Sublethal Irradiation Research Articles

AG-221, an Oral, Selective, First-in-Class, Potent IDH2-R140Q Mutant Inhibitor, Induces Differentiation in a Xenotransplant Model

Somatic point mutations in the active site of IDH (isocitrate dehydrogenase) 1 and IDH2 genes are observed in acute myeloid leukemia (AML). These mutations lead to the production and accumulation of R-2-hydroxyglutarate (2-HG) in the tumor blast cells as well as in the plasma of patients. High levels of 2-HG have been shown to inhibit alpha-ketoglutarate-dependent dioxygenases, including epigenetic regulators (i.e. histone or DNA demethylases), resulting in altered cellular differentiation. Ex vivo experiments in primary cells from AML IDH2 mutant patients have demonstrated that IDH2 mutant inhibitors are able to revert this phenotype. In order to test the biological activity of AG-221, an oral, reversible and selective inhibitor of mutated IDH2 currently in phase I trials, we developed primary human AML xenograft models.Primary blast cells from 3 AML patients with normal karyotype (n=3), NPM1 mutation (n=3) and FLT3ITD (n=1) were injected into immunodeficient NOD SCID IL2R gamma null (NSG) mice by intrafemoral injection after sub-lethal irradiation. Medullar and peripheral tumoral engraftment was monitored by flow cytometry (using species-specific antibodies) and serum 2-HG measurements. Engraftment of IDH2-R140Q human blast cells was not lethal, but these AML cells persisted over time. Twelve months after injection, IDH2-R140Q blast cell-engrafted mice were randomized into two groups, for treatment with AG-221 (30 mpk) or vehicle (0.5% methylcellulose / 0.2% Tween 80, in water) (n=10 mice; 5 mice per condition). Treatments were administered by oral gavage for 38 days twice per day (BID). Peripheral blood was collected at multiple time points for the determination of pharmacokinetics (PK) and pharmacodynamics (PD), and the assessment of the differentiation effects by AG-221 on human tumor cells.PK/PD analyses showed good plasma exposure of AG-221 and reduction of 2-HG. Furthermore, AG-221 administration also induced a burst of proliferation of human blasts followed by myeloid differentiation starting at day 20 in peripheral blood as measured by the expression of CD11b, CD14, CD15, CD24 and cell morphology. No effects on proliferation or differentiation were seen in the absence of AG-221 administration. Histological analyses of hematopoietic organs in treated animals showed a decrease of infiltrating human cells, as well as obvious morphological changes in the human cell population in AG-221-treated animals compared with vehicle-treated animals. Flow cytometry confirmed the differentiation of the human cells in the spleen and bone marrow of the AG-221-treated mice. Furthermore, cells undergoing differentiation retained the R140Q mutation, demonstrating the differentiating effect of the compound.In conclusion, AG-221 reduced serum 2-HG levels and triggered differentiation of leukemic blast cells engrafted in NSG mice. These results are consistent with what has been observed clinically in IDH2 mutant AML patients treated with AG-221 in a phase I dose escalation trial. DisclosuresQuivoron:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la recherche contre le Cancer (ARC): Grant, Grant Other; AGIOS: Grant Other. David:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la Recherche contre le Cancer (ARC): Grant, Grant Other; Association Laurette Fugain: Grant, Grant Other; AGIOS: Grant Other. Straley:Agios Pharmaceuticals: Employment, Stockholder Other. Travins:Agios Pharmaceuticals: Employment, Stockholder Other. Kim:Agios Pharmaceuticals: Employment, Stockholder Other. Chen:Agios Pharmaceuticals: Employment, Stockholder Other. Zhu:Agios Pharmaceuticals: Employment, Stockholder Other. Bawa:AGIOS: Grant Other. Bernard:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Ligue Nationale contre le cancer (LNCC): Grant, Grant Other; AGIOS: Grant Other. Yang:Agios Pharmaceuticals: Employment, Stockholder Other. Agresta:Agios Pharmaceuticals: Employment, Stockholder Other. de Botton:AGIOS: Grant Other. Yen:Agios: Employment. Penard-Lacronique:Institut National de la Santé Et de la Recherche Médicale (INSERM): Grant Other; Association Laurette Fugain: Grant, Grant Other; Institut National du Cancer (INCa): Grant, Grant Other; Association pour la recherche contre le Cancer (ARC): Grant, Grant Other; AGIOS: Grant Other.

Presence of Endogenous TCR Antigen in Vivo Attenuates Efficacy of Anti-CD19 Targeted CAR T Cell Therapy

