Syngeneic Transplantation Model Research Articles

Stem cell transplantation impairs dendritic cell trafficking and herpesvirus immunity.

Long-term survivors after hematopoietic stem cell transplantation are at high risk of infection, which accounts for one-third of all deaths related to stem cell transplantation. Little is known about the cause of inferior host defense after immune cell reconstitution. Here, we exploited a murine syngeneic BM transplantation (BMT) model of late infection with murine gammaherpesvirus 68 (MHV-68) to determine the role of conventional DC (cDC) trafficking in adaptive immunity in BMT mice. After infection, the expression of chemokine Ccl21 in the lung is reduced and the migration of cDCs into lung draining lymph nodes (dLNs) is impaired in BMT mice, limiting the opportunity for cDCs to prime Th cells in the dLNs. While cDC subsets are redundant in priming Th1 cells, Notch2 functions in cDC2s are required for priming increased Th17 responses in BMT mice, and cDC1s can lessen this activity. Importantly, Th17 cells can be primed both in the lungs and dLNs, allowing for increased Th17 responses without optimum cDC trafficking in BMT mice. Taken together, impaired cDC trafficking in BMT mice reduces protective Th1 responses and allows increased pathogenic Th17 responses. Thus, we have revealed a previously unknown mechanism for BMT procedures to cause long-term inferior immune responses to herpes viral infection.

Effect of Static Cold Storage on Skeletal Muscle after Vascularized Composite Tissue Allotransplantation.

Prolonged cold ischemia associated with static cold storage (SCS) results in higher incidence of acute and chronic allograft rejection in solid organ transplantations. Deleterious effects of SCS on vascularized composite tissue allograft were studied with limited data on muscle structure and function. The aim of this study is to evaluate the long-term impact of SCS on muscle metabolism, structure, and force generation using a syngeneic rat hindlimb transplantation model. Sixty-five male Lewis rats (250 ± 25 g) were distributed into five groups, including naive control, sciatic nerve denervation/repair, immediate transplantation, transplantation following static warm storage for 6 hours at room temperature, and transplantation following SCS for 6 hours at 4°C. Sciatic nerves were repaired in all transplantations. Muscle samples were taken for histology and metabolomics analysis following electromyography and muscle force measurements at 12 weeks after transplantation. All cold-preserved limbs remained viable at 12 weeks, whereas animals receiving limbs preserved in room temperature had no survivors. The SCS transplantation group showed a 73% injury score, significantly higher than groups receiving immediate transplants without cold preservation (50%, p < 0.05). A significant decline in muscle contractile force was also demonstrated in comparison to the immediate transplantation group (p < 0.05). In the SCS group, muscle energy reserves remained relatively well preserved in surviving fibers. SCS extends allograft survival but fails to preserve muscle structure and force.

Abstract LB-014: Antibody targeting soluble NKG2D ligand sMIC sensitizes metastatic prostate tumor and other MIC+ tumors to PD1/PD-L1 blockade therapy in pre-clinical models

Abstract Targeting programmed cell death-1 (PD-1) has produced a survival benefit for patients diagnosed with many types of malignancies. However, the clinical survival benefit in metastatic prostate cancer is only marginal to-date. Currently, the understanding of why metastatic prostate cancer is resistant to PD-1 blockade therapy remains to be defined. Among the many factors that negatively impact response to PD-1 blockade treatment, tumor-derived soluble NKG2D ligands have been reported to hinder the clinical response to immune checkpoint blockade in melanoma patients. Metastatic prostate cancer cells release high levels of the circulating soluble NKG2D ligand, soluble MHC I Chain related molecule (sMIC), a molecule that is absent in classical rodent tumor model. Here, we tested the hypothesis that targeting sMIC sensitizes metastatic prostate tumors to PD-1 blockade therapy. Using a well-described double transgenic TRAMP/MIC mouse model that closely resembles the onco-immune dynamics of human metastatic prostate cancer, we demonstrate that an antibody targeting sMIC significantly enhances the response of prostate tumors to PD-1/PD-L1 blockade therapy. Mechanistically, targeting sMIC enriches NK cell and CD8 T cell infiltration into tumors, equips CD8 T cells with enhanced NKG2D and CD28 dual co-stimulation, and increases the expression of PD-L1+ on prostate tumor cells. Combination of the antibody targeting sMIC and a PD-1/PD-L1 blockade cooperatively augments intra-tumoral NK and antigen-specific CD8 T cell anti-tumor immunity. These findings were validated in syngeneic prostate tumor transplant models and other models of other cancer types. Our pre-clinical study provides proof-of-concept and uncovered novel mechanisms of a viable combination immunotherapy for metastatic prostate cancer and other MIC+ cancers. Citation Format: Jennifer D. Wu, Jinyu Zhang, Pablo Larrocha, Payal Dhar. Antibody targeting soluble NKG2D ligand sMIC sensitizes metastatic prostate tumor and other MIC+ tumors to PD1/PD-L1 blockade therapy in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-014.

