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XLS file - 75KB, Details of signature and expression analyses.

PDF file - 126KB, Analyses of two patients with detectable SF3B1 mutations in CD19+, CD34+ and CD14+ fractions.

Loss-of-function mutations in ten-eleven translocation-2 (TET2) are recurrent events in acute myeloid leukemia (AML) as well as in preleukemic hematopoietic stem cells (HSCs) of age-related clonal hematopoiesis. TET3 mutations are infrequent in AML, but the level of TET3 expression in HSCs has been found to decline with age. We examined the impact of gradual decrease of TET function in AML development by generating mice with Tet deficiency at various degrees. Tet2f/f and Tet3f/f mice were crossed with mice expressing Mx1-Cre to generate Tet2f/wtTet3f/fMx-Cre+ (T2ΔT3), Tet2f/fTet3f/wtMx-Cre+ (ΔT2T3), and Tet2f/fTet3f/fMx-Cre+ (ΔT2ΔT3) mice. All ΔT2ΔT3 mice died of aggressive AML at a median survival of 10.7 weeks. By comparison, T2ΔT3 and ΔT2T3 mice developed AML at longer latencies, with a median survival of ∼27 weeks. Remarkably, all 9 T2ΔT3 and 8 ΔT2T3 mice with AML showed inactivation of the remaining nontargeted Tet2 or Tet3 allele, respectively, owing to exonic loss in either gene or stop-gain mutations in Tet3. Recurrent mutations other than Tet3 were not noted in any mice by whole-exome sequencing. Spontaneous inactivation of residual Tet2 or Tet3 alleles is a recurrent genetic event during the development of AML with Tet insufficiency.

International audience; Aberrant NF-κB activation is a hallmark of most B-cell malignancies. Recurrent inactivating somatic mutations in the NFKBIE gene, which encodes IκBε, an inhibitor of NF-κB-inducible activity, are reported in several B-cell malignancies with highest frequencies in chronic lymphocytic leukemia and primary mediastinal B-cell lymphoma, and account for a fraction of NF-κB pathway activation. The impact of NFKBIE deficiency on B-cell development and function remains, however, largely unknown. Here, we show that Nfkbie-deficient mice exhibit an amplification of marginal zone B cells and an expansion of B1 B-cell subsets. In germinal center (GC)-dependent immune response, Nfkbie deficiency triggers expansion of GC B-cells through increasing cell proliferation in a B-cell autonomous manner. We also show that Nfkbie deficiency results in hyperproliferation of a B1 B-cell subset and leads to increased NF-κB activation in these cells upon Toll-like receptor stimulation. Nfkbie deficiency cooperates with mutant MYD88 signaling and enhances B-cell proliferation in vitro. In aged mice, Nfkbie absence drives the development of an oligoclonal indolent B-cell lymphoproliferative disorders, resembling monoclonal B-cell lymphocytosis. Collectively, these findings shed light on an essential role of IκBε in finely tuning B-cell development and function.

International audience; The TET2 gene encodes an α-ketoglutarate-dependent dioxygenase able to oxidize 5-methylcytosine into 5-hydroxymethylcytosine, which is a step toward active DNA demethylation. TET2 is frequently mutated in myeloid malignancies but also in B- and T-cell malignancies. TET2 somatic mutations are also identified in healthy elderly individuals with clonal hematopoiesis. Tet2-deficient mouse models showed widespread hematological differentiation abnormalities, including myeloid, T-cell, and B-cell malignancies. We show here that, similar to what is observed with constitutive Tet2-deficient mice, B-cell-specific Tet2 knockout leads to abnormalities in the B1-cell subset and a development of B-cell malignancies after long latency. Aging Tet2-deficient mice accumulate clonal CD19+ B220low immunoglobulin M+ B-cell populations with transplantable ability showing similarities to human chronic lymphocytic leukemia, including CD5 expression and sensitivity to ibrutinib-mediated B-cell receptor (BCR) signaling inhibition. Exome sequencing of Tet2-/- malignant B cells reveals C-to-T and G-to-A mutations that lie within single-stranded DNA-specific activation-induced deaminase (AID)/APOBEC (apolipoprotein B messenger RNA editing enzyme, catalytic polypeptide-like) cytidine deaminases targeted motif, as confirmed by the lack of a B-cell tumor in compound Tet2-Aicda-deficient mice. Finally, we show that Tet2 deficiency accelerates and exacerbates T-cell leukemia/lymphoma 1A-induced leukemogenesis. Together, our data establish that Tet2 deficiency predisposes to mature B-cell malignancies, which development might be attributed in part to AID-mediated accumulating mutations and BCR-mediated signaling.

