Mesenchymal stem cells (MSCs) play an important role in the proliferation and differentiation of hematopoietic stem cells (HSCs) in the bone marrow via cell-to-cell contact, as well as secretion of cytokines and microvesicles (MVs). In this study, we investigated the effect of mesenchymal stem cell-derived microvesicles (MSC-MVs) on erythroid differentiation of umbilical cord blood-derived CD34+ cells.Methods: In this descriptive study, CD34+ cells were cultured with mixture of SCF (10 ng/ml) and rhEPO (5 U/ml) cytokines in complete IMDM medium as positive control group. Then, in MV1- and MV2-groups, microvesicles at 10 and 20 µg/ml concentration were added. After 72 hours, erythroid specific markers (CD71 and CD235a) and genes (HBG1, GATA1, FOG1 and NFE2) were assessed by flow cytometry and qRT-PCR, respectively.Results: The expression of specific markers of the erythroid lineages (CD71 and GPA) in the presence of different concentration of microvesicles were lower than that of the control group (P<0.001). Also, the expression of specific genes of the erythroid lineages (NFE2, FOG1, GATA1, and HBG1) was investigated in comparison to the internal control (GAPDH). Among all of them, HBG1 and FOG1 genes were significantly decreased to the control group (P<0.0001) but GATA1 and NFE2 gene expressions was not significant.Conclusion: The results of this study showed that MSC-MVs decrease the erythroid differentiation of umbilical cord blood-derived CD34+ cells. Therefore, MSC-MVs play a key role in the regulation of normal erythropoiesis.

Platelet-predominate gene expression and reticulated platelets in nonvalvular atrial fibrillation: Effect of pulmonary veins isolation.

Reticulated platelet (RP) content is increased in nonvalvular atrial fibrillation (NVAF). The purpose of this study was to determine if platelet content, morphology, and RP proportion are modulated by platelet genes. Expression of six platelet-predominate genes impacting platelet formation and release, platelet count, and RP content was assessed in NVAF patients before and 3-4 months after pulmonary veins isolation (PVI) and compared to normal sinus rhythm (NSR) controls. RNA from isolated platelets was reverse-transcribed assayed against selected genes utilizing real-time qPCR, and expressed as mean cycle threshold (ΔCt) using beta-2-microglobulin as endogenous control. RP content was assessed by flow cytometry. A fourfold lower expression of CFL1 gene coding for nonmuscle cofilin (7.8 ± 0.9vs. 5.7 ± 1.6, P<0.001) and twofold lower expression of four other genes were associated with similar platelet counts but fourfold higher (28.7+7.0vs. 6.7+5.4, P<0.001) RP content (%) in 97 NVAF cases compared to 51 NSR controls. Three to 4 months after PVI, RP decreased by 28%, while CFL1 gene expression increased over twofold but TUBA4A gene expression decreased almost twofold; NFE2 and MYL6 gene expression remained unchanged. NVAF is associated with notable downregulation of genes directing platelet production and size but increased RP content. PVI impacts the expression of many of these genes, implying a direct relationship between atrial fibrillation and platelet biogenesis.

The effect of histone deacetylase inhibitors on AHSP expression.

Alpha-hemoglobin stabilizing protein (AHSP) is a molecular chaperone that can reduce the damage caused by excess free α-globin to erythroid cells in patients with impaired β-globin chain synthesis. We assessed the effect of sodium phenylbutyrate and sodium valproate, two histone deacetylase inhibitors (HDIs) that are being studied for the treatment of hemoglobinopathies, on the expression of AHSP, BCL11A (all isoforms), γ-globin genes (HBG1/2), and some related transcription factors including GATA1, NFE2, EKLF, KLF4, and STAT3. For this purpose, the K562 cell line was cultured for 2, 4, and 6 days in the presence and absence of sodium phenylbutyrate and sodium valproate. Relative real-time qRT-PCR analysis of mRNA levels was performed to determine the effects of the two compounds on gene expression. Expression of all target mRNAs increased significantly (p < 0.05), except for the expression of BCL11A, which was down-regulated (p < 0.05) in the cells treated with both compounds relative to the levels measured for untreated cells. The findings indicated that sodium valproate had a more considerable effect than sodium phenylbutyrate (p < 0.0005) on BCL11A repression and the up-regulation of other studied genes. γ-Globin and AHSP gene expression continuously increased during the culture period in the treated cells, with the highest gene expression observed for 1 mM sodium valproate after 6 days. Both compounds repressed the expression of BCL11A (-XL, -L, -S) and up-regulated GATA1, NFE2, EKLF, KLF4, STAT3, AHSP, and γ-globin genes expression. Moreover, sodium valproate showed a stronger effect on repressing BCL11A and escalating the expression of other target genes. The findings of this in vitro experiment could be considered in selecting drugs for clinical use in patients with β-hemoglobinopathies.

