Sublethal Irradiation Research Articles

Calcium/calmodulin-dependent kinase kinase 2 regulates hematopoietic stem and progenitor cell regeneration

Hematopoietic stem and progenitor cells (HSPCs) are predominantly quiescent in adults, but proliferate in response to bone marrow (BM) injury. Here, we show that deletion of Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) promotes HSPC regeneration and hematopoietic recovery following radiation injury. Using Camkk2-enhanced green fluorescent protein (EGFP) reporter mice, we found that Camkk2 expression is developmentally regulated in HSPC. Deletion of Camkk2 in HSPC results in a significant downregulation of genes affiliated with the quiescent signature. Accordingly, HSPC from Camkk2 null mice have a high proliferative capability when stimulated in vitro in the presence of BM-derived endothelial cells. In addition, Camkk2 null mice are more resistant to radiation injury and show accelerated hematopoietic recovery, enhanced HSPC regeneration and ultimately a prolonged survival following sublethal or lethal total body irradiation. Mechanistically, we propose that CaMKK2 regulates the HSPC response to hematopoietic damage by coupling radiation signaling to activation of the anti-proliferative AMP-activated protein kinase. Finally, we demonstrated that systemic administration of the small molecule CaMKK2 inhibitor, STO-609, to irradiated mice enhanced HSPC recovery and improved survival. These findings identify CaMKK2 as an important regulator of HSPC regeneration and demonstrate CaMKK2 inhibition is a novel approach to promoting hematopoietic recovery after BM injury.

INKT cell activation plus T-cell transfer establishes complete chimerism in a murine sublethal bone marrow transplant model.

Transplant tolerance induction makes it possible to preserve functional grafts for a lifetime without immunosuppressants. One powerful method is to generate mixed hematopoietic chimeras in recipients by adoptive transfer of donor-derived bone marrow cells (BMCs). In our murine transplantation model, we established a novel method for mixed chimera generation using sublethal irradiation, CD40-CD40L blockade, and invariant natural killer T-cell activation. However, numerous BMCs that are required to achieve stable chimerism makes it difficult to apply this model for human transplantation. Here, we show that donor-derived splenic T cells could contribute to not only the reduction of BMC usage but also the establishment of complete chimerism in model mice. By cotransfer of T cells together even with one-fourth of the BMCs used in our original method, the recipient mice yielded complete chimerism and could acquire donor-specific skin-allograft tolerance. The complete chimeric mice did not show any remarks of graft versus host reaction in vivo and in vitro. Inhibition of the apoptotic signal resulted in increase in host-derived CD8+ T cells and chimerism brake. These results suggest that donor-derived splenic T cells having veto activity play a role in the depletion of host-derived CD8+ T cells and the facilitation of complete chimerism.

Life history traits and previous exposure predict resistance to UV irradiation in the freshwater cnidarianHydra oligactis

Abiotic stress is an important source of mortality for cnidarians and is likely to be a major factor shaping their life histories. In freshwater hydra, the ability to withstand exogenous sources of stress varies between species and populations, but little is known about the factors responsible for this variation. Here, we investigated resistance to UV irradiation in Hydra oligactis, a common temperate freshwater cnidarian. We collected polyps from 12 populations and propagated these asexually under standard conditions in the laboratory to obtain 69 laboratory clonal lines with a total of 324 polyps of different age. We measured the size of polyps and recorded their budding rate. In addition, a subset of animals was exposed to hormetic treatment, where experimental animals received a short, sublethal irradiation 2 d before testing their resistance to a higher dose. We investigated how life history traits (age, size, and budding rate), hormetic treatment, and the interaction between life history traits and hormetic treatment relate to the ability of hydra polyps to tolerate high doses of UV irradiation. In multivariate models controlling for the effect of other variables, stress tolerance was positively related to age (lower tolerance in freshly detached buds compared to adult hydra) and size (higher tolerance in polyps with a large body column). Budding rate was negatively associated with stress tolerance. Hormetic treatment increased resistance to UV irradiation, but we found no evidence for an interaction between hormetic response and any of the life history traits, suggesting that the ability to upregulate physiological defense mechanisms after exposure to mild stress does not depend on the life history background of the individuals.

PO-0989: Sub-lethal radiation allows an efficient antitumor therapy with engineered T-cells in RIP-Tag2 mice

PO-0989: Sub-lethal radiation allows an efficient antitumor therapy with engineered T-cells in RIP-Tag2 mice

Akt1/NFκB signaling pathway activation by a small molecule DMA confers radioprotection to intestinal epithelium in xenograft model

Normal tissue protection and recovery of radiation-induced damage are of paramount importance for development of radioprotector. Radioprotector which selectively protects normal tissues over cancerous tissues improves the therapeutic window of radiation therapy. In the present study, small bisbenzimidazole molecule, DMA (5-(4-methylpiperazin-1-yl)-2-[2′-(3,4-dimethoxy-phenyl)-5′-benzimidazolyl]-benzimidazole) was evaluated for in vivo radioprotective effects to selectively protect normal tissue over tumor with underlying molecular mechanism. Administration of single DMA dose prior to radiation has enhanced survival of Balb/c mice against sublethal and supralethal total body irradiation. DMA ameliorated radiation-induced damage of normal tissues such as hematopoietic (HP) and gastrointestinal tract (GI) system. Oxidative stress marker Malondialdehyde level was decreased by DMA whereas it maintained endogenous antioxidant status by increasing the level of reduced glutathione, glutathione reductase, glutathione-s-transferase, superoxide dismutase and total thiol content in hepatic tissue of irradiated mice. Mechanistic studies revealed that DMA treatment prior to radiation leads to Akt1/NFκB signaling which reduced radiation-induced genomic instability in normal cells. However, these pathways were not activated in tumor tissues when subjected to DMA treatment in similar conditions. Abrogation of Akt1 and NFκB genes resulted in no radioprotection by DMA and enhanced apoptosis against radiation. Plasma half-life of DMA was 3.5h and 2.65h at oral and intravenous dose respectively and 90% clearance was observed in 16h. In conclusion, these data suggests that DMA has potential to be developed as a safe radioprotective agent for radiation countermeasures and an adjuvant in cancer therapy.