Adoptive therapy using T cells genetically engineered to express chimeric antigen receptors (CAR) has proven extremely effective against acute lymphoblastic leukemia (ALL) in clinical trials with the use of anti-CD19 CAR T cells. Most CAR T cell protocols use autologous T cells, which are then activated, transduced with the anti-CD19 CAR, and expanded ex-vivo before infusion back into the patient. This approach minimizes the risk of graft-versus-host disease (GVHD) even in allogeneic transplant recipients, due to tolerization of the donor T cell repertoire in the recipient. However, many patients have heavy disease burden and lymphopenia due to previous treatments, which makes the isolation of healthy T cells difficult. Thus, centers are exploring the potential of allogeneic T cell donors and the possibility of universal T cell donors for CAR-based therapy including the use of virus-specific T cells. In these cases, in addition to the chimeric receptor specificity, the transduced T cell population will also have reactivity against target antigens through the endogenous TCR. However, little is known about the impact of signaling of the endogenous TCR on CAR T cell activity, particularly in vivo. To test this, we used a syngeneic transplantable ALL murine model, E2aPBx, in which CD19 CAR T cells can effectively eradicate ALL. CD4 (Marilyn) and CD8 (Matahari) T cells from syngeneic HY-TCR transgenic donors specific for the minor histocompatibility male antigen, HY, were used as CAR T cell donors to control for endogenous TCR reactivity. Splenic T cells isolated from Matahari, Marilyn, or B6 mice were activated ex-vivo using anti-CD3/anti-CD28 beads, with the addition of IL2 and IL7. T cells were transduced with a retroviral vector expressing a murine CAR composed of anti-CD19 scfv/CD28/CD3ζ on days two and three. CAR T cells are evaluated in vitro by CD107a degranulation assay and INF gamma ELISA. In response to HY peptide alone or HY+CD19- line M39M, transduced CD8 HY (Matahari) cells produced IFN gamma and expressed CD107a whereas transduced CD4 HY (Marilyn) cells only produced IFN gamma. Interestingly, in response to CD19+HY- ALL, both Matahari and Marilyn expressed CD107a and produced IFN gamma indicating that CD4 T cells can acquire CD8-like lytic activity when stimulated through a CAR receptor. When CD19 CAR transduced Marilyns and Mataharis were stimulated in the presence of HY and CD19, CD8 Mataharis had an attenuated effect against CD19, suggesting that the presence of antigen activated TCR adversely affects the potency of the CAR receptor. Efficacy of the HY and polyclonal CAR T cells were next tested in-vivo in male and female B6 mice. Mice were given 1E6 E2aPBx ALL leukemia cells on day 1, and received 500 rads sub-lethal total body irradiation on day 4 as a lymphodepleting regimen. On day 5, mice were given a low (1E5) or high (5E6) dose of CAR T cells. There was a statistically significant (p=0.0177) improvement in the survival of female versus male mice after treatment with the CD4+ HY specific anti-CD19 CAR T cells, and female mice that received HY anti-CD19 CAR T cells survived longer than untreated control females (p=0.01). Remarkably, the survival of male mice that received HY anti-CD19 CAR T cells was statistically worse than untreated control males (p=0.008). This suggests that the presence of TCR antigen negatively impacts the function of CAR T cells. Furthermore, in a separate experiment using an equally mixed population of Marilyn (CD4+) and Matahari (CD8+) HY specific T cells, males has a statistically significantly (p=0.0116) worse survival compared to females after receiving 5E5 HY specific T cells. In conclusion, simultaneous stimulation through both CAR and TCR results in attenuated cytokine production and degranulation by CD8 T cells. In vivo, in the presence of the endogenous TCR antigen, both CD4 and CD8 CAR T cells are less potent at eradicating leukemia. These have implications for the development of universal donors for CAR T cell therapy. DisclosuresNo relevant conflicts of interest to declare.

Ex Vivo Expansion and Characterization of Human Umbilical Cord Blood CD34+ Stem Cells

Background: Human umbilical cord blood (hUCB) has been considered as an alternative source of hematopoietic stem cells (HSCs) for allogeneic cell transplantation owing to its easy availability and less stringent requirement for HLA matching. However, the absolute number of HSCs in hUCB is much smaller than that in bone marrow or mobilized peripheral blood. Therefore, there is a need to develop a reliable and efficient approach for the expansion of HSCs of hUBC in vitro.Method: CD34+ cells were purified from hUCB using magnetic-activated cell sorting system and cultured in IMDM basal medium supplemented with cytokine cocktails. CD34+ cell population was identified by flow cytometry after in vitro expansion. Nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice were randomly allocated into four groups (n=4 for each group). Twenty-four hours after exposure to sub-lethal irradiation of 60Co, CD34+ cells obtained directly from hUCB or from in vitro expansion were transplanted into NOD/SCID mice. Four groups of mice were injected with saline, 0.4 million unexpanded CD34+ cells, 0.4 million in vitro expanded CD34+ cells, and 2.9 million in vitro expanded CD34+ cells, respectively. Multi-lineage differentiation was assessed using antibodies against a panel of cell surface markers (e.g., CD34, CD45, CD11, CD19, CD15, CD71, CD66, CD41a, and CD42b) at 1, 3, and 8 weeks post transplantation. Circulation peripheral blood was also collected for various analyses.Result: Human CD34+ cells were increased about 7.3 folds after 4 days’ culture in vitro. For in vivo mouse study, no cells expressing human cell surface marker were detected in mice injected with saline (negative control). One week after injection with CD34+ cells, human surface markers including CD34, CD45, and CD15 were easily detectable in all three groups. Three weeks after cell injection, CD34+ cells were decreased below 1% in group 2 and 3 but not in group 4. On the other hand, CD45+ and CD15+ populations were increased in groups 2, 3, and 4. There was a strong correlation between the numbers of injected CD34+ cells and differentiated cell populations. This trend was also observed with cells expressing megakaryocytic marker CD41 and erythroid progenitor cell marker CD71 in mouse groups 2 to 4. Eight weeks post transplantation, human CD34+ cells were hardly detected in any groups. However, peripheral blood and bone marrow cells expressing human cell surface markers of various hematopoietic lineages were detectable in groups 2-4. These results indicated that CD34+ cells from hUCB either before or after expansion in vitro are capable of differentiation into hematopoietic cells of various lineages in vivo. Our study also indicates that in vitro expanded CD34+ cells are successfully engrafted to bone marrow of NOD/SCID mice.Conclusion: An efficient experimental method has been developed for the expansion of hUBC CD34+ cells in vitro and these cells can be potentially explored for various applications in the clinic. DisclosuresJiang:Biopharmagen.corp: Employment. Ren:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment.

