�����ƽ��

The only cure of HIV has been achieved in a small number of people who received a hematopoietic stem cell transplant (HSCT) comprising allogeneic cells carrying a rare, naturally occurring, homozygous deletion in the CCR5 gene. The rarity of the mutation and the significant morbidity and mortality of such allogeneic transplants precludes widespread adoption of this HIV cure. Here, we show the application of CRISPR/Cas9 to achieve >90% CCR5 editing in human, mobilized hematopoietic stem progenitor cells (HSPC), resulting in a transplant that undergoes normal hematopoiesis, produces CCR5 null T cells, and renders xenograft mice refractory to HIV infection. Titration studies transplanting decreasing frequencies of CCR5 edited HSPCs demonstrate that <90% CCR5 editing confers decreasing protective benefit that becomes negligible between 54% and 26%. Our study demonstrates the feasibility of using CRISPR/Cas9/RNP to produce an HSPC transplant with high frequency CCR5 editing that is refractory to HIV replication. These results raise the potential of using CRISPR/Cas9 to produce a curative autologous HSCT and bring us closer to the development of a cure for HIV infection. Subject terms: HIV infections, CRISPR-Cas9 genome editing, Retrovirus, Translational research

Epigenetic modulators of the histone deacetylase (HDAC) family control key biological processes and are frequently dysregulated in cancer. There is superior activity of HDAC inhibitors (HDACi) in patients with myeloproliferative neoplasms (MPNs) that carry the Janus kinase-2 point mutant JAK2 V617F . This constitutively active tyrosine kinase activates signal-transducer-and-activator-of-transcription (STAT) transcription factors to promote cell proliferation and inflammatory processes. We reveal that the inhibition of HDAC1/HDAC2 with the clinically advanced HDACi romidepsin, the experimental HDACi entinostat and MERCK60, and genetic depletion of HDAC1/HDAC2 induce apoptosis and long-term growth arrest of primary and permanent MPN cells in vitro and in vivo. This treatment spares normal hematopoietic stem cells and does not compromise blood cell differentiation. At the molecular level, HDAC1 and HDAC2 control the protein stability of SIAH2 through acetylation. Genetic knockout experiments show that SIAH2 accelerates the proteasomal degradation of JAK2 V617F in conjunction with the E2 ubiquitin-conjugating enzyme UBCH8. SIAH2 binds to the surface-exposed SIAH degron motif VLP1002 in the catalytic domain of JAK2 V617F . At the functional level, SIAH2 knockout MPN cells are significantly less sensitive to HDACi. Global RNA sequencing verifies that JAK-STAT signaling is a prime target of SIAH2. Moreover, HDAC1 is an adverse prognostic factor in patients with acute myeloid leukemia ( n = 150, p = 0.02), being a possible complication of MPNs. These insights reveal a previously unappreciated link between HDAC1/HDAC2 as key molecular targets, the still undefined regulation of cytoplasmic-to-nuclear signaling by HDACs, and how HDACi kill JAK2 V617F -positive cells from MPN patients and mice with JAK2 V617F in vitro and in vivo. Subject terms: Haematological cancer, Oncogenes, Target identification, Haematopoietic stem cells

Low dose methotrexate (LD‐MTX) remains the gold standard in rheumatoid arthritis (RA) therapy. Multiple mechanisms on a variety of immune cells contribute to the anti‐inflammatory effects of LD‐MTX. Inflammatory signaling is deeply implicated in hematopoiesis by regulating hematopoietic stem and progenitor cell (HSPC) fate decisions; raising the question of whether HSPC are also modulated by LD‐MTX. This is the first study to characterize the effects of LD‐MTX on HSPC. CD34 + HSPC were isolated from healthy donors' non‐mobilized peripheral blood. Resting and/or cycling HSPCs were treated with LD‐MTX [dose equivalent to that used in RA patients]. Flow cytometry was performed to assess HSPC viability, cell cycle, surface abundance of reduced folate carrier 1 (RFC1), proliferation, reactive oxygen species (ROS) levels, DNA double‐strand breaks, p38 activation, and CD34 + subpopulations. HSPC clonogenicity was tested in colony‐forming cell assays. Our results indicate that in cycling HSPC, membrane RFC1 is upregulated and, following LD‐MTX treatment, they accumulate more intracellular MTX than resting HSPC. In cycling HSPC, LD‐MTX inhibits HSPC expansion by promoting S‐phase cell‐cycle arrest, increases intracellular HSPC ROS levels and DNA damage, and reduces HSPC viability. Those effects involve the activation of the p38 MAPK pathway and are rescued by folinic acid. The effects of LD‐MTX are more evident in CD34 + CD38High progenitors. In non‐cycling HSPC, LD‐MTX also reduces the proliferative response while preserving their clonogenicity. In summary, HSPC uptake LD‐MTX differentially according to their cycling state. In turn, LD‐MTX results in reduced proliferation and the preservation of HSPC clonogenicity.