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Acquired von Willebrand syndrome (aVWS) has been reported in patients with congenital heart diseases associated with shear stress caused by significant blood flow gradients. Its etiology and impact on intraoperative bleeding during pediatric cardiac surgery have not been systematically studied. This single-center, prospective, observational study investigated appropriate diagnostic tools of aVWS compared with multimer analysis as diagnostic criterion standard and aimed to clarify the role of aVWS in intraoperative hemorrhage. A total of 65 newborns and infants aged 0 to 12 months scheduled for cardiac surgery at our tertiary referral center from March 2018 to July 2019 were included in the analysis. The glycoprotein Ib M assay (GPIbM)/von Willebrand factor antigen (VWF:Ag) ratio provided the best predictability of aVWS (area under the receiver operating characteristic curve [AUC], 0.81 [95% CI, 0.75-0.86]), followed by VWF collagen binding assay/VWF:Ag ratio (AUC, 0.70 [0.63-0.77]) and peak systolic echocardiographic gradients (AUC, 0.69 [0.62-0.76]). A cutoff value of 0.83 was proposed for the GPIbM/VWF:Ag ratio. Intraoperative high-molecular-weight multimer ratios were inversely correlated with cardiopulmonary bypass (CPB) time (r = −0.57) and aortic cross-clamp time (r = −0.54). Patients with intraoperative aVWS received significantly more fresh frozen plasma (P = .016) and fibrinogen concentrate (P = .011) than those without. The amounts of other administered blood components and chest closure times did not differ significantly. CPB appears to trigger aVWS in pediatric cardiac surgery. The GPIbM/VWF:Ag ratio is a reliable test that can be included in routine intraoperative laboratory workup. Our data provide the basis for further studies in larger patient cohorts to achieve definitive clarification of the effects of aVWS and its potential treatment on intraoperative bleeding.

Purpose Veno-occlusive disease (VOD) is a serious complication of hematopoietic stem cell transplantation (HSCT) with a high incidence in pediatric patients. This study aimed to detect signs of hypofibrinolysis using thrombelastography. Methods In this prospective single-center study, thrombelastographic measurements (EX and TPA tests) were taken during HSCT to detect signs of impaired coagulation, clot formation, or hypofibrinolysis. Results Of 51 patients undergoing allogeneic and autologous HSCT, five (9.8%) developed VOD and received defibrotide treatment. Thrombelastography measurements were also obtained from 55 healthy children as a control group. The results show that clot lysis was prolonged in VOD patients compared to other HSCT patients and control group (lysis time, TPA test: day + 14 to + 21: VOD: 330 ± 67 s vs. HSCT: 246 ± 53 s; p = 0.0106; control: 234 ± 50 s; control vs. VOD: p = 0.0299). The maximum lysis was reduced in HSCT patients compared to controls (EX test: control: 8.3 ± 3.2%; HSCT: day 0 to + 6: 5.3 ± 2.6%, p p p Conclusion These results suggest that HSCT patients exhibit reduced fibrinolytic capacities and patients diagnosed with VOD show signs of hypofibrinolysis. This prospective study shows that fibrinolysis can be assessed in a rapid and accessible way via thrombelastography. Thrombelastography might be a parameter to support the diagnosis of a VOD and to serve as a follow-up parameter after the diagnosis of a VOD.

Anti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCRβ chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon γ), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CAR-T cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL, TCR-KO-CAR-T cells clearly controlled CD19+ leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched third-party adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.

Background: CD123 is frequently expressed on hematologic malignancies including 96-98% of AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) showed a promising approach anti-AML activity, however T-cell persistence was limited. Interleukin-15 (IL15) has emerged as a candidate immunomodulator as it enhances T-cell expansion and persistence, and induces long-lasting memory T-cells. To improve the efficacy and persistence of CD123-ENG T-cells we developed IL15 expressing CD123-ENG T-cells. Here, we report on the characterization and efficacy of IL15-secreting CD123-ENG T cells in vitro and in vivo models of adult AML. Methods/Results: A cDNA encoding IL15 was cloned into retroviral vectors encoding CD123-ENG or CD19-ENG (CD123-ENG.IL15; CD19-ENG.IL15). ENG T-cells were generated from human peripheral blood mononuclear cells (PBMCs) from normal donors or T-cells from AML patients by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells and T-cells expressing CD123 (CD123-ENG T-cells) served as controls. IL15 production of CD19-ENG.IL15 and CD123-ENG.IL15 T cells was confirmed by ELISA (144-159 pg/ml vs 38 and 46 pg/ml of NT and CD123-ENG T cells, p Conclusion: We here demonstrated that transgenic expression of IL15 in CD123-ENG T-cells results in improved expansion and persistence, and anti-AML activity. These results warrant further exploration of IL15-modified CD123-targeted T-cells as immunotherapy for AML. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:EMD Serono: Honoraria; Inmatics: Membership on an entity's Board of Directors or advisory committees; ASSISI fundation of Memphis: Research Funding; TESSA Therapeutics: Other: Research Collaboration; ViraCyte: Consultancy; NHLBI: Research Funding; America Lebanese Syrian Associated Charities: Research Funding; California Institute for Regenerative Medicine: Research Funding; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; Sanofi: Honoraria; Tidal: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy. Velasquez:St. Jude: Patents & Royalties: Patent Applications in the Fields of Cell and Gene Therapy ; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees. Andreeff:Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstaZeneca: Consultancy; Amgen: Consultancy; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; CPRIT: Research Funding; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding.

1. Background Chemotherapy-induced nausea and vomiting (CINV) is a feared and burdensome complication after emetogenic chemotherapy (EC), especially in pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). Antiemetic prophylaxis (AEP) is thus a substantial factor in the transplantation management. Guidelines recommend a comprehensive AEP with NK1R-antagonists (neurokinin-1 receptor), 5-HT3R-antagonists (5-hydroxytryptamine-3 receptor), and corticosteroids. The water-soluble aprepitant derivative fosaprepitant (NK1R-antagonist) was shown to be highly effective in adult patients with moderately to highly EC when combined with granisetron (5-HT3R-antagonist). There is very little experience with fosaprepitant in pediatric patients. 2. Patients and Methods In this single-center retrospective study, 80 pediatric patients during allogeneic HSCT who received AEP either with intravenous fosaprepitant and granisetron (fosaprepitant group; FG) or a standard prophylaxis regimen with granisetron two times per day(historical control group; CG) were analyzed for safety and efficacy of the prophylaxis regimen. Efficacy of the two AEP regimen was evaluated comparing the vomiting frequency, percentages of patients vomiting and the need for rescue medication (dimenhydrinate) during the acute ( 3. Results A total of 80 pediatric patients with a median age of 9 years (range 2-17 years) who underwent allogeneic HSCT between 2015 and 2018 were consecutively enrolled and analyzed. Patients were transplanted for the treatment of acute leukemia (acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), ALL-relapse, AML-relapse; total: 36.5% of the patients), myelodysplastic syndromes (4.8% of the patients) solid tumors and lymphoma (neuroblastoma, brain tumor, non-Hodgkin's lymphoma, Hodgkin's lymphoma; 44.9% of the patients), and non-malignant hematopoietic diseases (thalassemia major, sickle cell anemia; 13.8% of the patients). The patients received allogeneic grafts from matched unrelated donors (48.4%), matched family donors (16.2%) and mismatched family donors (35.5%). Distribution of the patient characteristics was not significantly different in both study groups (p>0.05). The patients of the FG (n=40; HSCT between 2017 and 2018) received a single dose of fosaprepitant (3.5-4 mg per kg bodyweight (mg/kg); maximum 150 mg; intravenous (IV) infusion) directly before starting the myeloablative conditioning and as PRN (pro re nata) medication every five days after HSCT (same dosage; IV infusion), as well as granisetron (2x20 µg/kg per day; starting on the first day of the conditioning; max. 3 mg; IV infusion 0.05). When compared to the patients of the CG, significantly less patients of the FG experienced vomiting in both, the acute (p 4. Conclusions The prophylaxis regimen with fosaprepitant in combination with granisetron was safe and more effective in comparison to the standard prophylaxis regimen with granisetron only in pediatric patients undergoing allogeneic HSCT with a myeloablative conditioning. However, larger prospective trials are needed to evaluate these findings. 5. Keywords Chemotherapy-induced nausea and vomiting; antiemetic prophylaxis; fosaprepitant; pediatric patients 6. Acknowledgments This study was supported by the Stefan-Morsch-Stiftung, Birkenfeld, Germany. Disclosures Schlegel: Miltenyi Biotec: Patents & Royalties, Research Funding. Seitz:Miltenyi Biotec: Patents & Royalties, Research Funding. Lang:Miltenyi Biotec: Patents & Royalties, Research Funding. Handgretinger:Miltenyi Biotec: Patents & Royalties: Co-patent holder of TcR alpha/beta depletion technologies, Research Funding.

