simplified-intrafemoral-injections-using-live-mice-allow-for

Simplified Intrafemoral Injections Using Live Mice Allow for Continuous Bone Marrow Analysis

Despite the complexity of hematopoietic cell transplantation in humans, researchers commonly perform intravenous or intrafemoral (IF) injections in mice. ...

Stanford University School of Medicine

Graft-versus-host disease (GVHD), mediated by donor-derived alloreactive T cells, is a major cause of nonrelapse mortality in allogeneic hematopoietic stem cell transplantation. Its therapy is not well-defined. We established allele-specific anti-human leukocyte antigen (HLA) monoclonal antibodies (ASHmAbs) that specifically target HLA molecules, with steady death of target-expressing cells. One such ASHmAb, against HLA-A*02:01 (A2-kASHmAb), was examined in a xenogeneic GVHD mouse model. To induce fatal GVHD, non-irradiated NOD/Shi-scid/IL-2Rγ(null) mice were injected with healthy donor human peripheral blood mononuclear cells, some expressing HLA-A*02:01, some not. Administration of A2-kASHmAb promoted the survival of mice injected with HLA-A*02:01-expressing peripheral blood mononuclear cells (p < 0.0001) and, in humanized NOD/Shi-scid/IL-2Rγ(null) mice, immediately cleared HLA-A*02:01-expressing human blood cells from mouse peripheral blood. Human peripheral blood mononuclear cells were again detectable in mouse blood 2 to 4 weeks after A2-kASHmAb administration, suggesting that kASHmAb may be safely administered to GVHD patients without permanently ablating the graft. This approach, different from those in existing GVHD pharmacotherapy, may open a new door for treatment of GVHD in HLA-mismatched allogeneic hematopoietic stem cell transplantation.

Effective treatment against severe graft-versus-host disease with allele-specific anti-HLA monoclonal antibody in a humanized mouse model.

The discovery of induced pluripotent stem cells (iPSCs) has created promising new avenues for therapies in regenerative medicine. However, the tumorigenic potential of undifferentiated iPSCs is a major safety concern for clinical translation. To address this issue, we demonstrated the efficacy of suicide gene therapy by introducing inducible caspase-9 (iC9) into iPSCs. Activation of iC9 with a specific chemical inducer of dimerization (CID) initiates a caspase cascade that eliminates iPSCs and tumors originated from iPSCs. We introduced this iC9/CID safeguard system into a previously reported iPSC-derived, rejuvenated cytotoxic T lymphocyte (rejCTL) therapy model and confirmed that we can generate rejCTLs from iPSCs expressing high levels of iC9 without disturbing antigen-specific killing activity. iC9-expressing rejCTLs exert antitumor effects in vivo. The system efficiently and safely induces apoptosis in these rejCTLs. These results unite to suggest that the iC9/CID safeguard system is a promising tool for future iPSC-mediated approaches to clinical therapy.

A Safeguard System for Induced Pluripotent Stem Cell-Derived Rejuvenated T Cell Therapy.

Aging is linked to functional deterioration and hematological diseases. The hematopoietic system is maintained by hematopoietic stem cells (HSCs), and dysfunction within the HSC compartment is thought to be a key mechanism underlying age-related hematopoietic perturbations. Using single-cell transplantation assays with five blood-lineage analysis, we previously identified myeloid-restricted repopulating progenitors (MyRPs) within the phenotypic HSC compartment in young mice. Here, we determined the age-related functional changes to the HSC compartment using over 400 single-cell transplantation assays. Notably, MyRP frequency increased dramatically with age, while multipotent HSCs expanded modestly within the bone marrow. We also identified a subset of functional cells that were myeloid restricted in primary recipients but displayed multipotent (five blood-lineage) output in secondary recipients. We have termed this cell type latent-HSCs, which appear exclusive to the aged HSC compartment. These results question the traditional dogma of HSC aging and our current approaches to assay and define HSCs.

Large-Scale Clonal Analysis Resolves Aging of the Mouse Hematopoietic Stem Cell Compartment.

Serum albumin has long been an essential supplement for ex vivo hematopoietic and immune cell cultures. However, serum albumin medium supplements represent a major source of biological contamination in cell cultures and often cause loss of cellular function. As serum albumin exhibits significant batch-to-batch variability, it has also been blamed for causing major issues in experimental reproducibility. We recently discovered the synthetic polymer polyvinyl alcohol (PVA) as an inexpensive, Good Manufacturing Practice-compatible, and biologically inert serum albumin replacement for ex vivo hematopoietic stem cell cultures. Importantly, PVA is free of the biological contaminants that have plagued serum albumin-based media. Here, we describe that PVA can replace serum albumin in a range of blood and immune cell cultures including cell lines, primary leukemia samples, and human T lymphocytes. PVA can even replace human serum in the generation and expansion of functional chimeric antigen receptor (CAR) T cells, offering a potentially safer and more cost-efficient approach for this clinical cell therapy. In summary, PVA represents a chemically defined, biologically inert, and inexpensive alternative to serum albumin for a range of cell cultures in hematology and immunology.

Use of polyvinyl alcohol for chimeric antigen receptor T-cell expansion.