Abstract 3779: Clonal tracing reveals different mechanisms of resistance to immune checkpoint blockade

Abstract Immune checkpoint blockade (ICB) therapy has improved patient survival in a variety of cancers, but its resistance presents a major clinical challenge. It remains unclear whether therapeutic resistance is driven predominantly by refractory clones of cancer cells or by immunosuppressive microenvironment. To address this, we utilized syngeneic transplantation model and performed clonal tracing of cancer cells to monitor the response to ICB. Different cancer clones displayed variable sensitivity to ICB treatment, reflecting intratumoral heterogeneity in therapy response. Furthermore, different recipients manifested distinct resistance mechanisms. ICB-responders developed resistance through selection and expansion of pre-existing ICB-resistant cancer clones, whereas non-responders presented with irresponsive microenvironment rather than dominance of resistant cancer clones. Extrapolation of the tumor-infiltrating immune repertoire revealed that higher IgG and lower IgA strongly correlated with better ICB response. This biomarker was further validated in multiple clinical cohorts. Our approach discriminates different sources of resistance to ICB, and identifies humoral immunity as biomarker of ICB response. Citation Format: Shengqing Gu, Xihao Hu, Xiaoqing Wang, Peng Jiang, Ziyi Li, Nicole Traugh, Xia Bu, Xiaofang Xing, Gordon J. Freeman, Myles Brown, Xiaole S. Liu. Clonal tracing reveals different mechanisms of resistance to immune checkpoint blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3779.

Structural and functional consequences of the STAT5BN642H driver mutation

Hyper-activated STAT5B variants are high value oncology targets for pharmacologic intervention. STAT5BN642H, a frequently-occurring oncogenic driver mutation, promotes aggressive T-cell leukemia/lymphoma in patient carriers, although the molecular origins remain unclear. Herein, we emphasize the aggressive nature of STAT5BN642H in driving T-cell neoplasia upon hematopoietic expression in transgenic mice, revealing evidence of multiple T-cell subset organ infiltration. Notably, we demonstrate STAT5BN642H-driven transformation of γδ T-cells in in vivo syngeneic transplant models, comparable to STAT5BN642H patient γδ T-cell entities. Importantly, we present human STAT5B and STAT5BN642H crystal structures, which propose alternative mutation-mediated SH2 domain conformations. Our biophysical data suggests STAT5BN642H can adopt a hyper-activated and hyper-inactivated state with resistance to dephosphorylation. MD simulations support sustained interchain cross-domain interactions in STAT5BN642H, conferring kinetic stability to the mutant anti-parallel dimer. This study provides a molecular explanation for the STAT5BN642H activating potential, and insights into pre-clinical models for targeted intervention of hyper-activated STAT5B.

High mobility group box 1 secretion blockade results in the reduction of early pancreatic islet graft loss

Pancreatic islet transplantation has been known as the best cure for patients suffering from severe type 1 diabetes mellitus (T1DM). Despite meaningful advances in human allogeneic islet transplantation field, significant amounts of islet loss in early post-transplantation periods is still a big concern for clinicians. One of the major factors determining the fate of the islets is the danger-associated molecular patterns (DAMPs) secreted by activated immune cells or islets themselves under hypoxic stress. High mobility group box 1 (HMGB1) protein is one of the best characterized DAMP molecules associated with islets. HMGB1 is known to be passively released by transplanted murine islet cells after taking damages from cytokines, reactive oxygen species, and other DAMPS, and the released HMGB1 harms neighboring islet cells by interacting with receptors expressed on murine islets such as toll-like receptor 2 (TLR2) and TLR4, thereby forming a vicious cycle. Here, we show that a small molecule inhibitor inflachromene (ICM) was capable of blocking the secretion of HMGB1 from murine islet cells during the normoxic and hypoxic post-isolation period. Notably, the treatment of ICM during the islet isolation process resulted in decreased HMGB1 levels during the subsequent cell culture. ICM's in vivo efficacy was evaluated in murine syngeneic islet transplantation model, and it significantly reduced the serum and graft level of HMGB1. Ultimately, the intraperitoneal administration of ICM prevented the loss of marginal-mass islet grafts and reversed the diabetes in mice.