INTRODUCTION Evolving techniques have made possible the direct detection, physical isolation, and study of AML minimal residual disease (MRD) after treatment. This could allow for better identification of therapeutic vulnerabilities in AML. Prior studies have focused on cells that initiate leukemia in mouse models, known as leukemia-initiating cells (LIC), generally with a foundational CD34+CD38- immunophenotype. LIC are typically derived from diagnostic samples of untreated patients. Such stem-like cells do not necessarily represent the residual fraction of AML after treatment. Relapse may originate from non-LIC, and the presence of phenotypically and molecularly defined MRD is now firmly established as a critical prognostic factor for patients. High-risk AML is characterized by relapse, despite morphologic complete remission with initial therapy in most cases. RNA-sequencing was performed on pre- and post-treatment AML subpopulations, including MRD, from high-risk patients, to determine differences in gene expression. METHODS Matched primary AML samples were collected from marrow and peripheral blood of patients with high-risk AML (including patients with unfavorable karyotype and/or TP53 mutation) at diagnosis and after treatment. Mononuclear cells were flow-sorted for bulk (CD45dim) and LIC (Lin-CD34+CD38-CD123+) from diagnostic samples. Post-treatment samples were sorted for bulk mononuclear cells (MNC) and MRD, based on difference-from-normal/MRD immunophenotype specific for each patient as determined from established 20-marker clinical flow cytometry analysis. RNA was isolated using low-input methodology, and RNA-sequencing was performed using Illumina HiSeq 2000. Gene expression was assessed using GO-Elite, and differences between patients and subpopulations were assessed using rank product method. RESULTS Gene expression in MRD was analyzed by RNA-sequencing in comparison to diagnostic samples in eight patients with high-risk AML. Four patients had unfavorable karyotype, including two with TP53 mutations. Patients had additional high-risk features, such as FLT3-ITD or RUNX1 mutations, or secondary/therapy-related AML. Treatment consisted of chemotherapy (6/8) or hypomethylating agents (2/8), with or without other targeted drugs. Residual leukemia was detected in post-treatment samples in all study patients. Significant differences in gene expression were detected between MRD and other sorted populations, including diagnostic bulk AML and LIC. Relevant MRD pathways included those with strong interactions with the microenvironment. Anti-apoptotic mechanisms, cytoskeletal, and cell adhesion related genes, WNT/beta-catenin signaling, and TGFbeta signaling ranked among the most relevant processes in AML MRD subpopulations (Figure 1A, GO-Elite interactome of highly expressed genes in AML MRD). To identify potentially critical and unique MRD-specific genes, rank product method was applied using 1) the most highly expressed genes in AML MRD, 2) the most differential expressed genes between MRD and bulk AML at diagnosis, and 3) the most differentially expressed genes between MRD and bulk MNC after treatment. Among the top 50 scoring genes using this approach (Figure 1B), 16 genes were among the top 5% of genes expressed in MRD among all patients and 20 genes have cell surface gene-products (shown in yellow). Several potential leukemia- and cancer-related genes of interest were identified (shown in bold). CONCLUSIONS Key differences exist between the gene expression profiles of post-treatment MRD from high-risk AML patients, in comparison to other populations and subpopulations of sorted cells before and after treatment. The highlighted differences suggest that MRD relies on specific intrinsic gene expression changes and microenvironmental interactions, and therefore may be targetable after elimination of bulk AML with initial therapy. Accessible surfacesome targets are among top hits. Disclosures Konopleva: cellectis: Research Funding; Immunogen: Research Funding; abbvie: Research Funding; Stemline Therapeutics: Research Funding. Andreeff:Astra Zeneca: Research Funding; Amgen: Consultancy, Research Funding; Jazz Pharma: Consultancy; Celgene: Consultancy; Reata: Equity Ownership; SentiBio: Equity Ownership; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Acute myeloid leukemia (AML) cells are relatively resistant to tumor necrosis factor α–related apoptosis-inducing ligand (TRAIL). We previously reported that triptolide, a potent anticancer agent from a Chinese herb, decreases XIAP in leukemic cells. We evaluated the combination of triptolide and TRAIL and found synergistic promotion of apoptosis in AML cells. XIAP-overexpressing U937 cells (U937XIAP) were more resistant to TRAIL than U937neo cells, and inhibition of XIAP with the small-molecule inhibitor 1396-11 enhanced TRAIL-induced apoptosis, implying XIAP as a resistance factor in AML. Furthermore, triptolide increased DR5 levels in OCI-AML3, while the DR5 increase was blunted in p53-knockdown OCI-AML3 and p53-mutated U937 cells, confirming a role for p53 in the regulation of DR5. In support of this finding, disruption of MDM2-p53 binding with subsequent increase in p53 levels by nutlin3a increased DR5 levels and sensitized OCI-AML3 cells to TRAIL. The combination of 1396-11 plus nutlin3a plus TRAIL was more effective than either the 1396-11 and TRAIL or nutlin3a and TRAIL combinations in OCI-AML3 cells, further supporting the role of triptolide as a sensitizer to TRAILinduced apoptosis in part by independent modulation of XIAP expression and p53 signaling. Thus, the combination of triptolide and TRAIL may provide a novel strategy for treating AML by overcoming critical mechanisms of apoptosis resistance.

Triptolide, a diterpenoid isolated from the Chinese herb Tripterygium wilfordii Hook.f, has shown antitumor activities in a broad range of solid tumors. Here, we examined its effects on leukemic cells and found that, at 100 nM or less, it potently induced apoptosis in various leukemic cell lines and primary acute myeloid leukemia (AML) blasts. We then attempted to identify its mechanisms of action. Triptolide induced caspase-dependent cell death accompanied by a significant decrease in XIAP levels. Forced XIAP overexpression attenuated triptolide-induced cell death. Triptolide also decreased Mcl-1 but not Bcl-2 and Bcl-XL levels. Bcl-2 overexpression suppressed triptolide-induced apoptosis. Further, triptolide induced loss of the mitochondrial membrane potential and cytochrome C release. Caspase-9 knock-out cells were resistant, while caspase-8–deficient cells were sensitive to triptolide, suggesting criticality of the mitochondrial but not the death receptor pathway for triptolide-induced apoptosis. Triptolide also enhanced cell death induced by other anticancer agents. Collectively, our results demonstrate that triptolide decreases XIAP and potently induces caspase-dependent apoptosis in leukemic cells mediated through the mitochondrial pathway at low nanomolar concentrations. The potent antileukemic activity of triptolide in vitro warrants further investigation of this compound for the treatment of leukemias and other malignancies.