Longterm Multilineage Human Hematopoietic Repopulation of Untreated KIT-Mutant Immunodeficient Mice

Background: Assessment of the growth and differentiation of human hematopoietic cells in immunodeficient mice has become vital to much basic and translational research. Historically, the primary focus has been to quantify different types of repopulating cells, based on the diversity and longevity of their clonal outputs in transplanted mice, assuming the results would be relevant to clinical transplants in humans. With the development of mice that lack B, T and NK cells on a variety of backgrounds and that have normal life expectancies but differences in cellular DNA repair ability, an increasing application is to use mice repopulated with human cells to interrogate and perturb mechanisms controlling normal, genetically modified and malignant cell behavior. We have previously shown that C57Bl6 mice homozygous for the W41 mutation of c-kit are fertile with a normal lifespan but have a functionally compromised hematopoietic stem cell (HSC) population. This enables single transplanted syngeneic HSCs to be detected at high frequency in these mice when they have been given a sublethal irradiation dose. Importantly, the HSC-derived clones produced in these mice display the same growth, self-renewal and differentiation abilities as in myeloablated recipients that require a co-transplant of normal mouse bone marrow (BM) cells to support their survival. We now report the development and improved repopulation by human cord blood (CB) CD34+ cells of mice that have the same genetically determined B, T, NK immunodeficiency as NOD/Rag1-/--IL2Rγc-/- (NRG) into which a homozygous W41 gene has been introduced.This was achieved by crossing and backcrossing the progeny of NRG x C57Bl6-W41/W41 matings and selecting mice that were homozygous for the Sirpα allele of the NOD mouse, the null Rag1 and null IL2Rgamma chain, genes of the NRG mouse and the W41 gene to obtain all of these on an otherwise mixed NODxC57Bl/6 background (NRG-W41 mice). The NRG mouse was chosen because the Rag1 KO has no effect on the radiosensitivity of other tissues as is the case with the scid (S) gene in the NSG mouse. As a result, use of the NRG mouse allows exploitation of the radioprotective effect of a reduced irradiation dose rate and hence delivery of a selectively higher dose to the HSCs of the host.Results: Initial studies showed that parental NRG mice given 900 cGy split or spread continuously over 3 hrs show similar repopulation by human CD34+ CB cells as NSG mice given 315 cGy, but are more robust with consistent longterm survival. We then performed a pilot experiment using the same transplant design (2x104 CD34+ CB cells/mouse) to compare chimerism obtained in NRG-W41 mice given an estimated “equivalent” radiosensitizing regimen of 150 cGy. The levels of multiple lineages of human cells measured in the BM and spleen 20 weeks post-transplant revealed these were greatly increased in the NRG-W41 mice (>95% human CD45+ cells in the BM vs 40% in NRG mice). Kinetic analysis of human cells in the blood also showed an enhanced output of human myeloid and B-lymphoid cells over time (5-fold higher in the NRG-W41 mice after >3 weeks). Particularly notable was the selectively increased (20-fold) and sustained output of human glycophorin A+ (GPA+) erythroid cells in the NRG-W41 mice (5% human GPA+ cells in the BM of 20-week NRG-W41 mice given 150 cGy and 5x104 CD34+ CB cells/mouse vs 0.25% in the BM of the matched NRG mice given 900 cGy). A similar marked increase (20-fold) was seen on the level of circulating human platelets (SSClowCD41+ CD61+ cells) in comparable groups of transplanted NRG-W41 and NRG mice. We then investigated the extent of repopulation achievable in untreated NRG-W41 recipients. We therefore transplanted mice of both strains with 5x104 CD34+CB cells each and have now followed the levels of human cells in their circulation and BM for up to 20 weeks. Human cells were barely detectable at 3 weeks post-transplant in either strain, but then in the unirradiated NRG-W41 mice only, their levels (all lineages) increased to close to those attained in NRG mice given 900 cGy.Conclusion: NRG-W41 mice support robustly enhanced and long term generation in vivo of a wide range of human hematopoietic cell types including erythrocytes and platelets, with high levels of chimerism achieved even in unirradiated primary recipients transplanted with relatively low numbers of human CD34+ CB cells. DisclosuresNo relevant conflicts of interest to declare.

Sub-lethal UV-C radiation induces callose, hydrogen peroxide and defence-related gene expression in Arabidopsis thaliana.

