Implications of stress-induced gene expression for hematopoietic stem cell aging studies.

A decline in hematopoietic stem cell (HSC) function is believed to underlie hematological shortcomings with age; however, a comprehensive molecular understanding of these changes is currently lacking. Here we provide evidence that a transcriptional signature reported in several previous studies on HSC aging is linked to stress-induced changes in gene expression rather than aging. Our findings have strong implications for the design and interpretation of HSC aging studies.

FOXO1 and FOXO3 Cooperatively Regulate Innate Lymphoid Cell Development.

Natural killer (NK) cells play roles in viral clearance and early surveillance against malignant transformation, yet our knowledge of the underlying mechanisms controlling their development and functions remain incomplete. To reveal cell fate-determining pathways in NK cell progenitors (NKP), we utilized an unbiased approach and generated comprehensive gene expression profiles of NK cell progenitors. We found that the NK cell program was gradually established in the CLP to preNKP and preNKP to rNKP transitions. In line with FOXO1 and FOXO3 being co-expressed through the NK developmental trajectory, the loss of both perturbed the establishment of the NK cell program and caused stalling in both NK cell development and maturation. In addition, we found that the combined loss of FOXO1 and FOXO3 caused specific changes to the composition of the non-cytotoxic innate lymphoid cell (ILC) subsets in bone marrow, spleen, and thymus. By combining transcriptome and chromatin profiling, we revealed that FOXO TFs ensure proper NK cell development at various lineage-commitment stages through orchestrating distinct molecular mechanisms. Combined FOXO1 and FOXO3 deficiency in common and innate lymphoid cell progenitors resulted in reduced expression of genes associated with NK cell development including ETS-1 and their downstream target genes. Lastly, we found that FOXO1 and FOXO3 controlled the survival of committed NK cells via gene regulation of IL-15Rß (CD122) on rNKPs and bone marrow NK cells. Overall, we revealed that FOXO1 and FOXO3 function in a coordinated manner to regulate essential developmental genes at multiple stages during murine NK cell and ILC lineage commitment.

FOXO Dictates Initiation of B Cell Development and Myeloid Restriction in Common Lymphoid Progenitors.

The development of B cells relies on an intricate network of transcription factors critical for developmental progression and lineage commitment. In the B cell developmental trajectory, a temporal switch from predominant Foxo3 to Foxo1 expression occurs at the CLP stage. Utilizing VAV-iCre mediated conditional deletion, we found that the loss of FOXO3 impaired B cell development from LMPP down to B cell precursors, while the loss of FOXO1 impaired B cell commitment and resulted in a complete developmental block at the CD25 negative proB cell stage. Strikingly, the combined loss of FOXO1 and FOXO3 resulted in the failure to restrict the myeloid potential of CLPs and the complete loss of the B cell lineage. This is underpinned by the failure to enforce the early B-lineage gene regulatory circuitry upon a predominantly pre-established open chromatin landscape. Altogether, this demonstrates that FOXO3 and FOXO1 cooperatively govern early lineage restriction and initiation of B-lineage commitment in CLPs.

The Concerted Action of E2-2 and HEB Is Critical for Early Lymphoid Specification.

The apparition of adaptive immunity in Gnathostomata correlates with the expansion of the E-protein family to encompass E2-2, HEB, and E2A. Within the family, E2-2 and HEB are more closely evolutionarily related but their concerted action in hematopoiesis remains to be explored. Here we show that the combined disruption of E2-2 and HEB results in failure to express the early lymphoid program in Common lymphoid precursors (CLPs) and a near complete block in B-cell development. In the thymus, Early T-cell progenitors (ETPs) were reduced and T-cell development perturbed, resulting in reduced CD4 T- and increased γδ T-cell numbers. In contrast, hematopoietic stem cells (HSCs), erythro-myeloid progenitors, and innate immune cells were unaffected showing that E2-2 and HEB are dispensable for the ancestral hematopoietic lineages. Taken together, this E-protein dependence suggests that the appearance of the full Gnathostomata E-protein repertoire was critical to reinforce the gene regulatory circuits that drove the emergence and expansion of the lineages constituting humoral immunity.

