Acceleration of hematopoietic reconstitution with a synthetic cytokine (SC-55494) after radiation-induced bone marrow aplasia

The synthetic cytokine (Synthokine) SC-55494 is a high-affinity interleukin-3 (IL-3) receptor ligand that stimulates greater in vitro multilineage hematopoietic activity than native IL-3, while inducing no significant increase in inflammatory activity relative to native IL-3. The aim of this study was to investigate the in vivo hematopoietic response of rhesus monkeys receiving Synthokine after radiation-induced marrow aplasia. Administration schedule and dose of Synthokine were evaluated. All animals were total-body irradiated (TBI) with 700 cGy 60Co gamma radiation on day 0. Beginning on day 1, cohorts of animals (n = 5) received Synthokine subcutaneously (SC) twice daily with 25 micrograms/kg/d or 100 micrograms/kg/d for 23 days or 100 micrograms/kg/d for 14 days. Control animals (n = 9) received human serum albumin SC once daily at 15 micrograms/kg/d for 23 days. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (NEUT; absolute neutrophil count [ANC] 500/microL) and thrombocytopenia (THROM; platelet count 20,000/microL) were assessed. Synthokine significantly (P .05) reduced the duration of THROM versus the HSA-treated animals regardless of dose or protocol length. The most striking reduction was obtained in the animals receiving 100 micrograms/kg/d for 23 days (THROM = 3.5 v 12.5 days in HSA control animals). Although the duration of NEUT was not significantly altered, the depth of the nadir was significantly lessened in all animal cohorts treated with Synthokine regardless of dose versus schedule length. Bone marrow progenitor cell cultures indicated a beneficial effect of Synthokine on the recovery of granulocyte-macrophage colony-forming units that was significantly higher at day 24 post-TBI in both cohorts treated at 25 and 100 micrograms/kg/d for 23 days relative to the control animals. Plasma pharmacokinetic parameters were evaluated in both normal and irradiated animals. Pharmacokinetic analysis performed in irradiated animals after 1 week of treatment suggests an effect of repetitive Synthokine schedule and/or TBI on distribution and/or elimination of Synthokine. These data show that the Synthokine, SC55 94, administered therapeutically post-TBI, significantly enhanced platelet recovery and modulated neutrophil nadir and may be clinically useful in the treatment of the myeloablated host.

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Humanized Sc(Fv)2 Minibody against C-Mpl Successfully Increased Platelet Number in Monkey and Increased Engraftment of Human Umbilical Cord Blood Cells in NOD/SCID Mouse.

Blood ◽

10.1182/blood.v112.11.2322.2322 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2322-2322

Author(s):

Takashi Yoshikubo ◽

Yoshihiro Matsumoto ◽

Masahiko Nanami ◽

Takayuki Sakurai ◽

Hiroyuki Tsunoda ◽

...

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Progenitor Cells ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Platelet Recovery ◽

Cynomolgus Monkeys ◽

Cd34 Cells

Abstract Thrombopoietin (TPO, the ligand for c-mpl) is a key factor for megakaryopoiesis. Several clinical trials of TPO have been conducted for thrombocytopenia without much success due to, in part, the production of neutralized antibodies against endogenous TPO, which causes thrombocytopenia. To overcome this problem, we previously demonstrated that mouse type minibody against c-mpl, with an amino acid sequence totally different from TPO, showed megakaryopoiesis and increased platelet numbers in monkey. This time, using CDR grafting, we generated a humanized sc(Fv)2VB22B minibody (huVB22B) against c-mpl for therapeutic use. The new minibody showed almost the same activity in vitro as TPO and the mouse type minibody, confirmed by both a human megakaryocyte cell (CD41+) differentiation assay and a proliferation assay with TPO-dependent cell line, M-07e. Single sc or iv administration of huVB22B to cynomolgus monkeys showed a dose-dependent increase in platelet numbers. Pharmacokinetic analysis showed that the plasma half-life (T1/2) of huVB22B at iv and sc administration to cynomolgus monkeys was 7–8 h and 11–16 h, respectively. After administration of huVB22B, the platelets of these monkeys increased and showed functional aggregation in response to ADP in vitro. Repeated administration of huVB22B (0.2, 2 and 20mg/kg/week) revealed that the increase in platelet level in cynomolgus monkeys was maintained for a month. Very slight reticular fibers in bone marrow were detected in a high dose group (20mg/ kg). No overt changes were detected by toxicity evaluations including clinical pathology and histopathology in 0.2 and 2mg/kg groups. No neutralized activities in plasma were observed during these experiments. Next, we examined the activities of huVB22B on human bone marrow-derived CD34-positive cells (BM-CD34+) and umbilical cord blood-derived CD34-positive cells (UCB-CD34+) in vitro. BM-CD34+ and UCB-CD34+ cells were cultured with huVB22B in serum free medium. HuVB22B induced differentiation of CD41+ cells from BM-CD34+ or UCB-CD34+ cells in a similar dose-dependent manner. However, UCB-CD34+ cells showed greater proliferation in response to huVB22B compared to BM-CD34+ cells. We then examined the in vivo activities of huVB22B on UCB CD34+ cells by treating NOD/SCID mice transplanted with human UCB-CD34+ cells with huVB22B and examining the bone marrow cells of the mice. The results showed that, compared with the control, administration of huVB22B showed an increase in the number of human hematopoietic progenitor cells (CD34+), lymphoid lineage cells (CD19+), and myeloid lineage cells (CD33+) in addition to human CFU-Meg cells (CD41+). These results suggest that c-mpl stimulation in vivo after transplantation might increase engraftment of progenitor cells in the bone marrow microenvironment and subsequently induce differentiation to multilineage cells. Umbilical cord blood transplantation faces frequent complications including a low-level stem/progenitor cell engraftment and delayed platelet recovery. Our results suggest that c-mpl stimulation might be used to increase the engraftment of UCB stem/progenitor cells and shorten the time of platelet recovery following UCB transplantation.

