The combination of CHK1 inhibitor with G-CSF overrides cytarabine resistance in human acute myeloid leukemia.

Cytarabine (AraC) represents the most effective single agent treatment for AML. Nevertheless, overriding AraC resistance in AML remains an unmet medical need. Here we show that the CHK1 inhibitor (CHK1i) GDC-0575 enhances AraC-mediated killing of AML cells both in vitro and in vivo, thus abrogating any potential chemoresistance mechanisms involving DNA repair. Importantly, this combination of drugs does not affect normal long-term hematopoietic stem/progenitors. Moreover, the addition of CHK1i to AraC does not generate de novo mutations and in patients' samples where AraC is mutagenic, addition of CHK1i appears to eliminate the generation of mutant clones. Finally, we observe that persistent residual leukemic cells are quiescent and can become responsive to the treatment when forced into cycle via granulocyte colony-stimulating factor (G-CSF) administration. This drug combination (AraC+CHK1i+G-CSF) will open the doors for a more efficient treatment of AML in the clinic.

Increased Vascular Permeability in the Bone Marrow Microenvironment Contributes to Disease Progression and Drug Response in Acute Myeloid Leukemia.

The biological and clinical behaviors of hematological malignancies can be influenced by the active crosstalk with an altered bone marrow (BM) microenvironment. In the present study, we provide a detailed picture of the BM vasculature in acute myeloid leukemia using intravital two-photon microscopy. We found several abnormalities in the vascular architecture and function in patient-derived xenografts (PDX), such as vascular leakiness and increased hypoxia. Transcriptomic analysis in endothelial cells identified nitric oxide (NO) as major mediator of this phenotype in PDX and in patient-derived biopsies. Moreover, induction chemotherapy failing to restore normal vasculature was associated with a poor prognosis. Inhibition of NO production reduced vascular permeability, preserved normal hematopoietic stem cell function, and improved treatment response in PDX.

Nuclear Factor Erythroid 2 Regulates Human HSC Self-Renewal and T Cell Differentiation by Preventing NOTCH1 Activation.

Nuclear factor erythroid-derived 2 (NF-E2) has been associated with megakaryocyte maturation and platelet production. Recently, an increased in NF-E2 activity has been implicated in myeloproliferative neoplasms. Here, we investigate the role of NF-E2 in normal human hematopoiesis. Knockdown of NF-E2 in the hematopoietic stem and progenitor cells (HSPCs) not only reduced the formation of megakaryocytes but also drastically impaired hematopoietic stem cell activity, decreasing human engraftment in immunodeficient (NSG) mice. This phenotype is likely to be related to both increased cell proliferation (p21-mediated) and reduced Notch1 protein expression, which favors HSPC differentiation over self-renewal. Strikingly, although NF-E2 silencing in HSPCs did not affect their myeloid and B cell differentiation in vivo, it almost abrogated T cell production in primary hosts, as confirmed by in vitro studies. This effect is at least partly due to Notch1 downregulation in NF-E2-silenced HSPCs. Together these data reveal that NF-E2 is an important driver of human hematopoietic stem cell maintenance and T lineage differentiation.

HDAC7 is a repressor of myeloid genes whose downregulation is required for transdifferentiation of pre-B cells into macrophages.

B lymphopoiesis is the result of several cell-commitment, lineage-choice, and differentiation processes. Every differentiation step is characterized by the activation of a new, lineage-specific, genetic program and the extinction of the previous one. To date, the central role of specific transcription factors in positively regulating these distinct differentiation processes to acquire a B cell-specific genetic program is well established. However, the existence of specific transcriptional repressors responsible for the silencing of lineage inappropriate genes remains elusive. Here we addressed the molecular mechanism behind repression of non-lymphoid genes in B cells. We report that the histone deacetylase HDAC7 was highly expressed in pre-B cells but dramatically down-regulated during cellular lineage conversion to macrophages. Microarray analysis demonstrated that HDAC7 re-expression interfered with the acquisition of the gene transcriptional program characteristic of macrophages during cell transdifferentiation; the presence of HDAC7 blocked the induction of key genes for macrophage function, such as immune, inflammatory, and defense response, cellular response to infections, positive regulation of cytokines production, and phagocytosis. Moreover, re-introduction of HDAC7 suppressed crucial functions of macrophages, such as the ability to phagocytose bacteria and to respond to endotoxin by expressing major pro-inflammatory cytokines. To gain insight into the molecular mechanisms mediating HDAC7 repression in pre-B cells, we undertook co-immunoprecipitation and chromatin immunoprecipitation experimental approaches. We found that HDAC7 specifically interacted with the transcription factor MEF2C in pre-B cells and was recruited to MEF2 binding sites located at the promoters of genes critical for macrophage function. Thus, in B cells HDAC7 is a transcriptional repressor of undesirable genes. Our findings uncover a novel role for HDAC7 in maintaining the identity of a particular cell type by silencing lineage-inappropriate genes.

C/EBPα bypasses cell cycle-dependency during immune cell transdifferentiation.

