Jae-Hung Shieh

Mobilization of endothelial and hematopoietic stem and progenitor cells by adenovector-mediated elevation of serum levels of SDF-1, VEGF, and angiopoietin-1.

Abstract: The chemokine stroma‐derived factor‐1 (SDF‐1) is produced within the bone marrow and mediates chemokinesis and chemotaxis on a variety of cell types that express the CXCR4 receptor. SDF‐1‐responsive cell types include monocytes and macrophages, B and T lymphocytes, platelets and megakaryocytes, and CD34+ cells, including both hematopoietic progenitors and stem cells. We have used intravenous injection of a replication‐incompetent adenovector expressing the SDF‐1 gene to elevate serum levels of SDF‐1 in Balb/c and SCID mice. Within 3 to 5 days there was a marked leukocytosis, predominantly involving monocytes, and a three‐fold increase in platelets. In addition, AdSDF‐1 mobilized CFU‐GM, CFU‐s, and cells with long‐term repopulating potential. We have identified a bone marrow‐derived, circulating endothelial stem cell characterized by expression of the VEGFR2 (Flk‐1/KDR). This cell exhibits a chemotactic and chemokinetic response to SDF‐1 and VEGF. We have elevated serum levels of VEGF165 using intravenous adenovector gene delivery and compared this to an adenovector expressing angiopoietin‐1 alone or in combination with VEGF. VEGF elevation was associated with rapid mobilization of hematopoietic stem and progenitor cells and a population of Flk‐1‐positive endothelial progenitors. In contrast angiopoietin induced a delayed mobilization of endothelial and hematopoietic progenitors. The combination of VEGF and angiopoietin produced a more prolonged elevation of these progenitors in the circulation with increased proliferation of capillaries and expansion of sinusoidal spaces in the marrow.

Recombination Signatures Distinguish Embryonic Stem Cells Derived by Parthenogenesis and Somatic Cell Nuclear Transfer

Parthenogenesis and somatic cell nuclear transfer (SCNT) are two methods for deriving embryonic stem (ES) cells that are genetically matched to the oocyte donor or somatic cell donor, respectively. Using genome-wide single nucleotide polymorphism (SNP) analysis, we demonstrate distinct signatures of genetic recombination that distinguish parthenogenetic ES cells from those generated by SCNT. We applied SNP analysis to the human ES cell line SCNT-hES-1, previously claimed to have been derived by SCNT, and present evidence that it represents a human parthenogenetic ES cell line. Genome-wide SNP analysis represents a means to validate the genetic provenance of an ES cell line.

NUP98 dysregulation in myeloid leukemogenesis.

Abstract:  Nucleoporin 98 (NUP98) is a component of the nuclear pore complex that facilitates mRNA export from the nucleus. It is mapped to 11p15.5 and is fused to a number of distinct partners, including nine members of the homeobox family as a consequence of leukemia‐associated chromosomal translocations. NUP98‐HOXA9 is associated with the t(7;11)(p15;p15) translocation in acute myeloid leukemia (AML), myelodysplastic syndrome, and blastic crisis of chronic myeloid leukemia. Expression of NUP98‐HOXA9 in murine bone marrow resulted in a myeloproliferative disease progressing to AML by 7–8 months. Transduction of NUP98 fusion genes into human CD34+ cells confers a proliferative advantage in long‐term cytokine‐stimulated and stromal cocultures and in NOD‐SCID engrafted mice, associated with a five‐ to eight‐fold increase in hematopoietic stem cells. NUP98‐HOXA9 expression inhibited erythroid and myeloid differentiation but enhanced serial progenitor replating. NUP98‐HOXA9 upregulated a number of homeobox genes of the A and B cluster as well as MEIS1 and Pim‐1, and downmodulated globin genes and C/EBPα. The HOXA9 component of the NUP98‐HOXA9 fusion protein was protected from cullin‐4A–mediated ubiquitination and subsequent proteasome‐dependent degradation. In NUP98‐HOX–transduced CD34+ cells and cells from AML patients with t(7;11)(p15;p15) NUP98 was no longer associated with the nuclear pore complex but formed intranuclear aggregation bodies. Analysis of NUP98 allelic expression in AML and myelodysplastic syndrome showed loss of heterozygosity observed in 29% of the former and 8% of the latter. This was associated with poor prognosis.

