EB10, an Anti-FLT3 Monoclonal Antibody, Prolongs Survival and Reduces NOD/SCID Engraftment of AML Cell Lines and Primary Blasts.

Abstract The FLT3 receptor is a potential target in AML due to its role in leukemogenesis and its high degree of expression on blasts from approximately 90% of acute myeloid leukemia (AML) patients. In addition, mutant forms of FLT3, including internal tandem duplications (ITD) in the juxtamembrane region and point mutations in the kinase domain, constitutively activate FLT3 signaling. ITD mutations in particular are also associated with poor prognosis. A number of small molecule tyrosine kinase inhibitors (TKI) against FLT3 are currently in clinical trials and have shown some clinical activity. However, TKIs have various limitations, including their lack of specificity, which may produce toxicities, and can select for drug resistant cells. In an attempt to overcome some of these limitations and to generate new agents which might cooperate in targeting FLT3, we generated a fully humanized phage display monoclonal antibody (EB10). This antibody is capable of inhibiting both ligand-activated wild-type and, to a lesser degree, ligand-independent mutant FLT3 signaling. When EB10 is used to treat cells expressing activated FLT3, inhibition of downstream pathways including STAT5, AKT and MAPK are also frequently seen. EB10 treatment of cells expressing FLT3 in the presence of NK cells leads to antibody-dependent cell-mediated cytotoxicity (ADCC). EB10 treatment of NOD/SCID mice injected with FLT3 expressing AML cell lines or with primary AML blasts significantly prolongs survival and/or reduces engraftment of leukemic cells. EB10 proved efficacious in vivo against cells even when in vitro EB10 treatment did not significantly reduce FLT3 signaling. This indicates that ADCC may be the primary mechanism mediating cytotoxicity as opposed to direct FLT3 inhibition. In contrast to the effects on AML cell lines and primary samples, EB10 treatment did not significantly reduce NOD/SCID engraftment of normal human CD34+ hematopoietic stem cells. Anti-FLT3 antibodies, like EB10, may be a promising therapeutic agent that can specifically target malignant cells with limited toxicities against normal hematopoietic stem cells and should be considered for clinical trials.

Download Full-text

Tanespimycin Reverses Bortezomib-Induced Inhibition of Granulopoiesis.

Blood ◽

10.1182/blood.v114.22.3846.3846 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3846-3846 ◽

Cited By ~ 2

Author(s):

Bruce D. Car ◽

Oliver P. Flint ◽

Jan Oberdoerster ◽

Jennifer Price ◽

William R. Foster ◽

...

Keyword(s):

Stem Cells ◽

Clinical Trials ◽

Hematopoietic Stem Cells ◽

Colony Formation ◽

Mononuclear Cells ◽

Cultured Cells ◽

Healthy Human ◽

Hematopoietic Stem ◽

Drug Concentrations

Abstract Abstract 3846 Poster Board III-782 INTRODUCTION Tanespimycin, an Hsp90 inhibitor, is in phase 3 clinical trials with bortezomib for the treatment of multiple myeloma (MM). Neutropenia and thrombocytopenia are commonly observed during bortezomib treatment in patients with MM. However, in a phase 1/2 study of tanespimycin + bortezomib in patients with MM, the incidence and severity of neutropenia was low (Richardson ASCO 2009). Here we present the in vitro effects of tanespimycin and bortezomib on hematopoiesis and granulopoiesis in cell culture systems using mononuclear cells from healthy subjects and cryopreserved hematopoietic stem cells. METHODS: Mononuclear cells were separated from fresh whole healthy human bone marrow cells and cultured for up to 14 days in suspension in the presence of tanespimycin (1 pM–300 μM) and bortezomib (1 nM–300 μM). Committed hematopoietic progenitor growth (CFC-GEMM [granulocyte/erythroid/macrophage/megakaryocyte], BFU-E [erythroid], GM-CFC [granulocyte/macrophage], MkCFC [megakaryocyte]) of cultured cells was assessed using intracellular ATP bioluminescence in the HemoGenix HALO® system, which couples specific hematopoietic lineage growth conditions with a surrogate for cell count. In addition, cryopreserved healthy human hematopoietic stem cells were cultured in semisolid methylcellulose and colonies (CFC-GEMM, CFU-E, BFU-E) were microscopically enumerated. RESULTS: When bortezomib and tanespimycin were given alone in suspension cultures, IC50 values for bortezomib-induced cytotoxicity and tanespimycin-induced cytotoxicity were approximately 1 nM to 2 nM and 100 nM, respectively, for all hematopoietic lineages. In semisolid methylcellulose cultures, 1 nM to 10 nM bortezomib induced a concentration-dependent inhibition of granulocyte-macrophage colony formation from 2% to 46% and of erythroid colony formation from 8% to 35%. While tanespimycin alone at a concentration of 10 nM had little or no effect on erythropoiesis or granulocytopoiesis, when bortezomib (1 nM or 3 nM) and tanespimycin (10 nM) were cocultured, tanespimycin completely reversed bortezomib-induced inhibition of erythropoiesis and granulopoiesis. More severe erythropoietic inhibition at 10 nM bortezomib was not reversed by tanespimycin, while granulopoietic inhibition was mitigated approximately 23%. At concentrations of melphalan (1 μM) and camptothecin (3 nM) that inhibit in vitro hematopoiesis approximately 50% and 90%, respectively, 10 nM tanespimycin had no effect. CONCLUSION: At clinically relevant drug concentrations in hematopoietic cultures, tanespimycin reverses bortezomib-induced inhibition of granulopoiesis, consistent with the low frequency of neutropenia observed in clinical trials of tanespimycin + bortezomib. This suggests tanespimycin may prevent bortezomib-induced apoptosis of granulocyte-macrophage progenitors. Disclosures: Flint: Bristol-Myers Squibb: Employment, Equity Ownership. Oberdoerster:Bristol-Myers Squibb: Employment. Price:Bristol-Myers Squibb: Employment. Foster:Bristol-Myers Squibb: Employment. Gemzik:Bristol-Myers Squibb: Employment. Berman:Bristol-Myers Squibb: Employment.