Expression of xCT and activity of system xc− are regulated by NRF2 in human breast cancer cells in response to oxidative stress

Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate by activating key transcription factors. One such master regulator, the redox sensitive transcription factor NF E2 Related Factor 2 (NRF2), controls the expression of cellular defense genes including those encoding intracellular redox-balancing proteins involved in glutathione (GSH) synthesis. Under basal conditions, Kelch-like ECH-associated protein 1 (KEAP1) targets NRF2 for ubiquitination. In response to oxidative stress, NRF2 dissociates from KEAP1, entering the nucleus and binding to the antioxidant response element (ARE) in the promoter of its target genes. Elevated reactive oxygen species (ROS) production may deplete GSH levels within cancer cells. System xc−, an antiporter that exports glutamate while importing cystine to be converted into cysteine for GSH synthesis, is upregulated in cancer cells in response to oxidative stress. Here, we provided evidence that the expression of xCT, the light chain subunit of system xc−, is regulated by NRF2 in representative human breast cancer cells. Hydrogen peroxide (H2O2) treatment increased nuclear translocation of NRF2, also increasing levels of xCT mRNA and protein and extracellular glutamate release. Overexpression of NRF2 up-regulated the activity of the xCT promoter, which contains a proximal ARE. In contrast, overexpression of KEAP1 repressed promoter activity and decreased xCT protein levels, while siRNA knockdown of KEAP1 up-regulated xCT protein levels and transporter activity. These results demonstrate the importance of the KEAP1/NRF2 pathway in balancing oxidative stress in breast cancer cells through system xc−. We have previously shown that xCT is upregulated in various cancer cell lines under oxidative stress. In the current investigation, we focused on MCF-7 cells as a model for mechanistic studies.

The Cell Cycle Regulators CDK4, CDK6 and Cyclin D3 As Well As the Histone Methyltransferases MLL2 and MLL4 Constitute NF-E2 Target Genes That Participate in a Novel Regulatory Loop

Background: Expression of the transcription factor and epigenetic modulator “nuclear factor erythroid-2” (NF-E2) is aberrantly elevated in patients with Myeloproliferative Neoplasms (MPN). We have shown that NF-E2 overexpression in a murine model causes a MPN phenotype. In addition, we have identified in-del mutations in the NF-E2 gene in MPN patients, which, despite having lost the ability to bind DNA, nonetheless increase the activity of wild type NF-E2. These mutants, which are acquired secondary to the JAK2V617F mutation, confer a proliferative advantage beyond that provided by the JAK2V617F alone. In all patients analyzed, cells carrying both mutations displayed clonal dominance, vastly outnumbering those carrying JAK2V617Falone. Mutant NF-E2 causes an increase in the number of cells entering S-phase mediated by increased expression of the cell cycle regulators CDK4, CDK6 and CyclinD3 that promote the G1-S transition. However, it is not known whether these cell cycle regulators constitute direct NF-E2 target genes or whether the proliferative effect is indirect. Moreover, additional downstream pathways by which this augmented transcription factor activity exerts its effects remain unknown.Aims: We therefore investigated whether CDK4, CDK6 and Cyclin D3 constitute direct NF-E2 target genes. Moreover, we identified additional novel NF-E2 targets in an effort to delineate the molecular mechanism by which increased NF-E2 activity contributes to MPN pathophysiology.Methods: We used ChIP and luciferase reporter assays as well as qRT-PCR and Western Blot to identify and characterize novel NF-E2 targets.Results: Our data show that both the cell cycle regulators CDK4, CDK6 and CyclinD3 as well as the histone methyltransferases MLL2 and MLL4 constitute direct NF-E2 target genes, as the transcription factor binds these genes in vivo.ChIP analysis of HEL cells shows NF-E2 binding to a site in the CDK4 promoter, four sites in the CDK6 and the Cyclin D3 locus each, to the MLL4 promoter as well as to three sites in the MLL2 locus, establishing these five genes as direct NF-E2 targets. Activity of NF-E2 on the MLL4 promoter was confirmed by reporter gene assays. Expression of both the cell cycle regulators and the methyltransferases was statistically significantly elevated in bone marrow from NF-E2 overexpressing mice. Most importantly, quantitative RT-PCR showed significantly increased mRNA expression of all five target genes in primary cells of patients with polycythemia vera (PV) compared to healthy controls. Hence, the expression of the cell cycle regulators and the epigenetically active enzymes is augmented in PV patients. Corresponding to the increased methyltransferase activity, primary cells from PV patients showed a significant elevation in global H3K4m1 levels, a chromatin mark conferred by the MLL proteins.The MLL2 protein has been previously shown to interact with NF-E2. NF-E2 serves as a scaffold and is required both for recruitment of MLL2 to the beta-globin locus as well as for chromatin remodeling at these sites. Here we show that MLL2 binds the CDK6 locus at the same sites as NF-E2, strongly suggesting that NF-E2 likewise acts to direct MLL2 to the CDK6 gene.Our data establish a novel interaction network where NF-E2 both regulates the expression levels of MLL2 while at the same time modulating its epigenetic activity at NF-E2 target genes, which include critical cell cycle regulators.Summary / Conclusions: Here we show that the cell cycle regulators CDK4, CDK6 and Cyclin D3 as well as the histone methyltransferases MLL2 and MLL4 constitute novel NF-E2 target genes. We propose that elevated NF-E2 levels in MPN patients contribute to disease pathophysiology by participating in a novel regulatory loop, which both enhances transcription and alteres histone methylation leading to increased proliferation. These data provide a molecular basis for pre-clinical investigation into the effects of CDK 4/6 inhibitors as well as of histone methyltransferase inhibitors on MPN cell biology. DisclosuresNo relevant conflicts of interest to declare.