Exposure of plants to UV-C irradiation induces gene expression and cellular responses that are commonly associated with wounding and pathogen defence, and in some cases can lead to increased resistance against pathogen infection. We examined, at a physiological, molecular and biochemical level, the effects of and responses to, sub-lethal UV-C exposure on Arabidopsis plants when irradiated with increasing dosages of UV-C radiation. Following UV-C exposure plants had reduced leaf areas over time, with the severity of reduction increasing with dosage. Severe morphological changes that included leaf glazing, bronzing and curling were found to occur in plants treated with the 1000 J·m(-2) dosage. Extensive damage to the mesophyll was observed, and cell death occurred in both a dosage- and time-dependent manner. Analysis of H₂O₂ activity and the pathogen defence marker genes PR1 and PDF1.2 demonstrated induction of these defence-related responses at each UV-C dosage tested. Interestingly, in response to UV-C irradiation the production of callose (β-1,3-glucan) was identified at all dosages examined. Together, these results show plant responses to UV-C irradiation at much lower doses than have previously been reported, and that there is potential for the use of UV-C as an inducer of plant defence.

Radiation persistently promoted oxidative stress, activated mTOR via PI3K/Akt, and downregulated autophagy pathway in mouse intestine

While acute effects of toxic radiation doses on intestine are well established, we are yet to acquire a complete spectrum of sub-lethal radiation-induced chronic intestinal perturbations at the molecular level. We investigated persistent effects of a radiation dose (2Gy) commonly used as a daily fraction in radiotherapy on oxidants and anti-oxidants, and autophagy pathways, which are interlinked processes affecting intestinal homeostasis. Six to eight weeks old C57BL/6J mice (n=10) were exposed to 2Gy γ-ray. Mice were euthanized two or twelve months after radiation, intestine surgically removed, and flushed using sterile PBS. Parts of the intestine from jejunal–ilial region were fixed, frozen, or used for intestinal epithelial cell (IEC) isolation. While oxidant levels and mitochondrial status were assessed in isolated IEC, autophagy and oxidative stress related signaling pathways were probed in frozen and fixed samples using PCR-based expression arrays and immunoprobing. Radiation exposure caused significant alterations in the expression level of 26 autophagy and 17 oxidative stress related genes. Immunoblot results showed decreased Beclin1 and LC3-II and increased p62, PI3K/Akt, and mTOR. Flow cytometry data showed increased oxidant production and compromised mitochondrial integrity in irradiated samples. Immunoprobing of intestinal sections showed increased 8-oxo-dG and nuclear PCNA, and decreased autophagosome marker LC3-II in IEC after irradiation. We show that sub-lethal radiation could persistently downregulate anti-oxidants and autophagy signaling, and upregulate oxidant production and proliferative signaling. Radiation-induced promotion of oxidative stress and downregulation of autophagy could work in tandem to alter intestinal functions and have implications for post-radiation chronic gastrointestinal diseases.

Successful Transplantation of Kidney Allografts in Sensitized Rats After Syngeneic Hematopoietic Stem Cell Transplantation and Fludarabine

Current methods to remove donor-specific HLA antibody (DSA) from sensitized patients remain imperfect. We tested novel approaches to desensitization using an animal model of allogeneic sensitization with skin grafts from dark agouti (DA) to Lewis rats. At the peak IgG alloantibody response we transplanted DA kidneys into nephrectomized Lewis recipients (n = 6) and all died within 10 days from antibody-mediated rejection (AMR). Allogeneic hematopoietic stem cell transplants (HSCT) from DA donors failed to engraft after lethal or sub-lethal irradiation. Sensitized rats given lethal irradiation plus syngeneic green fluorescent protein (GFP) + HSCT had repopulation of blood, spleen, thymus and lymph nodes by GFP+ cells. At 2 months after HSCT, serum DSA levels were reduced 60-70% and DSA (IgG) production in cultured splenocytes was also significantly decreased. However, there was only a modest improvement in graft survival from an average of 6.5 to 13.9 (n = 9) days. Adding seven daily doses of fludarabine to the preconditioning regimen resulted in long-term survival (>90 days) in 7 out of 10 rat kidney allografts. We conclude that syngeneic HSCT performed after preconditioning with irradiation and fludarabine can reduce DSA, prevent DSA rebound and AMR, enabling successful transplantation in animals with strong antibody reactivity to the donor MHC.

Abstract 1731: Dual inhibition of EGFR and HER2 in sublethally irradiated Lewis lung carcinoma cells suppresses MMP-9 production and cell invasiveness

