Lysine acetyltransferase Tip60 is required for hematopoietic stem cell maintenance.

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.Cancer Research UK, Wellcome Trust, National Institutes of Health, Singapore state fundin

Rap1 regulates hematopoietic stem cell survival and affects oncogenesis and response to chemotherapy

Khattar, E., Maung, K.Z.Y., Chew, C.L. et al. Rap1 regulates hematopoietic stem cell survival and affects oncogenesis and response to chemotherapy. Nat Commun 10, 5349 (2019). https://doi.org/10.1038/s41467-019-13082-

An Inhibitory Role for Human CD96 Endodomain in T Cell Anti-Tumor Responses

Immune checkpoint blockade (ICB) therapy involves the inhibition of immune checkpoint regulators which reverses their limitation of T cell anti-tumor responses and results in long-lasting tumor regression. However, poor clinical response or tumor relapse was observed in some patients receiving such therapy administered via antibodies blocking the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or the programmed cell death 1 (PD-1) pathway alone or in combination, suggesting the involvement of additional immune checkpoints. CD96, a possible immune checkpoint, was previously shown to suppress natural killer (NK) cell anti-tumor activity but its role in human T cells remains controversial. Here, we demonstrate that CRISPR/Cas9-based deletion of CD96 in human T cells enhanced their killing of leukemia cells in vitro. T cells engineered with a chimeric antigen receptor (CAR) comprising human epidermal growth factor receptor 2 (EGFR2/HER2)-binding extracellular region and intracellular regions of CD96 and CD3ζ (4D5-96z CAR-T cells) were less effective in suppressing the growth of HER2-expressing tumor cells in vitro and in vivo compared with counterparts bearing CAR that lacked CD96 endodomain (4D5-z CAR-T cells). Together, our findings implicate a role for CD96 endodomain in attenuating T cell cytotoxicity and support combination tumor immunotherapy targeting multiple rather than single immune checkpoints

An Inhibitory Role for Human CD96 Endodomain in T Cell Anti-Tumor Responses

Immune checkpoint blockade (ICB) therapy involves the inhibition of immune checkpoint regulators which reverses their limitation of T cell anti-tumor responses and results in long-lasting tumor regression. However, poor clinical response or tumor relapse was observed in some patients receiving such therapy administered via antibodies blocking the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) or the programmed cell death 1 (PD-1) pathway alone or in combination, suggesting the involvement of additional immune checkpoints. CD96, a possible immune checkpoint, was previously shown to suppress natural killer (NK) cell anti-tumor activity but its role in human T cells remains controversial. Here, we demonstrate that CRISPR/Cas9-based deletion of CD96 in human T cells enhanced their killing of leukemia cells in vitro. T cells engineered with a chimeric antigen receptor (CAR) comprising human epidermal growth factor receptor 2 (EGFR2/HER2)-binding extracellular region and intracellular regions of CD96 and CD3ζ (4D5-96z CAR-T cells) were less effective in suppressing the growth of HER2-expressing tumor cells in vitro and in vivo compared with counterparts bearing CAR that lacked CD96 endodomain (4D5-z CAR-T cells). Together, our findings implicate a role for CD96 endodomain in attenuating T cell cytotoxicity and support combination tumor immunotherapy targeting multiple rather than single immune checkpoints

Hematopoiesis specific loss of Cdk2 and Cdk4 results in increased erythrocyte size and delayed platelet recovery following stress.

Mouse knockouts of Cdk2 and Cdk4 are individually viable whereas the double knockouts are embryonic lethal due to heart defects, and this precludes the investigation of their overlapping roles in definitive hematopoiesis. Here we use a conditional knockout mouse model to investigate the effect of combined loss of Cdk2 and Cdk4 in hematopoietic cells. Cdk2(fl/fl)Cdk4(-/-)vavCre mice are viable but displayed a significant increase in erythrocyte size. Cdk2(fl/fl)Cdk4(-/-)vavCre mouse bone marrow exhibited reduced phosphorylation of the retinoblastoma protein and reduced expression of E2F target genes such as cyclin A2 and Cdk1. Erythroblasts lacking Cdk2 and Cdk4 displayed a lengthened G1 phase due to impaired phosphorylation of the retinoblastoma protein. Deletion of the retinoblastoma protein rescued the increased size displayed by erythrocytes lacking Cdk2 and Cdk4, indicating that the retinoblastoma/Cdk2/Cdk4 pathway regulates erythrocyte size. The recovery of platelet counts following a 5-fluorouracil challenge was delayed in Cdk2(fl/fl)Cdk4(-/-)vavCre mice revealing a critical role for Cdk2 and Cdk4 in stress hematopoiesis. Our data indicate that Cdk2 and Cdk4 play important overlapping roles in homeostatic and stress hematopoiesis, which need to be considered when using broad-spectrum cyclin-dependent kinase inhibitors for cancer therapy.info:eu-repo/semantics/publishe

