MNDA controls the expression of MCL-1 and BCL-2 in chronic lymphocytic leukemia cells.

The myeloid nuclear differentiation antigen (MNDA) is a stress-induced protein that promotes degradation of the anti-apoptotic factor MCL-1 and apoptosis in myeloid cells. MNDA is also expressed in normal lymphoid cells and in B-cell clones isolated from individuals with chronic lymphocytic leukemia (CLL), a disease characterized by abnormal apoptosis control. We found that MNDA expression levels inversely correlate with the amount of the anti-apoptotic proteins MCL-1 and BCL-2 in human CLL samples. We report that in response to chemotherapeutic agents that induce genotoxic stress, MNDA exits its typical nucleolar localization and accumulates in the nucleoplasm of CLL and lymphoid cells. Then, MNDA binds chromatin at Mcl1 and Bcl2 genes and affects the transcriptional competence of RNA polymerase II. Our data also reveal that MNDA specifically associates with Mcl1 and Bcl2 (pre-) mRNAs and favors their rapid turnover as a prompt response to genotoxic stress. We propose that this rapid dynamic tuning of RNA levels, which leads to the destabilization of Mcl1 and Bcl2 transcripts, represents a post-transcriptional mechanism of apoptosis control in CLL cells. These results provide an explanation of previous clinical data and corroborate the finding that higher MNDA expression levels in CLL are associated with a better clinical course.

High-sensitivity C-reactive protein is associated with clonal hematopoiesis of indeterminate potential.

Clonal hematopoiesis of indeterminate potential (CHIP) is predictive of hematological cancers and cardiovascular diseases, but the etiology of CHIP initiation and clonal expansion is unknown. Several lines of evidence suggest that proinflammatory cytokines may favor mutated hematopoietic stem cell expansion. To investigate the potential link between inflammation and CHIP, we performed targeted deep sequencing of 11 genes previously implicated in CHIP in 1887 subjects aged >70 years from the Montreal Heart Institute Biobank, of which 1359 had prior coronary artery disease (CAD), and 528 controls did not. We assessed association of CHIP with log transformed high-sensitivity C-reactive protein (hs-CRP), a validated biomarker of inflammation. CHIP was identified in 427 of the 1887 subjects (22.6%). CHIP mutations were more frequently identified in DNMT3A (11.6%) and TET2 (6.1%), with a higher proportion of TET2 mutations occurring in controls than in patients with CAD (9.0% vs 4.9%, P < .001). CHIP carriers had 21% higher hs-CRP levels compared with their noncarrier counterparts (eß = 1.21, 95% confidence interval [CI]: 1.08 to 1.36; P = .001). A similar effect was observed in the subgroup of patients with known CAD (eß = 1.22, 95% CI: 1.06 to 1.41; P = .005). These findings confirm the association between inflammation and CHIP. This association may open investigational avenues aimed at documenting mechanisms linking inflammation to clonal progression and ultimately supports prevention interventions to attenuate CHIP's impact on cardiovascular disease and cancer.

NPM and NPM-MLF1 interact with chromatin remodeling complexes and influence their recruitment to specific genes.

Nucleophosmin (NPM1) is frequently mutated or subjected to chromosomal translocation in acute myeloid leukemia (AML). NPM protein is primarily located in the nucleus, but the recurrent NPMc+ mutation, which creates a nuclear export signal, is characterized by cytoplasmic localization and leukemogenic properties. Similarly, the NPM-MLF1 translocation product favors the partial cytoplasmic retention of NPM. Regardless of their common cellular distribution, NPM-MLF1 malignancies engender different effects on hematopoiesis compared to NPMc+ counterparts, highlighting possible aberrant nuclear function(s) of NPM in NPMc+ and NPM-MLF1 AML. We performed a proteomic analysis and found that NPM and NPM-MLF1 interact with various nuclear proteins including subunits of the chromatin remodeling complexes ISWI, NuRD and P/BAF. Accordingly, NPM and NPM-MLF1 are recruited to transcriptionally active or repressed genes along with NuRD subunits. Although the overall gene expression program in NPM knockdown cells is similar to that resulting from NPMc+, NPM-MLF1 expression differentially altered gene transcription regulated by NPM. The abnormal gene regulation imposed by NPM-MLF1 can be characterized by the enhanced recruitment of NuRD to gene regulatory regions. Thus, different mechanisms would orchestrate the dysregulation of NPM function in NPMc+- versus NPM1-MLF1-associated leukemia.

Lineage restriction analyses in CHIP indicate myeloid bias for TET2 and multipotent stem cell origin for DNMT3A.