TET2 somatic mutations occur in ∼10% of diffuse large B-cell lymphomas (DLBCL) but are of unknown significance. Herein, we show that TET2 is required for the humoral immune response and is a DLBCL tumor suppressor. TET2 loss of function disrupts transit of B cells through germinal centers (GC), causing GC hyperplasia, impaired class switch recombination, blockade of plasma cell differentiation, and a preneoplastic phenotype. TET2 loss was linked to focal loss of enhancer hydroxymethylation and transcriptional repression of genes that mediate GC exit, such as PRDM1. Notably, these enhancers and genes are also repressed in CREBBP-mutant DLBCLs. Accordingly, TET2 mutation in patients yields a CREBBP-mutant gene-expression signature, CREBBP and TET2 mutations are generally mutually exclusive, and hydroxymethylation loss caused by TET2 deficiency impairs enhancer H3K27 acetylation. Hence, TET2 plays a critical role in the GC reaction, and its loss of function results in lymphomagenesis through failure to activate genes linked to GC exit signals. Significance: We show that TET2 is required for exit of the GC, B-cell differentiation, and is a tumor suppressor for mature B cells. Loss of TET2 phenocopies CREBBP somatic mutation. These results advocate for sequencing TET2 in patients with lymphoma and for the testing of epigenetic therapies to treat these tumors. See related commentary by Shingleton and Dave, p. 1515. This article is highlighted in the In This Issue feature, p. 1494

Decitabine (DAC) is a hypomethylating agent indicated as front-line therapy for de novo or secondary acute myeloid leukemia (AML) in newly diagnosed patients aged 65 years or older unfit for standard induction chemotherapy (Kantarjian et al., 2012, Malik & Cashen, 2014, Nieto et al., 2016, He et al., 2017). Its mechanism of action at the DNA level mostly results in inhibition of cell proliferation. Cellular differentiation can also be involved in some extent in a fraction of the leukemic cell population, as reported in initial pharmacological studies (Creusot et al., 1982; Pinto et al., 1984). Overall survival advantage is nevertheless limited to several more months and the next challenge is to combine DAC with other drugs to improve it further (Kubasch & Platzbecker, 2018). Inecalcitol (INE: 14epi-,19nor-,23yne-,1,25dihydroxy-cholecalciferol) is a vitamin D receptor agonist characterized by potent anti-proliferative and pro-differentiating general properties on cancer cells and by a low calcemic potential (Okamoto et al., 2012; Ma et al., 2013; Medioni et al. 2014), and especially on AML cell lines (AACR 2017, 2018). INE is currently being tested in combination with DAC in this category of elderly AML patients unfit for standard chemotherapy. The aim of the present report was to look for synergies in vitro between DAC and INE on four non-APL human AML cell lines (MOLM-13, U-937, THP-1, OCI-AML2) both on inhibition of proliferation and induction of differentiation. After 72 hours of incubation, cells were counted and labeled for CD11b and CD14 at the cell surface as biomarkers of monocytic/macrophagic differentiation. The range of DAC concentrations had to adapted to each cell line to avoid maximal cytotoxicity: 1.2 nM to 100 nM on MOLM-13, 3 nM to 250 nM on U-937 and THP-1, and 31 nM to 500 nM on OCI-AML2. The same range of 0.12 to 10 nM INE concentrations was tested on each cell line. Each concentration of INE was tested in combination with each concentration of DAC. Synergy was calculated as the excess over the highest single agent (HSA) using the open source Combenefit software (Di Veroli et al., 2016). The highest concentration of DAC alone (MOLM-13: 100 nM, U-937 and THP-1: 250 nM; OCI-AML2: 500 nM) induced a decrease in cell count of 30% of THP-1 cells, 50% of OCI-AML2 cells, 65% of U-937 cells and 80% of MOLM-13 cells. The highest concentration of INE alone (10 nM) induced a decrease in cell count of 20% of U-937 and THP-1 cells, 60% of OCI-AML2 cells and 70% of MOLM-13 cells. The antiproliferative effects of DAC and INE were at least additive in all combinations tested. Significant HSA synergy indexes were found for the decrease in cell number in all four cell lines, ranging from 12% to 23% depending on cell lines and combinations of concentrations. The highest concentration of DAC alone had no (U-937, THP-1) or limited activity ( In summary, DAC exerted more antiproliferative activity than INE which was more potent to induce monocytic/macrophagic differentiation of four non-APL human AML cell lines. The combination of DAC and INE systematically resulted in a synergy to inhibit cell proliferation, and the strong stimulation of cell differentiation induced by INE alone was in some cases boosted by DAC. These in vitro results provide the mechanistic basis for the potential interest of treating elderly AML patients with INE in addition to DAC in the ongoing double-blind placebo-controlled Phase II clinical trial (NCT02802267). Disclosures Mouly: Hybrigenics: Employment. Rousseau:Hybrigenics: Employment. Planquette:Hybrigenics: Employment, Equity Ownership, Patents & Royalties: inventor, but no royalties. Delansorne:Hybrigenics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: inventor, but no royalties.