Abstract 506: H2O2 upregulates xCT in human breast cancer cells via NRF-2

Abstract Cancer cells adapt to high levels of oxidative stress in order to survive and proliferate, making the transcription factors involved in antioxidant defense regulation targets of interest. The transcription factor NF E2 Related Factor 2 (NRF-2) regulates cellular defense genes including those encoding intracellular redox-balancing proteins such as enzymes involved in glutathione metabolism. Glutathione in particular is an important intracellular antioxidant molecule. NRF-2 binds to the Antioxidant Response Element (ARE) in the promoter of its target genes. Under basal conditions, KEAP1 acts as an inhibitor that targets NRF-2 for ubiquitination. During oxidative stress, NRF-2 dissociates from KEAP1 and enters the nucleus to bind to the ARE sequence. It is hypothesized that the elevated Reactive Oxygen Species may be depleting the glutathione levels within the cancer cell. System xc- is a cysteine/glutamate antiporter that exports glutamate while importing cysteine to synthesize glutathione. In response to oxidative stress the cells increase system xc- activity in order to provide cysteine for glutathione synthesis. There is evidence that expression of xCT the specific subunit of System X is regulated by NRF-2. However this has not yet been demonstrated in human cancer cells, which is the focus of this project. Basal expression of NRF-2, Keap1 and xCT was characterized in two breast cancer cell lines (MDA MB 231 and MCF-7) compared to normal human breast epithelial 184B5 cells. Compared to 184B5 cells, Keap-1, NRF-2 and xCT were upregulated in MDA MB 231 cells, but down regulated in MCF-7 cells. MCF-7 cells were treated with H202 resulting in NRF-2 protein accumulation in the nucleus and decreased levels in the cytosol. Under the same treatment conditions, Keap-1 protein decreased in the nucleus and increased in the cytosol. With H202 treatment, xCT mRNA levels increased in MCF-7 cells. We have cloned approximately 2.6 kilobase (-2329 to +278 bp) of the human xCT promoter region from genomic DNA isolated from MDA MB 231 breast cancer cells. We found that the xCT promoter was responsive to treatment with H2O2, which increased transcriptional activity. This is consistent with results described for the mouse promoter, which is known to contain an antioxidant response element that can be bound by NRF-2 during oxidative stress. Co transfection of MDA MB 231 with the xCT promoter and an NRF-2 expression vector also increased promoter activity. This data suggests that under oxidative stress, NRF-2 is localized to the nucleus and transcriptionally upregulates xCT. This is the first study in which the regulation of xCT has been linked to oxidative stress via NRF-2 in human breast cancer cells. This work was supported by the Canadian Institutes for Health Research. Citation Format: Eric Habib, Katja Linher-Melville, Gurmit Singh. H2O2 upregulates xCT in human breast cancer cells via NRF-2. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 506. doi:10.1158/1538-7445.AM2014-506

Sox17 as a candidate regulator of myeloid restricted differentiation potential.