Cytotoxicity Assessment and Apoptosis-related Gene Profiling of Antibody Treated Acute Myeloid Leukemia (AML) and Acute Lymphocytic Leukemia (ALL) Cancerous Cell Lines.

Acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) are common acute leukemia in adults and children, respectively. In these malignancies, chemotherapy is the main treatment strategy that fails in many cases and is usually associated with adverse effects on healthy cells. In this regard, the development of new therapies is essential. Monoclonal antibodies directed to the cell surface markers of leukemic blasts may have promising consequences with minimal toxic effects on normal cells. Since cluster of differentiation 45Ra (CD45Ra) and CD123 antigens, two considered surface markers of leukemic blasts in AML and ALL respectively, are overexpressed on AML and ALL blasts, CD34+ leukemic progenitors, and AML-LSCs in comparison with normal hematopoietic stem cells (HSCs), they were selected to be targeted; using specific monoclonal antibodies. In this project, CD45Ra+ cells and CD123+ cells were targeted by anti-CD45Ra and/or anti-CD123 monoclonal antibodies. Cytotoxicity effect and cell death induction was determined by 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Changes in the expression profile of MCL1, cMyc, Survivin, Id1, and PIM1 genes were assessed by real-time PCR. Statistical analysis of the results showed effective antibody-mediated cytotoxicity and induction of apoptosis in KG1α (CD45Ra+) and Nalm6 (CD123+) cell lines. Also, a significant change in the expression level of some of the apoptosis-related genes was observed. According to the results of this study, it can be concluded that an effective targeting of AML and ALL cancerous cell lines can be performed by anti-CD45Ra and anti-CD123 monoclonal antibodies through their effector functions and apoptosis induction.

The histone chaperone NAP1L3 is required for haematopoietic stem cell maintenance and differentiation.

Nucleosome assembly proteins (NAPs) are histone chaperones with an important role in chromatin structure and epigenetic regulation of gene expression. We find that high gene expression levels of mouse Nap1l3 are restricted to haematopoietic stem cells (HSCs) in mice. Importantly, with shRNA or CRISPR-Cas9 mediated loss of function of mouse Nap1l3 and with overexpression of the gene, the number of colony-forming cells and myeloid progenitor cells in vitro are reduced. This manifests as a striking decrease in the number of HSCs, which reduces their reconstituting activities in vivo. Downregulation of human NAP1L3 in umbilical cord blood (UCB) HSCs impairs the maintenance and proliferation of HSCs both in vitro and in vivo. NAP1L3 downregulation in UCB HSCs causes an arrest in the G0 phase of cell cycle progression and induces gene expression signatures that significantly correlate with downregulation of gene sets involved in cell cycle regulation, including E2F and MYC target genes. Moreover, we demonstrate that HOXA3 and HOXA5 genes are markedly upregulated when NAP1L3 is suppressed in UCB HSCs. Taken together, our findings establish an important role for NAP1L3 in HSC homeostasis and haematopoietic differentiation.

The chromatin-remodeling factor CHD4 is required for maintenance of childhood acute myeloid leukemia.

Epigenetic alterations contribute to leukemogenesis in childhood acute myeloid leukemia and therefore are of interest for potential therapeutic strategies. Herein, we performed large-scale ribonucleic acid interference screens using small hairpin ribonucleic acids in acute myeloid leukemia cells and non-transformed bone marrow cells to identify leukemia-specific dependencies. One of the target genes displaying the strongest effects on acute myeloid leukemia cell growth and less pronounced effects on nontransformed bone marrow cells, was the chromatin remodeling factor CHD4 Using ribonucleic acid interference and CRISPR-Cas9 approaches, we showed that CHD4 was essential for cell growth of leukemic cells in vitro and in vivo Loss of function of CHD4 in acute myeloid leukemia cells caused an arrest in the G0 phase of the cell cycle as well as downregulation of MYC and its target genes involved in cell cycle progression. Importantly, we found that inhibition of CHD4 conferred anti-leukemic effects on primary childhood acute myeloid leukemia cells and prevented disease progression in a patient-derived xenograft model. Conversely, CHD4 was not required for growth of normal hematopoietic cells. Taken together, our results identified CHD4 as a potential therapeutic target in childhood acute myeloid leukemia.

