ALBERT

Beschreibung: Key Points C/EBPα is needed for transition from stem/progenitor cells to common dendritic cell progenitors. C/EBPα is dispensable in later stages of dendritic cell maturation.

Beschreibung: The homeobox transcription factor Cdx2 is one of the most frequent ectopically expressed proto-oncogenes in human AML and when retrovirally expressed causes AML in mice (Rawat et. al. PNAS 2004 and Blood 2008). As the leukemogenic potential of Cdx2 was dependent on its N-terminal transactivation domain, we now extended structure-function analyses by inactivating the evolutionarily conserved MAPK phosphorylation sites (S60, S99, S108, S156, S60-S99-108 (3S), and S60-S99-108-156 (4S)) in Cdx2. Peripheral blood analysis of the mice, transplanted with bone marrow cells retrovirally transduced with different Cdx2 serine mutants (n=5) after six weeks, revealed that all the Cdx2 serine mutants showed significant growth advantage over non-transduced carrier cells. However, assessment of engraftment after 16 weeks showed that mice transplanted with BM expressing the S99, the S108 mutants, the 3S and 4S failed to develop leukemic engraftment in contrast to wild type Cdx2, indicating that the S99 and S108 serine sites are critical for leukemic transformation. Furthermore, mice transplanted with BM expressing Cdx2 wild type (n=24), the S60 (n=3) or S156 mutant (n=5) developed AML after median latency of 120, 90 and 167 days post transplantation, respectively. In contrast S99, S108 and 3S mice developed AML after very long latency of 328 (5/5 mice), 341(3/5 mice) and 336 (3/5 mice) days post transplantation, respectively. Interestingly, 4S mice (n=5), did not develop any disease up to an observation time of 400 days, indicating that the transforming potential of Cdx2 was dependent on multiple N-terminal serines. As it was shown that the transcriptional activity of Cdx2 is dependent on the phosphorylation status of N-terminal serines in non-hematopoietic tissue we tested the phosphorylation status in hematopoietic cells: murine cells retrovirally transduced with Cdx2 and human AML cell lines positive for CDX2 expression showed a phosphorylated form of CDX2 and an activated Erk1/2 pathway, in contrast to AML cell lines negative for CDX2 expression. Based on this we tested whether inhibition of the MAPK pathway would impair the transforming potential of Cdx2. When Cdx2 transduced BM cells were incubated with MEK1/2 inhibitors (PD98059, U0126) for 14 days in liquid culture, viability of the cells was reduced by 78% (n=3, p

Beschreibung: Recently, we and others have shown that protein-protein interactions play an important role in the pathogenesis of leukemia, and that the transcription factor C/EBPα is a key player in granulopoiesis and leukemogenesis. In the present study, we sought to identify C/EBPα interacting proteins. A glutathione-S-transferase-C/EBPα fusion protein was used to pull down interacting proteins from U937 nuclear extracts. These proteins were analyzed by 2-D gel electrophoresis, 1-D nano LC and identified by MALDI or MALDI-TOF/TOF. Several novel C/EBPα interactors were identified including known proteins like Rb, hnRNP and E2F4. Two novel interactions, one of cell cycle regulator protein MCM5 and the other with the MYST domain histone acetyl transferase TIP60 with C/EBPα were further confirmed by using pull-down and co-immunoprecipitation experiments. TP60 was able to markedly enhance C/EBPα mediated transcription in reporter gene assays, suggesting that TIP60 is a co-activator of C/EBPα. This co-activator function of TIP60 was dependent on an intact histone acetyl transferase domain and on the C/EBPα DNA binding domain. TIP60 was found to be associated with the human C/EBPα promoter in-vivo in a chromatin immunoprecipitation assay with a concomitant increase in histone H3 and H4 acetylation. Furthermore, we observed a lower expression of TIP60 mRNA in U937 CD11b− compared to retinoic acid induced U937 CD11b+ cells suggesting that higher TIP60 expression is associated with myeloid differentiation. There was also a marked correlation between the expression levels of TIP60 and C/EBPa in normal bone marrow, chronic myeloid leukemia and acute myeloid leukemia samples with t(8;21), inv(16) and t(15;17). This finding further confirms the functional synergism between C/EBPα and TIP60 and suggests that TIP60 might be an important player in leukemogenesis.