Kinetin riboside preferentially induces apoptosis by modulating Bcl-2 family proteins and caspase-3 in cancer cells

Here, we demonstrate that kinetin riboside (KR), a cytokinin analog, induces apoptosis in HeLa and mouse melanoma B16F-10 cells. KR disrupted the mitochondrial membrane potential and induced the release of cytochrome c and activation of caspase-3. Bad were upregulated while Bcl-2 was down-regulated under KR exposure. A tumor growth in mice was dramatically suppressed by KR. In contrast, human skin fibroblast CCL-116 and bovine primary fibroblast cells show resistances to KR and no significant changes in Bad, Bcl-XL, and cleaved PARP were observed. Our data suggest that KR selectively induces apoptosis in cancer cells through the classical mitochondria dependent apoptosis pathway.Accepted versio

Hematopoietic stem cell enhancer: a powerful tool in stem cell biology

There has been considerable interest in identifying a cis-regulatory element that targets gene expression to stem cells. Such an element, termed stem cell enhancer, holds the promise of providing important insights into the transcriptional programs responsible for inherent stem cell-specific properties such as selfrenewal capacity. The element also serves as a molecular handle for stem cell-specific marking, transgenesis and gene targeting, thereby becoming invaluable to stem cell research. A series of candidate enhancers have been identified for hematopoietic stem cells (HSCs). This review summarizes currently known HSC enhancers with emphasis on an intronic enhancer in the Runx1 gene which is critical for the generation and maintenance of HSCs. The element, named eR1 (+24m), is active specifically in HSCs, but not in progenitors, and is hence the most definitive HSC enhancer

Cyclin A2 regulates erythrocyte morphology and numbers

<p>Cyclin A2 is an essential gene for development and in haematopoietic stem cells and therefore its functions in definitive erythropoiesis have not been investigated. We have ablated cyclin A2 in committed erythroid progenitors in vivo using erythropoietin receptor promoter-driven Cre, which revealed its critical role in regulating erythrocyte morphology and numbers. Erythroid-specific cyclin A2 knockout mice are viable but displayed increased mean erythrocyte volume and reduced erythrocyte counts, as well as increased frequency of erythrocytes containing Howell-Jolly bodies. Erythroblasts lacking cyclin A2 displayed defective enucleation, resulting in reduced production of enucleated erythrocytes and increased frequencies of erythrocytes containing nuclear remnants. Deletion of the Cdk inhibitor p27^Kip1 but not Cdk2, ameliorated the erythroid defects resulting from deficiency of cyclin A2, confirming the critical role of cyclin A2/Cdk activity in erythroid development. Loss of cyclin A2 in bone marrow cells in semisolid culture prevented the formation of BFU-E but not CFU-E colonies, uncovering its essential role in BFU-E function. Our data unveils the critical functions of cyclin A2 in regulating mammalian erythropoiesis.</p

Disruption of Runx1 and Runx3 Leads to Bone Marrow Failure and Leukemia Predisposition due to Transcriptional and DNA Repair Defects

The RUNX genes encode transcription factors involved in development and human disease. RUNX1 and RUNX3 are frequently associated with leukemias, yet the basis for their involvement in leukemogenesis is not fully understood. Here, we show that Runx1;Runx3 double-knockout (DKO) mice exhibited lethal phenotypes due to bone marrow failure and myeloproliferative disorder. These contradictory clinical manifestations are reminiscent of human inherited bone marrow failure syndromes such as Fanconi anemia (FA), caused by defective DNA repair. Indeed, Runx1;Runx3 DKO cells showed mitomycin C hypersensitivity, due to impairment of monoubiquitinated-FANCD2 recruitment to DNA damage foci, although FANCD2 monoubiquitination in the FA pathway was unaffected. RUNX1 and RUNX3 interact with FANCD2 independently of CBFβ, suggesting a nontranscriptional role for RUNX in DNA repair. These findings suggest that RUNX dysfunction causes DNA repair defect, besides transcriptional misregulation, and promotes the development of leukemias and other cancers

Stem cell exhaustion due to Runx1 deficiency is prevented by Evi5 activation in leukemogenesis

The RUNX1/AML1 gene is the most frequently mutated gene in human leukemia. Conditional deletion of Runx1 in adult mice results in an increase of hematopoietic stem cells (HSCs), which serve as target cells for leukemia; however, Runx1−/− mice do not develop spontaneous leukemia. Here we show that maintenance of Runx1−/− HSCs is compromised, progressively resulting in HSC exhaustion. In leukemia development, the stem cell exhaustion was rescued by additional genetic changes. Retroviral insertional mutagenesis revealed Evi5 activation as a cooperating genetic alteration and EVI5 overexpression indeed prevented Runx1−/− HSC exhaustion in mice. Moreover, EVI5 was frequently overexpressed in human RUNX1-related leukemias. These results provide insights into the mechanism for maintenance of pre-leukemic stem cells and may provide a novel direction for therapeutic applications