Mice deficient of Myc super-enhancer region reveal differential control mechanism between normal and pathological growth.

The gene desert upstream of the MYC oncogene on chromosome 8q24 contains susceptibility loci for several major forms of human cancer. The region shows high conservation between human and mouse and contains multiple MYC enhancers that are activated in tumor cells. However, the role of this region in normal development has not been addressed. Here we show that a 538 kb deletion of the entire MYC upstream super-enhancer region in mice results in 50% to 80% decrease in Myc expression in multiple tissues. The mice are viable and show no overt phenotype. However, they are resistant to tumorigenesis, and most normal cells isolated from them grow slowly in culture. These results reveal that only cells whose MYC activity is increased by serum or oncogenic driver mutations depend on the 8q24 super-enhancer region, and indicate that targeting the activity of this element is a promising strategy of cancer chemoprevention and therapy.

Niche-mediated depletion of the normal hematopoietic stem cell reservoir by Flt3-ITD-induced myeloproliferation.

Although previous studies suggested that the expression of FMS-like tyrosine kinase 3 (Flt3) initiates downstream of mouse hematopoietic stem cells (HSCs), FLT3 internal tandem duplications (FLT3 ITDs) have recently been suggested to intrinsically suppress HSCs. Herein, single-cell interrogation found Flt3 mRNA expression to be absent in the large majority of phenotypic HSCs, with a strong negative correlation between Flt3 and HSC-associated gene expression. Flt3-ITD knock-in mice showed reduced numbers of phenotypic HSCs, with an even more severe loss of long-term repopulating HSCs, likely reflecting the presence of non-HSCs within the phenotypic HSC compartment. Competitive transplantation experiments established that Flt3-ITD compromises HSCs through an extrinsically mediated mechanism of disrupting HSC-supporting bone marrow stromal cells, with reduced numbers of endothelial and mesenchymal stromal cells showing increased inflammation-associated gene expression. Tumor necrosis factor (TNF), a cell-extrinsic potent negative regulator of HSCs, was overexpressed in bone marrow niche cells from FLT3-ITD mice, and anti-TNF treatment partially rescued the HSC phenotype. These findings, which establish that Flt3-ITD-driven myeloproliferation results in cell-extrinsic suppression of the normal HSC reservoir, are of relevance for several aspects of acute myeloid leukemia biology.

Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model.

X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

Lymphomyeloid contribution of an immune-restricted progenitor emerging prior to definitive hematopoietic stem cells.

In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the innate immune system are formed early in development, whereas lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we demonstrate that the embryonic lymphoid commitment process initiates earlier than previously appreciated, prior to emergence of definitive HSCs, through establishment of a previously unrecognized entirely immune-restricted and lymphoid-primed progenitor. Notably, this immune-restricted progenitor appears to first emerge in the yolk sac and contributes physiologically to the establishment of lymphoid and some myeloid components of the immune-system, establishing the lymphomyeloid lineage restriction process as an early and physiologically important lineage-commitment step in mammalian hematopoiesis.

FLT3-ITDs instruct a myeloid differentiation and transformation bias in lymphomyeloid multipotent progenitors.

Whether signals mediated via growth factor receptors (GFRs) might influence lineage fate in multipotent progenitors (MPPs) is unclear. We explored this issue in a mouse knockin model of gain-of-function Flt3-ITD mutation because FLT3-ITDs are paradoxically restricted to acute myeloid leukemia even though Flt3 primarily promotes lymphoid development during normal hematopoiesis. When expressed in MPPs, Flt3-ITD collaborated with Runx1 mutation to induce high-penetrance aggressive leukemias that were exclusively of the myeloid phenotype. Flt3-ITDs preferentially expanded MPPs with reduced lymphoid and increased myeloid transcriptional priming while compromising early B and T lymphopoiesis. Flt3-ITD-induced myeloid lineage bias involved upregulation of the transcription factor Pu.1, which is a direct target gene of Stat3, an aberrantly activated target of Flt3-ITDs, further establishing how lineage bias can be inflicted on MPPs through aberrant GFR signaling. Collectively, these findings provide new insights into how oncogenic mutations might subvert the normal process of lineage commitment and dictate the phenotype of resulting malignancies.