Abstract Background: Radiotherapy (RT) is an essential component in multi-modality cancer management. While RT yields effective local disease control, distant failures remain great challenges that hinder cancer cure. Tumor cells that survive sublethal irradiation become invasive and metastasize, through up-regulation of matrix metalloproteinase-9 (MMP-9) and remodeling of extracellular matrix. Studies have shown that RT induces MMP-9 production in tumor cells by activating NF-κB through PI3K/Akt and MAPK/ERK pathways. However, clinical applications of inhibitors targeting these pathways are lacking due to toxicity and low efficacy. EGFR and HER2 are canonical upstream signaling molecules that activate these pathways. Additionally, EGFR and HER2, when stimulated with ligands, have been found to induce MMP-9 expression. In this study, we aim to determine the roles of EGFR and HER2 in RT-induced MMP-9 expression, tumor cell invasiveness and metastasis using EGFR inhibitor, erlotinib and EGFR/HER2 inhibitor, afatinib. Methods and Materials: We used western blot analysis to determine EGFR, HER2, and Akt phosphorylations in irradiated Lewis lung carcinoma (LLC) cells (7.5 Gy) pretreated with erlotinib or afatinib. We then used RT-PCR, western blot assay, ELISA, and gelatin zymography to evaluate MMP-9 expression in irradiated LLC cells pretreated with erlotinib or afatinib. Clonogenic assays estimated irradiated LLC cell survival treated with erlotinib or afatinib. Matrigel-coated Boyden chamber assay was used to evaluate invasion capability of irradiated LLC cells. Lastly, tissue western blot analyses of EGFR/HER2/Akt phosphorylations and MMP-9 were performed in LLC tumor grown on the thigh of C57BL/6 mice treated with combined RT and either drug. Results: RT induced EGFR, HER2, and Akt phosphorylations as well as increased MMP-9 expression in LLC cells. Pretreatment with afatinib suppressed RT-induced MMP-9 mRNA and protein expression more significantly than erlotinib. Afatinib inhibited RT-induced invasiveness of LLC cells more than erlotinib did. Tissue western blot analysis of irradiated LLC ectopic xenografts showed more inhibition of RT-induced in vivo MMP-9 expression by afatinib than erlotinib. Afatinib was a stronger inhibitor than erlotinib for RT-induced MMP-9 up-regulation and cell invasiveness in LLC cells and ectopic xenografts. However, clonogenic assay of LLC cells showed that neither erlotinib nor afatanib sensitized LLC cells to RT in survival. Conclusion: Our results suggest that sublethal irradiation induces MMP-9 expression through EGFR and HER2 activation. Dual inhibition of EGFR and HER2 kinase better suppress RT-induced MMP-9 expression and abolish tumor cell invasiveness. Afatinib may serve as an orally bioavailable agent that ameliorates distant metastasis from inadequately irradiated primary lung tumor. Citation Format: Wei-Lin Liu, Tommy Wei-Hsien Hou, Jason Chia-Hsien Cheng. Dual inhibition of EGFR and HER2 in sublethally irradiated Lewis lung carcinoma cells suppresses MMP-9 production and cell invasiveness. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1731. doi:10.1158/1538-7445.AM2014-1731

Abstract 636: Sub-lethal irradiation of diverse human carcinoma cells imparts enhanced and sustained expression of important modulators of effector CTL activity

Abstract Current cancer immunotherapy (CIT) approaches can efficiently introduce anti-tumor effector T-cells in patients, however, tumor cells can be immunosuppressive and directly inhibitory to T-cell activity. Previous data suggests that human tumor cells surviving radiation are phenotypically modulated making them better targets for CTLs. We examined several tumor cell lines (colorectal, breast and prostate) for their response to various doses of radiation (0-10Gy). Experiments quantified changes in the expression of genes that could result in altered effector CTL activity (OX40L, 41BBL, PD-L1, ICOSL and CD70) against tumor cells. Following irradiation, changes in expression of effector costimulatory molecules was examined in surviving and proliferating tumor cells. In human tumor cells expressing similar levels of major histocompatibility complex class I (MHC-I) and tumor associated antigens (TAA) following radiation, differences in CTL activity correlated with modulation of two positive costimulators, OX40L and 41BBL. The genes upregulated in our study (41BBL and OX40L) provide positive signals to killer T-cells that can attack tumor cells. Increased expression occurred when radiation was delivered either as a single dose or in fractions and could be observed as long as 7-days post-irradiation. We also found that radiation could alter gene expression in tumor cells via epigenetic mechanisms such as histone modifications of specific promoters. Furthermore we discovered that altered expression of OX40L and 41BBL (but not CD70, ICOSL or PD-L1) correlated with enhanced killing of irradiated tumor cells by both CEA and MUC-specific CTLs in cytotoxicity assays and this lysis was reversed by costimulatory blocking or gene-knockdown. We also observed enhanced activation and survival of CTLs exposed to irradiated tumor cell lines as well as exposed to ex vivo irradiated CD326+ tumor cells isolated from patient tumors. Overall, the results of our studies suggest that radiation can be used to make human tumors more amenable to effector CTL attack. Significance: The full role of IR as an independent immune-enhancer, in the absence of cell death or in radio-resistant tumors, remains unclear. Our data addresses a critical barrier to progress in improving combination radiation-cancer immunotherapy (RT-CIT) strategies by identifying mechanisms responsible for increased killing of irradiated tumor cells by effector CTLs. This “immunogenic modulation” of tumor cells is a mechanism different from ICD (requiring death of tumor cells and subsequent antigen processing and presentation by dendritic cells) that could be invoked independently, or to complement ICD approaches. This approach represents an alternate way of triggering important T-cell signal pathways that does not require the use of agonist antibodies. Citation Format: Anita Kumari, Orpha Rachel Mott, Ercan Cacan, Susanna F. Greer, Charlie T. Garnett. Sub-lethal irradiation of diverse human carcinoma cells imparts enhanced and sustained expression of important modulators of effector CTL activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 636. doi:10.1158/1538-7445.AM2014-636

Abstract LB-40: Inducible in vivo silencing of Brd4 identifies potential toxicities of sustained BET protein inhibition