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Phosphorylation of the Mdm2 oncoprotein by the c-Abl tyrosine kinase regulates p53 tumor suppression and the radiosensitivity of mice

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1611798114 ◽

2016 ◽

Vol 113 (52) ◽

pp. 15024-15029 ◽

Cited By ~ 5

Author(s):

Michael I. Carr ◽

Justine E. Roderick ◽

Hong Zhang ◽

Bruce A. Woda ◽

Michelle A. Kelliher ◽

...

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Tumor Suppression ◽

Bone Marrow Failure ◽

Genotoxic Stress ◽

Whole Body ◽

Atm Kinase ◽

Radiation Induced

The p53 tumor suppressor acts as a guardian of the genome by preventing the propagation of DNA damage-induced breaks and mutations to subsequent generations of cells. We have previously shown that phosphorylation of the Mdm2 oncoprotein at Ser394 by the ATM kinase is required for robust p53 stabilization and activation in cells treated with ionizing radiation, and that loss of Mdm2 Ser394 phosphorylation leads to spontaneous tumorigenesis and radioresistance in Mdm2S394A mice. Previous in vitro data indicate that the c-Abl kinase phosphorylates Mdm2 at the neighboring residue (Tyr393) in response to DNA damage to regulate p53-dependent apoptosis. In this present study, we have generated an Mdm2 mutant mouse (Mdm2Y393F) to determine whether c-Abl phosphorylation of Mdm2 regulates the p53-mediated DNA damage response or p53 tumor suppression in vivo. The Mdm2Y393F mice develop accelerated spontaneous and oncogene-induced tumors, yet display no defects in p53 stabilization and activity following acute genotoxic stress. Although apoptosis is unaltered in these mice, they recover more rapidly from radiation-induced bone marrow ablation and are more resistant to whole-body radiation-induced lethality. These data reveal an in vivo role for c-Abl phosphorylation of Mdm2 in regulation of p53 tumor suppression and bone marrow failure. However, c-Abl phosphorylation of Mdm2 Tyr393 appears to play a lesser role in governing Mdm2-p53 signaling than ATM phosphorylation of Mdm2 Ser394. Furthermore, the effects of these phosphorylation events on p53 regulation are not additive, as Mdm2Y393F/S394A mice and Mdm2S394A mice display similar phenotypes.

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In Vivo Expansion of Megakaryocyte Progenitors in CD26−/ − Mice: Implications for Novel Post-Transplant Therapeutics.

Blood ◽

10.1182/blood.v110.11.1188.1188 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1188-1188

Author(s):

Shannon Kidd ◽

Carlos E. Bueso-Ramos ◽

Laura A. Paganessi ◽

Henry Fung ◽

Stephanie Gregory ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Flow Cytometric Analysis ◽