Daratumumab (DARA), the first monoclonal antibody directed against CD38, received accelerated approval in 2015 and regular approval in 2016 by the Food and Drug Administration for the treatment of relapsed/refractory multiple myeloma (MM) (Bhatnagar et al., 2017); its therapeutic indications were recently extended to first-line newly diagnosed MM patients in combination with standards of care following a positive clinical trial in this setting (Mateos et al., 2018). Therefore, DARA has brought extensive evidence that targeting CD38 is a highly successful strategy against MM (Frerichs et al., 2018; Plesner & Krejcik, 2018; Touzeau & Moreau, 2017). Interestingly, the pretreatment levels of CD38 expression on MM cells were significantly higher in patients who achieved at least partial response (PR) as compared to patients who achieved less than PR (Nijhof et al., 2016); furthermore, low level of CD38 expression has recently been identified as an adverse prognostic factor with high significance (p < 0.001) among MM patients (Arana et al., 2018). Besides MM, there are growing preclinical hints that DARA can be efficacious against acute myeloid leukemia (AML) cell lines (Fatehchand et al., 2016; Buteyn et al., 2018) or even T-cell acute lymphoblastic leukemia cells obtained from children patients (Bride et al., 2018), provided that enough CD38 molecules are present at the surface of the leukemic cells. Inecalcitol (INE: 14epi-,19nor-,23yne-,1,25dihydroxy-cholecalciferol), a vitamin D receptor agonist characterized by potent anti-proliferative and pro-differentiating properties and by a low calcemic potential (Okamoto et al., 2012; Ma et al., 2013; Medioni et al. 2014), has been shown to stimulate CD38 expression levels in 37 out of 38 MM cell lines (ASH 2017) and in 10 out of 11 AML cell lines (ASH 2016, AACR 2018). The next question was to know if this increase in CD38 expression induced by INE resulted in an increase in DARA-induced antibody-dependent cell cytotoxicity (ADCC). We have tested two MM (MM1.S, L-363) and seven AML (HL-60, U-937, THP-1, Molm-13, UOC-M1, OCI-AML2, Kasumi-1) cell lines representative of various levels of spontaneous CD38 cell surface density, as measured in thousands of CD38 molecules per cell by fluorescent anti-CD38 labeling (HIT2 clone) followed by quantitative flow cytometry using Cellquant calibrator beads (BioCytex). They ranged from very high CD38-positive (MM.1S, 51.0 ± 1.6 and UOC-M1, 46.5 ± 4.9) to high (OCI-AML2, 16.6 ± 1.5), low (L-363, 7.1 ± 0.1 and THP-1, 7.0 ± 0.2), very low (Molm-13, 2.1 ± 1.0 and Kasumi-1, 1.1 ± 1.2) and CD38-negative (HL-60, U-937). After exposure to 10 nM INE or vehicle for 72h, DARA-induced ADCC was assessed using either the calcein staining of target cells exposed to healthy donors peripheral blood mononuclear cells (PBMC) as effector cells, or the ADCC Reporter Bioassays Kit from Promega (G7018). After control vehicle incubation, DARA-induced ADCC was concentration-related in the range of 0.01 to 1 µg/mL in all cell lines and achieved a maximum ranging from 7% in THP-1, although a CD38-negative cell line, to respectively 27% and 28% in MM1.S and UOC-M1, the two highest CD38-positive cell lines. However, the correlation between maximal DARA-induced ADCC and CD38 surface density did not reach statistical significance (Spearman's rank correlation r = 0.6134, p = 0.09, n = 9). After incubation with 10 nM INE, the initially CD38-negative U-937 and HL-60 cells were found to express low (7.9 ± 0.3) and high (25.6 ± 7.3) surface CD38 levels, respectively. All the CD38-positive cell lines have shown a marked increase in the number of surface CD38 molecules: MM1.S (228 ± 12), OCI-AML2 (76,7 ± 6.5), UOC-M1 (68.8 ± 3.1), L-363 (63.9 ± 1.6), Molm-13 (38.2 ± 8.9), THP-1 (17.9 ± 0.9) and Kasumi-1 (15.7 ± 3.3) (paired Student's t-test, p = 0.02, n = 7). DARA-induced ADCC was concentration-related in the same 0.01 to 1 µg/mL range. Maximal DARA-induced ADCC was increased by 10 nM INE in all cell lines (paired Student t-test, p = 0.0025, n = 9), ranged from 20% in the lowest CD38-expressing cell line U-937 to 42% in the highest CD38-expressing cell line MM1.S, and was significantly correlated with the number of CD38 molecules/cell (Spearman's rank correlation r = 0.8333, p < 0.01, n = 9). Altogether, these results suggest that inecalcitol could represent a therapeutic agent of choice to enhance the clinical response of MM and AML patients to anti-CD38 antibodies such as daratumumab. Disclosures Mouly: Hybrigenics: Employment. Planquette:Hybrigenics: Employment, Equity Ownership, Patents & Royalties: inventor, but no royalties. Rousseau:Hybrigenics: Employment. Delansorne:Hybrigenics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: inventor, but no royalties.