A New Face Subunit Transplant Model in Mice, Containing Skin, Mandible, and Oral Mucosa for Future Face Vascularized Composite Allotransplantation Studies.

In immunologic research, mice have advantages over other animals, such as low costs, easy handling, suitable life cycle, and adequate laboratory resources. However, vascularized composite allotransplantation surgery using mice is not popular, partly because of technical difficulties and high mortality rates. The authors' goal was to demonstrate a face transplantation model in mice that includes skin, mandible, and oral mucosa. The authors developed a new syngeneic face transplantation model composed of skin, mandible, teeth, and oral mucosa in C57BL/6 mice. The following assessment included measuring the length of the right incisor on the transplanted mandibles, computed tomographic scan in one mouse for mandibular structure evaluation, and histologic examination of different tissue samples in another mouse for viability evaluation. The authors performed five consecutive transplantations. The donor vessels were the common carotid artery (approximately equal to 0.4 mm) and the anterior facial vein (approximately equal to 0.2 mm), and the recipients were the common carotid artery and the posterior facial vein (approximately equal to 0.4 mm). The mean operative time was 80 minutes for the donor and 123 minutes for the recipient. There were neither flap failures nor animal deaths. The follow-up was 6 months. The right incisor of the transplant grew at different rates in all cases. Histologic samples showed viability in all tissues, including mandibular bone marrow. Computed tomography demonstrated normal structure of the transplanted bone. The authors' syngeneic partial face transplantation model in mice, which included skin, oral mucosa, and mandible with teeth, should be useful for future face allotransplantation research, as the myriad of tissues it provides, of different immunomodulatory functions, is similar to that in the clinical scenario.

α2,6-Sialylation promotes immune escape in hepatocarcinoma cells by regulating T cell functions and CD147/MMP signaling.

Altered glycosylation is a common feature of cancer cells and plays an important role in tumor progression. β-Galactoside α2-6-sialyltransferase 1 (ST6Gal-I) is the critical sialyltransferase responsible for the addition of α2-6-sialic acid to the terminal N-glycans on the cell surface. However, the functions and mechanism of ST6Gal-I in tumor immune escape remain poorly understood. Here, we found that ST6Gal-I overexpression promoted hepatocarcinoma cell proliferation, migration, and immune escape by increasing the levels of CD147, MMP9, MMP2, and MMP7. When CD8+ T cells were co-cultured with cell lines expressing different levels of ST6Gal-I, we found that ST6Gal-I upregulation inhibited the T cell proliferation and increased the secretion of IL-10 and TGF-β1, while secretion of IFN-γ and TNF-α was diminished. In a syngeneic tumor transplant model, ST6Gal-I upregulated Hca-P. In addition, Hepa1-6 cells formed significantly larger tumors and suppressed intratumoral penetration by CD8+ T cells. In combination, these results suggest that ST6Gal-I promotes the immune escape of hepatocarcinoma cells in the tumor microenvironment and highlight the importance of assessing ST6Gal-I status for immunotherapies.

A Novel Injury Site-Targeted Complement Inhibitor Which Protects against Lung Transplant Ischemia Reperfusion Injury

Purpose Self-reactive natural Abs initiate complement activation and injury following ischemia and reperfusion of certain tissues, but their role in post-ischemic lung injury is unknown. Here, we investigated the expression of two previously identified post-ischemic neoepitopes, namely a modified annexin IV epitope recognized by B4 IgM mAb, and an epitope consisting of a subset of phospholipids recognized by C2 IgM mAb, and additionally determined their role in lung transplant ischemia reperfusion injury (IRI). Methods Lungs from C57Bl/6 mice were orthotopically transplanted into Rag1-/- (antibody-deficient) mice reconstituted with either B4 and C2 mAbs. Results Rag1-/- mice were protected from lung IRI, but reconstitution with either B4 or C2 natural IgM mAbs restored lung IRI. The C2 mAb was more effective than the B4 mAb at restoring injury, as determined by histological and micro-CT analysis. Based on this information, we developed and characterized a therapeutic strategy that exploited the post-ischemia recognition system of natural antibodies. We constructed a single-chain antibody (scFv) derived from C2 mAb and linked it to Crry, an inhibitor of C3 activation (C2scFv-Crry). In models of syngeneic (B6-B6) and allogeneic (Balb/c-B6) lung transplant models in which recipients contain a full natural antibody repertoire, a single post-transplant injection of C2scFv-Crry blocked the binding of endogenous IgM to the graft, inhibited complement activation, and significantly reduced graft inflammation and injury as determined by histology, μCT, and immunhistochemical analysis. The severity of IRI is associated with the onset of acute rejection. Conclusion We identified pathophysiologically important epitopes expressed within the lung after lung ischemia and reperfusion post transplantation. We further describe a novel translatable strategy for complement inhibition that is specifically targeted to the transplanted lung, and that has several advantages over currently available approaches that rely on systemic complement inhibition. Significantly, the C2 epitope has been identified in both mouse and man, increasing the translational relevance of the approach.