Severe Thrombocytopenia ◽

Hematopoietic Stem ◽

Platelet Recovery ◽

Post Transplant ◽

Complete Blood Counts ◽

And Function

Abstract Hematopoietic stem cell transplantation (HSCT) is a successful treatment option for patients with malignant or non-malignant severe hematologic diseases. Readily available umbilical cord blood (CB) has emerged as an important donor source that has a lower histocompatability requirement and carries a reduced risk of graft vs. host disease. However, a frequent complication of CB use is delayed hematopoietic recovery, in particular long lasting severe thrombocytopenia (median of 117 days to platelet recovery, COBLT 2005). To overcome this clinically, effective strategies for enhancing megakaryopoiesis, thrombopoiesis, or both are needed. We have previously described the importance of CD26 (dipeptidylpeptidase IV) in engraftment (Christopherson, KW 2nd, et al, Science2004. 305:1000–3). However, the involvement of CD26 in megakaryopoiesis has not been investigated. We hypothesized that CD26 acts to suppress megakaryopoiesis and that removal of CD26 activity would result in expansion of the megakaryocyte progenitor population in vivo. To test this hypothesis, we evaluated megakaryocyte (MK) development in the context of development of other mature blood cells in CD26 deficient (CD26−/ −) mice as compared to control C57BL/6 mice. Histological analysis of formalin fixed paraffin embedded tissue sections and peripheral blood cytopsins revealed an increased presence of MK in the bone marrow, spleen, thymus and peripheral blood. However, complete blood counts (CBC) suggest no difference in white blood cell, neutrophil, lymphocyte, monocyte, eosinophil, basophil, and platelet counts. Flow cytometric analysis also revealed no significant changes in CD3, CD4, or CD8 T-cells; B220 B-cells; and Gr-1/Mac-1 granulocytes/neutrophils in the bone marrow, spleen, thymus, or peripheral blood as appropriate. There was also no change in the percentage of peripheral blood CD41 MK but there was a increase from 0.09% in C57BL/6 BM to 0.26% CD41+Sca-1+c-kit− MK progenitors in the CD26−/ − BM (P≤0.05). Methylcellulose based myeloid progenitor assays did also reveal respective CFU-GM, BFU-E, and CFU-GEMM per femur values (mean±SEM) of 24480±1426, 1448±154.86, and 852±41 for C57BL/6 BM cells; 24451±1342, 974.29±81, and 1634±177 for CD26−/ − BM cells. This represents a 92% increase in CFU-GEMM and corresponding 30% decrease in BFU-E in the CD26−/ − mouse BM (P≤0.01, n=7 mice/group). Collagen based megakaryocyte progenitor assays (CFU-MK) revealed a 25% increase from 4455.00±207.62 colonies/femur in C57BL/6 BM to 5555.00±608.12 in CD26−/ − BM (P≤0.05, n=3 mice/group). These results establish a basis on which to propose that CD26 may act to regulate early events in megakaryocyte progenitor formation and function. They also suggest that the use of CD26 inhibitors may have a beneficial effect on improved megakaryocyte progenitor function and/or reconstitution post-transplant.

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Improved Survival and Recovery of Hematopoiesis Following Lethal Radiation by Chloroquine-Mediated Activation of ATM

Blood ◽

10.1182/blood.v116.21.2228.2228 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2228-2228

Author(s):

Yiting Lim ◽

Mohammad Hedayati ◽

Akil Merchant ◽

Yonggang Zhang ◽

Theodore DeWeese ◽

...

Keyword(s):

