DOI: 10.1002/pbc.26591 Pediatric Blood & Cancer The American Society of Pediatric Hematology/Oncology ABSTRACTS 2017 ASPHO ABSTRACTS Plenary Paper # 4001 Transgenic Expression of IL15 Improves Antiglioma Activity of IL13R2-CAR T Cells Giedre Krenciute, Brooke Prinzing, Zhongzhen Yi, Irina Balyasnikova, Gianpietro Dotti, Stephen Gottschalk Baylor College of Medicine, Houston, Texas, United States Background: Diffuse intrinsic pontine glioma (DIPG) and glioblastoma (GBM) are the most aggressive primary brain tumors in children. Immunotherapy with T cells expressing chimeric antigen receptors (CARs) specific for the DIPG and GBM antigens is an attractive approach to improve outcomes. IL13R2 is expressed at a high frequency in DIPG and GBM but not in normal brain, making it a promising target for CAR T-cell immunotherapy. We recently generated the first scFv-based CAR that is specific for IL13R2 and demonstrated that it had potent anti-GBM activity in preclinical models. However, CAR T-cell persistence was limited, resulting in recurrence of IL13R2-positive GBMs. Objectives: The goal of this project is to evaluate if transgenic expression of IL-15, a cytokine that is critical for T-cell proliferation and survival, enhances persistence and anti-tumor activity of IL13R2-CAR.CD28. T cells. Design/Method: We generated IL13R2-CAR.CD28. T cells expressing IL-15 (IL13R2- CAR.IL15 T cells) by double transducing T cells with retroviruses containing expression cassettes encoding i) IL13R2-CAR.CD28. or ii) IL-15, tNGFR, and iC9 separated by 2A sequences. We determined the effector function of IL13R2-CAR.IL15 T cells in vitro using standard assays, and in the U373 GBM xenograft model. Results: Double transduction of CD3/CD28-activated T cells resulted in T-cell lines that expressed both transgenes in 45–50% of T cells. At a baseline, IL13R2-CAR.IL15 T cells produced on average 69.5 pg/ml of IL15. Production was significantly increased after CD3 or antigen-specific T-cell stimulation (176.7 pg/ml; n = 6; p<0.001). IL13R2-CAR.IL15 T cells were as efficient as IL13R2-CAR T cells in killing IL13R2-positive GBMs in vitro. After intratumoral injection into U373 glioma-bearing mice IL13R2-CAR.IL15 T cells persisted significantly longer than IL13R2-CAR T cells (p<0.05). This resulted in a significant increase in progression free (84 vs 49 days; p = 0.008) and overall survival (p = 0.02) of treated mice. Up to date, 4/10 IL13R2-CAR.IL15 T-cell treated mice remain glioma free with a follow up of at least 80 days. 3/5 examined, recurring U373 gliomas post IL13R2-CAR.IL15 T-cell ther- apy had downregulated IL13R2 expression, indicating immune escape. While all recurring GBMs expressed the glioma-associated antigen EphA2, 1/5 GBMs had also downregulated the expression of the glioma-associated antigen HER2. Conclusion: Here we demonstrate that transgenic expression of IL15 enhances the in vivo persistence of IL13R2-CAR T cells resulting in improved anti-glioma activity. However, enhanced T-cell persistence resulted in the development of antigen-specific and -unspecific loss variants highlighting the need to target multiple glioma-associated antigens in tumors with heterogeneous antigen expression such as GBM and DIPG. Plenary Paper # 4002 P2Y12 Receptor Function and In Vivo Platelet Response to Cangrelor in Neonates with Cyanotic Congenital Heart Disease Thomas Diacovo, Elisabeth Kaza, Matthew Egalka, Hairu Zhou, Jianchun Chen, Richard Li, Scott Diamond, Julie Vincent, Emile Bacha Columbia University, New York, New York, United States Background: Neonates with cyanotic congenital heart disease (CHD) palliated with a systemic-to-pulmonary artery shunt are at risk for thrombosis in the early post-operative period. Thus, there is an urgent need to identify a target and therapy suitable for thromboprophylaxis during this vulnerable period. Objectives: This study sought to determine whether the P2Y12 receptor on platelets from neonatal cardiac patients supports in vivo thrombus formation and whether cangrelor, a direct acting and reversible receptor antagonist, can reduce clot size. Design/Method: Blood samples from patients with CHD (neonates to 18 years) and healthy adults were collected to assess platelet reactivity and response to cangrelor in laser injured arterioles of avatar mice that support human platelet-mediated thrombosis. Drug effect was also evaluated in whole blood using a microfluidic device with collagen as a thrombogenic surface. EC50 and IC50 values for ADP and cangrelor, respectively, were determined by light transmission aggregometry (LTA). Results: Platelets from neonates with CHD formed thrombi of similar size to those from older patients and adults, with cangrelor reducing clot size by >45%. P2Y12 receptor response to ADP and cangrelor was also nearly identical based on EC50 and IC50 values, respectively. The potency of cangrelor was further established in a microfluidic assay yielding results comparable to LTA. Conclusion: Platelets isolated from neonatal patients with CHD at the time of surgical repair / palliation have a robust response to ADP and are as amenable to P2Y12 inhibition with can- grelor as their adult counterparts. Moreover, our findings appear to be independent of age and type of cardiac lesion. Unique to this study was our ability to establish the