Exploiting Immunogenic Modulation in Chordoma: Sublethal Radiation Increases EGFR Expression and Sensitizes Tumor Cells to Cetuximab

Chordoma is a rare primary spinal neoplasm for which standard therapy consists of surgery and radiation. However, the high dose of radiation required to kill chordoma is not always possible given their proximity to the brain and spinal cord. Cancer cells surviving radiation often have phenotypic changes making them more susceptible to immunotherapy, a process termed immunogenic modulation. Among these phenotypic changes is the upregulation of surface markers for which FDA approved monoclonal antibodies exist, such as trastuzumab (Anti‐HER2), rituximab (anti‐CD20), and cetuximab (anti‐EGFR). Upregulation of monoclonal antibody targets have previously been shown to increase natural killer (NK) cell lysis by antibody dependent cell‐mediated cytotoxicity (ADCC) in both chordoma and other carcinoma types. It is known that approximately 80% of chordomas express Epidermal Growth Factor Receptor (EGFR), though to varying degrees of positivity. We hypothesized that sublethal radiation of chordoma cells would increase EGFR expression and enhance the therapeutic effects of cetuximab. Three distinct chordoma cell lines were exposed to sublethal doses of radiation and changes in surface EGFR expression were monitored via flow cytometry. ADCC assays were then performed using NK cells from normal donors and cetuximab. We show here that chordoma cells surviving low dose ionizing radiation express increased EGFR levels. The functional consequence of this upregulation is significantly decreased proliferation compared to either radiation or cetuximab alone. ADCC assays also show increased killing in cells that have been irradiated compared to those which have not. While both radiotherapy and cetuximab have had mixed results as monotherapies for chordoma, our results suggest that cetuximab in combination with radiation will produce a more complete response than either therapy alone.Support or Funding InformationNational Cancer Institute

GM-CSF and IL-4 Fusion Cytokine Induces B Cell-Dependent Hematopoietic Regeneration

Hematopoietic stem cells (HSCs) have the capacity to self-renew and differentiate into hematopoietic cells and have been utilized to replace diseased bone marrow for patients with cancers and blood disorders. Although remarkable progress has been made in developing new tools to manipulate HSCs for clinic use, there is still no effective method to expand HSCs invivo for quick repopulation of hematopoietic cells following sublethal irradiation. We have recently described a novel synthetic cytokine that is derived from the fusion of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4; named as GIFT4), and we have now discovered that GIFT4 fusokine promotes long-term hematopoietic regeneration in a B cell-dependent manner. We found that GIFT4 treatment triggered a robust expansion of endogenous bone marrow HSCs and multipotent progenitors invivo. Delivery of GIFT4 protein together with B cells rescued lethally irradiated mice. Moreover, adoptive transfer of autologous or allogeneic GIFT4-treated B cells (GIFT4-B cells) enhanced long-term hematopoietic recovery in radiated mice and prevented the mice from irradiation-induced death. Our data suggest that GIFT4 as well as GIFT4-B cells could serve as means to augment HSC engraftment in the setting of bone marrow transplantation for patients with hematological malignancy.

CIRCULATING HEMATOPOIETIC PROGENITOR CELLS IN PATIENTS AFFECTED BY CHORNOBYL ACCIDENT

To investigate functional activity of circulating hematopoietic progenitor cells from peripheral blood and bone marrow of cleanup workers in early and remote periods after the accident at Chornobyl nuclear power plant (CNPP). The assessment of the functional activity of circulating hematopoietic progenitor cells was performed in samples of peripheral blood and bone marrow of 46 cleanup workers, who were treated in the National Scientific Center for Radiation Medicine of the Academy of Medical Sciences of Ukraine alongside with 35 non radiated patients, who served as a control. Work was performed by culturing peripheral blood and bone marrow mononuclear cells in the original gel diffusion capsules, implanted into the peritoneal cavity of CBA mice. It was shown that hematopoietic progenitor cells could be identified in the peripheral blood of liquidators of CNPP accident. At the same time the number of functionally active progenitor cells of the bone marrow was significantly decreased and during the next 10 years after the accident, counts of circulating progenitor cells in the peripheral blood as well as functionally active hematopoietic cells in bone marrow returned to normal levels. It was shown that hematopoietic progenitor cells are detected not only in the bone marrow but also in the peripheral blood of liquidators as a consequence of radiation exposure associated with CNPP accident. This article is a part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".

Towards 'Off-the-Shelf ' Universal Chimeric Antigen Receptor (CAR) T Cells: Mouse Anti-3rd Party Central Memory CD8 Veto Cells Prolong Functional Engraftment of Allogeneic Genetically Modified T Cells