Our earlier work has shown that pre-B cells can be converted into macrophage-like cells by overexpression of the transcription factor C/EBPα or C/EBPß with high efficiency. Using inducible pre-B cell lines, we have now investigated the role of cell division during C/EBP-induced reprogramming. The majority of cells reprogrammed by C/EBPα incorporated BrdU before arresting at G(0), and all C/EBPß-induced cells incorporated the compound. This contrasts with reports from other systems where transdifferentiating cells essentially do not divide. Although inhibition of DNA synthesis led to an impairment of C/EBPα-induced transdifferentiation, sorted G(0)/G(1) and G(2)/M fractions showed no significant differences in their reprogramming kinetics. In addition, knocking-down p53 did not accelerate the transdifferentiation frequency, as it has been described for reprogramming of induced pluripotent (iPS) cells. Time-lapse experiments showed that, after C/EBPα induction, approximately 90% of cells divide once or twice, while 8% do not divide at all before acquiring a macrophage phenotype, supporting our BrdU incorporation results. Importantly, the non-dividing cell subset expressed the highest levels of C/EBPα and was the fastest in differentiating, suggesting that high levels of C/EBPα accelerate both the switching process and the cells' growth arrest. Our data show that traversing the cell cycle is not strictly required for pre-B cell to macrophage conversion and provides new evidence for the notion that the mechanisms of transcription factor induced transdifferentiation and iPS cell reprogramming differ.

Pre-B cell to macrophage transdifferentiation without significant promoter DNA methylation changes.

Transcription factor-induced lineage reprogramming or transdifferentiation experiments are essential for understanding the plasticity of differentiated cells. These experiments helped to define the specific role of transcription factors in conferring cell identity and played a key role in the development of the regenerative medicine field. We here investigated the acquisition of DNA methylation changes during C/EBPα-induced pre-B cell to macrophage transdifferentiation. Unexpectedly, cell lineage conversion occurred without significant changes in DNA methylation not only in key B cell- and macrophage-specific genes but also throughout the entire set of genes differentially methylated between the two parental cell types. In contrast, active and repressive histone modification marks changed according to the expression levels of these genes. We also demonstrated that C/EBPα and RNA Pol II are associated with the methylated promoters of macrophage-specific genes in reprogrammed macrophages without inducing methylation changes. Our findings not only provide insights about the extent and hierarchy of epigenetic events in pre-B cell to macrophage transdifferentiation but also show an important difference to reprogramming towards pluripotency where promoter DNA demethylation plays a pivotal role.

CCAAT/enhancer binding protein alpha (C/EBP(alpha))-induced transdifferentiation of pre-B cells into macrophages involves no overt retrodifferentiation.

Earlier work has shown that pre-B cells can be converted into macrophages by the transcription factor CCAAT/enhancer binding protein α at very high frequencies. Using this system, we performed a systematic analysis of whether during transdifferentiation the cells transiently reactivate progenitor-restricted genes or even retrodifferentiate. A transcriptome analysis of transdifferentiating cells showed that most genes are up- or down-regulated continuously, acquiring a macrophage phenotype within 5 d. In addition, we observed the transient reactivation of a subset of immature myeloid markers, as well as low levels of the progenitor markers Kit and FMS-like tyrosine kinase 3 and a few lineage-inappropriate genes. Importantly, however, we were unable to observe the reexpression of cell-surface marker combinations that characterize hematopoietic stem and progenitor cells, including c-Kit and FMS-like tyrosine kinase 3, even when CAAT/enhancer binding protein α was activated in pre-B cells under culture conditions that favor growth of hematopoietic stem and progenitor cells or when the transcription factor was activated in a time-limited fashion. Together, our findings are consistent with the notion that the conversion from pre-B cells to macrophages is mostly direct and does not involve overt retrodifferentiation.

Fibroblast-derived induced pluripotent stem cells show no common retroviral vector insertions.

Several laboratories have reported the reprogramming of mouse and human fibroblasts into pluripotent cells, using retroviruses carrying the Oct4, Sox2, Klf4, and c-Myc transcription factor genes. In these experiments the frequency of reprogramming was lower than 0.1% of the infected cells, raising the possibility that additional events are required to induce reprogramming, such as activation of genes triggered by retroviral insertions. We have therefore determined by ligation-mediated polymerase chain reaction (LM-PCR) the retroviral insertion sites in six induced pluripotent stem (iPS) cell clones derived from mouse fibroblasts. Seventy-nine insertion sites were assigned to a single mouse genome location. Thirty-five of these mapped to gene transcription units, whereas 29 insertions landed within 10 kilobases of transcription start sites. No common insertion site was detected among the iPS clones studied. Moreover, bioinformatics analyses revealed no enrichment of a specific gene function, network, or pathway among genes targeted by retroviral insertions. We conclude that Oct4, Sox2, Klf4, and c-Myc are sufficient to promote fibroblast-to-iPS cell reprogramming and propose that the observed low reprogramming frequencies may have alternative explanations.