KBM5 cells, derived from a patient with blast crisis Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML), and imatinib-resistant KBM5 (KBM5-STI571) cells were found to express high levels of survivin. Inhibition of Bcr-Abl by imatinib significantly decreased survivin expression and cell viability in KBM5, but much less so in KBM5-STI571 cells. Inhibition of MEK, downstream of the Bcr-Abl signaling cascade decreased survivin expression and cell viability in both KBM5 and KBM5-STI571 cells. In addition, down-regulation of survivin by a survivin antisense oligonucleotide (Sur-AS-ODN) inhibited cell growth and induced maximal G2M block at 48 hours, whereas cell death was observed only at 72 hours in both KBM5 and KBM5-STI571 cells as shown by annexin V staining. Further, the combination of Sur-AS-ODN and imatinib induced more cell death in KBM5 cells than did either treatment alone. Down-regulating survivin also decreased colony-forming units (CFUs) in blast crisis CML patient samples. Our data therefore suggest that survivin is regulated by the Bcr-Abl/MAPK cascade in Ph+ CML. The facts that down-regulating survivin expression induced cell-growth arrest and subsequent cell death regardless of the cell response to imatinib and enhanced the sensitivity to imatinib suggest the potential therapeutic utility of this strategy in patients with CML, both imatinib sensitive and resistant.

We tested the effects of small-molecule XIAP antagonists based on a polyphenylurea pharmacophore on cultured acute myelogenous leukemia (AML) cell lines and primary patient samples. X-linked inhibitor of apoptosis protein (XIAP) antagonist N-[(5R)-6-[(anilinocarbonyl)amino]-5-((anilinocarbonyl){[(2R)-1-(4-cyclohexylbutyl)pyrrolidin-2-yl]methyl}amino)hexyl]-N-methyl-N′-phenylurea (1396-12), but not a structurally related control compound, induced apoptosis of primary leukemia samples with a lethal dose (LD50) of less than 10 μM in 16 of 27 (60%) samples. In contrast, XIAP antagonist 1396-12 was not lethal to the normal hematopoietic cells in short-term cytotoxicity assays. Response of primary AML specimens to XIAP inhibitor correlated with XIAP protein levels, with higher levels of XIAP associated with sensitivity. The XIAP antagonist 1396-12 induced activation of downstream caspases 3 and 7 prior to the activation of upstream caspase 8 and caspase 9. Apoptosis induction was also independent of B-cell lymphoma protein-2 (Bcl-2) or caspase 8, indicative of a downstream effect on apoptotic pathways. Thus, polyphenylurea-based XIAP antagonsists directly induce apoptosis of leukemia cells and AML patient samples at low micromolar concentrations through a mechanism of action distinct from conventional chemotherapeutic agents.

Cysteine-rich 61 (Cyr61, CCN1) and connective tissue growth factor (CTGF, CCN2) are growth factor-inducible immediate-early gene products found in blood vessel walls and healing cutaneous wounds. We previously reported that the adhesion of endothelial cells, platelets, and fibroblasts to these extracellular matrix-associated proteins is mediated through integrin receptors. In this study, we demonstrated that both Cyr61 and CTGF are expressed in advanced atherosclerotic lesions of apolipoprotein E-deficient mice. Because monocyte adhesion and transmigration are important for atherosclerosis, wound healing, and inflammation, we examined the interaction of THP-1 monocytic cells and isolated peripheral blood monocytes with Cyr61 and CTGF. THP-1 cells and monocytes adhered to Cyr61- or CTGF-coated wells in an activation-dependent manner and this process was mediated primarily through integrin alpha(M)beta(2). Additionally, expression of alpha(M)beta(2) on human embryonic kidney 293 cells resulted in enhanced cell adhesion to Cyr61. Consistent with these data, a GST-fusion protein containing the I domain of the integrin alpha(M) subunit bound specifically to immobilized Cyr61 or CTGF. We have also investigated the requirement of cell surface heparan sulfate proteoglycans (HSPGs) as coreceptors for monocyte adhesion to Cyr61. Pretreatment of monocytes with heparin or heparinase I resulted in partial inhibition of cell adhesion to Cyr61. However, monocytes, but not fibroblasts, were capable of adhering to a Cyr61 mutant deficient in heparin binding activity. Collectively, these results show that activated monocytes adhere to Cyr61 and CTGF through integrin alpha(M)beta(2) and cell surface HSPGs. However, unlike fibroblast adhesion to Cyr61, cell surface HSPGs are not absolutely required for this adhesion process.

The blood contains high concentrations of circulating extracellular vesicles (EVs), and their levels and contents are altered in several disease states, including cardiovascular disease. However, the function of circulating EVs, especially the microRNAs (miRNAs) that they contain, are poorly understood. We sought to determine the effect of secreted vesicles produced by quiescent endothelial cells (ECs) on monocyte inflammatory responses and to assess whether transfer of microRNAs occurs between these cells. We observed that monocytic cells cocultured (but not in contact) with ECs were refractory to inflammatory activation. Further characterization revealed that endothelium-derived EVs (EC-EVs) suppressed monocyte activation by enhancing immunomodulatory responses and diminishing proinflammatory responses. EVs isolated from mouse plasma also suppressed monocyte activation. Importantly, injection of EC-EVs in vivo repressed monocyte/macrophage activation, confirming our in vitro findings. We found that several antiinflammatory microRNAs were elevated in EC-EV–treated monocytes. In particular, miR-10a was transferred to monocytic cells from EC-EVs and could repress inflammatory signaling through the targeting of several components of the NF-κB pathway, including IRAK4. Our findings reveal that ECs secrete EVs that can modulate monocyte activation and suggest that altered EV secretion and/or microRNA content may affect vascular inflammation in the setting of cardiovascular disease.