We recently demonstrated that mouse anti-3rd party central memory CD8 veto cells (Tcm) enable engraftment of fully mis-matched T cell depleted bone marrow (TDBM) in sublethally irradiated (5.5 Gy TBI) recipients (Eran et al. Blood 2013). Here we found that Tcm generated from (host x donor)F1 or fully allogeneic donors were able to engraft and sustain their presence in such sub-lethally irradiated mice in the absence of TDBM transplants. Limiting dilution analysis revealed that the reactivity of host T cells (HTC) against donor spleen cells was significantly diminished in the chimeric mice treated with Tcm, whereas reactivity against 3rd party was comparable betweenTcm treated and non-treated mice (Fig. 1). This specific immune tolerance indicated that vetoTcm could potentially enable the use of 'off-the-shelf' allogeneic CAR T cells or any other genetically modified T cells from the same donor.To further investigate this possibility, we tested the ability of Tcmveto cells to enable engraftment of CD8 T cells from OT1 mice, expressing a transgenic T cell receptor (TCR) directed againstovalbumin (OVA) residues 257-264 in the context of H2Kb MHC-I. To that end, 1x106 OT1 CD8 T cells (H2b) were infused into Balb/c (H2Dd) recipients sublethally irradiated with 5.25 Gy TBI in the presence or absence of different doses of veto Tcm. As shown in Fig. 2, when tested at 2 months post-transplant, OT1 cells could not be detected in the peripheral blood of untreated recipient mice, while they were readily detectable in mice receiving 2x106 Tcm (0.78±0.36), reaching a higher level (1.18± 0.61) upon infusion of 5x106Tcm.To evaluate the functionality of the engrafted OT1 T cells in the chimeric mice, we developed and calibrated a new murine model, using a melanoma B16-cell line of C57BL background that expresses the ovalbumin peptide and the tdTomatomarker (B16-OVA-tdTomato) (Fig 3A). In this model, we tried to mimic a state of minimal residual disease by injecting a small number of B16-OVAtdTomato melanoma cells (0.25x106) into syngeneic C57BL mice; we then treated the mice with sublethal irradiation (6Gy TBI) to simulate treatment of the tumor, but also to clear out space for the T-cell adoptive transfer. Finally, we injected 1x106 naïve OT-1 cells generated on a (Balb x-OT1-CD45.1+RAG2-) F1 background in the presence or absence of Balb/c veto Tcm (5x106), and followed the development of the tumor. We found thatBalb/c vetoTcm prolonged the engraftment of the transgenic donor-derived F1 OT1 CD8 T cells inBalb/c recipients, thereby enabling significantly improved control of tumor growth when tested at day 20 post-transplant ( p<0.05). In contrast non OVA-expressing tumor cells grew at the same rate in all treatment groups, thereby demonstrating the specificity of the anti-tumor effect (Fig.3B).Previous studies demonstrated that Tcmare depleted of GVH reactivity by virtue of their culture with 3rd party stimulators under cytokine starvation. Our present results demonstrate that suchTcm veto cells can pave the way for functional engraftment of CD8 T cells with genetically modified specificity. Taken together, these results provide a proof of concept for the potential clinical use of such veto cells to enable therapy with 'off-the-shelf ' allogeneic CAR T cells. The relative ease of growing humanTcm in large numbers over a short period of time (9-12 days) suggests that it could be possible to harvest byleukapheresis from a single donor, a sufficient number of cells, to support CAR therapy for more than 50 allogeneic recipients.* N.O.G and R.G equally contributed [Display omitted] [Display omitted] [Display omitted] DisclosuresOr Geva:Yeda LTD: Patents & Royalties. Gidron:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.

Response and Resistance to Bromodomain Inhibition in AML Driven By Hyperactive Ras Signaling

Background: Acute myeloid leukemia (AML) is an aggressive hematologic cancer characterized by clonal proliferation of hematopoietic stem and progenitor cells that exhibit impaired differentiation. Event free survival for patients with AML remains poor despite intensive myelosuppressive therapies and improvements in supportive care measures. This underscores the need for novel, biologically based therapies. Somatic mutations that deregulate epigenetic programs (e.g. DNMT3A, TET2, IDH1/2, EZH2, ASXL1) and signal transduction pathways (e.g., FLT3, NRAS, KRAS) frequently coexist in AML. While the former class of mutations is hypothesized to promote a chromatin state that is permissive for AML development and essential for leukemia maintenance, experimental data also suggest that signaling mutations play a central role in driving leukemic growth in vivo. Thus, simultaneously targeting the abnormal epigenetic landscape and aberrant signaling pathways in AML is a rational new therapeutic approach. Recent publications identified the bromodomain and extraterminal (BET) proteins, an important class of epigenetic reader proteins, as particularly promising therapeutic targets in AML. While these studies support the therapeutic potential of BET inhibition in AML, they have limitations. These include their dependence on exogenous overexpression of oncogenes, failure to inform potential combination therapeutic strategies, and a reliance on monoclonal in vitro systems that do not recapitulate the inherent genetic heterogeneity of human cancers.Methods: We previously generated a heterogeneous collection of murine AMLs by infecting Nras, Kras, and Nf1 mutant mice with the MOL4070 retrovirus, which exhibit distinct retroviral integrations that are maintained upon transplantation into sublethally irradiated recipient mice. We first established 15 mg/kg/day as the maximally tolerated dose of PLX51107, a selective and potent BET inhibitor, in sublethally irradiated mice in a C57Bl/6 x 129sv/J strain background. We performed pharmacokinetic analysis, which demonstrated excellent drug exposure at doses of 10 and 15 mg/kg/day. We next treated cohorts of recipient mice with PLX51107 (10 mg/kg/day) and in combination with the MEK inhibitor PD0325901 (PD901; 1.5 mg/day). Mice that appeared ill were euthanized and underwent full pathological examination. Despite continuous drug treatment, all recipient mice eventually succumbed to progressive AML.Results: We enrolled eight AMLs, including four with a Nras(G12D) mutation, two with a Kras(G12D) mutation, and two with Nf1 inactivation. Recipient mice received 450 cGy of sublethal irradiation followed by 2x10E6 leukemia cells via tail vein injection. Recipient mice were randomized to receive vehicle (n = 4 for each AML), PLX51107 (n = 5), or PLX51107+PD901 (n = 5). PLX51107 markedly extended the survival of recipients transplanted with Nras(G12D) AMLs 6695, 6606, and 6613 that was further enhanced by PD901 (Fig. 1A). Whereas, PD901 resulted in a 1.5-fold increase in survival over vehicle-treated mice, PLX51107 alone resulted in a 4-fold increase in survival and PLX51107+PD901 in a nearly 6-fold increase in survival in this cohort of Nras(G12D) AMLs. Surprisingly, the response to PLX51107 was blunted in Kras(G12D) and Nf1 inactivated AMLs compared to Nras(G12D) AMLs. The observation of novel MOL4070 integration sites in relapsed AMLs provided definitive evidence of clonal evolution (Fig. 1B). Importantly, we went on to show that drug-treated clones emerging at relapse demonstrate intrinsic drug resistance by re-transplanting these leukemias into secondary recipients and re-treating them in vivo (Fig. 1C).Conclusion: PLX51107 shows impressive efficacy in a panel of primary AMLs treated in vivo that is further enhanced by PD901. The differential response between Nras(G12D) and Kras(G12D)/Nf(-/-) AMLs leads to the intriguing and unexpected hypothesis that the type of hyperactive Ras signaling mutation may influence the response to BET inhibition in AML. We are interrogating relapsed AMLs to identify and functionally validate candidate mechanisms underlying drug response and resistance through the use of established strategies to directly compare untreated and relapsed leukemias. Ongoing studies include assessing retroviral integrations and performing Western blotting, whole exome sequencing, RNA-seq, and ChIP-seq. [Display omitted] DisclosuresPowell:Plexxikon: Employment. Bollag:Plexxikon Inc.: Employment.