International audience; TEN-ELEVEN-TRANSLOCATION-2 (TET2) and DNA-METHYLTRANSFERASE-3A (DNMT3A), both encoding proteins involved in regulating DNA methylation, are mutated in hematological malignancies affecting both myeloid and lymphoid lineages. We previously reported an association of TET2 and DNMT3A mutations in progenitors of patients with angioimmunoblastic T-cell lymphomas (AITL). Here, we report on the cooperative effect of Tet2 inactivation and DNMT3A mutation affecting arginine 882 (DNMT3A(R882H)) using a murine bone marrow transplantation assay. Five out of eighteen primary recipients developed hematological malignancies with one mouse developing an AITL-like disease, two mice presenting acute myeloid leukemia (AML)-like and two others T-cell acute lymphoblastic leukemia (T-ALL)-like diseases within 6 months following transplantation. Serial transplantations of DNMT3A(R882H) Tet2(-/-) progenitors led to a differentiation bias toward the T-cell compartment, eventually leading to AITL-like disease in 9/12 serially transplanted recipients. Expression profiling suggested that DNMT3A(R882H) Tet2(-/-) T-ALLs resemble those of NOTCH1 mutant. Methylation analysis of DNMT3A(R882H) Tet2(-/-) T-ALLs showed a global increase in DNA methylation affecting tumor suppressor genes and local hypomethylation affecting genes involved in the Notch pathway. Our data confirm the transformation potential of DNMT3A(R882H) Tet2(-/-) progenitors and represent the first cooperative model in mice involving Tet2 inactivation driving lymphoid malignancies.

Summary Fanconi anemia (FA) is an inherited DNA repair deficiency syndrome. FA patients undergo progressive bone marrow failure (BMF) during childhood, which frequently requires allogeneic hematopoietic stem cell transplantation. The pathogenesis of this BMF has been elusive to date. Here we found that FA patients exhibit a profound defect in hematopoietic stem and progenitor cells (HSPCs) that is present before the onset of clinical BMF. In response to replicative stress and unresolved DNA damage, p53 is hyperactivated in FA cells and triggers a late p21 Cdkn1a -dependent G0/G1 cell-cycle arrest. Knockdown of p53 rescued the HSPC defects observed in several in vitro and in vivo models, including human FA or FA-like cells. Taken together, our results identify an exacerbated p53/p21 "physiological" response to cellular stress and DNA damage accumulation as a central mechanism for progressive HSPC elimination in FA patients, and have implications for clinical care.