Beschreibung: Hematopoietic stem cells (HSCs) have the potential to replenish the blood system for the lifetime of the organism. Their 2 defining properties, self-renewal and differentiation, are tightly regulated by the epigenetic machineries. Using conditional gene-knockout models, we demonstrated a critical requirement of lysine acetyltransferase 5 (Kat5, also known as Tip60) for murine HSC maintenance in both the embryonic and adult stages, which depends on its acetyltransferase activity. Genome-wide chromatin and transcriptome profiling in murine hematopoietic stem and progenitor cells revealed that Tip60 colocalizes with c-Myc and that Tip60 deletion suppress the expression of Myc target genes, which are associated with critical biological processes for HSC maintenance, cell cycling, and DNA repair. Notably, acetylated H2A.Z (acH2A.Z) was enriched at the Tip60-bound active chromatin, and Tip60 deletion induced a robust reduction in the acH2A.Z/H2A.Z ratio. These results uncover a critical epigenetic regulatory layer for HSC maintenance, at least in part through Tip60-dependent H2A.Z acetylation to activate Myc target genes.

Beschreibung: FL is the most common indolent nodal lymphoma and considered incurable for the majority of patients. Virtually all patients eventually develop resistant disease, and up to 45% histologic transformation (tFL) with poor outcome. Alterations associated with progression to tFL include increased copy number variations (e.g. CDKN2A deletions, MYC amplification), acquisition of additional translocations (e.g. MYC or BCL6), and higher mutation load (including TP53). However the underlying molecular mechanisms promoting this genomic instability remain elusive. Interestingly, histologic transformation occurs at a remarkably constant rate of ~3% per year implying a rather stochastic process. We previously described convergent evolution for disruptive ARID1A mutations in FLs of a donor and the recipient patient following an allogeneic bone marrow transplantation (Weigert, Canc Discovery 2012). The independent acquisition of ARID1Aloss in two separate hosts after transplantation of an identical FL ancestor clone suggested (i) a later acquired event in the molecular ontogeny and (ii) functional relevance. ARID1A promotes the formation of SWI/SNF nucleosome remodeling complexes containing BRG1 or BRM, which catalyze disruption of DNA-histone contacts, thereby controlling chromatin condensation and DNA accessibility. Nucleosome-remodelling has been linked to DNA damage response, including nucleotide and base excision repair, homologous recombination and non-homologous end-joining (NHEJ). We thus hypothesized that ARID1Ahaplodeficiency might contribute to genetic and genomic instability in FL. In a cohort of 305 FL specimens, we identified 44 ARID1A mutations in 43 patients (14%). The majority of these mutations were disruptive (66%; 19 nonsense and 10 frame shift mutations), 6 cases harbored splice site mutations. MutSigCV analysis (Lawrence, Nature 2013) indicated that ARID1A was significantly mutated in FL, i.e. more often than expected by chance given background mutation processes. Consistent with our hypothesis, the number of non-synonymous mutations in 104 targeted genes was higher in ARID1A mutated FLs (5.6+0.34, mean+sem) compared to ARID1A wild-type (wt) FLs (4.7+0.12; p=0.0099), and similar compared to 17 FLs with TP53 mutations (5.6+0.12). Of note, ARID1A and TP53 mutations were mutually exclusive. For functional studies, we confirmed deleterious ARID1A alterations in 6 lymphoma cell lines (Namalwa, Karpas422, SUPHD-1, SU-DHL5, WSU-FSCCL, U-OH1). Immunoblotting showed decreased ARID1A protein expression in all 6 lines compared to 5 ARID1A wt lymphomas. Tet-induced re-expression of ARID1A in two tested ARID1A mutant cell lines (Namalwa and SUP-HD1) reduced cell proliferation compared to tet-induced control (LacZ), but had no effect in ARID1A wt OCI-Ly1. As a proof-of-principle experiment we assayed the impact of ARID1A loss on DNA double strand break (DSB) repair efficiency in genetically well defined mouse embryo fibroblasts (MEFs), avoiding uncontrolled bias introduced by the genetic and genomic complexity in lymphoma cell lines. Early passage MEFs from either wt or conditional ARID1A knock-out mice (ARID1Af/f; Gao, PNAS 2008) were transiently transfected or retrovirally transduced with self-excising Cre-recombinase (Silver&Livingston, Mol Cell 2001) or control, and irradiated with 2 Gy after 36 hrs. Cre-induced ARID1A loss was confirmed by Western blot. To assay NHEJ, the predominate DSB-repair pathway used by cells in G1 phase, we stained cells for γH2A.X and 53BP1. In 3 independent and blinded experiments, evaluation of 〉100 cells per condition demonstrated significantly more double-positive cells (〉6 foci/cell) in ARID1A-/- MEFs (43+14%) compared to both ARID1Af/fMEFs (9+1%) and Cre-exposed wt MEFs (15+5%) at 16 hrs post irradiation, whereas there was no significant difference for ARID1Af/for wt MEFs with or without Cre at baseline (all 50%). This data indicates that ARID1A loss slows the repair kinetics of NHEJ and delays the clearance of DSB. We conclude that ARID1A is recurrently and significantly mutated in FL. Most mutations are disruptive leading to functionally relevant ARID1A protein haplodeficiency. ARID1A loss impairs NHEJ, which might sensitize tumors to DNA damaging agents and irradiation, but also contribute to genetic and genomic instability promoting histologic transformation. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Follicular Lymphoma (FL) is the second most common non-Hodgkin lymphoma and remains an incurable disease for the majority of patients who present with advanced stage disease. Virtually all patients subsequently acquire treatment resistance and a third of patients ultimately develop histologically transformed disease with aggressive clinical course and poor prognosis. Thus, there is an unmet clinical need to decipher the underlying biology of this disease, and identify dysregulated pathways that may serve as therapeutic targets. In a cohort of 258 FL specimens, we identified 33 cases with STAT6 mutations (12.8%), including 15 mutations at D419. Overall, 34 out of 35 (97.1%) of STAT6 mutations were within the DNA-binding domain. MutSigCV analysis (Lawrence, Nature 2013) indicated that STAT6 was significantly mutated in FL, i.e., more commonly mutated than expected based on background mutation rate. Previously, STAT6 mutations, including D419 hotspot mutations, have been reported in other lymphomas, including primary mediastinal B-cell lymphoma (Ritz et al., Blood 2009) and FL (Yildiz et al., Blood 2015). STAT6 proteins are transcription factors that are mainly activated by interleukin-4 (IL-4). A recent study identified a subset of IL-4 producing follicular helper T cells to be involved in the survival of FL B cells (Ame-Thomas et al., Blood 2015). We compared the expression levels of selected STAT6 target genes in FL patient samples with or without STAT6 mutations. Gene expression data (nCounter, Nanostring) was available for 138 patient samples with known STAT6 mutation status. Thereof, 13 cases had STAT6 mutations, including six at D419. Among the STAT6 target genes were CD23 (and its soluble form, sCD23) and SOCS1. Both showed significantly higher expression (P