Nr5a2 promotes cancer stem cell properties and tumorigenesis in nonsmall cell lung cancer by regulating Nanog.

Lung cancer has the highest mortality rate due to late diagnosis and high incidence of metastasis. Cancer stem cells (CSCs) are a subgroup of cancer cells with self‐renewal capability similar to that of normal stem cells (NSCs). While CSCs may play an important role in cancer progression, mechanisms underlying CSC self‐renewal and the relationship between self‐renewal of the NSCs and CSCs remain elusive. The orphan nuclear receptor Nr5a2 is a transcriptional factor, and a regulator of stemness of embryonic stem cells and induced pluripotent stem cells. However, whether Nr5a2 regulates the self‐renewal of lung CSCs is unknown. Here, we showed the diagnostic and prognostic values of elevated Nr5a2 expression in human lung cancer. We generated the mouse LLC‐SD lung carcinoma CSC cellular model in which Nr5a2 expression was enhanced. Using the LLC‐SD model, through transient and stable siRNA interference of Nr5a2 expression, we provided convincing evidence for a regulatory role of Nr5a2 in the maintenance of lung CSC self‐renewal and stem cell properties in vitro. Further, using the syngeneic and orthotopic lung transplantation model, we elucidated augmented cancer biological properties associated with Nr5a2 promotion of LLC‐SD self‐renewal. More importantly, we revealed that Nr5a2’s regulatory role in promoting LLC‐SD self‐renewal is mediated by transcriptional activation of its direct target Nanog. Taken together, in this study, we have provided convincing evidence in vitro and in vivo demonstrating that Nr5a2 can induce lung CSC properties and promote tumorigenesis and progression through transcriptional up‐regulation of Nanog.

Conditioning for Hematopoietic Stem Cell Transplantation Using Antibody-Drug Conjugate Targeting CD45 Permits Engraftment across Immunologic Barriers

INTRODUCTION: Hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for benign and malignant hematologic diseases. It also holds great promise as treatment for infectious and autoimmune disease and for autologous gene therapy. However, due to the toxicity of conditioning regimens for HSCT, it is generally reserved for the most life-threatening diagnoses, such as acute leukemia. Targeted approaches to conditioning may improve the safety of HSCT and extend its applicability to other diseases. Recently, conditioning regimens were described consisting of treatment with anti-CD45.2 or anti-cKit antibodies conjugated to the ribosome-inhibiting protein saporin (CD45-SAP and cKit-SAP, respectively) (Palchaudhuri et al (2016), Nat Biotechnol, Czechowicz et al (2016), ASH Abstract). These were reported to deplete murine hematopoietic stem cells (HSCs) and permit engraftment of syngeneic HSCs with minimal toxicity. However, the ability of CD45-SAP and cKit-SAP to permit HSCT across immunologic barriers without chemotherapy or radiation, or to target malignant cells expressing these receptors, are potential applications that were not explored.METHODS: We compared CD45-SAP and cKit-SAP conditioning in syngeneic (B6 to B6) and allogeneic (CB6F1 to B6) murine transplantation models. Donor chimerism in peripheral blood (PB) and complete blood counts (CBCs) were assessed monthly during these experiments. Successful HSC engraftment was confirmed by serial transplantation experiments. Human CD45-SAP and cKit-SAP conjugates were also tested for their ability to target MOLM13 AML cells using XTT viability assays, and in targeting cord-blood derived HSCs using colony forming cell assays.RESULTS: We confirmed that CD45-SAP dosed at 75 µg per mouse could effectively deplete B6 mice of HSCs and condition them for syngeneic HSCT as reported (Palchaudhuri et al (2016), Nat Biotechnol), with normal-range CBCs and stable donor PB chimerism of approximately 80% at 6 months post-transplant. Donor-derived HSC in CD45-SAP treated mice successfully engrafted upon transplantation to secondary B6 recipients. cKit-SAP dosed at 10 µg per mouse was sufficient for HSC depletion, but robust syngeneic engraftment required dosing at 50 µg per mouse. Notably, CD45-SAP treatment combined with CD4+and CD8+ T cell depletion permitted haploidentical transplantation from CB6F1 donors to B6 recipients, with normal-range CBCs and stable donor PB chimerism of approximately 70% at 3 months post-transplant. Finally, human CD45-SAP conjugates could successfully target MOLM13 leukemia cells and cord-blood derived HSCs in vitro.CONCLUSIONS: Transplantation in mice across immunological barriers is achievable using CD45-SAP conditioning, with high-level donor chimerism without chemotherapy or radiation. Furthermore, human CD45-SAP conjugates can successfully target human leukemia cell lines and primary HSCs in vitro. Further studies are underway in three areas. First, by combining T and NK cell depletion with CD45-SAP conditioning, we are attempting full-mismatch HSCT in mice. Second, using humanized mouse models, we will assess the ability of CD45-SAP to target human leukemia cells and HSCs in vivo. Finally, we are exploring using HSCT with CD45-SAP conditioning as a treatment in mouse models of autoimmune disease. DisclosuresRettig:Amphivena Therapeutics: Research Funding; Novimmune: Research Funding.