In vitro Stimulation with WT1 Peptide-Loaded Epstein-Barr Virus-Positive B Cells Elicits High Frequencies of WT1 Peptide-Specific T Cells with In vitro and In vivo Tumoricidal Activity

The Wilms tumor protein (WT1) is overexpressed in most acute and chronic leukemias. To develop a practicable, clinically applicable approach for generation of WT1-specific T cells and to comparatively evaluate the immunogenicity of WT1 in normal individuals, we sensitized T cells from 13 HLA-A0201+ and 5 HLA-A2402+ donors with autologous EBV-transformed B cells or cytokine-activated monocytes, loaded with the HLA-A0201-binding WT1 peptides 126–134RMFPNAPYL or 187–195SLGEQQYSV or a newly identified HLA-A2402-binding WT1 peptide 301–310RVPGVAPTL. WT1-specific T cells were regularly generated from each donor. T cells sensitized with peptide-loaded EBV-transformed B cells generated higher numbers of WT1-specific T cells than peptide-loaded cytokine-activated monocytes. Contrary to expectations, the frequencies of WT1 peptide-specific T cells were equivalent to those generated against individual highly immunogenic HLA-A0201-binding EBV peptides. Each of these T-cell lines specifically killed WT1+ leukemias and solid tumors in an HLA-restricted manner but did not lyse autologous or HLA-matched normal CD34+ hematopoietic progenitor cells or reduce their yield of colony-forming unit-granulocyte-macrophage (CFU-GM), burst-forming unit erythroid (BFU-E), or mixed colonies (CFU-mix). Furthermore, WT1 peptide-specific T cells after adoptive transfer into nonobese diabetic-severe combined immunodeficient mice bearing subcutaneous xenografts of WT1+ and WT1− HLA-A0201+ leukemias preferentially accumulated in and induced regressions of WT1+ leukemias that expressed the restricting HLA allele. Such cells are clinically applicable and may prove useful for adoptive cell therapy of WT1+ malignant diseases in humans.

Improved ex vivo expansion of adult hematopoietic stem cells by overcoming CUL4-mediated degradation of HOXB4.

Direct transduction of the homeobox (HOX) protein HOXB4 promotes the proliferation of hematopoietic stem cells (HSCs) without induction of leukemogenesis, but requires frequent administration to overcome its short protein half-life (∼1 hour). We demonstrate here that HOXB4 protein levels are post-translationally regulated by the CUL4 ubiquitin ligase, and define the degradation signal sequence (degron) of HOXB4 required for CUL4-mediated destruction. Additional HOX paralogs share the conserved degron in the homeodomain and are also subject to CUL4-mediated degradation, indicating that CUL4 likely controls the stability of all HOX proteins. Moreover, we engineered a degradation-resistant HOXB4 that conferred a growth advantage over wild-type HOXB4 in myeloid progenitor cells. Direct transduction of recombinant degradation-resistant HOXB4 protein to human adult HSCs significantly enhanced their maintenance in a more primitive state both in vitro and in transplanted NOD/SCID/IL2R-γ(null) mice compared with transduction with wild-type HOXB4 protein. Our studies demonstrate the feasibility of engineering a stable HOXB4 variant to overcome a major technical hurdle in the ex vivo expansion of adult HSCs and early progenitors for human therapeutic use.

Imatinib resistance and microcytic erythrocytosis in a KitV558Δ;T669I/+ gatekeeper-mutant mouse model of gastrointestinal stromal tumor

Most gastrointestinal stromal tumors (GISTs) harbor a gain-of-function mutation in the Kit receptor. GIST patients treated with the tyrosine kinase inhibitor imatinib frequently develop imatinib resistance as a result of second-site Kit mutations. To investigate the consequences of second-site Kit mutations on GIST development and imatinib sensitivity, we engineered a mouse model carrying in the endogenous Kit locus both the KitV558Δ mutation found in a familial case of GIST and the KitT669I (human KITT670I) “gatekeeper” mutation found in imatinib-resistant GIST patients. Similar to KitV558∆/+ mice, KitV558∆;T669I/+ mice developed gastric and colonic interstitial cell of Cajal hyperplasia as well as cecal GIST. In contrast to the single-mutant KitV558∆/+ control mice, treatment of the KitV558∆;T669I/+ mice with either imatinib or dasatinib failed to inhibit oncogenic Kit signaling and GIST growth. However, this resistance could be overcome by treatment of KitV558∆;T669I/+ mice with sunitinib or sorafenib. Although tumor lesions were smaller in KitV558∆;T669I/+ mice than in single-mutant mice, both interstitial cell of Cajal hyperplasia and mast cell hyperplasia were exacerbated in KitV558∆;T669I/+ mice. Strikingly, the KitV558∆;T669I/+ mice developed a pronounced polycythemia vera-like erythrocytosis in conjunction with microcytosis. This mouse model should be useful for preclinical studies of drug candidates designed to overcome imatinib resistance in GIST and to investigate the consequences of oncogenic KIT signaling in hematopoietic as well as other cell lineages.