Bone Marrow ◽

Dose Rate ◽

Cell Lines ◽

Research Funding ◽

Bone Marrow Cells ◽

Bone Marrow Failure ◽

Chloroquine Treatment ◽

Radiation Induced

Abstract Abstract 2228 Irreversible bone marrow damage and impaired blood formation is the primary cause of death following exposure to high doses of radiation. Moreover, the rate at which radiation is delivered may have a profound impact on cytotoxicity; prolonged exposure at a low dose-rate (LDR; 9.4 cGy/hr) has been found to induce greater cell death than the same total dose given at a high dose-rate (HDR; 4500 cGy/hr). Few non-toxic agents are presently available that can offer substantial protection against radiation induced bone marrow failure and death, especially during LDR exposure. We previously demonstrated that chloroquine, a commonly used agent in the treatment of malaria and rheumatologic diseases, can prevent LDR radiation induced cytotoxicity of cell lines in vitro and studied its effects on hematopoiesis in vivo. We initially quantified the effects of LDR radiation on C57/B6 mice and found that 9 Gy delivered at 9.4 cGy/hr for 95.7 hrs induced death in 13/19 (68%) of animals at 15–35 days after radiation. The administration of syngeneic bone marrow cells (1 × 106 cells) immediately after LDR radiation completely rescued animals (10/10) demonstrating that bone marrow failure was responsible for LDR radiation induced death similar to HDR radiation. Next we treated mice with chloroquine (0.0594 mg/17g body weight, i.p.) 24 hrs and 4 hrs prior to exposure to LDR radiation and found that it significantly improved survival (80%, p < 0.05) compared to untreated animals exposed to LDR radiation (32%). We examined hematopoietic recovery following LDR radiation and found that the peripheral WBC was significantly greater in mice treated receiving chloroquine (3.4 × 106/ml vs 1.1 × 106/ml at day 16, p<0.05). Similarly, we found that in vivo chloroquine treatment significantly increased the recovery of bone marrow myeloid CFC (p=0.02), suggesting that it impacted myeloid progenitors. To further validate this finding, we transplanted bone marrow from LDR irradiated mice into lethally irradiated CD45 congenic recipient mice, and found a significant improvement in early engraftment (4.2% vs. 0.4% engraftment at 6 weeks post-transplant, p=0.015). Chloroquine has been found to protect cancer cell lines from LDR radiation in vitro by activating ATM, an essential DNA damage sensor. We examined the effect of chloroquine on ATM and treated unradiated lin- bone marrow cells with chloroquine in vitro (35 ug/ml, 2 hr). Compared to control cells, chloroquine treated cells expressed 2.5-fold more phosphorylated ATM suggesting that the activation of ATM by chloroquine abrogated the lethal effects of LDR radiation in hematopoietic progenitors. We confirmed that ATM was required for chloroquine-mediated radioprotection by studying ATM null mice. In contrast to wild type mice, chloroquine treatment failed to protect ATM null mice from LDR radiation (9 Gy total) with 8/13 (62%) and 9/13 (69%) of animals surviving in treated or non treated mice, respectively (p=0.86). These data suggest that chloroquine exerts a radioprotective effect from LDR radiation by activating ATM in vivo, and may represent a novel means of limiting acute bone marrow failure in the event of widespread environmental LDR radiation exposure. Disclosures: Matsui: Pfizer: Consultancy; Bristol-Meyers Squibb: Consultancy; Infinity Phamaceuticals: Consultancy, Patents & Royalties; Merck: Consultancy, Research Funding; Geron Corporation: Research Funding.

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A Small Molecule Inhibitor Targeting CREB and CBP Inhibits Proliferation of AML Cells In Vitro and In Vivo,

Blood ◽

10.1182/blood.v118.21.3607.3607 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3607-3607

Author(s):

Grace I Aldana-Masangkay ◽

Bryan Mitton ◽

Alan K Ikeda ◽

Kazunari Yamada ◽

Bingbing Li ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Small Molecule ◽

Binding Protein ◽

Small Molecule Inhibitor ◽

Pharmacokinetic Analysis ◽

Serum Drug Concentration ◽

Molecule Inhibitor

Abstract Abstract 3607 The cAMP Response Element Binding Protein, CREB, is a nuclear transcription factor that is a downstream target of signaling pathways regulating memory, glucose homeostasis, cell proliferation, differentiation, and survival. We previously demonstrated that CREB is overexpressed at both the protein and mRNA levels in leukemia blasts and in leukemia stem cells. AML patients who overexpress CREB in their bone marrow have an increased risk of relapse and decreased event-free survival. To determine whether CREB is sufficient for leukemogenesis, we created a transgenic mouse in which CREB is expressed under the control of a myeloid specific hMRP8 promoter. Bone marrow progenitors from CREB transgenic mice had higher proliferative potential and replating ability. These mice developed myeloproliferative disease after one year but not acute leukemia, suggesting that CREB is not sufficient to induce myeloid transformation. To determine whether CREB is necessary for leukemia cell proliferation, we transduced AML cells with lentiviral CREB shRNA. We observed that downregulation of CREB led to decreased AML cell proliferation and survival in vitro. Furthermore, our results demonstrated that CREB knockdown inhibits the growth of AML cells in vivo without affecting normal hematopoietic stem cell function. Together, these results strongly suggest that CREB acts as a proto-oncogene and is a potential target for AML therapy. CREB is activated through phosphorylation, leading to the recruitment of the histone acetyltransferase, CREB binding protein (CBP) and subsequent target gene expression. XX-650-23 (MW: 288.3) is a small molecule that was identified through in silico screening methods to inhibit the interaction between CREB and CBP. We tested the effects of the drug on various AML cell lines using MTT assays and trypan blue exclusion. The IC50 ranged from 700 nM to 2 μM after 72 hours of drug treatment. However, treatment of normal human bone marrow progenitors cultured in methylcellulose containing XX-650-23 at a concentration of 10 μM had no effect on colony numbers. We also tested the in vivo effects of XX-650-23 using xenograft NOD-SCID IL-2Rgamma null (NSG) mouse models. To assess toxicity, mice were treated with the drug at various concentrations, ranging from 10 to 20 mg/kg, by intraperitoneal injection (IP) once daily for 28 days. We did not observe any weight loss or hematologic, renal, hepatic, or cardiac toxicity in the mice. We also performed pharmacokinetic analysis to determine the half-life and stability of the drug. After 1 hour of treatment, the serum drug concentration was 33 nM. The estimated drug mean residence time was 7.5 hours. Plasma clearance divided by IP absorption fraction was 9.6 L/min/kg. The mice were treated daily with drug (17.5mg/kg IP) or vehicle control once MV4-11 cells reached a tumor size of 300mm3 or at the time cells were injected. Our results demonstrated significant inhibition of tumor growth with treatment of the drug started on the day of injection of cells compared to waiting until the tumor reached 300mm3. Together, these data suggest that a small molecule inhibitor targeting CREB and CBP interaction is a potential avenue for drug development. We are currently studying the mechanisms by which XX-650-23 inhibits AML cell proliferation and analyzing the effects of combining the drug with chemotherapy. Disclosures: No relevant conflicts of interest to declare.