CXCL5 as Regulator of Neutrophil Function in Cutaneous Melanoma

Chemokines mold the tumor microenvironment by recruiting distinct immune cell populations, thereby strongly influencing disease progression. Previously, we showed that CXCL5 expression is upregulated in advanced stages of primary melanomas, which correlates with the presence of neutrophils in the tumor. The analysis of neutrophil populations in various tissues revealed a distinct phenotype of tumor-associated neutrophils. Tumor-associated neutrophils expressed PD-L1, CXCR4, CCR5, Adam17, and Nos2 and were immunosuppressive in a T-cell proliferation assay. To investigate the impact of CXCL5 and neutrophils invivo, we established a syngeneic mouse tumor transplantation model using CXCL5-overexpressing and control melanoma cell lines. Growth behavior or vascularization of primary tumors was not affected by CXCL5 expression and neutrophils alone. However, in combination with Poly(I:C), tumor-associated neutrophils were able to attenuate induced antitumoral T-cell responses. CXCL5-overexpressing tumors had reduced lung metastasis compared with control tumors. Neutrophil depletion reversed this effect. Invitro, unstimulated lung-derived neutrophils had higher levels of reactive oxygen species compared with tumor-associated neutrophils, and CXCL5 stimulation further increased reactive oxygen species levels. In summary, in melanoma, neutrophils play a context-dependent role that is influenced by local or systemic factors, and interfere with therapies activating the acquired immune system. Actively switching neutrophils into antitumorigenic mode might be a new therapeutic strategy.

Cold Storage Injury to Rat Small-bowel Transplants-Beneficial Effect of a Modified HTK Solution.

The small bowel is prone to ischemic injury during transport before transplantation, an injury that endangers the recipient patient. The small-bowel mucosal microcirculation in particular appears to be highly sensitive to injury. Current preservation solutions such as histidine-tryptophan-ketoglutarate (HTK) solution provide some protection to the graft. However, these were developed decades ago and do not address several critical processes, such as hypoxia-induced membrane pores and free radical-mediated hypothermic injury. To protect the graft from cold ischemic injury, we implemented a modified HTK solution here, including glycine, alanine, and iron chelators in a heterotopic, syngeneic small-bowel transplantation model of the rat. The effects of the modified solution and its major components were compared against the conventional HTK solution using intravital microscopy in the early reperfusion period. The amino acid glycine, added to HTK solution, slightly improved mucosal perfusion. Both, the modified base solution (without iron chelators) and iron chelators increased functional capillary density of the mucosa during the early reperfusion period. The complete modified solution (with glycine, alanine, and iron chelators) significantly increased the perfusion index, functional capillary density of the mucosa, and red blood cell velocity in the grafts after reperfusion in comparison with the grafts preserved with HTK. The modified preservation solution improved the microcirculation of the transplants and needs detailed evaluation in further models of small-bowel transplantation.

Early TLR4 Blockade Attenuates Sterile Inflammation-mediated Stress in Islets During Isolation and Promotes Successful Transplant Outcomes.