Background: Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous disease with poor outcome. Targeted therapy of FMS-like tyrosine kinase-3 (FLT3)-mutated AML patients using small molecular inhibitors including sorafenib showed clinical success in reducing leukemia blasts in peripheral blood, but has limited effect on leukemic stem cells in the bone marrow (BM) microenvironment (1, 2). The BM is presumed to be the reservoir for leukemia stem cells (LSCs) which persist under targeted therapy and mediate disease relapse. The interaction of leukemic blasts with the BM microenvironment mediated by receptor-ligand axes such as CXCR4/SDF-1, E-selectin/HECA-452, and cell-cell contact have also been associated with drug resistance in FLT3 mutated AML (3-5). We recently reported that targeting CXCR4/E-selectin with the dual inhibitor GMI-1359 (GlycoMimetics, Inc., Rockville, MD) showed efficient mobilization of leukemia cells into the circulation and significant prolongation of survival of mice in FLT3-ITD mutant AML cells-xenografted murine model (Zhang et al., ASH Abstract #3790, 2015; Zhang et al., AACR Abstract #3284, 2016). Methods: In the present study, we further evaluated anti-leukemia effects of the dual CXCR4/E-selectin inhibitor GMI-1359 in a patient-derived xenograft (PDX) murine model. Primary FLT3 ITD mutated AML cells were isolated from BM aspirates and injected into irradiated NSG mice. Human CD45+/CD33+ cells were isolated from spleens of secondary engrafted mice after 3-6 months. The PDX leukemic cells were injected into irradiated NSG mice via tail vein (2x106 cells/mouse) and mice received either GMI-1359 (40mg/kg) or sorafenib (10mg/kg), or combination treatment starting at Day 18 after injection of leukemia cells. Vehicle treated mice served as controls. Leukemic cell burden was evaluated by flow cytometry (hCD45+ cells), histology and immunohistochemistry. Results: GMI-1359 treatment alone efficiently mobilized leukemic cells into blood circulation (absolute hCD45+ cells: 23.24k/UL vs. 9.06k/UL, respectively, in GMI-1359 vs. vehicle treatment groups after 71 days of treatment). Sorafenib alone or combined with GMI-1359 enhanced apoptosis induction and markedly reduced hCD45+ cells in circulation, BM and liver. The combination treatment profoundly reduced spleens leukemia cell infiltration (immunohistochemistry of CD45). A novel and unexpected finding of this study was that the combination of GMI-1359 with sorafenib significantly increased the regeneration of normal bone marrow cell populations (erythropoiesis, myelopoiesis and megakaryopoiesis) in the PDX model analyzed by histomorphology. Morphology and frequency of the hematopoiesis were identified in normal bone marrow of non-tumor bearing mice, suggesting a partial treatment response to the combination of GMI-1359 and sorafenib. In addition, GMI-1359 alone, sorafenib alone, or the combination treatment profoundly extended survival of PDX mice compared to vehicle treatment (98, 98 and 98 days vs 60.5 days, respectively (p = 0.04)). Conclusions: The dual CXCR4/E-selectin inhibitor GMI-1359 alone or in combination with the FLT3 ITD inhibitor sorafenib demonstrated anti-leukemia effects in a PDX model of FLT3 ITD mutated AML. Interestingly, combining GMI-1359 with sorafenib strikingly increased normal hematopoiesis in the bone marrow. The underlying mechanisms are under investigation. Disclosures Fogler: GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Background: Internal tandem duplication (ITD) mutation in Fms-like kinase 3 (FLT3) are observed in approximately 25% of newly diagnosed acute myeloid leukemia (AML) patients. FLT3 ITD is generally associated with poor survival outcome. A recent study has demonstrated that the TAM (Tyro3/MER/AXL) tyrosine kinase AXL is required for FLT3 inhibitor resistance in FLT3 ITD AML cells (Park et al Leukemia 2014). AXL expression is prognostic for poor outcome in AML (Ben-Batalla et al Blood 2013). These findings suggest that AXL is an excellent candidate for AML therapy particularly in patients with FLT3 ITD. ONO-9330547 (Ono Pharmaceutical Co, Osaka, Japan) is a highly specific AXL/MER inhibitor being developed for AML therapy. Here we report on the in vitro and in vivo effects of ONO-9330547 in AML models. Methods: FLT3 WT (HL60, OCI-AML3) and FLT3 ITD (Molm13, MV4;11) cells were treated with varying doses of ONO-9330547 and effect on cell viability and induction of apoptosis was assessed by flow cytometry using DAPI, Annexin V, and counting beads. Cell cycle was assessed by flow cytometry using EdU incorporation and FX Cycle Violet dye. Protein was isolated and reverse phase protein array (RPPA) analysis was performed on ONO-9330547 treated cells. RPPA results were validated by immunoblot analysis. RNA was collected and qRT-PCR performed. The drug was also tested on FLT3 ITD cells in an in vitro model of the AML microenvironment using co-culture with mesenchymal stromal cells (MSC). Finally, efficacy of ONO-9330547 in an in vivo AML xenograft model was tested using Molm13 cells expressing luciferase/GFP in NSG mice. Drug was given in feed at 0.013% (estimated ~ 20 mg/kg/day) and 0.004% (estimated ~ 6 mg/kg/day). Leukemia burden was assessed by IVIS imaging. Results: FLT3 ITD cell lines were highly sensitive to ONO-9330547. A dose of 10 nM drug induces apoptosis after 48 hours and 50 nM eliminates > 95% after 72 hours. MSC protect leukemia cells from ONO-9330547, but killing was still effective with higher doses. ONO-9330547 was effective arresting cell growth of Molm13 and MV4;11 cells. After 24 hours, 5 nM ONO-9330547 resulted in accumulation of cells in G1/G0. RPPA analysis of cells treated with the AXL inhibitor revealed suppression of known AXL targets (e.g. p-S6RP likely via AKT). RPPA also revealed novel targets including CDK1, p-RB, Cyclin B1, and PLK1. Immunoblot analysis also demonstrated that the drug suppressed expression of MCL-1 (predicted due to block of ERK and AKT signaling by the drug). Analysis of PLK1 gene expression by qRT-PCR revealed potent suppression of PLK1 mRNA by ONO-9330547. These findings suggest that inhibition of AXL results in block of CDK1 activity with concomitant loss of RB phosphorylation leading to suppression of PLK1 expression (presumably via RB association with E2F). This model is consistent with the observed effect of ONO-9330547 blocking cell entry into S phase. Finally, both low dose (~ 6 mg/kg) and high dose (~ 20 mg/kg) ONO-9330547 was effective reducing leukemia burden and significantly enhancing survival of mice bearing Molm13 leukemia cells. Interestingly, while mice receiving control feed displayed leukemia infiltrate in the liver, ONO-9330547 greatly reduced or prevented Molm13 cell infiltration of the liver. Conclusions: These results suggest that ONO-9330547 is effective killing AML cells with FLT3 ITD. The identification of p-RB and PLK1 as novel targets of the drug suggests for the first time that AXL regulates cell cycle in AML cells via CDK/RB/PLK1 axis. Finally, ONO-9330547 proved effective in in vivo AML xenograft models and the drug prevented AML infiltration of the liver. These results suggest that ONO-9330547 is a promising candidate for AML therapy particularly in AML patients with FLT3 ITD. Disclosures Yasuhiro: 3Ono Pharmaceutical Co. Ltd: Employment. Yoshizawa:3Ono Pharmaceutical Co. Ltd: Employment. Cortes:Ariad: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; Arog: Research Funding; Astellas: Research Funding; Ambit: Research Funding.

T cells are central players in adaptive immunity and T cell receptor (TCR) signaling and co-signaling govern immune defense, immune homeostasis and immune surveillance. Upon antigen encounter, the TCR becomes engaged and in concert with context-sensitive co-receptors, such as CD28, T cell activation, differentiation and function are achieved. Whereas TCR-dependent signaling has been subject to intense investigations, knowledge about CD28-dependent signal cascades is rather incomplete. In murine T cells, Rltpr was discovered to play an important role in CD28 co-signaling. We identified four patients from two consanguineous families presenting with generalized EBV-associated smooth muscle cell tumors and susceptibility to recurrent viral, bacterial, and fungal infections. All patients had homozygote loss-of-function mutations in RLTPR (c.489insG and c.871+1G>T respectively) and did not express RLTPR protein. Primary RLTPR-mutated T cells were characterized by deficiency to signal via CD28. In addition, the cells showed disturbed cytoskeletal microtubule network and defective migratory pattern with increased spontaneous motility in 2D and 3D, but reduced migratory persistence. To determine the protein interactome of human RLTPR, we designed CRISPR/Cas9 constructs and targeted the RLTPR locus in Jurkat cells. We derived five RLTPR-defective Jurkat clones, as determined by Western blot analysis and Sanger sequencing studies. Next, these clones were engineered via retroviral vectors to re-express a GFP-tagged human RLTPR protein. Jurkat T-cell clones expressing RLTPR with N-terminal-GFP, RLTPR with C-terminal-GFP or GFP only were stimulated with anti-CD3/CD28 or left unstimulated and lysed after 15 minutes. Upon pull-down using GFP-Trap, the RLTPR interactome was comprehensively investigated by nano liquid chromatography tandem mass spectrometry (LC-MS/MS) and analyzed using Maxquant. Confirmation by immunoprecipitation and western blotting was performed for selected proteins. In seven independent experiments, 903 different proteins were detected in total, 192 proteins repeatedly in at least half of the RLTPR-GFP samples. Quantitative analysis and statistical evaluation yielded 26 proteins significantly enriched in the RLTPR-GFP compared to the control samples (permutation-based q-values < 0.05). The interactome of RLTPR in genetically modified Jurkat cells primarily included cytoskeletal proteins such as capping proteins and (co)chaperones. In addition, RLTPR also interacted with a metabolic enzyme involved in nucleotide synthesis and with vesicle transport proteins involved in the initiation of TCR signaling. Known TCR or CD28 signaling molecules were not detected indicating an indirect contribution of RLTPR in these pathways. In summary, studying rare patients with EBV-associated smooth-muscle cell tumors has allowed us to define RLTPR-deficiency as a novel human primary immunodeficiency disorder and to define RLTPR as a central scaffolding molecule for CD28-mediated TCR co-signaling, cytoskeletal dynamics and metabolic signaling. Disclosures No relevant conflicts of interest to declare.