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Adipose-Derived Mesenchymal Stem Cells with Potential to Ameliorate Platelet Recovery and Enhance Megakaryopoiesis in a Mouse Model of Radiation-Induced Thrombocytopenia Via the PI3k/Akt Pathway

Blood ◽

10.1182/blood.v124.21.2931.2931 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2931-2931

Author(s):

Xiao-Hui Zhang ◽

Jia-Min Zhang ◽

Fei-Er Feng ◽

Qian-Ming Wang ◽

Xiao-Lu Zhu ◽

...

Keyword(s):

Bone Marrow ◽

Platelet Transfusion ◽

Treated Group ◽

Saline Group ◽

Akt Pathway ◽

Severe Thrombocytopenia ◽

Platelet Recovery ◽

Phosphorylated Akt ◽

Radiation Induced

Abstract *XHZ and JMZ contribute equally to this work # Co-correspondence to: Xiao-hui Zhang and Xiao-Jun Huang E-mail: [email protected] Introduction: Substantial damage to the bone marrow can be caused by exposure to radiation, which can then develop into pancytopenia, especially severe thrombocytopenia. Severe thrombocytopenia can be a major, life-threatening event in untreated individuals. Recent studies have highlighted the role that radiation-induced thrombocytopenia plays in radiation mortality. However, studies focused on mitigating radiation-induced thrombocytopenia have rarely been reported. Among all alternative therapies available to patients experiencing radiation-induced thrombocytopenia, platelet transfusion is most effective. As a supportive therapy, platelet transfusion cannot substantially reconstitute the damaged hematopoietic properties of the bone marrow. Adipose-derived mesenchymal stem cells (ADSCs) are capable of migrating to injured tissue sites for damage repair. Moreover, it was demonstrated that ADSCs could support hematopoiesis both in vitro and in vivo. However, the therapeutic effects of ADSCs in radiation-induced thrombocytopenia as well as the underlying mechanism remain unknown. In this study, we hypothesized that administration of ADSCs may mitigate thrombocytopenia after radiation exposure. We investigated the in vivo impact of ADSCs on megakaryopoiesis and platelet recovery, and whether this amelioration effect was mediated through the activation of PI3K/Akt pathway in irradiated mice. Methods: The mouse model of radiation-induced thrombocytopenia was established by first irradiating mice with a 4Gy dose. Then, 15 mice were immediately injected with suspended ADSCs (1×106cells in 0.3 ml) and another 15 mice with equivalent saline. Ten unirradiated mice served as a control group. Platelet counts and white blood cell (WBC) counts in the peripheral blood were detected every week. Total colony formation units (CFU), megakaryocyte colony formation units (MK-CFU) and CD41+ cells in the bone marrow were assessed 21 days after irradiation. Bone marrow cellularity was determined by hematoxylin and eosin (HE) staining, and apoptosis was detected by terminal-deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) assay. Western blots were performed with anti-phosphorylated Akt, anti-Bcl-2 and anti-bax antibodies. Results: ADSCs obtained from the inguinal adipose exhibited a fibroblast-like morphology. Cultured cells were positive for CD29 (99.79%) and CD90 (97.82%), but not CD34 (3.08%), CD45 (1.04%) and CD31 (3.08%). Radiation markedly reduced peripheral blood counts, with a nadir on day 7. Recovery of both platelets (546.33±62.99 vs 375.48±50.33×109/L, P<0.05) and WBCs (4.23±0.51 vs 2.46±3.10×109/L, P<0.05) were better in the ADSCs-treated group compared with the saline group, respectively, 21 days after irradiation. A significant increase in total CFU (34.55±4.21 vs 12.86±2.15, P<0.05) and MK-CFU (6.28±0.74 vs 2.54±0.39, P<0.05) after irradiation were observed in the ADSCs group compared with the saline group, respectively. Further, the proportion of CD41+ cells in the ADSCs group was significantly higher than that in the saline group (4.2%±0.54% vs 1.21%±0.11%, P<0.05). A radioprotective effect was shown in the ADSCs-treated group, especially in the megakaryocytic lineage, by HE staining. In the ADSCs group, the number of apoptotic cells was significantly lower than that in the saline group (3.52±0.42 vs 13.48±2.15 per field, P<0.05). Administration of ADSCs up-regulated protein expression of phosphorylated Akt and Bcl-2, whereas the expression of Bax, a protein related to apoptosis, was significantly lower in the ADSCs group. Conclusion:For the first time, we demonstrated that ADSCs were capable of promoting platelet recovery and improving megakaryopoiesis in irradiated mice. This protective function of ADSCs is likely to be mediated via the PI3K/Akt pathway, which would thus provide a new therapeutic alternative for mitigating radiation-induced thrombocytopenia. Disclosures No relevant conflicts of interest to declare.