In response to antigens and cytokines, mouse B cells undergo class-switch recombination (CSR) and differentiate into Ig-secreting cells. T-bet, a T-box transcription factor that is up-regulated in lymphocytes by IFN-γ or IL-27, was shown to regulate CSR to IgG2a after T cell–independent B-cell stimulations. However, the molecular mechanisms controlling this process remain unclear. In the present study, we show that inactivation of the Ets-1 transcription factor results in a severe decrease in IgG2a secretion in vivo and in vitro. No T-bet expression was observed in Ets-1–deficient (Ets-1−/−) B cells stimulated with IFN-γ and lipopolysaccharide, and forced expression of T-bet in these cells rescued IgG2a secretion. Furthermore, we identified a transcriptional enhancer in the T-bet locus with an activity in B cells that relies on ETS-binding sites. After IFN-γ stimulation of Ets-1−/− B cells, activated Stat1, which forms a complex with Ets-1 in wild-type cells, no longer binds to the T-bet enhancer or promotes histone modifications at this site. These results demonstrate that Ets-1 is critical for IgG2a CSR and acts as an essential cofactor for Stat1 in the regulation of T-bet expression in B cells.

Background Multiple myeloma is characterized by the accumulation of tumor plasma cells in the bone marrow. Despite therapeutic improvements brought by proteasome inhibitors such as bortezomib, myeloma remains an incurable disease. In a variety of human cancers, human immunodeficiency virus protease inhibitors (e.g. nelfinavir) effectively inhibit tumor progression, but their impact on myeloma is unknown. We assessed the in vitro and in vivo effects of nelfinavir on multiple myeloma.Design and Methods The effects of nelfinavir (1–10 μM) on proteasome activity, proliferation and viability of myeloma cell lines and plasma cells from patients were assessed by measuring PERK, AKT, STAT3 and ERK1/2 phosphorylation and CHOP expression with immunoblotting or flow cytometry. The in vivo effect was assessed in NOD/SCID mice injected with luciferase expressing human myeloma cell lines and treated with nelfinavir at a dose of 75 mg/kg/day. Tumor progression was evaluated using a bioluminescent system.Results Nelfinavir inhibited 26S chymotrypsin-like proteasome activity, impaired proliferation and triggered apoptosis of the myeloma cell lines and fresh plasma cells. It activated the pro-apoptotic unfolded protein response pathway by inducing PERK phosphorylation and CHOP expression. Cell death triggered by nelfinavir treatment correlated with decreased phosphorylation of AKT, STAT3 and ERK1/2. Nelfinavir enhanced the anti-proliferative activity of bortezomib, dexamethasone and histone deacetylase inhibitors and delayed tumor growth in a myeloma mouse model.Conclusions These results suggest that nelfinavir, used at a pharmacological dosage, alone or in combination, may be useful in the treatment of myeloma. Our data provide a preclinical basis for clinical trials using nelfinavir in patients with myeloma.

International audience; Regulatory T cells (T reg cells) constitute a population of CD4(+) T cells that limits immune responses. The transcription factor Foxp3 is important for determining the development and function of T reg cells; however, the molecular mechanisms that trigger and maintain its expression remain incompletely understood. In this study, we show that mice deficient for the Ets-1 transcription factor (Ets-1(-/-)) developed T cell-mediated splenomegaly and systemic autoimmunity that can be blocked by functional wild-type T reg cells. Spleens of Ets-1(-/-) mice contained mostly activated T cells, including Th2-polarized CD4(+) cells and had reduced percentages of T reg cells. Splenic and thymic Ets-1(-/-) T reg cells expressed low levels of Foxp3 and displayed the CD103 marker that characterizes antigen-experienced T reg cells. Thymic development of Ets-1(-/-) T reg cells appeared intrinsically altered as Foxp3-expressing cells differentiate poorly in mixed fetal liver reconstituted chimera and fetal thymic organ culture. Ets-1(-/-) T reg cells showed decreased in vitro suppression activity and did not protect Rag2(-/-) hosts from naive T cell-induced inflammatory bowel disease. Furthermore, in T reg cells, Ets-1 interacted with the Foxp3 intronic enhancer and was required for demethylation of this regulatory sequence. These data demonstrate that Ets-1 is required for the development of natural T reg cells and suggest a role for this transcription factor in the regulation of Foxp3 expression.