Download Full-text

5-Azacytidine depletes hematopoietic stem cells and synergizes with an anti-CD117 antibody to augment donor engraftment in immunocompetent mice

Blood Advances ◽

10.1182/bloodadvances.2020003841 ◽

2021 ◽

Author(s):

Andriyana K. Bankova ◽

Wendy Pang ◽

Brenda Josefina Velasco ◽

Janel R. Long-Boyle ◽

Judith A. Shizuru

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Hematopoietic Cell Transplantation ◽

Apoptotic Cell ◽

Donor Cell ◽

Leukemic Cells ◽

Hematopoietic Stem ◽

Immunocompetent Mice

Depletion of hematopoietic stem cells (HSC) is used therapeutically in many malignant and non-malignant blood disorders in the setting of a hematopoietic cell transplantation (HCT) to eradicate diseased HSC allowing donor HSC to engraft. Current treatments to achieve HSC elimination rely on modalities that cause DNA strand breakage (i.e., alkylators, radiation) resulting in multiple short-term and long-term toxicities, and sometimes even death. These risks have severely limited HCT utilization to patients with few to no co-morbidities, and excluded many others with diseases curable by HCT. 5-Azacytidine (AZA) is a widely used hypomethylating agent that is thought to preferentially target leukemic cells in myeloid malignancies. Here, we reveal a previously unknown effect of AZA on HSC. We show that AZA induces early HSC proliferation in vivo and exerts a direct cytotoxic effect on proliferating HSC in vitro. When used to pretreat recipient mice for transplant, AZA permitted low level donor HSC engraftment. Moreover, by combining AZA with a monoclonal antibody (mAb), targeting CD117 (c-Kit), a molecule expressed on HSC, more robust HSC-depletion and substantially higher levels of multilineage donor cell engraftment was achieved in immunocompetent mice. The enhanced effectiveness of this combined regimen correlated with increased apoptotic cell death in HSPC. Together, these findings highlight a previously unknown therapeutic mechanism for AZA which may broaden its utilization in clinical practice. Moreover, the synergy we show between AZA and anti-CD117 mAb is a novel strategy to eradicate abnormal HSC which can be rapidly tested in the clinical setting.

Download Full-text

Hypoxia-Adapted CML Cells Are More Primitive Population and Are Eradicated by Glyoxalase-1 Inhibitors.

Blood ◽

10.1182/blood.v114.22.2166.2166 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2166-2166 ◽

Cited By ~ 2

Author(s):

Miki Takeuchi ◽

Shinya Kimura ◽

Junya Kuroda ◽

Eishi Ashihara ◽

Makoto Kawatani ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Tyrosine Kinase ◽