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P53 Downregulation-Based Strategy to Protect Genomic Integrity from Radiation Therapy

Blood ◽

10.1182/blood-2020-139137 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 24-25

Author(s):

Hang Su ◽

Mei-Jun Long ◽

Joel E Michalek ◽

Michael Weil ◽

Chul S Ha

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Gene Deletion ◽

Bone Marrow Cells ◽

Genomic Integrity ◽

Normal Tissues ◽

Radiation Induced ◽

Marrow Cells

Background: Activation of p53 is one of major pathways by which DNA damaging agents (DDA) such as radiation and chemotherapy cause toxicity in normal tissues and it induces a cascade of events that eventually leads to cell senescence or cell death. We have reported that a brief pretreatment with low dose arsenic (LDA), by temporarily and reversibly downregulating p53 at the time of treatment with DDA, reduces the normal tissue toxicity without compromising tumor response to treatment. This protective effect is selective to normal tissues, as it requires functional p53. Though not every cancer cell has detectable p53 mutations, essentially every cancer cell has dysfunctional p53. Therefore most cancer cells will not be protected by this strategy. Genomic instability and inability to repair DNA damage from DDA in the hematopoietic stem cells have been attributed to the development of therapy-induced myelodysplastic syndrome (tMDS) and acute myeloid leukemia (AML). We have also been studying the effect of LDA on the genome in the setting of cancer therapy. We have reported that LDA pretreatment significantly reduces radiation-induced DNA double strand breaks (DSBs) and apoptosis in normal cells both in-vitro and in-vivo. Persistent DNA damage such as DSBs can trigger genomic instability and can be prevented by proper DNA repair. Our previous work using comet assay to quantify DNA damage after radiation has indicated that DNA repair capacity is enhanced by LDA pretreatment. A role for LDA in maintaining genomic integrity has been implicated in our in-vitro studies, where we found that LDA protected telomeres from enhanced erosion by DDA in Concanavalin A-activated normal human lymphocytes, and that LDA reduced spontaneous and radiation-induced mutations in mouse embryonic stem cells. Yet, whether this p53 downregulation-based strategy helps genome maintenance during cancer treatment using DDA has not been investigated in-vivo. CBA/Ca mice have 15-25% incidence of AML after 3 Gy of total body ionizing radiation (IR). About 95% of mice that develop radiation-induced AML (rAML) have a deletion on chromosome 2 encompassing the PU.1 gene. Since PU.1 deletion is a critical contributor to and a useful surrogate marker for leukemogenesis in the murine rAML model, we tested a hypothesis whether pretreatment with LDA before IR helps maintain genomic integrity by evaluating bone marrow cells for PU.1 gene deletion. Method: One hundred twenty mice were randomized into four groups: PBS+sham IR (control), LDA+sham IR, PBS+IR and LDA+IR. Prior to sham or 3 Gy of IR, CBA/Ca mice were injected with either PBS or LDA intraperitoneally at the dose of 0.4mg/kg for 3 days. At 7, 30 and 180 days after radiation, bone marrow cells were collected from femurs and fixed with Carnoy's Fixative. To assess the effect of LDA on PU.1 gene deletion, fluorescence in-situ hybridization (FISH) assay was performed. An ATTO550 labeled PU.1 probe was designed and used to detect deletions that occur in 2qE1 and involve the PU.1 gene locus, as well as two 6-FAM labeled probes for centromere and telomere respectively. Four to five hundred cells were analyzed for each mouse. Statistical significance was determined from a two-way analysis of variance in log units using SAS Version 9.4. Result: We successfully established the FISH assay that can specifically detect the PU.1 gene not only in metaphase cells but also in interphase cells. As shown in the figure, mice in the LDA+IR group have significantly fewer bone marrow cells exhibiting PU.1 gene deletion compared with PBS+IR group at all three time points examined (Day 7: 2±1.2% vs 3.7±2.6%, P=0.047; Day 30: 1.9±1.1% vs 3.2±1.9%, P=0.040; Day 180: 2.8±1.0% vs 5.6±3.5%, P=0.014). LDA treatment alone has a negligible effect on PU.1 loss as compared to the control group. Conclusion: Our result suggests that LDA pretreatment protects genomic integrity following IR treatment in-vivo. As the development of rAML is a multi-step process, the impact of LDA pretreatment on the actual incidence of secondary malignancy needs further validation in animal models. The genome-protective effect of LDA that we have revealed supports its potential use as a strategy to reduce the development of radiation-induced secondary malignances such as MDS and AML. Disclosures Ha: Protectum Oncology: Current Employment, Current equity holder in private company.