Laminin α4 Deficiency Results in Impaired Hematopoietic Recovery Accompanied with Altered Bone Marrow Niche after Myelosuppression

Myelosuppression is a life-threatening complication of anti-cancer therapy including irradiation. Rapid and complete hematopoietic recovery after therapy-induced myelosuppression is required for a successful treatment outcome. This process relies on efficient regeneration of hematopoietic stem cells (HSCs) and is tightly controlled by bone marrow (BM) microenvironment consisting of mesenchymal stem/progenitor cells, endothelial cells as well as secreted factors including cytokines and extracellular matrix proteins (ECM). However, the extrinsic factors critical for promoting the hematopoietic recovery remain poorly understood. Laminins are heterotrimetric ECM composed of α, β, and γ chains. Laminin α4 chain (LAMA4) is an active component for laminin-411 and -421, which are located in vascular basement membrane. LAMA4 plays an important role for HSC homing after transplantation via interaction with laminin receptor integrin α6 (Qian H et al., Blood 2006). However, the role of LAMA4 in normal hematopoiesis and HSC reconstitution after irradiation-induced myelosuppression is not known.In this study, we first detected Lama4 gene expression in BM endothelial cells (CD31+), mesenchymal stem cells (MSC: CD45-Ter119-CD31-CD44-Sca1+CD51+), and mesenchymal progenitor cells (MPC: CD45-Ter119-CD31-CD44-Sca1-CD51+) in young adult mice. By using Lama4 deficient (Lama4-/-) mice, we analyzed the functional role of LAMA4 on hematopoietic activity at steady state. We found the lower number of platelets (PLTs) (p = 0.03), and neutrophils (Gr1+CD11b+) (p = 0.03) in the peripheral blood (PB) of Lama4-/- mice, but a higher frequency of common myeloid progenitor (Lin-Sca1-Kit+CD34+FcRlow) (p < 0.01) in the Lama4-/- BM at steady state, indicating that LAMA4 plays a role in the maintenance of physiological hematopoiesis.The important role of LAMA4 in hematopoietic recovery was demonstrated by delayed and incomplete recoveries of mature red blood cells, PLTs, and Gr1+CD11b+ cells in PB following sublethal irradiation (7Gy). The impaired recovery of erythropoiesis was also indicated by the higher values of mean corpuscular hemoglobin and mean corpuscular volume in PB as well as the higher frequency of megakaryocyte-erythrocyte progenitor (Lin-Sca1-Kit+CD34-FcR-) (p < 0.01) and colony-forming unit-erythrocyte (CFU-E) (p = 0.03) in the BM of the Lama4-/- mice at 6 weeks after irradiation, suggesting blocked erythrocyte maturation. In keeping with the refractory neutropenia, the frequency of colony-forming unit-granulocyte-macrophage (CFU-GM) was lower in the Lama4-/- BM compared to that in the age- and gender-matched wild type mice (p = 0.04). These data indicate that LAMA4 is critical for multiple hematopoietic lineage reconstitution post irradiation.To investigate the cellular and molecular mechanisms underlying the critical role of LAMA4 in hematopoietic recovery after the irradiation, we characterized the BM niche by colony assay, flow cytometry immunophenotyping, quantitative real time PCR (qPCR), and histological analysis. The number of colony-forming unit-fibroblast (CFU-F) was comparable between wild type and Lama4-/- in steady state. Interestingly, the proportion of BM MPCs, a population containing osteoblast progenitors, was significantly lower in the Lama4-/- mice compared to that in the wild type controls at steady state (p < 0.01). qPCR analysis showed downregulation of Il6 (p < 0.05) in the MSC and Angpt1 (p = 0.02) in the MPC of the Lama4-/- mice post irradiation. These data suggest that Lama4 deficiency alters BM stromal cell composition and gene expressions, which may be related to the impaired hematopoietic reconstitution. The recovery of BM vascular structure is essential for efficient reconstitution of hematopoiesis. We observed uniquely dilated blood vessels in Lama4-/- BM at 6-week post irradiation. This might be caused by the lower Angpt1 expression in Lama4-/- MPC since Angpt1/Tie2 signaling is required for vascular regeneration (Kopp HG et al., Blood 2005, Zhou BO et al., eLife 2015). The functional consequences of this phenotype are still under investigation.Altogether, LAMA4 is required for rapid and complete hematopoietic recovery post irradiation-induced myelosuppression. Therapeutic strategies to upregulate Lama4 may facilitate the recovery of hematopoiesis following HSC transplantation under preconditioning using irradiation. DisclosuresNo relevant conflicts of interest to declare.

Neuropeptide Y Is Critical for Bone Marrow Stromal Cells and Hematopoietic Recovery Following Injury

Bone marrow suppression is the most common limiting side-effect of conventional cancer chemo/radio therapy and is the primary cause of morbidity/mortality after accidental exposure to a high dose of ionizing radiation. The mechanisms mediating radiation-induced hematopoietic stem and stromal cell dysfunction however are not well understood. Radiation therapy causes substantial sensory neuropathy in patients. Recent studies reveal that bone marrow cells are highly innervated by sympathetic nerve fibers and that chemotherapy induced nerve-damage can impair hematopoietic regeneration, suggesting a contribution of nerve fibers in the regulation of hematopoietic stem cell and stromal cell activities. Whether irradiation- mediated nerve injury is a crucial lesion that causes deficits in hematopoietic recovery is not known. We recently discovered that differential signaling from the neuropeptide Y (NPY) receptors on bone marrow endothelial cells regulates vascular permeability and stem cell egress. NPY is an important neurotransmitter of the sympathetic nervous system and the principal adreno-medullary hormone. In this study, we found that NPY is important for reconstitution of the bone marrow niche and hematopoietic regeneration following sublethal irradiation (650 cGy). The levels of NPY were significantly reduced in bone marrow of irradiated mice suggesting damage to nerve fibers. Treatment of wild-type mice with full length NPY (1µg/mouse/day) for 3 consecutive days after irradiation markedly reduced the loss of mesenchymal stem cells (CD45-Ter119-CD31-Nestin+PDGFR+CD51+), endothelial cells (CD45-Ter119-CD31+VE-cadherin+) and hematopoietic stem and progenitor cells (SLAM LSK and LSK) in the bone marrow and promoted faster hematopoietic recovery. In addition, pharmacological NPY treatment prevented irradiation mediated nerve fiber damage. In contrast, in NPY knockout mice, regeneration of CD45neg stromal cells, SLAM LSK and LSK cells after irradiation was significantly reduced compared to wild-type controls. This reduced hematopoietic recovery in NPY deficient mice following irradiation was associated with increased apoptosis/necrosis of stromal cells and hematopoietic stem and progenitor cells. We also examined whether NPY played an intrinsic or extrinsic role in stem cell homing. Wild-type or NPY deficient BM cells were transplanted into wild-type or NPY knockout recipients. Strikingly, the homing of wild-type donor cells into NPY deficient recipients or NPY knockout donor cells into wild-type recipients were both reduced. To explore whether NPY regulates human stem cells, we treated human cord blood CD34+ cells ex vivo with NPY for 3 days and evaluated cell expansion. Long-term culture assays demonstrated that NPY treatment enhanced the clonal expansion of CD34+ cells. In conclusion, our studies suggests that NPY plays both an intrinsic and extrinsic role in hematopoiesis and that NPY-mediated protection of the sympathetic nervous system within the bone marrow can facilitate stem cell niche regeneration and enhance regenerative hematopoiesis following irradiation/injury. DisclosuresNo relevant conflicts of interest to declare.

