Pten cell autonomously modulates the hematopoietic stem cell response to inflammatory cytokines

Summary: Pten negatively regulates the phosphatidylinositol 3-kinase (PI3K) pathway and is required to maintain quiescent adult hematopoietic stem cells (HSCs). Pten has been proposed to regulate HSCs cell autonomously and non-cell autonomously, but the relative importance of each mechanism has not been directly tested. Furthermore, the cytokines that activate the PI3K pathway upstream of Pten are not well defined. We sought to clarify whether Pten cell autonomously or non-cell autonomously regulates HSC mobilization. We also tested whether Pten deficiency affects the HSC response to granulocyte colony-stimulating factor (G-CSF) and interferon-α (IFNα) since these cytokines induce HSC mobilization or proliferation, respectively. We show that Pten regulates HSC mobilization and expansion in the spleen primarily via cell-autonomous mechanisms. Pten-deficient HSCs do not require G-CSF to mobilize, although they are hyper-sensitized to even low doses of exogenous G-CSF. Pten-deficient HSCs are similarly sensitized to IFNα. Pten therefore modulates the HSC response to inflammatory cytokines. : Magee and colleagues show that Pten suppresses HSC mobilization and extramedullary expansion primarily through cell-autonomous mechanisms. The authors also show that Pten-deficient HSCs are hyper-sensitive to mobilizing effects of G-CSF and interferon-α, even at low-cytokine concentrations. These findings suggest that a key function of Pten in HSCs is to blunt signal transduction downstream of inflammatory cytokines

The efficiency of murine MLL-ENL–driven leukemia initiation changes with age and peaks during neonatal development

MLL rearrangements are translocation mutations that cause both acute lymphoblastic leukemia and acute myeloid leukemia (AML). These translocations can occur as sole clonal driver mutations in infant leukemias, suggesting that fetal or neonatal hematopoietic progenitors may be exquisitely sensitive to transformation by MLL fusion proteins. To test this possibility, we used transgenic mice to induce one translocation product, MLL-ENL, during fetal, neonatal, juvenile and adult stages of life. When MLL-ENL was induced in fetal or neonatal mice, almost all died of AML. In contrast, when MLL-ENL was induced in adult mice, most survived for >1 year despite sustained transgene expression. AML initiation was most efficient when MLL-ENL was induced in neonates, and even transient suppression of MLL-ENL in neonates could prevent AML in most mice. MLL-ENL target genes were induced more efficiently in neonatal progenitors than in adult progenitors, consistent with the distinct AML initiation efficiencies. Interestingly, transplantation stress mitigated the developmental barrier to leukemogenesis. Since fetal/neonatal progenitors were highly competent to initiate MLL-ENL-driven AML, we tested whether Lin28b, a fetal master regulator, could accelerate leukemogenesis. Surprisingly, Lin28b suppressed AML initiation rather than accelerating it. This may explain why MLL rearrangements often occur before birth in human infant leukemia patients, but transformation usually does not occur until after birth, when Lin28b levels decline. Our findings show that the efficiency of MLLENL- driven AML initiation changes through the course of pre- and postnatal development, and developmental programs can be manipulated to impede transformation

BCL11A enhancer edited hematopoietic stem cells persist in rhesus monkeys without toxicity

Gene editing of the erythroid-specific BCL11A enhancer in hematopoietic stem and progenitor cells (HSPCs) from sickle cell disease (SCD) patients induces fetal hemoglobin (HbF) without detectable toxicity as assessed by mouse xenotransplant. Here, we evaluated autologous engraftment and HbF induction potential of erythroid-specific BCL11A enhancer edited HSPCs in four non-human primates. We utilized a single guide RNA (sgRNA) with identical human and rhesus target sequences to disrupt a GATA1 binding site at the BCL11A +58 erythroid enhancer. Cas9 protein and sgRNA ribonucleoprotein complex (RNP) was electroporated into rhesus HSPCs, followed by autologous infusion after myeloablation. We found that gene edits persisted in peripheral blood (PB) and bone marrow (BM) for up to 101 weeks similarly for BCL11A enhancer or control locus (AAVS1) targeted cells. Biallelic BCL11A enhancer editing resulted in robust gamma-globin induction, with the highest levels observed during stress erythropoiesis. Indels were evenly distributed across PB and BM lineages. Off-target edits were not observed. Non-homologous end-joining repair alleles were enriched in engrafting HSCs. In summary, we find that edited HSCs can persist for at least 101 weeks post-transplant, and biallelic edited HSCs provide substantial HbF levels in PB red blood cells, together supporting further clinical translation of this approach

Attenuation of Zinc Finger Nuclease Toxicity by Small-Molecule Regulation of Protein Levels

Zinc finger nucleases (ZFNs) have been used successfully to create genome-specific double-strand breaks and thereby stimulate gene targeting by several thousand fold. ZFNs are chimeric proteins composed of a specific DNA-binding domain linked to a non-specific DNA-cleavage domain. By changing key residues in the recognition helix of the specific DNA-binding domain, one can alter the ZFN binding specificity and thereby change the sequence to which a ZFN pair is being targeted. For these and other reasons, ZFNs are being pursued as reagents for genome modification, including use in gene therapy. In order for ZFNs to reach their full potential, it is important to attenuate the cytotoxic effects currently associated with many ZFNs. Here, we evaluate two potential strategies for reducing toxicity by regulating protein levels. Both strategies involve creating ZFNs with shortened half-lives and then regulating protein level with small molecules. First, we destabilize ZFNs by linking a ubiquitin moiety to the N-terminus and regulate ZFN levels using a proteasome inhibitor. Second, we destabilize ZFNs by linking a modified destabilizing FKBP12 domain to the N-terminus and regulate ZFN levels by using a small molecule that blocks the destabilization effect of the N-terminal domain. We show that by regulating protein levels, we can maintain high rates of ZFN-mediated gene targeting while reducing ZFN toxicity

Analysis of dd-ZFNs.