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Mechanism of Action of PLX-R18, a Placental-Derived Cellular Therapy for the Treatment of Radiation-Induced Bone Marrow Failure

Blood ◽

10.1182/blood.v126.23.2417.2417 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2417-2417 ◽

Cited By ~ 1

Author(s):

Racheli Ofir ◽

Lena Pinzur ◽

Akyüz Levent ◽

Zami Aberman ◽

Raphael Gorodetsky ◽

...

Keyword(s):

Bone Marrow ◽

Mechanism Of Action ◽

Blood Cells ◽

Treated Group ◽

Cell Counts ◽

Animal Survival ◽

Radiation Induced ◽

Human Cytokines

Abstract Bone marrow (BM) failure occurs in individuals who fail to produce sufficient red blood cells, white blood cells, or platelets. This may be a result of damage to hematopoietic stem cells by a congenital defect or by exposure to a noxious substance or factor. PLX-R18 are 3D-expanded placenta-derived cells, with biological properties including a profound capacity to protect and regenerate bone marrow. The cells secrete a broad array of cytokines including G-CSF, IL-6, MCP-1, MCP-3 and GRO that contribute to the reconstitution of the hematopoietic and immune systems. To assess their therapeutic potential, PLX-RAD cells were administered to C3H/HeN male mice intramuscularly, one and five days following 7.7 Gy total body irradiation. Body weight and animal survival were monitored for 3 weeks, then the animals were euthanized for BM and blood analysis. In the PLX-R18-treated group, 10 out of 11 (91%) animals survived, compared to only 4 out of 9 in the vehicle treated group (44%), (P < 0.05). Weight reduction after irradiation was also improved in the PLX-RAD treated mice where the average weight loss was less than 10% compared to almost 20% for vehicle-treated controls. Consistent with the improved survival, cell counts of all the three hematopoietic lineages were significantly increased in the BM and blood of PLX-R18 treated mice as compared to the control animals, and attained close to normal levels. Analysis of plasma in the irradiated treated animals detected the presence of critical, PLX-R18-derived (human) cytokines as well as alterations in the equivalent murine cytokines, suggesting a direct role of PLX-R18 secreted cytokines in animal survival. Interestingly, human cytokines were detected only after irradiation and were not present in the plasma of sham non-irradiated animals treated with PLX-R18. This may imply that the active in vivo cytokine secretion by PLX-R18 is a response to signals from the environment in the irradiated animals. Already by day 4-6 after irradiation, the number of colony forming progenitors in the BM of PLX-R18 treated animals was significantly higher than in control animals suggesting that earlier regeneration of BM induced by PLX-R18 secreted cytokines results in improved blood counts and increased survival. Studies in vitro demonstrated that PLX-R18-derived conditioned medium induced the formation of all colony types in a methyl cellulose colony formation assay as well as a 3.1 fold-increase in the number of migrating cells in a BM migration assay compared to the SDF-1-supplemented positive control. These results, in vivo and in vitro, reveal key clues to the resolution of the underlying mechanism of action. Administration of the novel cell product PLX-R18 markedly improved survival and recovery of the three hematopoietic blood lineages after radiation induced BM failure indicating potential as a highly effective therapy for general radiation-induced BM damage, or in a nuclear disaster scenario. Further clinical studies are planned. Disclosures Ofir: Pluristem ltd: Employment. Pinzur:Pluristem ltd: Employment. Aberman:Pluristem ltd: Employment. Gorodetsky:Pluristem ltd: Consultancy. Volk:Pluristem ltd: Consultancy.