Presymptomatic central nervous system (CNS) treatment in children with a late isolated first bone marrow (BM) relapse of acute lymphoblastic leukemia (ALL) was based on intermediate-dose systemic and intrathecal (IT) methotrexate (MTX) in the multicenter trial, ALL-REZ BFM 85. Because this was associated with an excess of overt second CNS relapses, cranial radiotherapy (cRT) plus prolonged triple IT therapy with MTX, cytarabine, and prednisone was instituted during the course of the subsequent trial, ALL-REZ BFM 87. Results of children with or without cRT, but otherwise identical chemotherapy, are presented here. Between April 1985 and March 1990, 93 children with their first late isolated BM relapse of ALL were entered on protocols ALL-REZ BFM 85M and ALL-REZ BFM 87. An intensive 6-month phase of multiagent chemotherapy that included 8 courses of systemic MTX (1 g/m2) plus IT MTX was followed by 2 years of conventional maintenance therapy with daily 6-thioguanine and biweekly MTX. Children with bone marrow transplantation excluded, 73 were in complete remission at the end of intensive polychemotherapy, 40 of whom received fractionated cRT plus triple IT therapy during the following 6 months; 11 did not receive cRT but prolonged triple IT; 22 received neither cRT nor prolonged triple IT. Except for a higher percentage of children who had received cRT in front-line protocols (29 of 33 v 20 of 40), the patient groups without or with salvage cRT were comparable. Of 33 children without salvage cRT, 26 relapsed, compared with 21 of 40 who had received cRT (P < .05). The difference was solely attributable to second relapses with CNS involvement (10 of 33 v 1 of 40; P < .01). Estimated 6-year event- free survival rates were .18 for children without cRT and .46 for children with cRT (P < .01). In patients without cRT, no impact of prolonged IT therapy could be shown. The data suggest that second CNS prophylaxis with cRT and prolonged triple IT therapy in children with late isolated BM relapse of ALL is effective in preventing CNS relapses, in reducing the overall relapse rate, and in increasing the overall survival rate.

For older patients with early unfavorable or advanced stage Hodgkin lymphoma (HL) the prognosis is much worse than for younger HL patients. We thus developed a new regimen, BACOPP (bleomycin, adriamycin, cyclophosphamide, vincristine, procarbazine, and prednisone), to improve both tolerability and efficacy of treatment for older HL patients. Between 2004 and 2005, 65 patients with early unfavorable or advanced stage HL aged between 60 and 75 years were enrolled in this phase 2 trial. Treatment consisted of 6 to 8 cycles of BACOPP. Residual tumor masses were irradiated. Primary endpoints were feasibility as determined by adherence to protocol and overall response rate. Secondary endpoints included toxicity, freedom from treatment failure, and progression free and overall survival. For the final analysis 60 patients (92%) were eligible; 75% of treatment courses were administered according to protocol. World Health Organization grade 3/4 toxicities occurred in 52 patients. Fifty-one patients (85%) achieved complete remission, 2 (3%) partial remission, and 4 (7%) developed progressive disease. With a median observation time of 33 months, 18 patients died (30%), including 7 treatment-associated deaths. Three patients died before response assessment. Thus, the BACOPP regimen is active in older HL patients but is compromised by a high rate of toxic deaths. This trial was registered at www.clinicaltrials.gov as #NCT00284271.

Merocyanine 540 (MC 540) is a photoactive dye used to purge bone marrow of tumor cells in autologous bone marrow transplantation. The effects of MC 540 on the lymphoid components in the marrow are unknown. This study evaluates the treatment of lymphocytes by MC 540 (15 micrograms/mL) and light (70 W/m2) on: (1) phytohemagglutinin and Con A- induced proliferation; (2) allogeneic mixed lymphocyte cultures (MLC); (3) the regulation of Ig synthesis by T cells; and (4) the ability of B cells to produce polyclonal Igs as measured by an enzyme-linked immunosorbent assay-plaque assay. The results show that MC 540 and light treatment reduced Con A-stimulated T-cell proliferation greater than 50% after 30 minutes and greater than 80% after 60 minutes of MC 540-sensitized photoirradiation. Ninety minutes of MC 540 and light exposure (designated treatment) inhibited MLC greater than 90%. In polyclonal Ig synthesis, T-cell helper activity could be abrogated by 90 minutes of treatment in cocultures containing untreated B cells. Purified B cells treated for 90 minutes cocultured with normal T cells did not produce Ig. Treatment of B cells completely inhibited Epstein- Barr virus-stimulated Ig synthesis. These data show that T- and B-cell immunity is suppressed by the MC 540-sensitized photoirradiation. Treatment of bone marrow with MC 540 and light may have profound effects on immune reconstitution in autologous marrow graft recipients. More provocative is the fact that the same immunomodulatory effects may be applicable to partially mismatched marrow transplant situations as a means of reducing graft-versus-host reactions.