Deregulation of the  gene family plays an important role in the pathogenesis of acute myeloid leukemia (AML). The BCL2 inhibitor, venetoclax, has received FDA approval for the treatment of AML. However, upfront and acquired drug resistance ensues due, in part, to the clinical and genetic heterogeneity of AML, highlighting the importance of identifying biomarkers to stratify patients onto the most effective therapies. By integrating clinical characteristics, exome and RNA sequencing, and inhibitor data from primary AML patient samples, we determined that myelomonocytic leukemia, upregulation of  and , as well as mutations of  and  conferred resistance to venetoclax and multiple venetoclax combinations. Venetoclax in combination with an MCL1 inhibitor AZD5991 induced synthetic lethality and circumvented venetoclax resistance.

Integrated analysis of patient samples identifies biomarkers for venetoclax efficacy and combination strategies in acute myeloid leukemia.

The conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is a key step in DNA demethylation that is mediated by ten-eleven translocation (TET) enzymes, which require ascorbate/vitamin C. Here, we report the 5hmC landscape of normal hematopoiesis and identify cell type-specific 5hmC profiles associated with active transcription and chromatin accessibility of key hematopoietic regulators. We utilized CRISPR/Cas9 to model TET2 loss-of-function mutations in primary human hematopoietic stem and progenitor cells (HSPC). Disrupted cells exhibited increased colonies in serial replating, defective erythroid/megakaryocytic differentiation, and in vivo competitive advantage and myeloid skewing coupled with reduction of 5hmC at erythroid-associated gene loci. Azacitidine and ascorbate restored 5hmC abundance and slowed or reverted the expansion of TET2-mutant clones in vivo. These results demonstrate the key role of 5hmC in normal hematopoiesis and TET2-mutant phenotypes and raise the possibility of utilizing these agents to further our understanding of preleukemia and clonal hematopoiesis.

The Cell Type-Specific 5hmC Landscape and Dynamics of Healthy Human Hematopoiesis and TET2-Mutant Preleukemia.

Isocitrate dehydrogenase 1 and 2 (IDH) are mutated in multiple cancers and drive production of (R)-2-hydroxyglutarate (2HG). We identified a lipid synthesis enzyme [acetyl CoA carboxylase 1 (ACC1)] as a synthetic lethal target in mutant IDH1 (mIDH1), but not mIDH2, cancers. Here, we analyzed the metabolome of primary acute myeloid leukemia (AML) blasts and identified an mIDH1-specific reduction in fatty acids. mIDH1 also induced a switch to b-oxidation indicating reprogramming of metabolism toward a reliance on fatty acids. Compared with mIDH2, mIDH1 AML displayed depletion of NADPH with defective reductive carboxylation that was not rescued by the mIDH1-specific inhibitor ivosidenib. In xenograft models, a lipid-free diet markedly slowed the growth of mIDH1 AML, but not healthy CD34+ hematopoietic stem/progenitor cells or mIDH2 AML. Genetic and pharmacologic targeting of ACC1 resulted in the growth inhibition of mIDH1 cancers not reversible by ivosidenib. Critically, the pharmacologic targeting of ACC1 improved the sensitivity of mIDH1 AML to venetoclax.

Dysregulated Lipid Synthesis by Oncogenic IDH1 Mutation Is a Targetable Synthetic Lethal Vulnerability.

Disease-initiating mutations in the transcription factor RUNX1 occur as germline and somatic events that cause leukemias with particularly poor prognosis. However, the role of RUNX1 in leukemogenesis is not fully understood, and effective therapies for RUNX1-mutant leukemias remain elusive. Here, we used primary patient samples and a RUNX1-KO model in primary human hematopoietic cells to investigate how RUNX1 loss contributes to leukemic progression and to identify targetable vulnerabilities. Surprisingly, we found that RUNX1 loss decreased proliferative capacity and stem cell function. However, RUNX1-deficient cells selectively upregulated the IL-3 receptor. Exposure to IL-3, but not other JAK/STAT cytokines, rescued RUNX1-KO proliferative and competitive defects. Further, we demonstrated that RUNX1 loss repressed JAK/STAT signaling and rendered RUNX1-deficient cells sensitive to JAK inhibitors. Our study identifies a dependency of RUNX1-mutant leukemias on IL-3/JAK/STAT signaling, which may enable targeting of these aggressive blood cancers with existing agents.

Article	Year
Effective treatment against severe graft-versus-host disease with allele-specific anti-HLA monoclonal antibody in a humanized mouse model. Experimental hematology\| PubMed ID: 25448490	2015
A Safeguard System for Induced Pluripotent Stem Cell-Derived Rejuvenated T Cell Therapy. Stem cell reports\| PubMed ID: 26321144	2015
Large-Scale Clonal Analysis Resolves Aging of the Mouse Hematopoietic Stem Cell Compartment. Cell stem cell\| PubMed ID: 29625072	2018
Use of polyvinyl alcohol for chimeric antigen receptor T-cell expansion. Experimental hematology\| PubMed ID: 31874780	2019
Integrated analysis of patient samples identifies biomarkers for venetoclax efficacy and combination strategies in acute myeloid leukemia. Nature cancer\| PubMed ID: 33123685	2020
The Cell Type-Specific 5hmC Landscape and Dynamics of Healthy Human Hematopoiesis and TET2-Mutant Preleukemia. Blood cancer discovery\| PubMed ID: 35532363	2022
Dysregulated Lipid Synthesis by Oncogenic IDH1 Mutation Is a Targetable Synthetic Lethal Vulnerability. Cancer discovery\| PubMed ID: 36355448	2023
RUNX1 loss renders hematopoietic and leukemic cells dependent on IL-3 and sensitive to JAK inhibition. The Journal of clinical investigation\| PubMed ID: 37581927	2023

Yusuke Nakauchi

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