Acute myeloid leukemia (AML) is a heterogeneous disease classified into 9 FAB subtypes according to the origin and/or maturity of the malignant cells: M0 undifferentiated, M1 minimally mature myeloblastic, M2 mature myeloblastic, M3 promyelocytic, M4 myelomonocytic, M4eos eosinophilic, M5 monocytic, M6 erythrocytic and M7 megakaryocytic. Overcoming this heterogeneity by the induction of a common surface antigen which would already be the target of an existing immunotherapy would be of great potential therapeutic interest to standardize a common immunotherapy approach to AML. Inecalcitol, a vitamin D receptor agonist characterized by a high anti-proliferative effect and a low calcemic potential, has been shown to stimulate CD38 expression at the surface of the M3 subtype HL-60 cell line and of the M5 subtype U-937, MOLM-13 and THP-1 cell lines (ASH 2016, AACR 2017). We now report the effect of inecalcitol on AML cell lines representative of the 7 other FAB subtypes: KG-1 (M0), UOC-M1 (M1), Kasumi-1 (M2), OCI-AML2 (M4), EOL-1 (M4eos), HEL-92.1.7 (M6) and CMK (M7). Inecalcitol induced CD38 at the surface of CD38- cell lines (HL-60, U-937 and EOL-1) and strongly increased CD38 density at the surface of already CD38+ cell lines (KG-1, UOC-M1, Kasumi-1, OCI-AML2, MOLM-13, THP-1 and CMK) in a concentration-dependent manner, but remained without effect on CD38- HEL-92.1.7 cells. We have quantified the number of CD38 molecules at the surface of all cell lines (except HEL-92.1.7 which was unresponsive to inecalcitol) using the CellQuant calibrator (Biocytex) analysis by flow cytometry. In basal vehicle-treated condition, the number (in thousands, mean ± sem, n=2 or 3) of CD38 molecules per CD38+ cell were: Kasumi-1, 1.5 ± 1.0; MOLM-13, 2.5 ± 0.6; KG-1, 2.7 ± 0.9; THP-1, 7.0 ± 0.2; CMK, 12.1 ± 1.5; OCI-AML2, 16.1 ± 1.2. With 10nM inecalcitol, a maximal increase in CD38 density was usually obtained within 3 days, or after 7 days for KG-1 and CMK: Kasumi-1, 16.1 ± 3.3 (x11); MOLM-13, 43.3 ± 0.3 (x17); KG-1, 15.4 ± 3.7 (x5.7); THP-1, 17.9 ± 0.6 (x2.3); CMK, 15.4 ± 3.3 (x1.3); OCI-AML2, 73.5 ± 3.5 (x4.6). After 3 days of exposure to 10 nM inecalcitol, CD38 molecules appeared at the surface of initially CD38- cells and became quantifiable: U-937, 7.5 ± 0.4 (n=3); HL-60, 24.4 ± 4.9 (n=3) and EOL-1 ranged from 5.5 to 72.7 (n=2). In conclusion, after exposure to inecalcitol, CD38 appeared at the surface of CD38- AML cell lines and the density of CD38 increased at the surface of CD38+ AML cell lines, altogether representative of all 9 FAB classification subtypes M0 to M7, except M6 (erythrocytic) which represents 5% of all AML cases. These results suggest that treatment by inecalcitol may render 95% of AML patients sensitive to highly sensitive to anti-CD38 immunotherapy such as daratumumab. Citation Format: Enguerran Mouly, Cécile Planquette, Emilie Rousseau, Rémi Delansorne. Inecalcitol respectively induces or increases CD38 expression at the surface of CD38- or CD38+ AML cell lines representative of all 9 FAB subtypes except M6 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1890.

In humans, the CD4 molecule is expressed on a subset of T-cells and at various levels on myeloid and lymphoid cells. The mechanisms regulating human CD4 gene expression are yet poorly understood. We speculated that the CD4 silencer, which operates in CD8+ T-cells to repress CD4 expression, could be responsible for CD4 repression in human lymphoid non-T-cells. To test this possibility, we used lentiviral vectors carrying CD4 regulatory sequences, with or without the silencer element, to express an eGFP reporter gene. We observed that (i) in the absence of the silencer element, eGFP expression was detected in CD34+-derived B- and NK-cells that otherwise lacked endogenous CD4 mRNA, indicating active repression of the CD4 regulatory sequences and (ii) the addition of the CD4 silencer could repress eGFP expression in these same cells, as well as in human B-cells generated in vivo in NOD/SCID mice. Collectively, our results suggest that beyond its well-characterized function in T-cells, the CD4 silencer also regulates CD4 gene expression in human lymphoid non-T-cells.