During the isolation process, pancreatic islets are exposed to an environment of sterile inflammation resulting in an upregulated inflammatory state before transplantation. Toll-like receptor 4 (TLR4) has been identified as a major mediator of sterile inflammation. Therefore, we sought to determine whether early TLR4 blockade would be effective in reducing the inflammatory burden in islets pretransplant. Islets from C57BL/6 mice were treated with a TLR4 antagonist during the pancreatic ductal perfusion and digestion steps of the isolation process. Islets were then analyzed for inflammation by RT-PCR and Western blot, and for viability and function in vitro. A syngeneic transplant model using a marginal mass of islets transplanted intraportally into mice with streptozotocin-induced diabetes was used to study transplant outcomes after early TLR4 blockade. Diabetic mice receiving 150 islets treated with early TLR4 blockade achieved euglycemia at a higher rate than mice receiving untreated islets (75% vs 29%; P < 0.05) and had improved long-term function (P < 0.05). Serum markers for islet damage and inflammation were significantly reduced posttransplant (P < 0.05). Both the expression of key inflammatory genes and the activation of mitogen-activated protein kinases were reduced by early TLR4 blockade. Islet viability was improved (P < 0.05) while preserving islet insulin secretory capacity postisolation. Early TLR4 blockade protects islets from sterile inflammation-mediated stress sustained during isolation and promotes positive transplant outcomes. Our findings support the use of early TLR4 blockade during clinical islet isolation procedures to reduce pretransplant inflammation and improve transplant outcomes.

Abstract IA23: Enhancer remodeling in response to MEK inhibition in TNBC

Abstract In contrast to acquired resistance to targeted therapeutics via genomic changes, adaptive resistance in cancer involves rapid cellular reprogramming at a nongenomic level. In triple-negative breast cancer (TNBC) a robust transcriptional adaptation to MEK inhibition (MEKi) by the MEK inhibitor, trametinib, is observed in both TNBC patients and preclinical models. This transcriptional adaptation includes upregulation of bypass signaling molecules, including receptor tyrosine kinases (RTKs), to reactivate cellular proliferation programs in the presence of the initial drug challenge. Rather than attempting to antagonize RTK upregulation using a secondary kinase inhibitor in conjunction with the initial, primary targeted therapeutic, we have employed a strategy to broadly inhibit MEKi-induced transcriptional adaptation through the use of BET bromodomain inhibitors. BET protein family bromodomains bind to acetylated lysine moieties of histone subunits or transcription factors to regulate transcriptional elongation via recruitment of P-TEFb, a RNA polymerase II regulatory complex containing CDK9 and Cyclin T1. In TNBC we found the combination of MEKi + BET bromodomain inhibition (BETi) resulted in durable tumor cell growth inhibition not obtained with trametinib alone, nor with different kinase inhibitor combinations. Importantly, we observed in vivo MEKi + BETi synergy in orthotopic SUM159PT xenograft and T11 and 2225 mouse orthotopic syngeneic transplant models of TNBC. This synergism is not limited to TNBC, as we have shown that MEKi + BETi results in durable growth suppression in HER2+ breast cancer. A central question is the mechanism of BETi efficacy in the context of suppressing adaptation to targeted therapeutics. We asked if BRD4 function at enhancers is critical for adaptive transcription in response to MEK1/2 inhibition by trametinib. We found vast chromatin remodeling in the form of de novo enhancer formation and remodeling in response to trametinib in TNBC cells. Enhancers with pronounced BRD4 density and co-occupied with prototypical enhancer marks (H3K27ac, MED1, H3K4me1) were rapidly (1-4 h) formed genome-wide, including at sites proximal to receptor tyrosine kinase loci including DDR1, KDR, FGFR2, and PDGFRB, each influential in TNBC adaptive resistance. Trametinib-responsive enhancers were remodeled across the genome, but with BETi the total number of enhancers remained near baseline. In fact, BETi was able to disrupt enhancers seeded in response to trametinib. We observed correlation of transcriptional induction of genes proximal to the enhancer density induced by trametinib and a corresponding correlative decrease in transcript levels of the cognate genes of the seeded enhancers with combination MEKi+BETi treatment. Does the enhancer paradigm provide a potential source of pharmacologic targets for attenuating adaptive transcription beyond that of BRD4? In addition to BETi, we found that pharmacologic perturbation of CBP/p300 acetyltransferase, capable of depositing acetylation at H3K27 of enhancers, or core P-TEFb constituent CDK9 abrogated adaptive RTK upregulation elicited by MEKi. BRD4 directly associates with the transcriptional regulator JMJD6, a JmjC family demethylase, and, accordingly, the use of a pan-JmjC family demethylase inhibitor or siRNA targeting JMJD6 diminished RTK adaption. Similar response abrogation was achieved with depletion of the histone H3 lysine 36 methyltransferase NSD3 or by depleting CDK7, whose transcriptional regulatory activity is conferred by phosphorylation of its substrates including CDK9 and the carboxy-terminal domain (CTD) of RNA polymerase II. While small-molecule inhibitors are currently not yet available or are pre-phase I for many of these purported strategies to block adaptive resistance, our preclinical data suggest that protein complexes of drug-induced enhancers may be a largely untapped frontier of epigenetic targets to block adaptive bypass resistance to targeted therapies. Citation Format: Jon Zawistowski, Samantha Bevill, Gary L. Johnson. Enhancer remodeling in response to MEK inhibition in TNBC [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr IA23.