YE-SENSITIZED photoirradiation is a relatively reD cent addition to the arsenal of techniques used for the elimination of tumor cells from autologous bone marrow (BM) grafts.’ One photosensitizer, merocyanine 540 (MC 540), has been characterized extensively in preclinical and is now being used in a phase I clinical trial for extracorporeal purging of autologous marrow grafts in patients with acute lymphocytic or nonlymphocytic leukemia, Hodgkin’s or non-Hodgkin’s lymphoma, or stage IV neuroblastoma.’o~” The preclinical evaluation of MC 540 has shown that MC 540 has a low acute systemic toxicity” and low mutagenic potential.” It preferentially photosensitizes normal erythroid progenitor cells, leukemiabymphoma cell^,'^*^^-^ and neuroblastoma cell^.'^^ By contrast, pluripotent hematopoietic progenitor cells as measured by day 12 colony-forming unit-spleen (CFU-S) or CFU-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM) are quite resistant to MC 540-sensitized ph~toirradiation~.’~ and substantial reductions of tumor cell concentrations can be achieved without causing excessive damage to the graft’s capacity to rescue lethally irradiated host~.’~~~~~~’~ In all of the reported animal experiments, survival was the end-point and the exact kinetics of hematopoietic and lymphopoietic reconstitution were not determined. The kinetics of the hematopoietic and lymphopoietic recovery may be influenced by T cells and accessory cells (ie, cells other than the pluripotent hematopoietic stem cell) and it is conceivable that the damage done to T cells and accessory cells by a given purging procedure may affect clinical transplantation. This report addresses the effects of MC 540-sensitized photoirradiation on the functions of T and B cells. Specifically, we ask whether treatment with MC 540sensitized photoirradiation: (1) modulates T-cell proliferative responses to mitogens; (2) alters the ability of T cells to respond to alloantigens; (3) modulates T-cell helper activity; and (4) affects the ability of B cells to produce polyclonal Ig. In this study, we show that treatment with MC 540 and white light is a potent inhibitor of T- and greater than 90%. In polyclonal lg synthesis, T-cell helper activity could be abrogated by 90 minutes of treatment in cocultures containing untreated B cells. Purified B cells treated for 90 minutes cocultured with normal T cells did not produce Ig. Treatment of B cells completely inhibited EpsteinBarr virus-stimulated lg synthesis. These data show that Tand B-cell immunity is suppressed by the MC 540-sensitized photoirradiation. Treatment of bone marrow with MC 540 and light may have profound effects on immune reconstitution in autologous marrow graft recipients. More provocative is the fact that the same immunomodulatory effects may be applicable to partially mismatched marrow transplant situations as a means of reducing graft-versus-host reactions. 0 1991 by The American Society of Hematology. B-lymphocyte functions at doses of dye and light comparable with or lower than those used in clinical trials. MATERIALS AND METHODS Viability after dye-mediatedphotoiradiation (DMP). Viability of the lymphocytes used for these assays was checked before culture and at 2, 6, and 24 hours after MC 540 DMP using trypan blue exclusion. Peripheral blood mononuclear cells (PBMC) were purified by Ficoll-hypaque density gradient centrifugation (F/H) of whole heparinized blood. T (E+) and non-T (E-) subpopulations were separated by rosetting with 2-aminoethylisothiuronium bromide hydrobromide-modified sheep red blood cells (RBC) as described.” The T-cell populations were greater than 95% E+ cells and the non-T-cell populations contained less than 5% E+ cells by rosetting. Unless otherwise mentioned, the initial lymphocytes placed into culture were viable based on trypan blue exclusion after treatment. RPMI 1640 supplemented with penicillinstreptomycin, 4 mmol/L glutamine, and 10% fetal bovine serum (FBS; Hyclone, Logan, UT) was used for culture except for the mixed lymphocyte cultures. Human AI3 serum (10%) was used in the mixed lymphocyte cultures. ’H-thymidine incorporation after Lymphocyte separation.

Bcr-Abl tyrosine kinase supports CML cell survival in part by regulating antiapoptotic Bcl-2 proteins such as Bcl-xL and Mcl-1. Tyrosine kinase inhibition, the front-line therapy for patients with chronic phase CML, is less effective in blast crisis (BC) patients and inactive against quiescent CML stem/progenitor cells. We reported that ABT-737, a dual Bcl-2/Bcl-xL inhibitor, induces apoptosis in BC CML cells including CD34+quiescent CML cells. ABT-199, a potent Bcl-2 specific inhibitor, has entered clinical trials for various hematological malignancies. We hypothesized that cooperative targeting of antiapoptotic Bcl-2 proteins using a combination of ABT-199 and tyrosine kinase inhibitors (TKIs) would exert enhanced activity against BC CML and CML stem/progenitor cells. Cells from patients (n=4) with TKI-resistant BC CML were treated with ABT-199, TKIs, and combinations. Although exerting low activity by itself, ABT-199 in combination with TKIs synergistically induced apoptosis (CI To evaluate the effect of these combinations on TKI-insensitive quiescent stem/progenitor CML cells, BC CML patient cells were stained with the cell division-tracking dye carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured with human bone marrow (BM)-derived mesenchymal stromal cells (MSCs). Once proliferating and quiescent cells were distinguishable by flow cytometry, cells were treated with ABT-199, TKIs, and their combinations for 48 hours with or without MSC co-culture. Apoptosis was measured in proliferating and quiescent progenitor cells, defined as the percentage of annexin V positivity in CD34+CFSEdim and CD34+CFSEbright cells, respectively. ABT-199 as a single agent decreased viability of CML cells cultured alone or co-cultured with MSCs in both proliferating (IC50=191±103nM and 194±64nM, respectively) and quiescent (IC50=221±75nM and 205±123nM, respectively) CD34+ CML cells. Combinations of ABT-199 with TKIs, including imatinib, nilotinib, dasatinib, or ponatinib, synergistically induced death (CI To further test the ability of ABT-199 and TKI combinations to eradicate CML stem cells, we used an inducible transgenic CML mouse model in which the BCR-ABL gene is expressed under control of a tet-regulated enhancer of the murine stem cell leukemia (Scl) gene, allowing targeted BCR-ABL expression in stem/progenitor cells. Once BM cells from transgenic Scl-tTa-BCR-ABL/GFP mice were engrafted in wild type recipient mice, the mice were treated with ABT-199, nilotinib, or both. At the end of a 3-week treatment period, each single agent alone, and even more so with the combinations, significantly decreased blood total GFP+ WBC (12.9±1.4, 5.2±0.3, 6.1±0.4, and 1.6±0.3 x106/ml in controls, ABT-199, nilotinib, and combination, respectively) and neutrophils (1.43±0.03, 0.49±0.06, 0.32±0.03, and 0.25±0.05 x106/ml in the respective groups). ABT-199 (P=0.02), and more so with the combination (P Conclusions: ABT-199 and TKIs cooperatively target antiapoptotic Bcl-2 family proteins. This combination is highly effective in killing bulk and CD34+38- CML cells and quiescent CD34+ CML stem/progenitor cells from BC CML patients in vitro and in suppressing leukemia and leukemia stem cells in vivo. This strategy has the potential to eradicate BC CML cells and CML stem/progenitor cells, neither of which are effectively targeted by TKIs alone. Disclosures Carter: AbbVie, Inc.: Research Funding. Leverson:AbbVie, Inc.: Employment. Konopleva:AbbVie, Inc: clinic trial Other.