Appropriate cancer care requires a thorough understanding of the natural history of the disease, including the cell of origin, the pattern of clonal evolution, and the functional consequences of the mutations. Using deep sequencing of flow-sorted cell populations from patients with chronic lymphocytic leukemia (CLL), we established the presence of acquired mutations in multipotent hematopoietic progenitors. Mutations affected known lymphoid oncogenes, including BRAF, NOTCH1, and SF3B1. NFKBIE and EGR2 mutations were observed at unexpectedly high frequencies, 10.7% and 8.3% of 168 advanced-stage patients, respectively. EGR2 mutations were associated with a shorter time to treatment and poor overall survival. Analyses of BRAF and EGR2 mutations suggest that they result in deregulation of B-cell receptor (BCR) intracellular signaling. Our data propose disruption of hematopoietic and early B-cell differentiation through the deregulation of pre-BCR signaling as a phenotypic outcome of CLL mutations and show that CLL develops from a pre-leukemic phase. Significance: The origin and pathogenic mechanisms of CLL are not fully understood. The current work indicates that CLL develops from pre-leukemic multipotent hematopoietic progenitors carrying somatic mutations. It advocates for abnormalities in early B-cell differentiation as a phenotypic convergence of the diverse acquired mutations observed in CLL. Cancer Discov; 4(9); 1088–1101. ©2014 AACR. See related commentary by Jiang and Elemento, p. 995 This article is highlighted in the In This Issue feature, p. 973

Gaucher disease (GD) is an autosomal recessive disorder characterized by lysosomal glucocerebrosidase (GBA) deficiency leading to hematological and skeletal manifestations. Mechanisms underlying these symptoms have not yet been elucidated. In vivo, bone marrow (BM) mesenchymal stem cells (MSCs) have important role in the regulation of bone mass and in the support of hematopoiesis, thus representing potential candidate that could contribute to the disease. GBA deficiency may also directly impair hematopoietic stem/progenitors cells (HSPCs) intrinsic function and induce hematological defect. In order to evaluate the role of BM stem cells in GD pathophysiology, we prospectively analyzed BM-MSCs and HSPCs properties in a series of 10 patients with type 1 GD. GBA activity was decreased in all tested cell subtypes. GD-MSCs had an impaired growth potential, morphological and cell cycle abnormalities, decreased capacities to differentiate into osteoblasts. Moreover, GD-MSCs secreted soluble factors that stimulated osteoclasts resorbing activities. In vitro and in vivo primitive and mature hematopoiesis were similar between patients and controls. However, GD-MSCs had a lower hematopoietic supportive capacity than those from healthy donors. These data suggest that BM microenvironment is altered in GD and that MSCs are key components of the manifestations observed in GD.

Interleukin (IL)-10 and glucocorticoids (GCs) inhibit the ability of antigen-presenting dendritic cells (DCs) to stimulate T lymphocytes. We show that induction of GILZ (GC-induced leucine zipper) is involved in this phenomenon. IL-10, dexamethasone (DEX), and transforming growth factor (TGF)β stimulate GILZ production in human immature DCs derived from monocytes and from CD34+ cells. GILZ is necessary and sufficient for DEX, IL-10, and TGFβ modulation of CD80, CD83, CD86, immunoglobulin-like transcript (ILT)-3, and B7-H1 expression by DCs, and alteration of DC functions. GILZ stimulates the production of IL-10 by immature DCs and prevents the production of inflammatory chemokines by CD40L-activated DCs. In contrast, GILZ does not prevent CD40 ligand-mediated inhibition of phagocytosis, indicating that it affects some but not all aspects of DC maturation. GILZ prevents DCs from activating antigen-specific T lymphocyte responses. Administration of GCs to patients stimulates GILZ expression in their circulating antigen-presenting cells, and this contributes to the weak lymphocyte responses of GC-treated patients. Thus, regulation of GILZ expression is an important factor determining the decision of DCs whether or not to stimulate T lymphocytes, and IL-10, GCs, and TGFβ share this mechanism for influencing DC functions and the balance between immune response and tolerance.