Recombinant Thrombomodulin Alpha Protects Kidney Graft from Damage after Long Cold Ischemia Time in a Rat Kidney Transplant Model

Objectives Thrombomodulin (TM) extensively expresses on the endothelial cells in the steady state and is known to prevent hypercoagulation via combining with thrombin and inactivating its procoagulant activity. TM also has anti-inflammatory effects. In inflammatory situation including ischemia reperfusion injury TM is known to decrease its expression on the endothelial cells. The purpose of this study is to investigate whether perfusate solution saturated with recombinant TM alpha (rTM) can protect the kidney grafts from the damage after long cold ischemia time. Methods We employed a rat syngeneic kidney transplant model using Lewis strain. Donor kidney grafts were preserved in a cold UW perfusate solution saturated with rTM (Group A, n=7), or not (Group B, n=8) for 24 hours. Then kidney transplantation (Lewis to Lewis) was performed. Blood and urine samples were sequentially collected (post operative day 1, 2, and 7) to measure creatinine clearance. Neutrophil gelatinase-associated lipocalin (NGAL), an acute kidney injury marker, was also measured by ELISA. Kidney graft samples in an acute phase (2 hours after operation) were collected to investigate the grade of tubular damage. TUNEL assay by immunofluorescence was performed. Immunohistochemistry (IHC) for TM was also performed. Results IHC revealed that the expression of TM on kidney grafts after 24-hour cold ischemia was extensively reduced compared with those after 0-hour ischemia. Creatinine clearance (ml/min/kg) in Group A was significantly ameliorated compared with that in Group B: mean (SD), 95% CI of Group A vs Group B; 0.68 (0.33), 0.41-0.96 vs 0.23 (0.13), 0.11-0.35 on POD 1; 1.22 (0.40), 0.86-1.59 vs 0.59 (0.30), 0.31-0.86 on POD 2; and 2.63 (1.41), 1.45-3.81 vs 1.17 (0.69), 0.47-1.87 on POD 7; p=0.02, two-way repeated measured ANOVA. Furthermore, serum levels of NGAL (mg/ml) in Group A were significantly lower than those in Group B: mean (SD), 95% CI of Group A vs Group B; 6.5 (1.7), 0.70-4.71 vs 9.1 (1.1), 7.8-10.4 on POD 1; and 4.3 (2.7), 1.5-7.1 vs 8.9 (1.8), 6.6-11.1 on POD 2; p=0.021, two-way repeated measured ANOVA. Fewer damaged tubular cells and apoptosis positive cells at a very acute phase were observed in Group A (Figure 1), compared with in Group B (Figure 2). Conclusion Perfusion with rTM significantly attenuates kidney graft damage after long cold ischemia time by abrogating apoptosis in tubular cells

Abstract 1411: Proteogenomic analysis of the syngeneic mouse gastric stem cell-like cancer cell line

Abstract Gastric cancer is the third main cause of cancer deaths worldwide. To understand molecular mechanism of gastric cancer incidence and metastasis, we generated NCC-S1M lacking Smad4, Trp53 and Cdh1, which is the unique syngeneic gastric cancer cell line transplant model in the scientific society. NCC-S1M showed cancer stem cell-like features and strong tumor-initiating potential. Using proteogenomic analysis of NCC-S1M, we elucidated the specific signaling pathways that control gastric cancer stem cell-like properties and immune checkpoint. Cd274, Ccne1 and Il1rl1 were overexpressed and their protein levels were higher than normal gastric tissues. Cd274 encodes PD-L1, which is one of the important immune checkpoint proteins. The growth rate of NCC-S1M allograft was reduced after treatment with anti-PD-1. When Smad4 was ectopically expressed in NCC-S1M, Cd274 expression was decreased. In addition, TGF-β treatment induced reduction in Cd274 expression of Smad4 overexpressed NCC-S1M cells even more comparing with normal Smad4 overexpressed NCC-S1M. The knockdown of Il1rl1 in NCC-S1M reduced tumorigenicity and in vivo chemoresistance. We demonstrated that NCC-S1M focally amplified Ccne1 gene through array comparative genomic hybridization. Ccne1 knocked down NCC-S1M showed reduced metastatic potential. The proteogenomic characterization of NCC-S1M explains that Smad4 is important for PD-L1 immune evasion and that Il1rl1 has a significant role in cancer stem cell-like properties. Furthermore, it could imply that Ccne1 regulates metastasis. Citation Format: Hanna Yang, Hark Kyun Kim. Proteogenomic analysis of the syngeneic mouse gastric stem cell-like cancer cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1411.