Post-transplant lymphoproliferative diseases (PTLD) are severe complications of immunosuppressive therapy after solid organ transplantation (SOT) in children. Among those, Hodgkin’s disease (HD) or Hodgkin(-like) PTLD is a rare entity, and systematic data on pathogenesis, treatment, and prognosis are lacking. Here, we report on 16 children and adolescents who were treated according to standard treatment guidelines from the German HD (GPOH-HD) study cente. All patients with HD after SOT reported to the German pediatric PTLD (Ped-PTLD) registry between 1999 and 2013 were included in this analysis (14 of 182 patients = 7.7% of pediatric PTLDs). Data were supplemented by information recorded in the GPOH-HD registry. In addition, 2 U.S. patients treated according to the respective GPOH-HD guidelines were included. Central pathology review was performed in 12/16 cases. Epstein-Barr virus (EBV) in tumor tissue was evaluated by EBER in situ hybridization, immunohistochemistry for LMP-1 or EBNA-2, and/or EBV-PCR. All patients were treated with chemotherapy according to the respective GPOH-HD recommendations (treatment group (TG)-1: 2xOEPA; TG-2: 2xOEPA+2xCOPP/COPDAC; TG-3: 2xOEPA+4xCOPP/COPDAC) tailored according to stage and LDH. Indication for involved field radiotherapy was based on the current study recommendations. Six patients had additional rituximab treatment, two as upfront monotherapy and four as addition to chemotherapy. Events recorded included disease progression, relapse, second malignancy, and death of any cause. Overall and event-free survival was calculated using log-rank test. 16 predominantly male (13/16) patients developed HD after SOT, among them 7 renal, 4 liver, and 5 heart transplant recipients. Median age at diagnosis of HD was 13.9 [range 4.3 – 19.4] years with a median duration from SOT to HD of 7.4 [0.75 – 14.3] years. Histopathology review was classified as classical Hodgkin’s disease in 10 patients (nodular sclerosis =1, mixed type =5, epitheloid-cell predominant =2, not further specified =2) and Hodgkin-PTLD in 6 patients. 8 of 10 patients with data evaluable had a negative EBV-serology at transplantation, while antibodies against EBV were detected in 15/15 patients at the time of HD diagnosis. Fifteen of sixteen tumors proved positive for EBV and/or EBV-gene products. As expected, all tumors expressed CD30 and 7 of 16 tumors demonstrated moderate to strong expression of CD20. Staging revealed 1 patient with stage I disease, 8 with stage II, 5 with stage III, and 2 patients with stage IV disease. Four patients presented with extranodal involvement, among them one with graft organ involvement, and three patients with involvement of the gastrointestinal tract. None of the patients had CNS disease. Patients were treated in TG-1 (6 patients), TG-2 (5 patients), TG-3 (4 patients), respectively. One patient received an individualized treatment scheme, and 2 of 16 patients (both TG-2) were assigned to consolidating involved field radiotherapy. Median follow up was 4.1 [0.3 – 9.2] years. One patient in TG-3 died of infection during COPDAC treatment; all others are alive at the time of abstract preparation. Overall survival (OS) at 2 and 5 years was 93%. In addition to the treatment-related death two patients experienced events, 1 relapse after 9 months and 1 secondary malignancy (polymorphic PTLD) after 1.2 years. Event-free survival after HD chemotherapy at 2 and 5 years was 79%. Rituximab monotherapy did not result in long-term remission, both patients relapsed after 0.4 and 2.7 years, respectively. None of the parameters examined (organ graft, stage, treatment group) was significantly associated with overall or event-free survival. In conclusion, HD is a rare type of PTLD in children arising late after SOT, which may lead to underrepresentation in the pediatric patient population. The analysis of 16 patients treated according to a uniform concept represents the largest series reported to date. Almost all cases were EBV-associated. Although tumors often express CD20 rituximab monotherapy does not seem to establish long-lasting remissions. In contrast, the chemotherapy regimens based on the German protocols for de novo Hodgkin’s disease present well tolerated and effective treatment strategies for HD after SOT in children. International efforts are warranted to improve understanding and treatment for this rare disease. Disclosures No relevant conflicts of interest to declare.

Background: Except for the ‘good risk’ groups identified by current cytogenetic and molecular criteria, patients with acute myelogenous leukemia (AML) largely relapse after attaining initial remissions, and outcomes of patients with early relapse is uniformly poor, highlighting the need for strategies to overcome resistance to agents used in frontline therapy. Autophagy is an ancestral adaptive mechanism to stress and can be triggered by exposure to chemotherapy. Autophagy is also believed to play an important role in tumor protection by the microenvironment. Autophagy inhibition increases chemosensitivity in several experimental models of solid tumors. The role of autophagy is not well studied in AML, but the hypoxic bone marrow niche is expected to induce autophagy in AML cells and render them chemo-resistant. Our reverse phase protein array (RPPA) analysis of over 500 newly diagnosed AML patient samples indicated that abnormal expression of several autophagy proteins in leukemic blasts (LKB1, BECLIN 1 and ATG7) is associated with poor prognosis in AML (Borthakur, G et al. ASH 2011# 2513), suggesting a therapeutic role for autophagy suppression. Atg7 is a key molecule in autophagy vesicle elongation with a role in two essential ubiquitin-like reactions (LC3 lipidation and Atg 5/12 conjugation), and its knockdown is expected to inhibit autophagy globally. Aim: To determine the effects of autophagy inhibition on chemosensitivity of AML cells and on stroma induced resistance. Results: We semi-quantitatively assessed ATG7 expression in AML cell lines (THP1, OCI-AML2 and 3, HL-60, MOLT-4, MOLM 13 and 16) by Western blot. HL-60 and MOLM13 cells had the lowest expression of Atg7 and were most sensitive to treatment with ara-C and idarubicin while OCI-AML3 and THP-1 had highest levels of Atg7 and were most resistant to treatment (p= .001-.004). In addition, high expression of Atg7 is associated with high levels of anti-apoptotic Mcl-1 in these lines. As available autophagy inhibitors are non-specific, we used lentiviral shRNA to knock down Atg7 and study the role of autophagy. Atg7 knockdown cells (ATG7-KD) were treated with ara-C and idarubicin for 48-96 hours. At all time points, apoptosis was significantly higher in ATG7-KD cells compared to cells transduced with a non-silencing scrambled control (ATG7-Scr) at 96 hrs: 39.6±2.4 vs 22.4±1.3, p=.002 for ara-C 2 µM and 47.9±2.6 vs 34.7±2.3, p=.0004 for idarubicin. We co-cultured OCI-AML3 (ATG7-KD and -Scr) cells with normal bone marrow derived mesenchymal stromal cells (MSCs) to mimic the bone marrow micro-environment. Sensitivity to ara-C and idarubicin was reduced by co-culture for both cell types, but ATG7-KD cells remained more sensitive than were ATG7-Scr cells. Western blot analysis confirmed increased p62 and decreased LC3 lipidation in ATG7-KD cells (LC3 II/I ratio= 0.15 vs 0.7 in ATG7-Scr), indicating a block in autophagy, and increased caspase 3 cleavage, indicating apoptosis. The higher expression of pro-apoptotic NOXA and BAX, and lower expression of anti-apoptotic MCL-1 and BCL-2, in ATG7-KD cells compare to ATG7-Scr indicating mitochondrial pathway apoptosis. Gene expression profiling on Illumina HT12 arrays was used to study OCI-AML3 ATG7-KD and -Scr cells at baseline and after 24 and 48 hr of araC 0.5 µM. Gene set enrichment analysis (GSEA) showed significantly lower baseline expression of interferon response genes and JAK-STAT pathway genes in OCI-AML3 ATG7-KD cells. Changes with araC treatment were largely similar, but the response to araC was different between ATG7-KD and –Scr cells for a small number of genes. In-vivo chemo-sensitivity experiments are in progress. Conclusion: Autophagy inhibition by genetic silencing of ATG7 increases chemosensitivity of AML cells, even in the presence of bone marrow stromal cells. ATG7-KD cells appear to be more primed for apoptosis compared to their controls. Concomitant inhibition of ATG7, a potentially drugable E1 ligase, appears to be a valid strategy to enhance sensitivity to front-line treatment agents in AML. Disclosures No relevant conflicts of interest to declare.

