The ability to expand hematopoietic stem and progenitor cells (HSPCs) ex vivo is critical to fully realize the potential of HSPC-based therapies. In particular, the application of clinically ...effective therapies, such as cord blood transplantation, has been impeded because of limited HSPC availability. Here, using 3D culture of human HSPCs in a degradable zwitterionic hydrogel, we achieved substantial expansion of phenotypically primitive CD34
cord blood and bone-marrow-derived HSPCs. This culture system led to a 73-fold increase in long-term hematopoietic stem cell (LT-HSC) frequency, as demonstrated by limiting dilution assays, and the expanded HSPCs were capable of hematopoietic reconstitution for at least 24 weeks in immunocompromised mice. Both the zwitterionic characteristics of the hydrogel and the 3D format were important for HSPC self-renewal. Mechanistically, the impact of 3D zwitterionic hydrogel culture on mitigating HSPC differentiation and promoting self-renewal might result from an inhibition of excessive reactive oxygen species (ROS) production via suppression of O
-related metabolism. HSPC expansion using zwitterionic hydrogels has the potential to facilitate the clinical application of hematopoietic-stem-cell therapies.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The small number of hematopoietic stem and progenitor cells in cord blood units limits their widespread use in human transplant protocols. We identified a family of chemically related small molecules ...that stimulates the expansion ex vivo of human cord blood cells capable of reconstituting human hematopoiesis for at least 6 months in immunocompromised mice. The potent activity of these newly identified compounds, UM171 being the prototype, is independent of suppression of the aryl hydrocarbon receptor, which targets cells with more-limited regenerative potential. The properties of UM171 make it a potential candidate for hematopoietic stem cell transplantation and gene therapy.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
FLT3, DNMT3A, and NPM1 are the most frequently mutated genes in cytogenetically normal acute myeloid leukemia (AML), but little is known about how these mutations synergize upon cooccurrence. Here we ...show that triple-mutated AML is characterized by high leukemia stem cell (LSC) frequency, an aberrant leukemia-specific GPR56highCD34low immunophenotype, and synergistic upregulation of Hepatic Leukemia Factor (HLF). Cell sorting based on the LSC marker GPR56 allowed isolation of triple-mutated from DNMT3A/NPM1 double-mutated subclones. Moreover, in DNMT3A R882-mutated patients, CpG hypomethylation at the HLF transcription start site correlated with high HLF mRNA expression, which was itself associated with poor survival. Loss of HLF via CRISPR/Cas9 significantly reduced the CD34+GPR56+ LSC compartment of primary human triple-mutated AML cells in serial xenotransplantation assays. HLF knockout cells were more actively cycling when freshly harvested from mice, but rapidly exhausted when reintroduced in culture. RNA sequencing of primary human triple-mutated AML cells after shRNA-mediated HLF knockdown revealed the NOTCH target Hairy and Enhancer of Split 1 (HES1) and the cyclin-dependent kinase inhibitor CDKN1C/p57 as novel targets of HLF, potentially mediating these effects. Overall, our data establish HLF as a novel LSC regulator in this genetically defined high-risk AML subgroup.
•HLF is overexpressed in normal karyotype AML triple mutated for NPM1, DNMT3A, and FLT3–internal tandem duplication (ITD).•Loss of HLF reduces the CD34+GPR56+ compartment, accelerates cell cycle progression, and decreases HES1 and CDKN1C expression.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
A cryptic inv(16)(p13.3q24.3) encoding the
fusion is associated with poor outcome in infants with acute megakaryocytic leukemia. We aimed to broaden our understanding of the pathogenesis of this ...fusion through transcriptome profiling.
Available RNA from children and young adults with
acute myeloid leukemia (AML;
= 1,049) underwent transcriptome sequencing (mRNA and miRNA). Transcriptome profiles for those with the
fusion (
= 24) and without (
= 1,025) were contrasted to define fusion-specific miRNAs, genes, and pathways. Clinical annotations defined distinct fusion-associated disease characteristics and outcomes.