ROS Production Triggers Anti-Leukemic Effects of Green Tea

Beneficial effects of green tea (GT) consumption have been described, including the ability to reduce cancer development. Polyphenols are the main chemical constituents of GT extract and have been identified as the most effective substances that can inhibit tumorigenesis. Acute myeloid leukemia is an aggressive hematologic malignancy and there is no sufficient evidence that supports a protective role of tea intake on its development. In this concern, the aim of this study was to investigate GT effects in acute promyelocytic leukemia (APL) mice.A total of 1 × 106 leukemic cells obtained from hCG-PML-RARa transgenic mice were injected in the tail vein of 12- to 16-week-old NOD.CB17-Prkdcscid/J mice, after 4-6 h of sublethal cobalt irradiation with 2 Gy. The hematologic counts were monitored weekly, and the following criteria were used for the diagnosis of leukemia: presence of at least 1% of blast in peripheral blood associated with leukocytosis above 30 000 cells/L, hemoglobin levels below 10 g/dL, and thrombocytopenia below 500 × 103 cells/L (He et al, 1997). Twelve days after transplantation, mice were then submitted to daily oral treatment (gavage) with 250 mg/kg/day GT or vehicle only (water) for 5 consecutive days and were sacrificed; bone marrow (BM) and spleens were collected to the assays.Treatment with GT significantly increased the mean number of apoptotic cells in the BM (29.4 ± 5.2 vs untreated 21.0 ± 2.1 %, P < 0.05) and spleen (13.9 ± 3.1 vs untreated 9.2 ± 1.9 % P < 0.05) of mice, evaluated by Annexin V-FITC/PI. GT induced an increase in the median fluorescence intensity (MFI) of cleaved caspase-3 in the BM (83.9 ± 3.6 vs untreated 72.6 ± 4.7, P < 0.05) and in the spleen (75.5 ± 28.2 vs untreated 55.8 ± 7.3, P < 0.01); cleaved caspase-8 in the BM (117.3 ± 9.9 vs untreated 89.1 ± 12.3, P < 0.005) and in the spleen (118.0 ± 31.5 vs untreated 81.5 ± 14.8, P < 0.001); and cleaved caspase-9 in the BM (138.2 ± 52.4 vs untreated 85.8 ± 12.9, P < 0.001) and in the spleen (121.7 ± 49.2 vs untreated 76.5 ± 21.9, P < 0.001) of leukemic mice.Moreover, GT treatment reduced the percentage of CD34+ hematopoietic progenitor cells (32.4 ± 2.3 vs untreated 41.0 ± 0.5 %) as well as of CD117+ cells (33.4 ± 3.7 vs untreated 44.2 ± 1.8 %). We then evaluated the phenotype of cells infiltrated in the spleen. Interestingly, we found that GT induces a decrease in the percentage of CD117+ (40.7 ± 0.3 vs leukemic 44.6 ± 0.9 %) and Gr-1 cells (60.8 ± 0.2 vs untreated 65.6 ± 0.5 %) present in the spleen.We then analyzed the effects of GT in the production of intracellular ROS in the BM subpopulations of CD34+, CD117+ and Gr-1+ cells from leukemic mice. Significant increases in the median fluorescence intensity (MFI) of intracellular ROS production by Gr-1 cells of GT-treated mice were observed (670 ± 103 vs untreated 428.5 ± 5.2). Interestingly, GT induced a reduction of MFI of intracellular ROS production in the CD34+ (167.5 ± 27.1 vs untreated 405.5 ± 73.3) and CD117+ (360 ± 142 vs untreated 1635 ± 40.4) cells.We then studied the expression and localization of CXCR4 and HIF-1α proteins. Studies have shown that ROS increases expression of CXCR4 in cancer and immune cells (Li et al, 2009; Lin et al, 2011; Chetram et al, 2011; 2013) through nuclear translocation of HIF-1α (Lee et al, 2002; Salmeen et al, 2003). Our results showed that GT decreased the MFI of CXCR4 in the leukemic mice (9028 ± 1367 vs untreated 4196 ± 970). Reduction of the nuclear translocation of HIF-1α in GT-treated mice was also observed, using the ImageStream imaging flow.In conclusion, GT treatment in APL mice induces apoptosis of cells in the BM and spleen, confirmed by activation of caspase-3, -8 and -9, probably by modulating the production of intracellular ROS in the leukemic cells. Although GT and its polyphenols are well known as antioxidants, there is evidence that some of the effects of these compounds may be related to induction of oxidative stress in immune cells, which might be responsible for the induction of apoptosis of tumor cells. These pro-oxidant properties may also induce endogenous antioxidant systems in normal tissues that offer protection against cancer. On the other hand, it is possible that, in leukemic cells, which has excessive ROS, the antioxidant effect of GT become more evident. Several potential mechanisms have been proposed including both antioxidant and pro-oxidant effects to polyphenol compounds, but questions remain concerning the relevance of these mechanisms to cancer prevention. DisclosuresTorello:Fundação de Amparo à Pesquisa do Estado de São Paulo - Fapesp: Research Funding; Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq: Research Funding. Shiraishi:University of Campinas: Employment.