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Model-Based Dose-Exposure-Response Assessment for Lead and Backup Spectinamide in a Mouse Model of Tuberculosis

10.21007/etd.cghs.2020.0520 ◽

2020 ◽

Author(s):

◽

Santosh Wagh ◽

Keyword(s):

Population Pharmacokinetic Analysis ◽

Pharmacokinetic Analysis ◽

Pharmacokinetic Parameters ◽

Population Pharmacokinetic ◽

Response Relationship ◽

Exposure Response ◽

Antibiotic Effect ◽

Post Antibiotic Effect

Despite decades of research, tuberculosis remains the oldest pathogen-based disease that is the leading cause of death from a single infectious agent. Among many anti-tubercular therapies under investigation, the semisynthetic compounds spectinamides are a promising novel class of anti-tuberculosis agents. One such lead candidate, spectinamide 1810, and backup spectinamide 1599 have demonstrated excellent efficacy, safety, and drug-like properties in various in vitro and in vivo assessments. The dose-ranging and dose fractionation studies were designed to characterize the dose-exposure-response relationship for lead and backup spectinamide in a mouse model of Mycobacterium tuberculosis infection. In this current study, we used 26 and 23 combinations of dose level and dosing frequency for the lead and backup spectinamide, respectively. The dedicated pharmacokinetic studies with a collection of series of blood samples were conducted in healthy animals. Population pharmacokinetic analysis was performed using non-linear mixed effect modeling to estimate pharmacokinetic parameters in healthy animals. The Bayesian principles were applied for reliable pharmacokinetic estimation in infected animals by using informed priors obtained from healthy animals. The individual pharmacokinetic parameters were obtained for infected animals through post-hoc estimation and subsequently used for pharmacokinetic/-pharmacodynamic (PK/PD) indices and mechanism-based PK/PD modeling. The obtained data on spectinamides’ plasma concentrations and counts of colony-forming units were analyzed using a PK/PD approach as well as classical anti-infective PK/PD indices. The population pharmacokinetic analysis results suggest that there is no difference in the pharmacokinetic parameters of lead and backup spectinamide in infected animals as compared to healthy animals. The PK/PD index analysis showed that the efficacy of spectinamide 1810 is largely driven by concentration (Cmax/MIC) and exposure (AUC/MIC) rather than a threshold minimum inhibitory level (T>MIC). Although similar results were obtained for spectinamide 1599 in previously performed in vitro experiments, in the present in vivo studies, spectinamide 1599 did not demonstrate the expected correlation between efficacy and PK/PD indices. Therefore, we could not identify major drivers for the efficacy of this compound. Additionally, a novel mechanism-based PK/PD model with consideration to post-antibiotic effect could adequately describe the exposure-response relationship for lead and backup spectinamide. This supports the idea that the in vitro observed post-antibiotic effect of these spectinamides can translate to the in vivo situation, as well. Altogether we suggest, the obtained results and pharmacometric model for the exposure-response relationship of lead and backup spectinamides provide a rational basis for dose selection for future efficacy studies of these compounds against Mycobacterium tuberculosis in mice and other animal species.

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The sialyltransferase ST6GAL1 protects against radiation-induced gastrointestinal damage

Glycobiology ◽

10.1093/glycob/cwz108 ◽

2020 ◽

Vol 30 (7) ◽

pp. 446-453 ◽

Cited By ~ 1

Author(s):

Patrick R Punch ◽

Eric E Irons ◽

Charles T Manhardt ◽

Himangi Marathe ◽

Joseph T Y Lau

Keyword(s):

Bone Marrow ◽

Marrow Transplant ◽

High Dose ◽

Wild Type ◽

Γ Irradiation ◽

Risk Of Death ◽

Radiation Induced ◽

Total Body

Abstract High-dose irradiation poses extreme risk of mortality from acute damage to the hematopoietic compartment and gastrointestinal tract. While bone marrow transplantation can reestablish the hematopoietic compartment, a more imminent risk of death is posed by gastrointestinal acute radiation syndrome (GI-ARS), for which there are no FDA-approved medical countermeasures. Although the mechanisms dictating the severity of GI-ARS remain incompletely understood, sialylation by ST6GAL1 has been shown to protect against radiation-induced apoptosis in vitro. Here, we used a C57BL/6 St6gal1-KO mouse model to investigate the contribution of ST6GAL1 to susceptibility to total body irradiation in vivo. Twelve gray total body ionizing γ-irradiation (TBI) followed by bone marrow transplant is not lethal to wild-type mice, but St6gal1-KO counterparts succumbed within 7 d. Both St6gal1-KO and wild-type animals exhibited damage to the GI epithelium, diarrhea and weight loss, but these symptoms became progressively more severe in the St6gal1-KO animals while wild-type counterparts showed signs of recovery by 120 h after TBI. Increased apoptosis in the GI tracts of St6gal1-KO mice and the absence of regenerative crypts were also observed. Together, these observations highlight an important role for ST6GAL1 in protection and recovery from GI-ARS in vivo.

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