Sry related high mobility group box 17 (Sox17), which is a marker of endodermal cells and a transcriptional regulator, has a critical role in the maintenance of fetal and neonatal hematopoietic stem cells (HSC). Sox17 has been identified as a key regulator of the development and differentiation of fetal hematopoietic progenitors from the aorta-gonad-mesonephros (AGM) region. The co-culture of Sox17-transduced hematopoietic progenitor cells (CD45(low) c-Kit(high) cells) from AGM regions on OP9 stromal cells gives rise to multipotential hematopoietic stem/progenitor cells. Here, we show that in a primary transplantation experiment, Sox17-transduction in CD45(low) c-Kit(high) cells of embryonic day (E) 10.5 AGM increased the absolute number of common myeloid progenitors (CMPs) in the bone marrow (BM) of recipient mice in comparison to that of granulocyte/macrophage progenitors (GMPs) and the megakaryocyte/erythroid progenitors (MEPs). When Sox17-transduced cells were cultured with OP9 stromal cells, Sox17-transduced GMPs (Sox17-GMPs), Sox17-transduced CMPs (Sox17-CMPs), and Sox17-transduced MEPs (Sox17-MEPs) were generated. Sox17-GMPs and Sox17-CMPs maintained their self-renewal capacity and the hematopoietic ability upon co-culture with the OP9 stromal cells for some passages. Moreover, Sox17-GMPs exhibited the increase in expression of c-Mpl and GATA-2 in comparison to GMPs of BM and Sox17-CMPs showed the increase in expression of c-Mpl, NF-E2, and β-globin genes in comparison to CMPs of BM. Furthermore, when Sox17-transduced cells were cultured in methylcellulose to examine the colony-forming ability, Sox17-GMPs and Sox17-CMPs maintained the formation of mixed colonies for some passages. Taken together, Sox17 is suggested to regulate the maintenance and differentiation of hematopoietic progenitors derived from AGM regions at midgestation, in particular myeloid progenitors.

Overexpression Of The Histone Demethylase JMJD1C In Polycythemia Vera Contributes To NF-E2 Overexpression Via Epigenetic Dysregulation and An Auto-Regulatory Loop

Expression of the transcription factor and epigenetic modulator “nuclear factor erythroid-2” (NF-E2) is aberrantly elevated in patients with Myeloproliferative Neoplasms (MPN). We have recently shown that NF-E2 overexpression in a murine model causes a phenotype similar to MPN in humans. This includes thrombocytosis, leukocytosis, expansion of the stem- and progenitor cell compartments as well as spontaneous transformation to acute leukemia. However, both the downstream pathways by which this transcription factor exerts its effects as well as the mechanisms leading to its overexpression in MPN patients remain incompletely characterized.We show here that the histone demethylases JMJD1C and JMJD2C constitute novel NF-E2 target genes. Using chromatin immunoprecipitation (ChIP) we demonstrate NF-E2 binding to the JMJD1C and JMJD2C loci. Both JMJD1C and JMJD2C protein levels are statistically significantly elevated in patients with polycythemia vera (PV) compared to healthy controls.JMJD1C and 2C proteins mediate the demethylation of histone H3K9, converting H3K9me2 via H3K9me to unmethylated H3K9. Consistent with increased demethylase activity, we observed statistically significantly decreased levels of global H3K9me and H3K9me2 in PV patients compared to healthy controls.We subsequently investigated whether the NF-E2 gene is regulated by epigenetic mechanisms and whether these are altered in PV patients. Indeed, using ChIP we demonstrate presence of the repressive H3K9me2 mark at three sites within the NF-E2 locus in healthy control granulocytes, where NF-E2 is expressed at low levels. These repressive histone marks are significantly reduced or completely absent in PV patients, which overexpress NF-E2. At the same time, binding of the repressive heterochromatin protein 1-alpha (HP1a) to the NF-E2 gene, while present in healthy controls, is significantly reduced in PV patients. HP1a chromatin binding is dependent on the presence of the H3K9me2 modification. Decreased levels of H3K9me2 and decreased HP1a binding are therefore both consistent with elevated NF-E2 expression in PV.We hypothesized that increased levels of the H3K9me2 demethylase JMJD1C in PV patients are responsible for the loss of this histone mark in the NF-E2 gene. Consistent with this model, we demonstrate significantly increased binding of JMJD1C to the NF-E2 locus in purified primary PV granulocytes.Our data demonstrate that the NF-E2 gene is regulated by epigenetic mechanisms and that these histone modifications are perturbed in PV patients. Moreover, NF-E2 participates in an auto-regulatory loop, by directly regulating transcription of the histone demethylase JMJD1C. Increased levels of NF-E2 thus cause increased JMJD1C expression. The JMJD1C protein, in turn, demethylates H3K9me2 residues in the NF-E2 locus, thereby further augmenting NF-E2 expression. Besides sustaining this novel autoregulatory loop, increased JMJD1C activity may contribute to MPN pathology by altering gene expression at additional loci.We sought to determine whether elevated NF-E2 levels could be normalized by pharmacological intervention. Treatment with the nucleoside analogue Decitabine reduced NF-E2 expression, and restored both the H3K9me2 marks and HP1a binding to the NF-E2 locus.These data provide a molecular rational for pre-clinical investigation of the effects of histone demethylase inhibitors and Decitabine on MPN cell biology and, subsequently, for a phase I trial investigating a combination of Decitabine and histone demethylase inhibitors in MPN patients. Disclosures:No relevant conflicts of interest to declare.