Venetoclax Synergizes with Radiation Therapy for Treatment of B-Cell Lymphomas

Introduction:An estimated 19,970 Americans died of non-Hodgkin lymphoma (NHL) in 2015, with diffuse large B-cell lymphoma (DLBCL) accounting for roughly 30% of newly diagnosed NHL. Our study focuses on three NHL subtypes: germinal center (GCB)-DLBCL, the most common DLBCL subtype; activated (ABC)-DLBCL, a particularly aggressive and high-risk subtype; and mantle cell lymphoma (MCL), considered incurable. Constitutive B-cell receptor signaling is implicated in the pathogenesis of ABC-DLBCL and MCL and may couple with aberrant apoptotic BCL-2 pathway proteins. The BCL-2 inhibitor venetoclax is a promising targeted agent that promotes apoptosis in a variety of NHL subtypes, but is almost never curative as a single agent. Radiotherapy promotes apoptosis by creating DNA strand breaks, and we hypothesized that the combination of radiotherapy and venetoclax would act synergistically in NHL to increase the probability of cures.Methods:We tested in vitro killing efficacy of sublethal 137Cesium irradiation combined with venetoclax in 15 cell lines, representing a diversity of NHL subtypes. Cells were treated with 137Cesium and venetoclax in 8 x 8 dose combination matrices, incubated 72-120 hrs, then assayed for viability with Celltiter-Glo (Promega). The degree of treatment antagonism, additivity, or synergism was determined using isobolographic analyses. For in vivo studies, we tested combinations of venetoclax with either 137Cesium total body irradiation (TBI), or CD20 pre-targeted radioimmunotherapy (PRIT), in threetumor models chosen for divergent single agent sensitivities. Tumor xenografts of Rec-1 (MCL), U2932 (ABC-DLBCL), and SU-DHL-6 (GCB-DLBCL) were produced by subcutaneous flank injection of 10 x 106 cells in male and female NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ (NRG) mice. When tumor volumes were 50 mm3, mice (n = 8-12/group) were treated with either venetoclax (100-200mg/kg daily for 10-30 days), diluent control, TBI (single dose, 6-10 Gy 137Cesium), or a combination of venetoclax and TBI. In PRIT studies, mice were coinjected with 300µg unlabeled streptavidin-conjugated anti-CD20 antibody (murine IgG2a) and 400µg HB8181 (IgG2a isotype control to block non-specific binding) in place of TBI. Twenty-one hours later, 5.8 nmol biotin-galactose "clearing agent" was administered, followed in 3 hours by 1.2 nmol DOTA-biotin labeled with 400, 800, or 1200 µCi of 90Y (14.8, 29.6, or 44.4 MBq, respectively).Results:In vitro, 10 of 15 lymphoma cell lines responded synergistically to combined radiotherapy and venetoclax, including GCB-DLBCL, ABC-DLBCL and MCL lines (p < .04 in 10 cell lines). In vivo, each of 3 lymphoma models responded synergistically to combination therapy. In mice bearing Rec-1 xenografts, venetoclax alone did not affect mean survival time (p = .32), 8 Gy TBI lengthened survival by 44% compared to controls (p < .0001), but TBI combined with venetoclax tripled survival time compared to controls (p < .0001, combination group > TBI alone). The SU-DHL-6 model produced similar results. In the U2932 model, tumors disappeared during venetoclax monotherapy, but recurred in all mice, such that mean survival time doubled compared to controls (p = .0001). Six Gy TBI had no effect (p = .73), but combining TBI with venetoclax tripled survival time compared to controls (p = .0003, combination group > venetoclax alone). Using PRIT in place of TBI produced yet greater efficacy. In Rec-1 bearing mice, venetoclax had no effect alone (p = .12), 800µCi PRIT lengthened survival time 111% beyond controls (p = .0001), while the combination extended survival 483% beyond controls and cured 40% (p = .001, combination group > PRIT alone). In the U2932 xenograft model, venetoclax alone doubled survival time compared to controls (p < .0001) and 800µCi PRIT alone doubled survival and cured 30% (Fig. 1, p < .0001). Combination treatments cured 100% (Fig. 1).Conclusion:In vitro and in vivo results support our hypothesis that radiotherapy combines effectively with venetoclax to treat NHL. Despite differences in single agent sensitivity, xenograft models of GCB-DLBCL, ABC-DLBCL and MCL all responded synergistically to combinations of either TBI or PRIT with venetoclax. PRIT combinations with venetoclax produced cures (Fig. 1) without detectable toxicity, and merit clinical preference. Ongoing studies examine predictive biomarkers and optimal treatment protocols for therapeutic efficacy. [Display omitted] DisclosuresGopal:Paid Consultancy- Gilead, Janssen, Seattle Genetics, Spectrum, Research funding- Gilead, Janssen, Pfizer, BMS, Merck, Teva, Takeda, Spectrum, Seattle Genetics: Consultancy, Honoraria, Research Funding.