Impact of gene dosage, loss of wild-type allele, and FLT3 ligand on Flt3-ITD-induced myeloproliferation.

Acquisition of homozygous activating growth factor receptor mutations might accelerate cancer progression through a simple gene-dosage effect. Internal tandem duplications (ITDs) of FLT3 occur in approximately 25% cases of acute myeloid leukemia and induce ligand-independent constitutive signaling. Homozygous FLT3-ITDs confer an adverse prognosis and are frequently detected at relapse. Using a mouse knockin model of Flt3-internal tandem duplication (Flt3-ITD)-induced myeloproliferation, we herein demonstrate that the enhanced myeloid phenotype and expansion of granulocyte-monocyte and primitive Lin(-)Sca1(+)c-Kit(+) progenitors in Flt3-ITD homozygous mice can in part be mediated through the loss of the second wild-type allele. Further, whereas autocrine FLT3 ligand production has been implicated in FLT3-ITD myeloid malignancies and resistance to FLT3 inhibitors, we demonstrate here that the mouse Flt3(ITD/ITD) myeloid phenotype is FLT3 ligand-independent.

Expression and role of FLT3 in regulation of the earliest stage of normal granulocyte-monocyte progenitor development.

Mice deficient in c-fms-like tyrosine kinase 3 (FLT3) signaling have reductions in early multipotent and lymphoid progenitors, whereas no evident myeloid phenotype has been reported. However, activating mutations of Flt3 are among the most common genetic events in acute myeloid leukemia and mice harboring internal tandem duplications within Flt3 (Flt3-ITD) develop myeloproliferative disease, with characteristic expansion of granulocyte-monocyte (GM) progenitors (GMP), possibly compatible with FLT3-ITD promoting a myeloid fate of multipotent progenitors. Alternatively, FLT3 might be expressed at the earliest stages of GM development. Herein, we investigated the expression, function, and role of FLT3 in recently identified early GMPs. Flt3-cre fate-mapping established that most progenitors and mature progeny of the GM lineage are derived from Flt3-expressing progenitors. A higher expression of FLT3 was found in preGMP compared with GMP, and preGMPs were more responsive to stimulation with FLT3 ligand (FL). Whereas preGMPs and GMPs were reduced in Fl(-/-) mice, megakaryocyte-erythroid progenitors were unaffected and lacked FLT3 expression. Notably, mice deficient in both thrombopoietin (THPO) and FL had a more pronounced GMP phenotype than Thpo(-/-) mice, establishing a role of FL in THPO-dependent and -independent regulation of GMPs, of likely significance for myeloid malignancies with Flt3-ITD mutations.

DNA methylation protects hematopoietic stem cell multipotency from myeloerythroid restriction.

DNA methylation is a dynamic epigenetic mark that undergoes extensive changes during differentiation of self-renewing stem cells. However, whether these changes are the cause or consequence of stem cell fate remains unknown. Here, we show that alternative functional programs of hematopoietic stem cells (HSCs) are governed by gradual differences in methylation levels. Constitutive methylation is essential for HSC self-renewal but dispensable for homing, cell cycle control and suppression of apoptosis. Notably, HSCs from mice with reduced DNA methyltransferase 1 activity cannot suppress key myeloerythroid regulators and thus can differentiate into myeloerythroid, but not lymphoid, progeny. A similar methylation dosage effect controls stem cell function in leukemia. These data identify DNA methylation as an essential epigenetic mechanism to protect stem cells from premature activation of predominant differentiation programs and suggest that methylation dynamics determine stem cell functions in tissue homeostasis and cancer.

FLT3 ligand and not TSLP is the key regulator of IL-7-independent B-1 and B-2 B lymphopoiesis.