RUNX1 and NF-E2 Transcriptional Upregulation Is Not Specific For Myeloproliferative Neoplasms But Is Seen In Those Polycythemic Disorders With Augmented HIFs Signaling

▪RUNX1 and NF-E2 are transcription factors that regulate hematopoietic stem cell homeostasis. It has been reported that increased RUNX1 expression in the granulocytes is present in all three classical myeloproliferative neoplasms (MPN): polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis (Wang et al, Blood 2010), and that elevated NF-E2 promotes erythropoietin (EPO)-independent erythroid maturation of hematopoietic stem cells in vitro (Bogeska et al, Stem Cells Transl Med 2013). A mouse model overexpressing the NF-E2 transgene in hematopoietic cells was reported to be a new model of myeloproliferative neoplasms (Kaufmann et al, J Exp Med 2012).Polycythemic states can be divided into primary polycythemias, characterized by intrinsically hyperproliferative erythroid progenitors that are hypersensitive to EPO, and secondary polycythemias, wherein erythroid progenitors respond normally to EPO but circulating EPO is elevated or inappropriately normal for the level of increased red cell mass. Some congenital disorders including those with mutations in the hypoxia sensing pathway may share features of both primary and secondary polycythemias. We considered the possibility that increased transcripts of RUNX1 and NF-E2 might be the feature of other primary polycythemic states as well.We report a study of 19 polycythemic patients with primary or secondary polycythemia with diverse etiologies including mutations in positive and negative regulators of hypoxia sensing pathway. RUNX1 and NF-E2 transcripts were quantitated in granulocytes and BFU-E colonies by qPCR. All primary polycythemic patients had erythroid progenitors hypersensitive to or independent to EPO; all secondary polycythemic subjects had normal erythroid progenitor response to EPO.RUNX1 and NF-E2 gene transcripts were increased in granulocytes and BFU-E colonies in all PV patients, two unrelated subjects with the VHLR200W homozygous mutation (Chuvash polycythemia), one polycythemic patient homozygous for the VHLP138L exon 2 mutation, and a patient with the HIF2αM535V gain-of-function mutation. We also found upregulated expression of RUNX1 and NF-E2 in granulocytes and BFU-Es from a polycythemic patient (with no detectable EPOR, JAK2V617F or JAK2 exon 12 mutations and low level of EPO < 1 mU/mL) who was heterozygous for a SNP in exon 3 (rs147341899) in the LNK gene. We examined transcripts of RUNX1 and NF-E2 genes in granulocytes from two Croatian polycythemic patients with a homozygous VHLH191D exon 3 mutation whose erythroid progenitors were not hypersensitive to EPO and whose RUNX1, but not NF-E2, transcript was increased. We found similar results in two compound heterozygotes for VHLT124A exon 2 and VHLL188V exon 3 mutations. These two polycythemic siblings had hypersensitive erythroid colonies, increased RUNX1 transcripts and decreased NF-E2 transcripts in granulocytes. RUNX1 and NF-E2 transcripts were normal in two subjects with primary polycythemia due to the EPOR gain-of-function EPORQ434Xmutation, and in five unrelated subjects with secondary polycythemia.We next examined granulocyte transcripts of HIF-regulated genes: TFRC, SLC2A1, VEGF, BNIP3 and HK1, and found them to be increased in all PV patients and all studied polycythemic patients with VHL, HIF2α or LNK mutations, but not in polycythemic EPORQ434Xpatients or five patients with secondary polycythemia. Increased transcripts of HIF regulated genes are compatible with the previously unappreciated Warburg effect in PV (see S. Sana's Abstract at this ASH meeting).We propose that increased expression of RUNX1 and NF-E2 is not specific for myeloproliferative neoplasms but also is not universal for primary polycythemic disorders. Therefore, increased expression of RUNX1 and NF-E2 do not seem to be underlying mechanism for MPNs development but rather represent factors associated with diverse primary polycythemia states with augmented HIF signaling.(Note: KK and LL contributed equally to this work.)This work was supported by 1P01CA108671-O1A2 (NCI) Myeloproliferative Disorders (MPD) Consortium (PI Ron Hoffman) project#1 (PI Prchal) and the Leukemia & Lymphoma Society. Work by KK, LL, MH and VD was in part supported by the European Structural Funds (project CZ.1.07/2.3.00/20.0164 and CZ.1.07/2.3.00/30.0041), grant LF_2013_010 and by Czech Science Foundation (Project-P301/12/1503). Disclosures:No relevant conflicts of interest to declare.