Abstract BET family proteins are novel therapeutic targets for cancer and inflammation and represent the first epigenetic readers against which small-molecule inhibitors have been developed. First generation BET inhibitors have shown some therapeutic efficacy in pre-clinical models, however the consequences of disrupting BET protein function in normal tissues are largely unknown. Using an inducible and reversible transgenic RNAi mouse model targeting the BET family protein Brd4, we show that acute suppression of Brd4 in adult animals has immediate and dramatic effects in multiple tissues. Brd4-depleted mice displayed epidermal hyperplasia, alopecia, and decreased cellular diversity and stem cell depletion in the small intestine. To test whether the loss of intestinal stem cells induced by Brd4 suppression would have consequences in combination with agents that induce acute gut toxicity, we subjected mice to a sub-lethal irradiation. Whereas control mice showed intestinal recovery 4-5 days following irradiation, Brd4-depleted mice showed dramatic and continued crypt-villus atrophy, implying that potent BET protein inhibition in combination with cytotoxic chemotherapy may show significantly enhanced toxicity in the clinic. These findings provide important insight into Brd4 function in normal tissues and importantly, predict several potential outcomes associated with potent and sustained inhibition of BET protein function. Citation Format: Jessica Bolden, Nilgun Tasdemir, Lukas Dow, Johan H. van Es, John E. Wilkinson, Zhen Zhao, Hans Clevers, Scott W. Lowe. Inducible in vivo silencing of Brd4 identifies potential toxicities of sustained BET protein inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-40. doi:10.1158/1538-7445.AM2014-LB-40

Abstract 632: Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing

Abstract Radiation therapy (RT) is used for local tumor control through direct killing of tumor cells. Radiation-induced cell death can trigger tumor antigen-specific immune responses, but these are often noncurative. Radiation has been demonstrated to induce immunogenic modulation (IM) in various tumor types by altering the biology of surviving cells to render them more susceptible to T cell-mediated killing. Little is known about the mechanism(s) underlying IM elicited by sub-lethal radiation dosing. We have examined the molecular and immunogenic consequences of radiation exposure in breast, lung, and prostate human carcinoma cells. Radiation induced secretion of ATP and HMGB1 in both dying and surviving tumor cells. In vitro and in vivo tumor irradiation induced significant upregulation of multiple components of the antigen-processing machinery and calreticulin cell-surface expression. Augmented CTL lysis specific for several tumor-associated antigens was largely dictated by the presence of calreticulin on the surface of tumor cells and constituted an adaptive response to endoplasmic reticulum stress, mediated by activation of the unfolded protein response. This study provides evidence that radiation induces a continuum of immunogenic alterations in tumor biology, from immunogenic modulation to immunogenic cell death. We also expand the concept of immunogenic modulation, where surviving tumor cells recovering from radiation-induced endoplasmic reticulum stress become more sensitive to CTL killing. These observations offer a rationale for the combined use of radiation with immunotherapy, including for patients failing RT alone. Citation Format: Sofia R. Gameiro, Momodou L. Jammeh, Max M. Wattenberg, Kwong Y. Tsang, Soldano Ferrone, James W. Hodge. Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 632. doi:10.1158/1538-7445.AM2014-632

Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1γ.

Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1β, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent phosphorylation of HP1γ may participate in the regulation of dynamic interaction between HP1γ and histone H3K9me3 to promote DNA damage repair. This HIPK2/HP1γ pathway may uncover a new functional aspect of HIPK2 as a tumor suppressor.

Whole-body irradiation increases the magnitude and persistence of adoptively transferred T cells associated with tumor regression in a mouse model of prostate cancer.

Adoptive immunotherapy has demonstrated efficacy in a subset of clinical and preclinical studies, but the T cells used for therapy often are rendered rapidly nonfunctional in tumor-bearing hosts. Recent evidence indicates that prostate cancer can be susceptible to immunotherapy, but most studies using autochthonous tumor models demonstrate only short-lived T-cell responses in the tolerogenic prostate microenvironment. Here, we assessed the efficacy of sublethal whole-body irradiation (WBI) to enhance the magnitude and duration of adoptively transferred CD8(+) T cells in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. We demonstrate that WBI promoted high-level accumulation of granzyme B (GzB, Gzmb)-expressing donor T cells both in lymphoid organs and in the prostate of TRAMP mice. Donor T cells remained responsive to vaccination in irradiated recipients, but a single round of WBI-enhanced adoptive immunotherapy failed to affect significantly the existing disease. Addition of a second round of immunotherapy promoted regression of established disease in half of the treated mice, with no progression observed. Regression was associated with long-term persistence of effector/memory phenotype CD8(+) donor cells. Administration of the second round of adoptive immunotherapy led to reacquisition of GzB expression by persistent T cells from the first transfer. These results indicate that WBI conditioning amplifies tumor-specific T cells in the TRAMP prostate and lymphoid tissue, and suggest that the initial treatment alters the tolerogenic microenvironment to increase antitumor activity by a second wave of donor cells.

Photic injury to cultured RPE varies among individual cells in proportion to their endogenous lipofuscin content as modulated by their melanosome content.