<p>Unless otherwise indicated, rates of gene targeting at day 3 were normalized to the rate of gene targeting achieved using 20 ng of the unmodified ZFNs without drug treatment as this was previously determined to be the conditions used to obtain optimal gene targeting with the unmodified ZFNs. The absolute rate of gene targeting using ZFN-1/ZFN-2 at 20 ng in <i>HEK</i>293 cells was about 20,000 GFP positive cells per million cells transfected. (A) Titration of transfected DNA of dd-ZFNs in the gene targeting assay with increasing amounts of transfected DNA. (B) Time-course experiment for length of exposure of 1000 nM Shield1 using 5 ng of dd-ZFNs. Hours are given relative to the time of transfection, where “0” is the time of transfection. (C) Drug dose response curve for Shield1 with 5 ng of dd-ZFNs. (D) Gene targeting in the absence and presence of 1000 nM Shield1 for given ZFN pairs at stated DNA concentrations in <i>HEK293</i> cells. (E) Gene targeting in the absence and presence of 1000 nM Shield1 for given ZFN pairs at stated DNA concentrations in 3T3 cells. The absolute rate of gene targeting using ZFN-1/ZFN-2 at 20 ng in 3T3 cells was about 20,000 GFP positive cells per million cells transfected (2%). (F) Gene targeting in the absence and presence of 1000 nM Shield1 for given ZFN pairs at stated DNA concentrations in HeLa cells per million cells transfected. The absolute rate of gene targeting using ZFN-1/ZFN-2 at 20 ng in HeLa cells was about 2,000 GFP positive cells per million cells transfected (0.2%). (G) Toxicity assay for all iterations of dd-modified and unmodified ZFNs tested in the gene targeting assay relative to Sce. A value of <100% indicates decreased cell survival as compared with Sce, and demonstrates a toxic effect. Statistical analysis was performed using the Student's T-test comparing ZFN-1/ZFN-2 at 20 ng with no drug treatment to dd-modified ZFNs treated with Shield1. “*” indicates a P-value of <.05 and “n.s.” indicates no statistical significance or a P-value of >.05. Error bars are the standard deviation in measurement of three samples.</p

Characterization of Ub-X-ZFNs that display drug-dependent stability.

<p>(A) Genetic fusion of a ubiquitin moiety to a ZFN with either a “VV” linker or an “R” linker. (B) Expression profile of unmodified and Ub-X-ZFN proteins in the presence and absence of 10 uM of the proteasome inhibitor MG132 from 18–22 hours post-transfection. <i>HEK</i>293FT cells were transiently transfected with vectors encoding either ZFN-1/ZFN-2, Ub-VV-ZFN-1/-2, or UB-R-ZFN-1/ZFN-2. ZFNs were detected using Western blot analysis with an anti-Flag antibody. ZFN-1/ZFN-2 and Ub-R-ZFN-1/ZFN-2 were approximately 37 kD and Ub-VV-ZFN-1/ZFN-2 were approximately 47 kD. The size difference between the Ub-X-ZFNs is due to the Ub-moiety being cleaved off when linked via an R-linker. β-actin serves as a loading control.</p

Characterization of dd-ZFNs that display Shield1-dependent stability.

<p>(A) Genetic fusion of a destabilization domain derived from an FKBP12 mutant to a ZFN. (B) Expression profile of unmodified and dd-ZFN proteins in the presence and absence of 1 uM of the Shield1 from 0–24 hours post-transfection. <i>HEK</i>293FT cells were transiently transfected with vectors encoding either ZFN-1/-2, dd-ZFN-1/-2. ZFNs were detected with an anti-Flag antibody. β-actin serves as a loading control. The molecular weight of the unmodified GFP-ZFNs was approximately 37 kD and for the dd-ZFNs approximately 50 kD.</p

Analysis of Ub-X-ZFNs.

<p>Unless otherwise indicated, rates of gene targeting at day 3 were normalized to the rate of gene targeting achieved using 20 ng of the unmodified ZFNs without drug treatment as this was previously determined to be the conditions used to obtain optimal gene targeting with the unmodified ZFNs. The absolute rate of gene targeting using ZFN-1/ZFN-2 at 20 ng in <i>HEK</i>293 cells was about 20,000 GFP positive cells per million cells transfected (about 2%). (A) Titration of transfected DNA of Ub-VV-ZFNs in the gene targeting assay with increasing amounts of DNA. (B) Titration of transfected DNA of Ub-R-ZFNs in the gene targeting assay with increasing amounts of DNA. (C and D) Gene targeting in the absence and presence of 10 uM MG132 for given ZFN pairs at stated DNA concentrations. (E) Toxicity assay for all iterations of Ub-modified and unmodified ZFNs tested in the gene targeting assay relative to Sce. A value of <100% indicates decreased cell survival as compared with Sce, and demonstrates a toxic effect. Statistical analysis was performed using the Student's T-test comparing ZFN-1/ZFN-2 at 20 ng with no drug treatment to Ub-modified ZFNs treated with MG132. “*” indicates a P-value of <.05 and “n.s.” indicates no statistical significance or a P-value of >.05. Error bars are the standard deviation for three samples.</p