Ph-Negative Myeloproliferative Neoplasms Exhibit Some Features Of Warburg Effect

Malignant cells produce energy by preferentially utilizing glycolytic pathways instead of oxidation of pyruvate in mitochondria under normal oxygen states, a metabolic alteration known as the Warburg effect. Hypoxia-inducible transcription factor 1 (HIF-1) plays a crucial role in this major shift. Some Warburg effect mediators are SLC2A1 (glucose transporter1), PDK1 (pyruvate dehydrogenase kinase, isozyme 1) and VEGF-A (vascular endothelial growth factor A). Whether the Warburg effect is exhibited by pre-malignant, clonal disorders such as Ph-negative myeloproliferative neoplasms (MPN) is not known. We investigated the presence of the Warburg effect and levels of other selected HIF-1 regulated genes in clonal granulocytes and platelets of patients with MPN, i.e. polycythemia vera (PV), essential thrombocythemia (ET), and primary or secondary myelofibrosis (MF).We collected blood samples from 21 PV, 9 ET, 9 MF patients and 14 healthy controls. Total RNA from granulocytes and platelets was isolated by Tri-Reagent and reverse transcribed using SuperScript III First-Strand Synthesis kit. Expression of HIF-1 target genes SLC2A1, PDK1, VEGF A, NFE-2 and RUNX2 was determined by qPCR using hydrolysis probes. Relative gene expression was calculated using Rest 2009 Qiagen software.We report upregulation of SLC2A1 in granulocytes and PDK1 transcripts in platelets and down regulation of PDK1 transcripts in granulocytes. HIF-1 regulated NFE-2 and RUNX1 genes transcripts were also upregulated in PV patients (see K. Kapralova abstract at this ASH meeting). The VEGF-A transcript was increased in granulocytes and platelets, and SLC2A1was increased in platelets of MPN patients, compared to controls, but this did not reach statistical significance.We then directly evaluated glucose transport in red blood cells (another feature of the Warburg effect) in some of the PV, ET and MF studied patients using Raman scattering (RS). RS is a process by which the energy of an impinging photon is shifted due to an interaction with a molecule. The energy shift of the scattered photon usually corresponds to a vibrational mode of the involved molecule. The RS spectrum is highly molecule specific, as each molecule has a distinctive set of vibrational modes. However, the RS cross-section is very small, usually requiring an enhancement mechanism. RS, as well as other optical processes like fluorescence, can be enhanced in the vicinity of metallic nanoparticles, giving rise to surface-enhanced Raman scattering (SERS) and metal-enhanced fluorescence (MEF).SERS and MEF spectroscopy measurements done on granulocytes isolated from MPN patients show that fluorescence contribution to the spectra is higher and shifted when compared to spectra from control subjects. When healthy granulocytes were incubated with various concentrations of deoxyglucose (the glucose analog not metabolized but actively transported), there was an increase of the fluorescence contribution with higher concentrations, as well as a shift in the spectrum. Together, these spectroscopic results confirm a higher glucose uptake by granulocytes from MPN patients.The increased expression of SLC2A1 and PDK1 genes and enhanced uptake of glucose in MPN patients suggest the presence of Warburg effect. The difference in regulation of transcripts of PDK1 in granulocytes and platelets may be due to tissue-specific differences of gene transcription as the HIF-1 activity is often content-specific. To validate the likely presence of the Warburg effect in MPN patients, we are now analyzing the expression of HIF-1-regulated genes in hematopoietic stem cells, as well as in reticulocytes and in the BFU-E colonies in JAK2V617Fpositive and negative MPN.Note: First and second authors contributed equally. Disclosures:Swierczek:University of Utah : No financial compensation, No financial compensation Patents & Royalties.