We analyzed DNA from polymorphonuclear (PMN) cells, monocytes, B cells, and T cells of 107 individuals with clonal hematopoiesis of indeterminate potential (CHIP) to perform lineage restriction analysis of different gene mutations. Three lineage categories were defined: myeloid (PMN with or without monocytes), myelolympho-B (myeloid and B cells), and multipotent (myeloid, B and T cells). Six individuals with aberrant patterns were excluded from analysis. Ninety-four had a single mutation (56 in DNMT3A, 24 in TET2, 7 in other genes [JAK2, ASXL1, CBL or TP53]). Fourteen had multiple mutations. The lineage restriction patterns of single DNMT3A- or TET2-mutated individuals were different. The proportion of myeloid restricted mutations was higher for TET2 (54.2%, 13 of 24) than for DNMT3A (23.2%, 13 of 56) (P < .05). It was similar for myelolympho-B category but with a 1.5 fold greater proportion of myeloid cells for TET2 individuals (P < .05). Importantly, 0% (0 of 24) of the individuals with TET2 mutation in the multipotent category in contrast to 35.7% (20 of 56) for DNMT3A (P < .01). The clone size predicted multipotent pattern for DNMT3A suggesting a time delay for extensive lineage clonal dominance. These distinctive features may be important in deciphering the transformation mechanisms of these frequent mutations.

DNMT3A and TET2 dominate clonal hematopoiesis and demonstrate benign phenotypes and different genetic predispositions.

Age-associated clonal hematopoiesis caused by acquired mutations in myeloid cancer-associated genes is highly prevalent in the normal population. Its etiology, biological impact on hematopoiesis, and oncogenic risk is poorly defined at this time. To gain insight into this phenomenon, we analyzed a cohort of 2530 related and unrelated hematologically normal individuals (ages 55 to 101 years). We used a sensitive gene-targeted deep sequencing approach to gain precision on the exact prevalence of driver mutations and the proportions of affected genes. Mutational status was correlated with biological parameters. We report a higher overall prevalence of driver mutations (13.7%), which occurred mostly (93%) in DNMT3A or TET2 and were highly age-correlated. Mutation in these 2 genes had some distinctive effects on end points. TET2 mutations were more age-dependent, associated with a modest neutropenic effect (9%, P = .012), demonstrated familial aggregation, and associated with chronic obstructive pulmonary disease. Mutations in DNMT3A had no impact on blood counts or indices. Mutational burden of both genes correlated with X-inactivation skewing but no significant association with age-adjusted telomere length reduction was documented. The discordance between the high prevalence of mutations in these 2 genes and their limited biological impact raise the question of the potential role of dysregulated epigenetic modifiers in normal aging hematopoiesis, which may include support to failing hematopoiesis.

The IKAROS interaction with a complex including chromatin remodeling and transcription elongation activities is required for hematopoiesis.

IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of Ik(NULL) hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation.

Direct protein interactions are responsible for Ikaros-GATA and Ikaros-Cdk9 cooperativeness in hematopoietic cells.

Ikaros (Ik) is a critical regulator of hematopoietic gene expression. Here, we established that the Ik interactions with GATA transcription factors and cyclin-dependent kinase 9 (Cdk9), a component of the positive transcription elongation factor b (P-TEFb), are required for transcriptional activation of Ik target genes. A detailed dissection of Ik-GATA and Ik-Cdk9 protein interactions indicated that the C-terminal zinc finger domain of Ik interacts directly with the C-terminal zinc fingers of GATA1, GATA2, and GATA3, whereas the N-terminal zinc finger domain of Ik is required for interaction with the kinase and T-loop domains of Cdk9. The relevance of these interactions was demonstrated in vivo in COS-7 and primary hematopoietic cells, in which Ik facilitated Cdk9 and GATA protein recruitment to gene promoters and transcriptional activation. Moreover, the oncogenic isoform Ik6 did not efficiently interact with Cdk9 or GATA proteins in vivo and perturbed Cdk9/P-TEFb recruitment to Ik target genes, thereby affecting transcription elongation. Finally, characterization of a novel nuclear Ik isoform revealed that Ik exon 6 is dispensable for interactions with Mi2 and GATA proteins but is essential for the Cdk9 interaction. Thus, Ik is central to the Ik-GATA-Cdk9 regulatory network, which is broadly utilized for gene regulation in hematopoietic cells.

Onset and inheritance of abnormal epigenetic regulation in hematopoietic cells.

Abnormal epigenetic regulation of gene expression contributes significantly to a variety of human pathologies including cancer. Deletion of hypersensitive site 2 (HS2) at the human beta-globin locus control region can lead to abnormal epigenetic regulation of globin genes in transgenic mice. Here, two HS2-deleted transgenic mouse lines were used as model to demonstrate that heritable alteration of chromatin organization at the human beta-globin locus in multipotent hematopoietic progenitors contributes to the abnormal expression of the beta-globin gene in mature erythroid cells. This alteration is characterized by specific patterns of histone covalent modifications that are inherited during erythropoiesis and, moreover, is plastic because it can be reverted by transient treatment with the histone deacetylase inhibitor Trichostatin A. Altogether, our results indicate that aberrant epigenetic regulation can be detected and modified before tissue-specific gene transcription, a finding which may lead to novel strategies for the prevention of chromatin-related pathologies.