The
fusion was restricted to infants <3 years old (
< 0.001), and the presence of this fusion was highly associated with adverse outcome (
< 0.001) across all morphologic classifications. Further, there was a striking paucity of recurrent cooperating mutations, and transduction of cord blood stem cells with this fusion was sufficient for malignant transformation.
positive cases displayed marked upregulation of genes with cell membrane/extracellular matrix localization potential, including
and
. Additionally, miRNA profiling revealed significant overexpression of mature
and
, which are intronic miRNAs transcribed from the
locus. Gene-set enrichment identified dysregulated Hippo, TGFβ, and hedgehog signaling, as well as
(CD56) interaction pathways. Therapeutic targeting of fusion-positive leukemic cells with CD56-directed antibody-drug conjugate caused significant cytotoxicity in leukemic blasts.
The
fusion defines a highly refractory entity limited to infants that appears to be sufficient for malignant transformation. Transcriptome profiling elucidated several highly targetable genes and pathways, including the identification of CD56, providing a highly plausible target for therapeutic intervention.
The CBFA2T3-GLIS2 (C/G) fusion is a product of a cryptic translocation primarily seen in infants and early childhood and is associated with dismal outcome. Here, we demonstrate that the expression of ...the C/G oncogenic fusion protein promotes the transformation of human cord blood hematopoietic stem and progenitor cells (CB HSPCs) in an endothelial cell coculture system that recapitulates the transcriptome, morphology, and immunophenotype of C/G acute myeloid leukemia (AML) and induces highly aggressive leukemia in xenograft models. Interrogating the transcriptome of C/G-CB cells and primary C/G AML identified a library of C/G-fusion-specific genes that are potential targets for therapy. We developed chimeric antigen receptor (CAR) T cells directed against one of the targets, folate receptor α (FOLR1), and demonstrated their preclinical efficacy against C/G AML using in vitro and xenograft models. FOLR1 is also expressed in renal and pulmonary epithelium, raising concerns for toxicity that must be addressed for the clinical application of this therapy. Our findings underscore the role of the endothelial niche in promoting leukemic transformation of C/G-transduced CB HSPCs. Furthermore, this work has broad implications for studies of leukemogenesis applicable to a variety of oncogenic fusion-driven pediatric leukemias, providing a robust and tractable model system to characterize the molecular mechanisms of leukemogenesis and identify biomarkers for disease diagnosis and targets for therapy.
To establish novel and effective treatment combinations for chronic myelomonocytic leukemia (CMML) preclinically, we hypothesized that supplementation of CMML cells with the human oncogene Meningioma ...1 (MN1) promotes expansion and serial transplantability in mice, while maintaining the functional dependencies of these cells on their original genetic profile. Using lentiviral expression of MN1 for oncogenic supplementation and transplanting transduced primary mononuclear CMML cells into immunocompromised mice, we established three serially transplantable CMML-PDX models with disease-related gene mutations that recapitulate the disease in vivo. Ectopic MN1 expression was confirmed to enhance the proliferation of CMML cells, which otherwise did not engraft upon secondary transplantation. Furthermore, MN1-supplemented CMML cells were serially transplantable into recipient mice up to 5 generations. This robust engraftment enabled an in vivo RNA interference screening targeting CMML-related mutated genes including NRAS, confirming that their functional relevance is preserved in the presence of MN1. The novel combination treatment with azacitidine and the MEK-inhibitor trametinib additively inhibited ERK-phosphorylation and thus depleted the signal from mutated NRAS. The combination treatment significantly prolonged survival of CMML mice compared to single-agent treatment. Thus, we identified the combination of azacitidine and trametinib as an effective treatment in NRAS-mutated CMML and propose its clinical development.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Herein we demonstrate that oncolytic herpes simplex virus-1 (HSV-1) potently activates human peripheral blood mononuclear cells (PBMCs) to lyse leukemic cell lines and primary acute myeloid leukemia ...samples, but not healthy allogeneic lymphocytes. Intriguingly, we found that UV light–inactivated HSV-1 (UV-HSV-1) is equally effective in promoting PBMC cytolysis of leukemic cells and is 1000- to 10 000-fold more potent at stimulating innate antileukemic responses than UV-inactivated cytomegalovirus, vesicular stomatitis virus, reovirus, or adenovirus. Mechanistically, UV-HSV-1 stimulates PBMC cytolysis of leukemic cells, partly via Toll-like receptor-2/protein kinase C/nuclear factor-κB signaling, and potently stimulates expression of CD69, degranulation, migration, and cytokine production in natural killer (NK) cells, suggesting that surface components of UV-HSV-1 directly activate NK cells. Importantly, UV-HSV-1 synergizes with interleukin-15 (IL-15) and IL-2 in inducing activation and cytolytic activity of NK cells. Additionally, UV-HSV-1 stimulates glycolysis and fatty acid oxidation–dependent oxygen consumption in NK cells, but only glycolysis is required for their enhanced antileukemic activity. Last, we demonstrate that T cell–depleted human PBMCs exposed to UV-HSV-1 provide a survival benefit in a murine xenograft model of human acute myeloid leukemia (AML). Taken together, our results support the preclinical development of UV-HSV-1 as an adjuvant, alone or in combination with IL-15, for allogeneic donor mononuclear cell infusions to treat AML.