Background Lentiviral gene transfer into hematopoietic cells has been mostly optimized with vectors carrying a single reporter gene. For many clinical applications, lentiviral vectors should contain more than one gene because transduced cells should be enriched by a selectable marker or killed for safety reasons after use. Thus, we compared various vectors containing a bicistronic cassette driven by different ubiquitous promoters for their ability to transduce human T-lymphocytes, CD34+-cells, and dendritic cells (DCs) derived from CD34+-cells or monocytes. Methods We designed HIV or SIV constructs containing a bicistronic cassette composed of two reporter genes (thy1/GFP) linked by an internal ribosome entry site sequence and driven by the cytomegalovirus (CMV) or elongation factor 1α (EF1α) promoters. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was or not inserted within the constructs, the Vpx accessory protein was or not used for SIV vectors. Target cells were infected at the same multiplicity of infection, transduction efficiency was analyzed both by flow cytometry and vector integration. Results For T-cells, HIV-based vectors/WPRE+ in which the thy1/GFP cassette was driven by the EF1α promoter were more efficient than SIV-based vectors. For CD34+-cells and CD34+-derived DCs, better thy1/GFP expression was achieved when the CMV promoter drove the cassette inserted into HIV-based vectors/WPRE+. Conversely, for monocyte-derived DCs, the cassette yielded better thy1/GFP expression when inserted into SIV-based vectors/WPRE+ and driven by the CMV or EF1α promoters, the use of Vpx significantly improving the expression levels. Conclusions Our results provide guidelines for improving the transduction of T-cells, CD34+-cells or DCs with lentiviral bicistronic vectors designed for clinical applications. Copyright © 2005 John Wiley & Sons, Ltd.

Achieving cell-specific expression of a therapeutic transgene by gene transfer vectors represents a major goal for gene therapy. To achieve specific expression of a transgene in CD4(+) cells, we have generated lentiviral vectors expressing the enhanced green fluorescent protein (eGFP) reporter gene under the control of regulatory sequences derived from the CD4 gene--a minimal promoter and the proximal enhancer, with or without the silencer. Both lentiviral vectors could be produced at high titers (more than 10(7) infectious particles per milliliter) and were used to transduce healthy murine hematopoietic stem cells (HSCs). On reconstitution of RAG-2-deficient mice with transduced HSCs, the specific vectors were efficiently expressed in T cells, minimally expressed in B cells, and not expressed in immature cells of the bone marrow. Addition of the CD4 gene-silencing element in the vector regulatory sequences led to further restriction of eGFP expression into CD4(+) T cells in reconstituted mice and in ex vivo-transduced human T cells. Non-T CD4(+) dendritic and macrophage cells derived from human CD34(+) cells in vitro expressed the transgene of the specific vectors, albeit at lower levels than CD4(+) T cells. Altogether, we have generated lentiviral vectors that allow specific targeting of transgene expression to CD4(+) cells after differentiation of transduced mice HSCs and human mature T cells. Ultimately, these vectors may prove useful for in situ injections for in vivo gene therapy of HIV infection or genetic immunodeficiencies.

The discovery that ten-eleven translocation (TET) proteins are α-ketoglutarate-dependent dioxygenases involved in the conversion of 5-methylcytosines (5-mC) to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine and 5-carboxycytosine has revealed new pathways in the cytosine methylation and demethylation process. The description of inactivating mutations in TET2 suggests that cellular transformation is in part caused by the deregulation of this 5-mC conversion. The direct and indirect deregulation of methylation control through mutations in DNA methyltransferase and isocitrate dehydrogenase (IDH) genes, respectively, along with the importance of cytosine methylation in the control of normal and malignant cellular differentiation have provided a conceptual framework for understanding the early steps in cancer development. Here, we review recent advances in our understanding of the cytosine methylation cycle and its implication in cellular transformation, with an emphasis on TET enzymes and 5-hmC. Ongoing clinical trials targeting the activity of mutated IDH enzymes provide a proof of principle that DNA methylation is targetable, and will trigger further therapeutic applications aimed at controlling both early and late stages of cancer development.