Beschreibung: Hematopoietic stem cells (HSCs) are maintained for their two defining properties, a self-renewal and a multi-lineage differentiation ability, under various epigenetic processes. One of the epigenetic processes essential for HSC is a lysine acetylation, which was demonstrated in several previous studies utilizing knockout mice of a gene encoding a lysine acetyltransferase (KAT), such as Cbp/p300, Moz, or Mof. The lysine acetyl transferase 5, Kat5 (also called Tip60), is a member of MYST KAT family, defined by a protein containing a C2HC-type zinc finger and an acetyl-CoA binding domain, consisting of five members: Tip60, Moz, Morf, Hbo1, and Mof. Tip60 regulates gene transcription, genome stability, cell growth and apoptosis and has been demonstrated to be essential for embryonic development. To assess the role of Tip60 in murine HSC, we generated Tip60 conditional knockout mice in two different Cre strains (Mx1-Cre and Vav-iCre), wherein Tip60 gene was successfully deleted in HSCs. Tip60 abrogation induced embryonic and adult lethality due to hematopoietic failure. Remarkably, HSCs were rapidly depleted upon Tip60 disruption, exhibiting robust apoptosis, aberrant cell cycle progression, and accumulated DNA damages. Tip60Δ/Δ fetal LSK cells transduced with retroviruses expressing wild-type Tip60 recovered a long-term hematopoiesis in transplantation experiments, whereas acetyl transferase defective Tip60 did not rescue the reconstitution to any detectable level, suggesting that the Tip60 acetyltransferase activity is essential for HSC function. Gene set enrichment analysis in RNA-seq demonstrated that Tip60 was significantly associated with genes involved in cell-cycle and DNA repair process, and Myc transcriptional factors target gene sets. A genome-wide chromatin profiling corroborated these findings, uncovering a strong similarity between Tip60 and c-Myc binding genomic regions. Considering similarities of HSC phenotype between our Tip60 conditional knockout mice and c-myc and N-myc double knockout mice (Laurenti, E. et al., Cell Stem Cell 2008), Tip60 maintains HSC by regulating Myc target genes. We next evaluated alteration in histone modifications evoked by Tip60 deletion, performing ChIP-seq analysis in Tip60f/f; Rosa26-CreERT2 and Tip60f/f fetal c-Kit+ cells that were treated with 4-OHT. Intriguingly, we found that acetylated H2A.Z was enriched at the Tip60-bound active chromatin and Tip60 deletion reduced the acetylation level of H2A.Z at Myc target genes promoters, whereas neither H3K27 acetylation level, active promoter / enhancer mark, nor H3K27 tri-methylation level, repressive mark, were altered. Collectively, our results demonstrate that Tip60 - H2A.Z could be an epigenetic axis critical for active transcription of Myc target genes to maintain murine HSC. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Key Points Inhibition of HSP90 targets multiple dependences in mantle cell lymphoma. Clinically available HSP90 inhibitors overcome ibrutinib resistance in vitro and in vivo.