Deguelin Induced Differentiation of NPM1-Mutant AML In Vivo and in Vitro

Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy due to abnormal proliferation of hematopoietic progenitor cells, with difficulty in treatment and high mortality. Nucleophosmin 1 (NPM1), a restricted nucleolar localization protein, shuttles between nucleus and cytoplasm. NPM1-mutant protein, aberrant cytoplasmic dislocation of nucleophosmin, occurs in about one third of AML. Deguelin exhibits significant inhibitory effects and induces apoptosis in a variety of cancer cell lines in vitro and in vivo. Our previous researches showed deguelin induced differentiation of NPMc+ AML cells potentially by targeting NPM1-mutant protein. Thus, we extend our investigations of deguelin-induced differentiation activity to an AML xenograft mouse model and clinical samples.After sub-lethal whole body radiation, OCI/AML3 cells were injected intravenously in NOD/SCID mice. 7 days later, either deguelin or vehicle (2%DMSO) was injected intraperitoneally every day. Kaplan-Meier estimates showed 1mg/kg/day deguelin extended the longest survival of mice. The percentage of CD11b+ cells was 17.73%±3.03% in control group, 41.33%±13.22% and 76.15%±10.25% after deguelin-treated for 7 days and 14 days (P < 0.05). Thus, we used 14 days in the following experiments. The percent of CD114+ cells in the vehicle- and deguelin-treated group were 13.29%±10.79% and 47.36%±21.66%, respectively (P < 0.05). The percentage of CD14+ cells in the vehicle- and deguelin-treated group were 22.86%±15.54% and 83.49%±8.06%, respectively (P < 0.05). Wright’s Giemsa staining of mouse bone marrow cells showed differentiated. The ratios of differentiated cells were 36.92%±4.99% and 77.01%±1.98% in control and deguelin group (P < 0.01). Median OS of vehicle-treated group was 23 days, while that of deguelin-treated group was 55 days (HR, 0.5818, 95% confidence interval [CI], 0.1623-1.001). Survival of deguelin-treated mice was significantly longer compared with mice injected with vehicle (P=0.0002). IHC and IF staining for mutated NPM1 protein showed bone marrow biopsy densities significantly reduced in deguelin-treated mice which was further confirmed by western blot assay (P < 0.05).We analyzed the toxicity of deguelin (1mg/kg/day for 14 days) to healthy NOD/SCID mice. They had steady movement and no rigidity posture treated with deguelin. HE staining indicated no pathological changes in striatum, substantia nigra pars compacta (SNpc), liver, kidney and heart in deguelin-treated mice. Striatum and SNpc, subjected to immunostaining with anti-tyrosine hydroxylase (TH) antibody, showed deguelin did not alter TH immunoreactivity in striatum and SNpc in vivo (P >0.05). Plasmatic levels of liver enzymes (AST and ALT), renal function (Scr and BUN) and cardiac enzymes (CK-MB and cTnT) in deguelin-treated group had not statistical difference comparing with control group (P>0.05). Thus, deguelin did not cause neurotoxicity, hepatotoxicity, nephrotoxicity and cardiotoxicity to mice.We collected 82 cases of patients with acute leukemia. 24 cases out of 68 AML patients carried mutated NPM1 gene (20 cases of type A; 1 case of type B; 2 cases of type D and 1 case of type I). Bone marrow mononuclear cells (BMMCs) of 6 AML patients were analyzed by FACS analysis. We analyzed patient (pt.) 1 to 4 with NPM1 mutation. FACS showed deguelin increased the percentage of CD11b+ and CD14+ cells of pt.1 in a dose-dependent manner. The percent of CD11b+ and CD14+ cells were 55% and 57.9% separately at 16 nM. Deguelin increased CD14 but CD11b expression of BMMCs of pt.2 in the concentration of 8 nM, 12 nM, 16 nM and 20 nM. The highest percentage of CD14+ cells was 85.5%. The expression of CD14 in cells from pt.3 increased after treating with 16 nM or 20 nM and the highest percentage of CD14+ cells was 49.4%. Deguelin induced differentiation of BMMCs from pt.4. The maximum percent of CD11b+ cells was 42.5% at 16 nM. Moreover, FACS analysis showed deguelin did not promote differentiation in BMMCs from pt.5 and 6 without NPM1 mutation. These results demonstrated deguelin induced differentiation of BMMCs cells from AML patients with NPM1 mutation in vitro. Taken together, mutated NPM1 protein may be one of targets of deguelin, which provides molecular basis for inducing differentiation in NPM1-mutant AML cells. DisclosuresNo relevant conflicts of interest to declare.

Comprehensive Analysis of Activation and Proliferation Kinetics and Effector Functions of Human Lymphocyte Subsets in Xenogeneic GvHD Model

To better understand the biology of graft-versus-host disease (GvHD) after hematopoietic stem cell transplantation (HSCT) and develop novel treatment strategies, accurate and clinically relevant experimental animal models are indispensable. The majority of our understanding of this potentially lethal complication is based on mouse models of bone marrow transplantation in major histocompatibility complex antigens (MHC) and/or minor histocompatibility antigen mismatched settings. These mouse models of GvHD provide us with an immeasurable wealth of information; however, many findings obtained from mouse models are not necessarily correlated with clinical GVHD in humans. In addition, these mouse models are not suitable for predicting the effectiveness of recent human-specific therapies such as monoclonal anti-human antibodies and adaptive cell immunotherapies. To overcome these limitations, xenogeneic GvHD models using severe immunodeficient mice are currently being used in worldwide and allow us to investigate in vivo human immune reactions.The major aim of our study is to clarify the precise mechanism of immune response in vivo and to determine the principle components responsible for xenogeneic GvHD. We first observed that immunodeficient NOG mice receiving either no or sublethal irradiation consistently showed gradual body weight loss and eventual severe GvHD following injection of human unmanipulated peripheral blood mononuclear cells (PBMCs). Histopathology performed at the late phase of GvHD showed extensive infiltration of human CD45+ mononuclear cells, most of which were T-cells, and tissue destruction in lungs, bone marrow, liver, and spleen, with a smaller number detected in gut and skin unlike in human GvHD. We identified the infiltration of human T-cells exclusively in lungs and spleen before the onset of GvHD. Flow cytometric analysis showed a marked reduce of naïve (CD45RA+CCR7+) and central memory (CD45RA-CCR7+) T-cells and an increase of effector memory (CD45RA-CCR7-) T-cells within CD4+ subset in lungs, suggesting that xenogeneic response by human cells in lungs occur preceding systemic inflammation. Of note, the distribution of human cells was similar between intraperitoneal and intravenous injection. These results indicated that the acute lung injury is not associated with cell trapping within mouse pulmonary microvasculature by intravenous delivery.We next subdivided human lymphocyte subsets by magnetic cell sorting before adoptive transfer to characterize human cells responsive for xenogeneic GvHD development. CD4+ T-cells mediated more rapid and severe GvHD than did an equal number of CD8+ T-cells, whereas neither natural killer cells nor γδT cells caused any symptoms of GvHD. All antigen presenting cells (APCs)-depleted PBMCs mediated GvHD as well, suggesting that human T-cells are activated independently of their own APCs in this model.CFSE cell division assay showed more rapid proliferation of CD4+ rather than CD8+ T-cells. It is a noteworthy that the proliferation of and the expression of early activation marker CD69 on CD8+ T-cells was accelerated in the presence of CD4+ T-cells, indicating that CD4+ T-cells are required for the rapid and more efficacious response of CD8+ T-cells. We also found a strong increase of human IL-2, IFN-γ, and TNF-α in serum of mice injected with whole T-cells. In contrast, we observed very low levels of these inflammatory cytokines in mice injected with isolated CD8+ T-cells alone.Taken together, these results suggest that the early activation of human naïve CD4 T-cells by recognition of foreign antigen on mouse APCs following cytokine production play a critical role in triggering systemic inflammation in xenogeneic GvHD model. This in vivo response was confirmed in vitro by demonstrating that human CD4 T-cells rapidly proliferated and expressed CD69 with significantly increase of IL-2 and IFN-γ in response to mouse dendritic cells. These findings are helpful for adequate evaluation of immune response and creation of optimal experimental conditions in xenogeneic GvHD model. DisclosuresKanda:Otsuka Pharmaceutical: Honoraria, Research Funding.