Hematopoietic Stem Cells ◽

Cell Lines ◽

Lactate Production ◽

Glucose Consumption ◽

Leukemic Cells ◽

Hematopoietic Stem ◽

Nog Mice

Abstract Abstract 2166 Poster Board II-143 Normal hematopoietic stem cells reside in the epiphyses of the bone marrow that is a low oxygenated area and are protected against ROS-induced DNA damages. Recent study showed that the hypoxic environment plays a crucial role not only in maintaining stem cells but also in tumorigenesis. Hypoxia induces dramatical changes in cell characteristics including cell cycle quiescence, self renewal potency, and shift in energy production from an aerobic to anaerobic pathway, and it induces resistance to a variety of cell death stimuli. Chronic myelogenous leukemia (CML) is a disorder of hematopoietic stem cells caused by the constitutive activation of the Bcr-Abl tyrosine kinase. Tyrosine kinase inhibitors (TKIs) have led to marked improvement in prognosis of CML patients. However, CML cells could not be eradicated completely by TKI alone because quiescent CML stem cells are less sensitive to such molecular target drugs. Therefore, we speculate that the adaptation of leukemic cells to hypoxia in the bone marrow niche alters their characteristics contributing to minimal residual disease. We first evaluated the oxygen levels of engrafted leukemic cells by pimonidazol (hypoxia specific marker) staining.We transplanted K562 cells to the bone marrow of NOD/SCID/gcnull (NOG) mice and found that those cells engrafted and survived in the epiphysis of the bone marrow where O2 concentrations are less than 1.3%. Then, we generated two hypoxia-adapted (HA) CML subclones from K562 and KCL22 by cultivating under 1.0% O2, and were denoted as K562/HA and KCL22/HA, respectively. Both cell lines survived and proliferated continuously for years under 1.0% O2 conditions, although their growth was slower than that of their parental counterparts under 20% O2 conditions. Interestingly, HA-CML cells exhibited several unique characteristics compared to their parental cells. First, these HA cells showed higher transplantation efficacy in NOG mice. The transplanted HA cells grow more rapidly in vivo than the parental cells and mice transplanted with HA cells died earlier. Next, the percentage of G0 fractions in K562 and K562/HA cells were 0.87 ± 0.58 % and 4.9 ± 2.1 %, respectively, indicating that K562/HA cells included more quiescent fractions than the parental K562. Hoechst staining analysis confirmed that HA cell lines include more SP (side population) fractions than their parental cells, indicating that HA cells contains more dormant cells. We next examined the signaling pathway of HA cell lines. Despite the unchanged levels of AKT, STAT, and ERK phosphrylation, BCR-ABL phsophrylation was suppressed in HA cells. Both of HA cell lines showed higher expression of b-catenin which is considered essential for survival and self-renewality of CML stem cells. Furthermore, HA cells were less sensitive to TKIs (imatinib, dasatinib, and bafetinib) and chemotherapeutic agents (daunorubicin and busulfan). Taken together, our HA cell lines have characteristics of more primitive CML cell populations resistant to cytotoxic agents. We next examined the energy metabolites such as adenosine triphosphate (ATP), glucose consumption, and lactate production in HA cells. The amounts of ATP in K562/HA and KCL22/HA cells decreased, whereas glucose consumption and lactate production increased compared with those in their parental cell lines. These findings indicate that ATP production of HA cells depends on glycolysis. Furthermore, we found higher expression and kinase activity of Glyoxalase-1 (Glo-I). Glo-I is an enzyme that detoxifies glycolysis-specific cytocidal byproducts in glycolytis system. Glo-I inhibitors such as S-p-bromobenzylglutathione cyclopentyl diester (BBGC), 2-crotonyloxymethyl-4,5,6-trihydroxycylohex-2-enone (COTC), and methyl-gerfelin were much more cytotoxic against HA-CML cells than their parental cells in vitro. Notably, when K562/HA-transplanted mice were treated with 100 mg/kg/day BBGC for 8 days, the treated mice survived longer than the untreated mice (Figure 1). These findings suggest that Glo-1 plays an important role in primitive CML cells survival under hypoxia. In conclusion, Glo-1 is a novel attractive target against hypoxia-adapted primitive CML cells in the bone marrow milieu. Investigation of hypoxia-specific pathways and roles on CML cells could develop novel therapeutic approach targeting TKIs resistance. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

New immortalized human stromal cell lines enhancing in vitro expansion of cord blood hematopoietic stem cells

International Journal of Molecular Medicine ◽

10.3892/ijmm.13.3.363 ◽

2004 ◽

Author(s):

Sergio De Angeli ◽

Rosa Di Liddo ◽

Sabrina Buoro ◽

Luana Toniolo ◽

Maria Conconi ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cord Blood ◽

Cell Lines ◽

Stromal Cell ◽

Hematopoietic Stem ◽

In Vitro Expansion

Download Full-text

Systematic analysis of the ability of stromal cell lines derived from different murine adult tissues to support maintenance of hematopoietic stem cells in vitro

Journal of Cellular Physiology ◽

10.1002/jcp.1041450307 ◽

1990 ◽

Vol 145 (3) ◽

pp. 434-443 ◽

Cited By ~ 26

Author(s):

Maribel Rios ◽

David A. Williams

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Cell Lines ◽

Stromal Cell ◽

Systematic Analysis ◽

Hematopoietic Stem ◽

Adult Tissues

Download Full-text

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200225091339 ◽

2020 ◽

Vol 15 ◽

Author(s):

Fatima Aerts-Kaya

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Ex Vivo ◽

Stress Conditions ◽

Stress Factors ◽

Hematopoietic Stem ◽

Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

Download Full-text