Phenotypically and functionally distinct progenitors and developmental pathways have been proposed to exist for fetally derived B-1 and conventional B-2 cells. Although IL-7 appears to be the primary cytokine regulator of fetal and adult B lymphopoiesis in mice, considerable fetal B lymphopoiesis and postnatal B cells are sustained in the absence of IL-7; in humans, B-cell generation is suggested to be largely IL-7-independent, as severe combined immune-deficient patients with IL-7 deficiency appear to have normal B-cell numbers. However, the role of other cytokines in IL-7-independent B lymphopoiesis remains to be established. Although thymic stromal lymphopoietin (TSLP) has been proposed to be the main factor driving IL-7-independent B lymphopoiesis and to distinguish fetal from adult B-cell progenitor development in mice, recent studies failed to support a primary role of TSLP in IL-7-independent fetal B-cell development. However, the role of TSLP in IL-7-independent adult B lymphopoiesis and in particular in regulation of B-1 cells remains to be established. Here we demonstrate that, rather than TSLP, IL-7 and FLT3 ligand are combined responsible for all B-cell generation in mice, including recently identified B-1-specified cell progenitors. Thus, the same IL-7- and FLT3 ligand-mediated signal-ing regulates alternative pathways of fetal and adult B-1 and B-2 lymphopoiesis.

Down-regulation of Mpl marks the transition to lymphoid-primed multipotent progenitors with gradual loss of granulocyte-monocyte potential.

Evidence for a novel route of adult hematopoietic stem-cell lineage commitment through Lin-Sca-1+Kit+Flt3hi (LSKFlt3hi) lymphoid-primed multipotent progenitors (LMPPs) with granulocyte/monocyte (GM) and lymphoid but little or no megakaryocyte/erythroid (MkE) potential was recently challenged, as LSKFlt3hi cells were reported to possess MkE potential. Herein, residual (1%-2%) MkE potential segregated almost entirely with LSKFlt3hi cells expressing the thrombopoietin receptor (Mpl), whereas LSKFlt3hiMpl- LMPPs lacked significant MkE potential in vitro and in vivo, but sustained combined GM and lymphoid potentials, and coexpressed GM and lymphoid but not MkE transcriptional lineage programs. Gradually increased transcriptional lymphoid priming in single LMPPs from Rag1GFP mice was shown to occur in the presence of maintained GM lineage priming, but gradually reduced GM lineage potential. These functional and molecular findings reinforce the existence of GM/lymphoid-restricted progenitors with dramatically down-regulated probability for committing toward MkE fates, and support that lineage restriction occurs through gradual rather than abrupt changes in specific lineage potentials.

Permissive roles of hematopoietin and cytokine tyrosine kinase receptors in early T-cell development.

Although several cytokines have been demonstrated to be critical regulators of development of multiple blood cell lineages, it remains disputed to what degree they act through instructive or permissive mechanisms. Signaling through the FMS-like tyrosine kinase 3 (FLT3) receptor and the hematopoietin IL-7 receptor alpha (IL-7Ralpha) has been demonstrated to be of critical importance for sustained thymopoiesis. Signaling triggered by IL-7 and thymic stromal lymphopoietin (TSLP) is dependent on IL-7Ralpha, and both ligands have been implicated in T-cell development. However, we demonstrate that, whereas thymopoiesis is abolished in adult mice doubly deficient in IL-7 and FLT3 ligand (FLT3L), TSLP does not play a key role in IL-7-independent or FLT3L-independent T lymphopoiesis. Furthermore, whereas previous studies implicated that the role of other cytokine tyrosine kinase receptors in T lymphopoiesis might not involve permissive actions, we demonstrate that ectopic expression of BCL2 is sufficient not only to partially correct the T-cell phenotype of Flt3l(-/-) mice but also to rescue the virtually complete loss of all discernable stages of early T lymphopoiesis in Flt3l(-/-)Il7r(-/-) mice. These findings implicate a permissive role of cytokine receptors of the hematopoietin and tyrosine kinase families in early T lymphopoiesis.