Fully Synthetic Granulocyte Colony-Stimulating Factor Enabled by Isonitrile-Mediated Coupling of Large, Side-Chain-Unprotected Peptides

Human granulocyte colony-stimulating factor (G-CSF) is an endogenous glycoprotein involved in hematopoiesis. Natively glycosylated and nonglycosylated recombinant forms, lenograstim and filgrastim, respectively, are used clinically to manage neutropenia in patients undergoing chemotherapeutic treatment. Despite their comparable therapeutic potential, the purpose of O-linked glycosylation at Thr133 remains a subject of controversy. In light of this, we have developed a synthetic platform to prepare G-CSF aglycone with the goal of enabling access to native and designed glycoforms with site-selectivity and glycan homogeneity. To address the synthesis of a relatively large, aggregation-prone sequence, we advanced an isonitrile-mediated ligation method. The chemoselective activation and coupling of C-terminal peptidyl Gly thioacids with the N-terminus of an unprotected peptide provide ligated peptides directly in a manner complementary to that with conventional native chemical ligation-desulfurization strategies. Herein, we describe the details and application of this method as it enabled the convergent total synthesis of G-CSF aglycone.

BO-1055, a novel DNA cross-linking agent with remarkable low myelotoxicity shows potent activity in sarcoma models

DNA damaging agents cause rapid shrinkage of tumors and form the basis of chemotherapy for sarcomas despite significant toxicities. Drugs having superior efficacy and wider therapeutic windows are needed to improve patient outcomes. We used cell proliferation and apoptosis assays in sarcoma cell lines and benign cells; γ-H2AX expression, comet assay, immunoblot analyses and drug combination studies in vitro and in patient derived xenograft (PDX) models. BO-1055 caused apoptosis and cell death in a concentration and time dependent manner in sarcoma cell lines. BO-1055 had potent activity (submicromolar IC50) against Ewing sarcoma and rhabdomyosarcoma, intermediate activity in DSRCT (IC50 = 2-3μM) and very weak activity in osteosarcoma (IC50 >10μM) cell lines. BO-1055 exhibited a wide therapeutic window compared to other DNA damaging drugs. BO-1055 induced more DNA double strand breaks and γH2AX expression in cancer cells compared to benign cells. BO-1055 showed inhibition of tumor growth in A673 xenografts and caused tumor regression in cyclophosphamide resistant patient-derived Ewing sarcoma xenografts and A204 xenografts. Combination of BO-1055 and irinotecan demonstrated synergism in Ewing sarcoma PDX models. Potent activity on sarcoma cells and its relative lack of toxicity presents a strong rationale for further development of BO-1055 as a therapeutic agent.

Hematopoietic growth factor receptors

The formation of the cellular constituents of the blood is regulated by a series of endogenous polypeptides with largely paracrine function. A number of these hematopoietic growth factors (HGFs), which include colony stimulating factors, interleukins, and erythropoietin, have been purified to homogeneity and cloned, which in turn has led to extensive investigations of their biochemical properties and biological effects and functions. The HGFs act on target cells by binding to receptors. The kinetics and, to an even larger extent, dynamics of the factor/receptor associations display several intriguing characteristics, most of which are still poorly understood. Herein, the biochemical characteristics of HGFs receptors as well as the binding properties, post-receptor binding events and receptor modulation resulting from the association of HGFs and their target cells are reviewed.

Circulating Hematopoietic Stem and Progenitor Cells in Aging Atomic Bomb Survivors

It is not yet known whether hematopoietic stem and progenitor cells (HSPCs) are compromised in the aging population of atomic bomb (A-bomb) survivors after their exposure nearly 70 years ago. To address this, we evaluated age- and radiation-related changes in different subtypes of circulating HSPCs among the CD34-positive/lineage marker-negative (CD34+Lin−) cell population in 231 Hiroshima A-bomb survivors. We enumerated functional HSPC subtypes, including: cobblestone area-forming cells; long-term culture-initiating cells; erythroid burst-forming units; granulocyte and macrophage colony-forming units; and T-cell and natural killer cell progenitors using cell culture. We obtained the count of each HSPC subtype per unit volume of blood and the proportion of each HSPC subtype in CD34+Lin− cells to represent the lineage commitment trend. Multivariate analyses, using sex, age and radiation dose as variables, showed significantly decreased counts with age in the total CD34+Lin− cell population and all HSPC subtypes. As for the proportion, only T-cell progenitors decreased significantly with age, suggesting that the commitment to the T-cell lineage in HSPCs continuously declines with age throughout the lifetime. However, neither the CD34+Lin− cell population, nor HSPC subtypes showed significant radiation-induced dose-dependent changes in counts or proportions. Moreover, the correlations of the proportions among HSPC subtypes in the survivors properly revealed the hierarchy of lineage commitments. Taken together, our findings suggest that many years after exposure to radiation and with advancing age, the number and function of HSPCs in living survivors as a whole may have recovered to normal levels.