Epigenetic Down-Regulation Of AML2 Expression By EZH2 Contributes To NF-E2 Overexpression In Myeloproliferative Neoplasms

The transcription factor NF-E2 is overexpressed in the vast majority of patients with Myeloproliferative Neoplasms (MPN). In Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) NF-E2 levels are elevated independent of the presence or absence of the JAK2V617Fmutation. We have recently shown that NF-E2 overexpression in a murine model leads to an MPN phenotype followed by spontaneous transformation to acute leukemia in a subset of mice.We have demonstrated that increased NF-E2 transcription is mediated by the transcription factor and proto-oncogene AML-1, which is overexpressed in MPN patients, again irrespective of the JAK2V617Fstatus. AML-1 binds to a conserved enhancer region located 3500 bp upstream of the NF-E2 transcriptional start site. Since AML-1 responsive genes are often also inversely regulated by the tumor suppressor AML-2 (RUNX3), we investigated whether NF-E2 expression is affected by AML-2 as well.Using chomatin immunoprecipitation assays (ChIP) we show that AML-2 binds the NF-E2 enhancer in vivo at a site distinct from but in proximity to the three AML-1 sites in the -3500bp region. AML-2 binding strongly represses transcription off the NF-E2 enhancer in reporter gene assays. This repression is abrogated by site directed mutagenesis of the AML-2 recognition sequence. Likewise, lentivirally induced AML-2 expression drastically reduces the amount of NF-E2 protein in erythroid and myeloid cells. These data clearly demonstrate that NF-E2 expression is directly regulated by AML-2. AML-2 thus serves as a repressor on the hematopoietic NF-E2 gene, a function previously noted mainly in solid tumors.In primary cells from patients with polycythemia vera (PV) AML-2 mRNA expression is significantly reduced. Moreover, ChIP assays demonstrate that in primary PV cells, significantly less AML-2 is bound to the NF-E2 enhancer than in healthy controls. Decreased repression by AML-2 thus cooperates with increased AML-1 induced transcription to elevate NF-E2 levels in PV patients.It has been demonstrated that AML-2 expression can be regulated by two distinct epigenetic mechanisms. For one, DNA methylation of the AML-2 promoter has been reported to silence AML-2 expression in gastric, colorectal and bladder cancers. On the other hand, aberrant histone methylation in the promoter region can also silence AML-2 expression. Here we show that DNA methylation of the AML-2 promoter is unaltered in PV patients. Rather, PV patients display aberrant histone methylation in the AML-2 promoter. Compared to healthy controls, H3K27me3 is significantly increased and H3K4me3 is significantly decreased in primary PV cells. This results in an inactive chromatin conformation on the AML-2 promoter in PV patients. Moreover, we show here that the histone-lysine-methyl-transferase “enhancer of zeste homologue 2” (EZH2), which confers the K3K27me3 mark, binds to the AML-2 promoter and decreases AML-2 expression. PV patients demonstrate significantly increased levels of EZH2 binding to the AML-2 promoterTreatment of primary PV cells and MPN cell lines with 2'-deoxy-5-azacytidine (DAC, Decitabine) ex vivo reverses the altered histone methylation, restoring the pattern found in healthy controls and decreases EZH2 binding to the AML-2 promoter. At the same time, Decitabine treatment induces AML-2 protein expression, decreases EZH2 expression and reduces the elevated NF-E2 levels. Moreover, a post-PV AML patient receiving Decitabine, displayed normalization of AML-2 promoter histone methylation and EZH2 binding on day 8 and day 15 of treatment.Taken together our data demonstrate that epigenetic silencing of AML-2 contributes to the elevated NF-E2 expression observed in MPN patients. Treatment with Decitabine restores physiological histone modifications to the AML-2 locus and decreases NF-E2 levels by reactivating AML-2 expression. These data provide a molecular rationale for extending the clinical investigation of epigenetic modifiers such as Decitabine or Azacitidine, currently used in MDS and AML, to patients with MPNs. A phase I study using Azacitidine in high risk PMF patients is currently being planned by the MPD-RC. Disclosures:No relevant conflicts of interest to declare.