Galectins are a family of b-galactoside binding proteins with effects on cell adhesion, apoptosis, cell cycle, and mRNA processing. Galectin-3 (LGALS3) is unique among galectins by having an N terminal region of roughly 130 amino acids that allows for multimerization and binding to other proteins independent of carbohydrate binding. In addition to promoting BCL2 gene expression and mitochondrial integrity, LGALS3 (along with LGALS1) positively regulates RAS signaling and thus stabilizes survival proteins dependent on ERK phosphorylation such as MCL-1. The pro-survival functions of LGALS3 and other galectins suggest that their targeting could be therapeutic for cancers including AML. Indeed, LGALS3 expression is a predictor of poor prognosis in acute myeloid leukemia (AML), as reported by Cheng and colleagues (Blood 2013) for patients with non-M3 AML and CN-AML. The modified pectin GCS-100 (La Jolla Pharmaceutical, San Diego, CA), now in a Phase II clinical trial for chronic kidney disease, binds and blocks the function of LGALS3. We report that GCS-100 suppresses the growth of AML cell lines OCI-AML3, THP-1, and HL60 in vitro as a single agent, at doses under the 250 ug/mL (i.e., within clinically-achievable concentrations). Short-term treatment of cells (i.e., < 6 hr) potently suppressed phosphorylation of AKT and ERK and reduced expression of BCL2 and MCL-1. Because LGALS3 positively regulates anti-apoptotic BCL2 family members, the Raz group has suggested targeting galectins to enhance efficacy of BH3 mimetic drugs (Harazano et al Cancer Metastasis Review 2013). We found that GCS-100 potently synergized with ABT-737 to kill OCI-AML3 cells: while 1 uM ABT-737 or 125 ug/mL GCS-100 reduced total viable cells by ~ 30% and induced apoptosis in < 20% of cells after 48 hr as single agents, their combination at those doses and time point reduced viable cells by ~ 94% and induced apoptosis in ~ 70% of cells. Suppression of LGALS3 by lentiviral shRNA reduced BCL2 gene expression as determined by qRT-PCR and augmented killing with ABT-737. Lentiviral suppression of LGALS3 protected cells from GCS-100 at doses of 250 ug/mL but reduction of the galectin failed to protect cells from higher doses of the drug (i.e., 500 ug/mL). This result suggests other galectins are likely inhibited at higher doses of the agent. We used gene expression profiling (GEP) on Illumina HT12v4 human whole-genome arrays to assess more broadly the molecular effects of inhibiting galectins in AML cell lines OCI-AML3 and THP-1 treated with 250 ug/mL or 500 ug/ml GCS-100 for 24 hr. Data were analyzed by Gene Set Enrichment Analysis (GSEA) using gene sets from the Molecular Signatures Database (www.broadinstitute.org/gsea/msigdb/). GSEA suggested that GCS-100 promotes differentiation and inhibits genes associated with proliferation. Multiple upregulated gene sets suggest that there may be a release of a differentiation block as a result of GCS-100 treatment. Furthermore, two gene sets suggest that GCS-100 behaves similar to a GSK3 inhibitor: Known pathways regulated by GSK3 in hematopoietic stem cells are mTOR and Wnt/beta Catenin. Inhibition of Wnt/beta Catenin can release a differentiation block. Consistent with GCS-100 promoting cell differentiation, lentiviral shRNA reduced LGALS3 protein > 90% in THP-1 cells and increased CD11b expression, suggesting increased differentiation, compared to cells with control shRNA. GCS-100 was tested in an in vitro model of the bone marrow microenvironment using BM-derived mesenchymal stromal cell (MSC). MSC can protect leukemia cells from a variety of clinically relevant chemotherapy drugs including AraC. GCS-100 was effective at killing AML cells despite the presence of MSC. Both THP-1 and OCI-AML3 cells exhibited > 80% and > 60% reduction of viable cells, respectively, despite the presence of MSC when treated with 250 ug/mL GCS-100 for 72 hours. In addition, GCS-100 was found to block adhesion of OCI-AML3 cells to MSC suggesting that GCS-100 could be effective in mobilizing AML cells. In summary, our findings suggest that GCS-100 can induce apoptosis in AML cells as a single agent or in combination with the BH3 mimetic ABT-737. The agent is effective even in the presence of MSC suggesting it could be efficacious in the leukemia niche. These findings suggest GCS-100 could be effective for AML therapy. Disclosures Rolke: La Jolla Pharmaceutical Company: Employment. Tidmarsh:La Jolla Pharmaceutical Company: Employment.

The multidrug resistance 1 (MDR1) gene product P-glycoprotein (P-gp) is frequently implicated in cross-resistance of tumors to chemotherapeutic drugs. In contrast, acute promyelocytic leukemia (APL) cells do not express MDR1 and are highly sensitive to anthracyclines. The combination of ATRA and the novel histone deacetylase inhibitor (HDACI) depsipeptide (FK228) induced P-gp expression and prevented growth inhibition and apoptosis in NB4 APL cells subsequently exposed to doxorubicin (DOX). ATRA/FK228 treatment after exposure to DOX, however, enhanced apoptosis. Both agents, ATRA or FK228, induced MDR1 mRNA. This effect was significantly enhanced by ATRA/FK228 administered in combination, due in part to increased H4 and H3-Lys9 acetylation of the MDR1 promoter and recruitment of the nuclear transcription factor Y alpha (NFYA) transcription activator to the CCAAT box. Cotreatment with specific P-gp inhibitor PSC833 reversed cytoprotective effects of ATRA/FK228. G1 cell-cycle arrest and p21 mRNA induction were also observed in response to ATRA/FK228, which may restrict DOX-induced apoptosis of cells in G2 phase. These results indicate that epigenetic mechanisms involving NF-YA transcription factor recruitment and histone acetylation are activated by ATRA and HDACI, induce MDR1 in APL cells, and point to the critical importance of mechanism-based sequential therapy in future clinical trials that combine HDAC inhibitors, ATRA, and anthracyclines.

Survivin and XIAP, members of the protein family known as the inhibitors of apoptosis, interfere with the activation of caspases, called the “cell death executioners.” We examined Survivin (n = 116) and XIAP (n = 172) expression in primary acute myeloid leukemia (AML) blasts and assessed the impact of their expression on prognosis. They were detected in all samples analyzed. However, no correlation was observed with cytogenetics, remission attainment, or overall survival of patients with AML. To investigate the importance of caspases in chemotherapy-induced apoptosis in AML, we treated OCI-AML3 cells with Ara-C, doxorubicin, vincristine, and paclitaxel, which induced caspase cleavage and apoptosis. Blocking of caspase activation by pan-caspase inhibitor abolished poly(adenosine diphosphate [ADP]-ribose) polymerase cleavage and DNA fragmentation but did not prevent chemotherapy-induced cell death and did not inhibit, or only partially inhibited, mitochondrial release of cytochrome c, Smac, apoptosis-inducing factor (AIF), or loss of mitochondrial membrane potential. Caspase inhibition also did not protect AML blasts from chemotherapy-induced cell death in vitro. These results suggest that expression levels of Survivin or XIAP have no prognostic impact in AML patients. Although anticancer drugs induced caspase cleavage and apoptosis, cell killing was caspase independent. This may partially explain the lack of prognostic impact of XIAP and Survivin and may suggest caspase-independent mechanisms of cell death in AML. (Blood. 2003;102:4179-4186)

Recent studies suggest that the Bcl-2 and mitogen-activated protein kinase (MAPK) pathways together confer an aggressive, apoptosis-resistant phenotype on acute myelogenous leukemia (AML) cells. In this study, we analyzed the effects of simultaneous inhibition of these 2 pathways. In AML cell lines with constitutively activated MAPK, MAPK kinase (MEK) blockade by PD184352 strikingly potentiated the apoptosis induced by the small-molecule Bcl-2 inhibitor HA14-1 or by Bcl-2 antisense oligonucleotides. Isobologram analysis confirmed the synergistic nature of this interaction. Moreover, MEK blockade overcame Bcl-2 overexpression-mediated resistance to the proapoptotic effects of HA14-1. Most importantly, simultaneous exposure to PD184352 significantly (P = .01) potentiated HA14-1–mediated inhibition of clonogenic growth in all primary AML samples tested. These findings show that the Bcl-2 and MAPK pathways are relevant molecular targets in AML and that their concurrent inhibition could be developed into a new therapeutic strategy for this disease.