Display of CD47 protein on pancreatic islet grafts improves engraftment following intraportal transplantation

Abstract Intraportal islet transplantation is a curative therapy for type 1 diabetes. However, early islet engraftment is hampered by a thrombotic and inflammatory reaction defined as an instant blood-mediated inflammatory reaction (IBMIR). The CD47 molecule is an important regulator of IBMIR, by blocking phagocytosis. We hypothesized that the transient display of the CD47 protein on the surface of islet grafts may prevent early islet destruction by IBMIR following intraportal transplantation. We generated a chimeric CD47 fused with a modified streptavidin (SA-CD47). SA-CD47 was transiently displayed on the surface of rat splenocytes that had been modified with biotin taking advantage of the strong affinity between biotin and streptavidin (SA). SA-CD47 protein was also effectively displayed on the surface of biotinylated islets. Importantly, in a syngeneic marginal mass intraportal islet transplantation model, 7/8 streptozotocin-diabetic C57BL/6 mice transplanted with 125 SA-CD47 engineered islets established euglycemia long term (&amp;gt;80 days). In marked contrast, only 1/7 mice achieved euglycemia when transplanted with SA-engineered islet grafts. The engrafted islets in the SA-CD47 group had a comparable response to naïve mice in the intraperitoneal glucose tolerance test. Flow cytometric and qRT-PCR analyses of liver tissues did not reveal significant changes in cellular and molecular immune responses between SA-CD47 and SA-engineered control groups, suggesting that inhibition of islet phagocytosis as underlying mechanism of islet engraftment and survival. Collectively, these results demonstrate the utility of SA-CD47 as a regulator of IBMIR for prevention of early islet graft loss following intraportal transplantation

Targeting CREB Pathway Suppresses Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is the most deadly subtype of lung cancer due to its dismal prognosis. We have developed a lentiviral vector-mediated SCLC mouse model and have explored the role of both the NF-κB and CREB families of transcription factors in this model. Surprisingly, induction of NF-κB activity, which promotes tumor progression in many cancer types including non-small cell lung carcinoma (NSCLC), is dispensable in SCLC. Instead, suppression of NF-κB activity in SCLC tumors moderately accelerated tumor development. Examination of gene expression signatures of both mouse and human SCLC tumors revealed overall low NF-κB but high CREB activity. Blocking CREB activation by a dominant-negative form of PKA (dnPKA) completely abolished the development of SCLC. Similarly, expression of dnPKA or treatment with PKA inhibitor H89 greatly reduced the growth of SCLC tumors in syngeneic transplantation models. Altogether, our results strongly suggest that targeting CREB is a promising therapeutic strategy against SCLC.Implications: Activity of the transcription factor CREB is elevated in SCLC tumors, which helps to maintain its neuroendocrine signature and cell proliferation. Our results highlight the importance of targeting the CREB pathway to develop new therapeutics to combat SCLC. Mol Cancer Res; 16(5); 825-32. ©2018 AACR.

Donor kidney injury molecule-1 promotes graft recovery by regulating systemic necroinflammation.

Ischemia-reperfusion injury during kidney transplantation predisposes to delayed graft function, rejection, and premature graft failure. Exacerbation of tissue damage and alloimmune responses may be explained by necroinflammation: an autoamplification loop of cell death and inflammation, which is mediated by the release of damage-associated molecular patterns (eg, high-mobility group box-1; HMGB1) from necrotic cells that activate both innate and adaptive immune pathways. Kidney injury molecule-1 (KIM-1) is a phosphatidylserine receptor that is upregulated on injured proximal tubular epithelial cells and enables them to clear apoptotic and necrotic cells. Here we show a pivotal role for clearance of dying cells in regulating necroinflammation in a syngeneic murine kidney transplant model. We found persistent KIM-1 expression in KIM-1+/+ kidney grafts posttransplantation. Compared to recipients of KIM-1+/+ kidneys, recipients of KIM-1-/- kidneys exhibited significantly more renal dysfunction, apoptosis and necrosis, tubular obstruction, and graft failure. KIM-1-/- grafts also had more inflammatory cytokines, infiltrating neutrophils, and macrophages compared to KIM-1+/+ grafts. Most significantly, passive release of HMGB1 from apoptotic and necrotic cells led to dramatically higher serum HMGB1 levels and increased proinflammatory macrophages in recipients of KIM-1-/- grafts. Our data identify an endogenous protective mechanism against necroinflammation in kidney grafts that may be of therapeutic relevance in transplantation.