in vivo efficacy of cangrelor using an avatar mouse model that permits real-time evaluation of human platelet interactions with the injured vessel wall, yielding a biological response consistent with P2Y12 inhibition: disruption of thrombus shell formation. We also demonstrate the value of a microflu- idic device that can serve as a PD biomarker, requiring significantly less blood to assess changes in platelet reactivity than LTA. This multi-analytic approach will be employed in the upcom- ing clinical trail at our institution that will assess the PK and PD properties of cangrelor in neonatal patients with CHD requiring palliation with a systemic-to-pulmonary artery shunt (NCT02765633). This study was supported by grants from the NIH National Heart (HD081281-01), American Heart Association (16CSA28260000), and The Medicine Company, Parsippany, NJ. Paper Session # 4003/Young Investigator Award Recipient Mechanism of Action and Combination Therapy Studies on the XPO1 Inhibitor Selinexor in Pediatric High-Grade Glioma and Diffuse Intrinsic Pontine Glioma Adam Green, John DeSisto, Patrick Flannery, Rakeb Lemma, Trinayan Kashyap, Andrew Kung, Yosef Landesman, Rajeev Vibhakar University of Colorado School of Medicine, Aurora, Colorado, United States Background: Pediatric high-grade gliomas (HGG) and diffuse intrinsic pontine gliomas (DIPG) account for the majority of pediatric brain tumor deaths and respond poorly to chemotherapy. We have shown that selinexor, an inhibitor of the nuclear transporter XPO1, is effective against HGG and DIPG in cell culture and patient-derived xenograft (PDX) models, but resistance to treatment develops. In addition, selinexor’s mechanism of action, in these dis- eases and in general, is poorly understood. We are now studying selinexor in national pediatric clinical HGG/DIPG trials, making these issues crucial to address. Selinexor increases nuclear levels of the NF-B inhibitor IKB- through XPO1 inhibition, which leads to NF-B inhibi- tion. IKB- levels are also regulated by proteasomal inhibition. NF-B transcriptional activity is thought to be upregulated by NGFR. Objectives: To determine the mechanism of action and effective combination therapies of selinexor in pediatric HGG and DIPG Design/Method: We conducted a proteomics study using tandem mass spectrometry of HGG cells treated with selinexor versus control. Using functional genomics techniques, we knocked down expression of NGFR, IKB-, and NF-B, studied the effects using qPCR and immunoflu- orescence (IF), and quantified the impact of knockdown on selinexor’s effectiveness. We screened HGG and DIPG cells with all FDA-approved chemotherapy agents. We subsequently conducted viability assays using the proteasome inhibitors bortezomib, carfilzomib, and mari- zomib, alone and in combination with selinexor and radiation therapy (RT). Results: Selinexor treatment of HGG cells induced the overexpression of NGFR in proteomic analyses, neurosphere culture (p<0.01), and PDX models (p<0.0005) versus control. shRNA knockdown of NGFR in HGG/DIPG cells increased HGG/DIPG proliferation rate (p<0.05) and neurosphere formation (p<0.0005) versus shNull. NGFR knockdown increased the nuclear expression of NF-B (p<0.0005) and induced selinexor resistance (p<0.05), while NF-B inhi- bition increased sensitivity to selinexor (p<0.05). Proteasome inhibitors showed synergy with selinexor in our screen. On validation assays, bortezomib and carfilzomib showed IC50 levels of 1–200 nM and synergy with selinexor across a range of HGG/DIPG lines, while marizomib showed IC50 levels of 2–5 uM and antagonism with selinexor. Selinexor, bortezomib, and RT showed synergy (CI 0.1). Conclusion: The mechanism of action of selinexor in pediatric HGG/DIPG appears to involve NF-B inhibition through the induction of IB-, and, surprisingly, NGFR, calling into ques- tion current understanding of this pathway. FDA-approved proteasome inhibitors, which are known to upregulate IB-, show potential as a synergistic combination with selinexor and RT. We are now testing the combination in PDX studies for future clinical trial application. Paper Session # 4004a/Young Investigator Award Recipient Regulation of c-kit Oncogene by Ikaros and Casein Kinase II (CK2) in T Cell Acute Lymphoblastic Leukemia (T-ALL) Chandrika Gowda, Malika Kapadia, Chunhua Song, Meixian Xiang, Yali Ding, Kimberly Payne, Sinisa Dovat Penn State University, Hershey, Pennsylvania, United States Background: The c-kit oncogene encodes CD117, the receptor for stem cell factor. CD117 and its ligand, stem cell factor, are essential for normal hematopoiesis. Expression of CD117 has been detected in 9% of T cell Acute Lymphoblastic Leukemai (T-ALL), and is associated with a very early T lineage phenotype, LMO2 overexpression, activating flt3 mutations and features of early T-cell precursor leukemia (ETP). Recent studies suggest that c-kit expression in T-ALL regulates engraftment potential and radio sensitivity of transformed T lineage cells Objectives: It has been hypothesized that expression of c-kit is necessary for leukemia stem cell survival and c-kit inhibitors have been tested for the treatment of human leukemia. The Pediatr Blood Cancer. 2017;e26591. © 2017 Wiley Periodicals, Inc. S1 of S103 wileyonlinelibrary.com/journal/pbc https://doi.org/10.1002/pbc.26591