•UV-inactivated HSV-1 activates Toll-like receptor signaling in NK cells to kill leukemic, but not normal, allogeneic cells.•UV-inactivated HSV-1 increases the therapeutic efficacy of allogeneic mononuclear cell infusions in a xenograft model of AML.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Pre-B-cell leukemia homeobox interacting protein 1 or human PBX1 interacting protein (PBXIP1/HPIP) is a co-repressor of pre-B-cell leukemia homeobox 1 (PBX1) and is also known to regulate estrogen ...receptor functions by associating with the microtubule network. Despite its initial discovery in the context of hematopoietic cells, little is yet known about the role of HPIP in hematopoiesis. Here, we show that lentivirus-mediated overexpression of HPIP in human CD34+ cells enhances hematopoietic colony formation in vitro, whereas HPIP knockdown leads to a reduction in the number of such colonies. Interestingly, erythroid colony number was significantly higher in HPIP-overexpressing cells. In addition, forced expression of HPIP in K562 cells, a multipotent erythro-megakaryoblastic leukemia cell line, led to an induction of erythroid differentiation. HPIP overexpression in both CD34+ and K562 cells was associated with increased activation of the PI3K/AKT pathway, and corresponding treatment with a PI3K-specific inhibitor, LY-294002, caused a reduction in clonogenic progenitor number in HPIP-expressing CD34+ cells and decreased K562 cell differentiation. Combined, these findings point to an important role of the PI3K/AKT pathway in mediating HPIP-induced effects on the growth and differentiation of hematopoietic cells. Interestingly, HPIP gene expression was found to be induced in K562 cells in response to erythroid differentiation signals such as DMSO and erythropoietin. The erythroid lineage-specific transcription factor GATA1 binds to the HPIP promoter and activates HPIP gene transcription in a CCCTC-binding factor (CTCF)-dependent manner. Co-immunoprecipitation and co-localization experiments revealed the association of CTCF with GATA1 indicating the recruitment of CTCF/GATA1 transcription factor complex onto the HPIP promoter. Together, this study provides evidence that HPIP is a target of GATA1 and CTCF in erythroid cells and plays an important role in erythroid differentiation by modulating the PI3K/AKT pathway.
HPIP is a pre-B-cell leukemia homeobox 1 (PBX1) interacting protein with unknown function in hematopoiesis.
The HPIP gene is a target of GATA1 and CTCF and regulates erythroid differentiation involving PI3K/AKT-dependent mechanisms.
HPIP is a novel downstream target of GATA1 and serves as an essential regulator of erythroid differentiation.
A new regulator of erythroid differentiation is discovered. This finding may help in better understanding erythropoiesis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Enhanced gene transfer efficiencies and higher yields of transplantable transduced human hematopoietic stem cells are continuing goals for improving clinical protocols that use stemcell-based gene ...therapies. Here, we examined the effect of the HSC agonist UM171 on these endpoints in both in vitro and in vivo systems. Using a 22-hr transduction protocol, we found that UM171 significantly enhances both the lentivirus-mediated transduction and yield of CD34+ and CD34+CD45RA- hematopoietic cells from human cord blood to give a 6-fold overall higher recovery of transduced hematopoietic stem cells, including cells with long-term lympho-myeloid repopulating activity in immunodeficient mice. The ability of UM171 to enhance gene transfer to primitive cord blood hematopoietic cells extended to multiple lentiviral pseudotypes, gamma retroviruses, and non-integrating lentiviruses and to adult bone marrow cells. UM171, thus, provides an interesting reagent for improving the ex vivo production of gene-modified cells and for reducing requirements of virus for a broad range of applications.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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