We determined whether photic stress differentially impairs organelle motility of RPE lipofuscin and melanin granules, whether lethal photic stress kills cells in proportion to lipofuscin abundance, and whether killing is modulated by melanosome content. Motility of endogenous lipofuscin and melanosome granules within the same human RPE cells in primary culture was quantified by real-time imaging during sublethal blue light irradiation. Cell death during lethal irradiation was quantified by dynamic imaging of the onset of nuclear propidium iodide fluorescence. Analyzed were individual cells containing different amounts of autofluorescent lipofuscin, or similar amounts of lipofuscin and a varying content of phagocytized porcine melanosomes, or phagocytized black latex beads (control for light absorbance). Lipofuscin granules and melanosomes showed motility slowing with mild irradiation, but slowing was greater for lipofuscin. On lethal irradiation, cell death was earlier in cells with higher lipofuscin content, but delayed by the copresence of melanosomes. Delayed death did not occur with black beads, suggesting that melanosome protection was due to properties of the biological granule, not simple screening. Greater organelle motility slowing of the more photoreactive lipofuscin granule compared to melanosomes suggests that lipofuscin mediates mild photic injury within RPE cells. With lethal light stress endogenous lipofuscin mediates killing, but the effect is cell autonomous and modulated by coincident melanosome content. Developing methods to quantify the frequency of individual cells with combined high lipofuscin and low melanosome content may have value for predicting the photic stress susceptibility of the RPE monolayer in situ.

Stochastic modeling of stress erythropoiesis using a two-type age-dependent branching process with immigration

The erythroid lineage is a particularly sensitive target of radiation injury. We model the dynamics of immature (BFU-E) and mature (CFU-E) erythroid progenitors, which have markedly different kinetics of recovery, following sublethal total body irradiation using a two-type reducible age-dependent branching process with immigration. Properties of the expectation and variance of the frequencies of both types of progenitors are presented. Their explicit expressions are derived when the process is Markovian, and their asymptotic behavior is identified in the age-dependent (non-Markovian) case. Analysis of experimental data on the kinetics of BFU-E and CFU-E reveals that the probability of self-renewal increases transiently for both cell types following sublethal irradiation. In addition, the probability of self-renewal increased more for CFU-E than for BFU-E. The strategy adopted by the erythroid lineage ensures replenishment of the BFU-E compartment while optimizing the rate of CFU-E recovery. Finally, our analysis also indicates that radiation exposure causes a delay in BFU-E recovery consistent with injury to the hematopoietic stem/progenitor cell compartment that give rise to BFU-E. Erythroid progenitor self-renewal is thus an integral component of the recovery of the erythron in response to stress.

Sublethal gamma irradiation affects reproductive impairment and elevates antioxidant enzyme and DNA repair activities in the monogonont rotifer Brachionus koreanus

To examine the effects of gamma radiation on marine organisms, we irradiated several doses of gamma ray to the microzooplankton Brachionus koreanus, and measured in vivo and in vitro endpoints including the survival rate, lifespan, fecundity, population growth, gamma ray-induced oxidative stress, and modulated patterns of enzyme activities and gene expressions after DNA damage. After gamma radiation, no individuals showed any mortality within 96h even at a high intensity (1200Gy). However, a reduced fecundity (e.g. cumulated number of offspring) of B. koreanus at over 150Gy was observed along with a slight decrease in lifespan. At 150Gy and 200Gy, the reduced fecundity of the rotifers led to a significant decrease in population growth, although in the second generation the population growth pattern was not affected even at 200Gy when compared to the control group. At sub-lethal doses, reactive oxygen species (ROS) levels dose-dependently increased with GST enzyme activity. In addition, up-regulations of the antioxidant and chaperoning genes in response to gamma radiation were able to recover cellular damages, and life table parameters were significantly influenced, particularly with regard to fecundity. DNA repair-associated genes showed significantly up-regulated expression patterns in response to sublethal doses (150 and 200Gy), as shown in the expression of the gamma-irradiated B. koreanus p53 gene, suggesting that these sublethal doses were not significantly fatal to B. koreanus but induced DNA damages leading to a decrease of the population size.

Protective effect of a laser-induced sub-lethal temperature rise on RPE cells from oxidative stress

Recently introduced new technologies that enable temperature-controlled laser irradiation on the RPE allowed us to investigate temperature-resolved RPE cell responses. In this study we aimed primarily to establish an experimental setup that can realize laser irradiation on RPE cell culture with the similar temperature distribution as in the clinical application, with a precise time/temperature history. With this setup, we conducted investigations to elucidate the temperature-dependent RPE cell biochemical responses and the effect of transient hyperthermia on the responses of RPE cells to the secondary-exposed oxidative stress. Porcine RPE cells cultivated in a culture dish (inner diameter = 30 mm) with culture medium were used, on which laser radiation (λ = 1940 nm, spot diameter = 30 mm) over 10 s was applied as a heat source. The irradiation provides a radially decreasing temperature profile which is close to a Gaussian shape with the highest temperature in the center. Power setting for irradiation was determined such that the peak temperature (Tmax) in the center of the laser spot at the cells reaches from 40 °C to 58 °C (40, 43, 46, 50, 58 °C). Cell viability was investigated with ethidium homodimer III staining at the time points of 3 and 24 h following laser irradiation. Twenty four hours after laser irradiation the cells were exposed to hydrogen peroxide (H2O2) for 5 h, followed by the measurement of intracellular glutathione, intracellular 4-hydroxynonenal (HNE) protein adducts, and secreted vascular endothelial growth factor (VEGF). The mean temperature threshold for RPE cell death after 3 h was found to be around 52 °C, and for 24 h around 50 °C with the current irradiation setting. A sub-lethal preconditioning on Tmax = 43 °C significantly induced the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, and decreased H2O2-induced increase of intracellular 4-HNE protein adducts. Although sub-lethal hyperthermia (Tmax = 40 °C, 43 °C, and 46 °C) caused a slight increase of VEGF secretion in 6 h directly following irradiation, secondary exposed H2O2-induced VEGF secretion was significantly reduced in the sub-lethally preheated groups, where the largest effect was seen following the irradiation with Tmax = 43 °C. In summary, the current results suggest that sub-lethal thermal laser irradiation on the RPE at Tmax = 43 °C for 10 s enhances cell defense system against oxidative stress, with increasing the GSH/GSSG ratio. Together with the results that the decreased amount of H2O2-induced 4-HNE in sub-lethally preheated RPE cells was accompanied by the lower secretion of VEGF, it is also strongly suggested that the sub-lethal hyperthermia may modify RPE cell functionality to protect RPE cells from oxidative stress and associated functional decrease, which are considered to play a significant role in the pathogenesis of age-related macular degeneration and other chorioretinal degenerative diseases.