Graft-Versus-Host Disease Following Liver Transplantation: Development of a High-Incidence Rat Model and a Selective Prevention Method.

Graft-versus-host disease (GvHD) following liver transplantation (LT) is a rare but serious complication with no presently available animal model and no preventive measures. To develop a rat model of GvHD after LT (LT-GvHD), we preconditioned hosts with sublethal irradiation plus reduction of natural killer (NK) cells with anti-CD8α mAb treatment, which invariably resulted in acute LT-GvHD. Compared with those in the peripheral counterpart, graft CD4+ CD25- passenger T cells showed lower alloreactivities in mixed leukocyte culture. Immunohistology revealed that donor CD4+ T cells migrated and formed clusters with host dendritic cells in secondary lymphoid organs, with early expansion and subsequent accumulation in target organs. For selectively preventing GvHD, donor livers were perfused ex vivo with organ preservation media containing anti-TCRαβ mAb. T cell-depleted livers almost completely suppressed clinical GvHD such that host rats survived for >100 days. Our results showed that passenger T cells could develop typical LT-GvHD if resistant cells such as host radiosensitive cells and host radioresistant NK cells were suppressed. Selective ex vivo T cell depletion prevented LT-GvHD without affecting host immunity or graft function. This method might be applicable to clinical LT in prediagnosed high-risk donor-recipient combinations and for analyzing immunoregulatory mechanisms of the liver.

Clonal fate mapping quantifies the number ofhaematopoietic stem cells that arise duringdevelopment.

Haematopoietic stem cells (HSCs) arise in the developing aorta during embryogenesis. The number of HSC clones born has been estimated through transplantation, but experimental approaches to assess the absolute number of forming HSCs in a native setting have remained challenging. Here, we applied single-cell and clonal analysis of HSCs in zebrafish to quantify developing HSCs. Targeting creERT2 in developing cd41:eGFP+ HSCs enabled long-term assessment of their blood contribution. We also applied the Brainbow-based multicolour Zebrabow system with drl:creERT2 that is active in early haematopoiesis to induce heritable colour barcoding unique to each HSC and its progeny. Our findings reveal that approximately 21 HSC clones exist prior to HSC emergence and 30 clones are present during peak production from aortic endothelium. Our methods further reveal that stress haematopoiesis, including sublethal irradiation and transplantation, reduces clonal diversity. Our findings provide quantitative insights into the early clonal events that regulate haematopoietic development.

Total-Body Irradiation Exacerbates Dissemination of Cutaneous Candida Albicans Infection.

Exposure to radiation, particularly a large or total-body dose, weakens the immune system through loss of bone marrow precursor cells, as well as diminished populations of circulating and tissue-resident immune cells. One such population is the skin-resident immune cells. Changes in the skin environment can be of particular importance as the skin is also host to a number of commensal organisms, including Candida albicans , a species of fungus that causes opportunistic infections in immunocompromised patients. In a previous study, we found that a 6 Gy sublethal dose of radiation in mice caused a reduction of cutaneous dendritic cells, indicating that the skin may have a poorer response to infection after irradiation. In this study, the same 6 Gy sublethal radiation dose led to a weakened response to a C. ablicans cutaneous infection, which resulted in systemic dissemination from the ear skin to the kidneys. However, this impaired response was mitigated through the use of interleukin-12 (IL-12) administered to the skin after irradiation. Concomitantly with this loss of local control of infection, we also observed a reduction of CD4+ and CD8+ T cells in the skin, as well as the reduced expression of IFN-γ, CXCL9 and IL-9, which influence T-cell infiltration and function in infected skin. These changes suggest a mechanism by which an impaired immune environment in the skin after a sublethal dose of radiation increases susceptibility to an opportunistic fungal infection. Thus, in the event of radiation exposure, it is important to include antifungal agents, or possibly IL-12, in the treatment regimen, particularly if wounds are involved that result in loss of the skin's physical barrier function.

Establishment of Mouse Model with Humanized Chronic Myeloid Leukemia

To establish a BALB/c nude mouse model with the huamanized chronic myeloid leukemia (CML) for the study of human CML. The BALB/c nude mice aged 4 weeks pretreated by splenectomy, the cyclophosphamide intraperitoneal injection and sublethal irradiation (SLI) were transplanted intravenously with bone marrow mononuclear cells from CML patients. The SLI-pretreated nude mice were divided into 2 groups: group A, in which the nude mice were injected with 0.3 ml PBS; group B, in which the nude mice were infused intravenously with 4.5×107 mononuclear cells from CML patients. Then the changes of body weight and appetite were observed, the hemogram and cell morphology were determined, the expressions of human CD13 and CD45 were detected by flow cytometry, the pathologic analysis of bone, liver and intestine were performed by biopsy, and the BCR/ABL fusion gene was detected by RT-PCR. The mice in group B displayed weakness, auantic, less foodintake and instabiligy of gait as time want on. The average survival time was 46.2±4.2 d (45-57 d). On the third week, the CD13+CD45+ cells accounted for 0.56±0.05% and 2.56±0.36% respectively in group A and B. While on the sixth week, the CD13+CD45+ cells accounted for 0.44±0.07% and 4.97±0.43% in A and B groups respectively, these results showed that cell count in B group was significantly higher than that in A group(P<0.05). Pathological examination showed that the leukemic cells were found in bone marrow of group B. The BCR/ABL fusion gene could be detected in bone marrow. BALB/c nude mouse model with huamanized chronic myeloid leukemia(CML) model has been established by pretreating mice with SLI. The survival time of mice in this model has been long, and the cost to establish the model is low.