Beschreibung: Hematopoietic stem cells are capable of perpetual self-renewal and multi-lineage differentiation, properties that are maintained throughout life by minimal cell cycle activity. Our work has focused on deciphering transcriptional driven differentiation versus self-renewal pathways in stem and progenitor cells. To this end, we have studied transcription factors that control the fate of hematopoietic stem cells by combining mouse models of activated self-renewal with models that can report transcription factor expression. We chose to study the Wnt pathway, activated in several types of leukemia, in combination with the ets family PU.1 transcription factor, vital to almost all myeloid and lymphoid lineages. PU.1 regulates a number of important myeloid specific genes that mediate differentiation to a specific cell fate. To understand the interaction of these pathways, we found that over-expression of Wnt signaling or beta-catenin, the downstream signaling component of the Wnt pathway, was able to inhibit PU.1-mediated differentiation in a PU.1-inducible cell line. There was little to no up-regulation of the myeloid markers Mac1 or Gr1 with activation of Wnt signaling upon induction with 4-hydroxy-tamoxifen (4-OHT). Additionally, many genes related to myeloid differentiation were not increased as compared to control-induced cultures. To understand how these interactions might function in vitro, we crossed a Cre-responsive activated beta-catenin (floxed allele Exon3) mouse to a PU.1-GFP knock-in mouse. From this model, we are able to see changes in PU.1 (GFP) expression in specific populations of hematopoietic progenitors upon activation of beta-catenin. Most importantly, in the LT-HSCs (defined by Lin- cKitHi Sca1+ CD150+ CD48-), we observed a significant increase in GFP (PU.1) intensity upon activation of active beta-catenin. Additionally, there was an increase in the total number of LT-HSCs, as defined by surface markers. LT-HSCs with active beta-catenin and GFP (PU.1) were found to be more in cycle and they express lower levels of transcription factors related to differentiation. These results demonstrate that when beta-catenin is activated, PU.1’s role is modified and the self-renewal program is enhanced at the expense of differentiation. Furthermore, activation of beta-catenin in the hematopoietic cells of mice has been shown to lead to impaired differentiation and eventual death. Even though active beta-catenin has been shown to be essential in several subtypes of myeloid leukemias using murine models, its over-expression is not sufficient to lead to leukemic development. However, heterozygous PU.1/GFP knock-in mice were crossed to the beta-catenin overexpression model, they rapidly developed leukemia post Cre induction. This is not observed in the PU.1/GFP knock-in mice in the absence of beta-catenin activation, suggesting that Wnt signaling adds to a block in differentiation needed for leukemic transformation. These mice show splenomegaly and increased myelocytic populations in the peripheral blood. The leukemia was transplantable to secondary mice and expressed high levels of GFP (PU.1) in the spleen, bone marrow and peripheral blood. These findings demonstrate that the interaction and crosstalk between these two pathways regulate hematopoietic stem cell fate. Future studies will focus on understanding how this interaction between transcription factor and self-renewal pathways becomes disrupted in leukemic stem cells. Disclosures No relevant conflicts of interest to declare.