Cytomegalovirus and early-life irradiation synergize to erode protective immunity in aging (VIR5P.1034)

Abstract Susceptibility to infectious disease increases with age, making it one of the leading causes of death in people over 65. The immune aging process may be accelerated by the presence of life-long latent infections, such as cytomegalovirus, that put a constant burden on the immune system. Theories of biological aging also suggest that immune cells may have defective functions in old age because of a lifetime of self-renewal, and DNA damage related senescence, among other factors. Sub-lethal whole-body irradiation results in dose-dependent death of immune cells throughout the body, forcing some peripheral self-renewal of surviving immune cells. We exposed mice to various sub-lethal irradiation doses (0Gy - 4Gy) in youth, and followed them for life. Some mice were also given life-long murine cytomegalovirus (MCMV) to reflect the most common latent virus in human populations. At the end of life, mice were vaccinated against, and then infected with, a potentially lethal dose of West Nile Virus (WNV). Neither MCMV, nor any dose of sub-lethal irradiation, independently altered survival following vaccination and WNV infection. However, mice that received a combination of 4Gy of irradiation in youth, and life-long MCMV infection, were less likely to survive WNV challenge. Mechanisms and implications for radiobiology, immune aging, and vaccine efficacy will be discussed.

The actin-bundling protein L-plastin regulates alveolar macrophage pro-inflammatory cascade (INM2P.426)

Abstract Regulation of the actin cytoskeleton is essential to multiple immune cell functions, including cell motility, adhesion, and activation. In lymphocytes, the actin-bundling protein L-plastin (LPL) plays critical roles in sustaining cell polarization and formation of the immunological synapse. We now show defects in the innate immune system that lead to profound susceptibility to pulmonary pneumococcal infection. LPL is required for generation of alveolar macrophages: there are fewer alveolar macrophages in the bronchoalveolar lavage fluid of LPL-/- mice, and regeneration of alveolar macrophages following sublethal irradiation is delayed when bone marrow is donated by LPL-/- mice. Activation of LPL-/- alveolar macrophages is also defective, with reduced upregulation of IL-1-beta and IL-6 following LPS stimulation. Reduced numbers and activation of alveolar macrophages in LPL-/- mice correlate with disrupted podosome formation. Reduced numbers and activation of alveolar macrophages would be expected to result in an inability to clear pulmonary pathogens. When challenged with pulmonary infection with pneumococcus, LPL-/- mice show a clearance defect within 3 hours, with a ten-fold increase in recoverable pneumococci compared to wild-type mice. LPL-/- mice thus offer a model system in which to detail how actin cytoskeletal integrity regulates macrophage development and activation.

Rescue of vaccine T cell memory following sublethal gamma irradiation (VAC3P.952)

Abstract Subsequent to sublethal γ radiation exposure, survivors reacquire immune competence, but lose vaccine immunity. Our goal is to rescue CD8+ T cell-mediated memory responses following γ radiation exposure. In a mouse model of Listeria monocytogenes (LM) infection, we found that rapid revaccination (rescue) of immunized mice following irradiation preserves CD8+ T cell mediated immunity. Rescued mice are able to survive a wild type LM challenge infection when the rescue vaccine strain, LM-deltaActA, is given 0-3 days following radiation exposure. A signature of rescue was revealed in gene profiling studies in which a group of cytokines and chemokines were uniquely up regulated in the spleens of LM-rescued animals as compared to mice irradiated without rescue. These included factors important in resistance to LM, such as proinflammatory factors CCL2, TNFα, and IL-6 and bacterocidal factors CXCL9, 10, and 11 as well as anti-apoptotic factors including IL-15 and IER3, which may have a role in rescue of memory CTL. RAW264.7, a macrophage cell line, produced a similar cytokine profile when irradiated and infected with LM, suggesting a probable myeloid source for the majority of these factors. Monocyte activation by the combination of irradiation and LM infection may thus create conditions of rescue by producing cytokines and chemokines that enhance clearance of LM and rescue CD8+ CTL mediating vaccine immunity.