MPN patients harbor recurrent truncating mutations in transcription factor NF-E2

The molecular etiology of myeloproliferative neoplasms (MPNs) remains incompletely understood, despite recent advances incurred through the discovery of several different mutations in MPN patients. We have recently described overexpression of the transcription factor NF-E2 in MPN patients and shown that elevated NF-E2 levels in vivo cause an MPN phenotype and predispose to leukemic transformation in transgenic mice. We report the presence of acquired insertion and deletion mutations in the NF-E2 gene in MPN patients. These result in truncated NF-E2 proteins that enhance wild-type (WT) NF-E2 function and cause erythrocytosis and thrombocytosis in a murine model. NF-E2 mutant cells acquire a proliferative advantage, witnessed by clonal dominance over WT NF-E2 cells in MPN patients. Our data underscore the role of increased NF-E2 activity in the pathophysiology of MPNs.

The Gene Encoding Nuclear Erythroid Factor 2 (NFE2) Is Recurrently Mutated in Acute Myeloid Leukemia

AbstractAbstract 1392 Background:Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the accumulation of various acquired (cyto)genetic aberrations in the leukemic blasts. Novel state-of-the-art sequencing technologies enable sequencing of complete disease genomes. Methods:We have used Complete Genomics (CG) next-generation sequencing to identify novel recurrent mutations in AML. We have selected a single AML case, WHO: AML with maturation, FAB: M2, karyotype 45X, -Y and a NPM1 mutation. Mutations in FLT3, CEBPA, ASXL1, IDH1, IDH2, NRAS, KRAS and DNMT3A were absent. By whole genome sequencing of the AML and its corresponding remission sample, we identified acquired mutations in the protein coding regions of 31 genes (CG somatic score >0.1), including NPM1 and PTPN11. Results:Interestingly, a frame-shift mutation in the protein coding region of the Nuclear Erythroid Factor 2 (NFE2) transcription factor gene was identified and acquisition of this mutation was confirmed by Sanger sequencing of both AML and remission samples. The complete NFE2 gene of the index AML patient was sequenced but no additional mutation was present, nor was the remaining allele affected by deletions and/or amplifications. The index mutation introduces a premature stop codon (PTC) in the NFE2 gene, upstream of the region encoding the bZIP domain.To investigate if NFE2 would be recurrently mutated in AML, we screened a cohort of 1139 AML cases by denaturing high performance liquid chromatography (dHPLC) analyses for mutations in a 350bp region surrounding the index mutation in the NFE2 gene. We identified NFE2 mutations in 5 additional cases of AML. Subsequently, we screened the complete NFE2 gene in 254 randomly selected AML cases by Roche 454 sequencing. This analysis revealed 8 NFE2 mutant cases in total. These results indicate that approximately 3.5% (8/254) of unselected primary AML cases carry NFE2 mutations. The acquisition of the NFE2 mutations was confirmed by Sanger sequencing of all NFE2mutant AML cases and their corresponding derived T cells.Frame shift mutations upstream of the NFE2 bZIP domain introducing PTCs were present in 6 out of all identified NFE2 mutant cases (n=13). The remaining cases carried non-synonymous NFE2 mutations and a single case an in-frame insertion/deletion. In our cohort of molecularly and clinically well-characterized cohort of AML patients the NFE2 mutations were not associated with any clinical characteristic or any other (cyto)genetic aberration. Discussion:It is currently unclear if the NFE2 mutations would lead to a gain-of-function or a loss-of-function. Nfe2-deficient mice lack circulating platelets and die of hemorrhage; their megakaryocytes showed no cytoplasmic platelet formation. In addition, NFE2 transgenic mice show MPNs, including thrombocytosis, and spontaneous transformation to acute myeloid leukemia. However, the NFE2 mutations did not associate with abnormal platelet counts in the affected AML cases, nor did the AML cases have consistent megakaryocyte abnormalities. Mutant NFE2 is currently functionally studied by introduction into various cell line models and mouse primary bone marrow. Conclusion:In conclusion, we have identified recurrent mutation in the transcription factor gene NFE2 in a subset of AML cases. The exact role of mutant NFE2 is currently being investigated. Disclosures:No relevant conflicts of interest to declare.