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Chimeric antigen receptor (CAR)–modified T-cell therapies targeting CD19 represent a new treatment option for patients with relapsed/refractory (R/R) B-cell malignancies. However, CAR T-cell therapy fails to elicit durable responses in a significant fraction of patients. Limited in vivo proliferation and survival of infused CAR T cells are key causes of failure. In a phase 1/2 clinical trial of CD19 CAR T cells for B-cell malignancies (#NCT01865617), low serum interleukin 15 (IL-15) concentration after CAR T-cell infusion was associated with inferior CAR T-cell kinetics. IL-15 supports T-cell proliferation and survival, and therefore, supplementation with IL-15 may enhance CAR T-cell therapy. However, the clinical use of native IL-15 is challenging because of its unfavorable pharmacokinetic (PK) and toxicity. NKTR-255 is a polymer-conjugated IL-15 that engages the entire IL-15 receptor complex (IL-15Rα/IL-2Rβγ) and exhibits reduced clearance, providing sustained pharmacodynamic (PD) responses. We investigated the PK and immune cell PDs in nonhuman primates treated with NKTR-255 and found that NKTR-255 enhanced the in vivo proliferation of T cells and natural killer cells. In vitro, NKTR-255 induced dose-dependent proliferation and accumulation of human CD19 CAR T cells, especially at low target cell abundance. In vivo studies in lymphoma-bearing immunodeficient mice demonstrated enhanced antitumor efficacy of human CD19 CAR T cells. In contrast to mice treated with CAR T cells alone, those that received CAR T cells and NKTR-255 had markedly higher CAR T-cell counts in the blood and marrow that were sustained after tumor clearance, without evidence of persistent proliferation or ongoing activation/exhaustion as assessed by Ki-67 and inhibitory receptor coexpression. These data support an ongoing phase 1 clinical trial of combined therapy with CD19 CAR T cells and NKTR-255 for R/R B-cell malignancies.

NKTR-255 is an investigational polyethylene glycol-modified recombinant human IL-15 (rhIL-15) receptor agonist, designed to improve the immunotherapeutic and anti-cancer benefit observed with rhIL-15 while circumventing the toxicities associated with this therapy. In preclinical studies, NKTR-255 has demonstrated enhanced proliferation and function of CD8Lay abstract Interleukin-15 (IL-15) is a protein that helps the body's natural immune system to defend itself against infections and diseases like cancer. This article discusses a clinical trial in patients with multiple myeloma or non-Hodgkin's lymphoma that evaluates a new investigational medicine, NKTR-255, a polymer-modified form of IL-15 that has been engineered to improve its ability to provide a sustained anti-tumor immune response. The trial will explore different doses of NKTR-255 to determine patient side effects and to find the highest acceptable dose that patients can tolerate. Based on this, a dose will be chosen that offers an optimal balance between having a positive anti-cancer effect and minimizing side effects. This dose will be tested further in patients who have had different treatments in the past. If the side effects are acceptable, this dose will be tested in a new trial in a large number of patients.

BackgroundNKTR-255 is an investigational IL-15Rα-dependent, polymer-conjugated, recombinant human IL-15 agonist designed to provide sustained pharmacodynamic (PD) responses without the need for daily dosing. NKTR-255 engages all IL-15 receptor binding complexes to expand, proliferate and activate natural killer (NK) and CD8+ T-cells. This Phase 1b/2 trial (NCT04616196) evaluates NKTR-255+cetuximab in highly refractory patients with head-and-neck squamous cell carcinoma (HNSCC) or colorectal cancer (CRC).MethodsSuccessive cohorts received escalating doses of NKTR-255 (q3w) +cetuximab (250mg/m< sup >2 weekly), 1 week after a loading dose of cetuximab alone. Safety (CTCAEv5.0; MTD/recommended Phase 2 dose [RP2D]) and efficacy (RECISTv1.1) were measured. PK/PD analyses were conducted, including assessment by flow cytometry/plasma cytokine analysis. Fold-change was calculated as treatment over baseline for NKTR-255 (baseline=1).ResultsAs of August 15, 2021, 12 patients had received ≥1 dose of NKTR-255+cetuximab; (37–70 years; 92% male; HNSCC n=4, CRC n=8; NKTR-255 1.5µg/kg n=7, NKTR-255 3.0µg/kg n=5). Patients had received a median 3.5 lines of prior therapy for metastatic disease. 11 patients had no response to the most recent prior therapy. Of the 12 patients, seven remain on treatment, with five not yet reaching first scan. RP2D has not yet been reached; dose escalation is ongoing.10 patients experienced an AE; one G5 AE occurred (due to progression). Seven patients reported NKTR-255-related AEs (all G1-2, except one G3 [which resolved in 24 hours]). Any-cause AEs in ≥20% were acneiform dermatitis, fatigue, and infusion-related reaction.Treatment-induced transient changes in inflammatory cytokines, including IFNy, MCP-1 and IL-6, at 1.5µg/kg (n=3) peaked 4 hours post-infusion and resolved by 24-48 hours. Mean T1/2 of NKTR-255 (1.5µg/kg dose, first cycle) was 27.8 hours.Dose-dependent expansion of NK and CD8+ T-cells was observed in peripheral blood. For NK cells, mean peak fold-change was ~4-fold and ~6-fold, and for CD8+ T-cells was ~2-fold and ~3-fold (1.5µg/kg and 3µg/kg dose levels, respectively). NK and CD8+ T-cells demonstrated increased Ki67+ proliferative ability.As of August 15, four patients in the 1.5µg/kg NKTR-255 dose cohort were response-evaluable: one CRC patient (4 prior metastatic treatment lines) reported a confirmed PR (–52%) after 3 cycles; two HNSCC patients reported SD.ConclusionsNKTR-255 was biologically active and led to expansion and proliferation of NK and CD8+ T-cells. Early dose-escalation data suggest that NKTR-255+cetuximab is safe and tolerable with preliminary anti-tumor activity. Updated data will be presented. NKTR-255, alone and in combination with daratumumab and rituximab, is also being evaluated in liquid tumors.AcknowledgementsThe authors thank the patients and their families involved in the trial.Trial RegistrationNCT04616196Ethics ApprovalThe study was approved by site IRBs.ConsentN/A

Background NKTR-255, an investigational IL-15Rα-dependent polymer-conjugated recombinant human IL-15 (rhIL-15) agonist, maintains the full spectrum of IL-15 biology and provides sustained pharmacodynamic (PD) responses without the need for daily dosing. NKTR-255 engages IL-15Rα and IL-2/IL-15Rβγ leading to natural killer(NK) and CD8+ T-cell expansion, proliferation and activation. In preclinical studies, NKTR-255 enhanced antibody-dependent cellular cytotoxicity(ADCC) of each of daratumumab, rituximab, trastuzumab and cetuximab, resulting in synergistic anticancer activity. This ongoing phase 1 trial(NCT04136756) evaluates NKTR-255 in patients with hematologic malignancies. Methods Heavily pretreated patients with relapsed/refractory multiple myeloma(MM) or non-Hodgkin lymphoma(NHL) received escalating doses of NKTR-255 intravenously q3w. Patients were observed for 3 weeks following the first NKTR-255 dose for dose-limiting toxicity(DLT). Preliminary safety, PK and PD were evaluated in all patients and bone marrow biopsy was evaluated in one patient. NKTR-255-mediated activation of the immune system was assessed by flow cytometry and plasma cytokine analysis. Results As of June 25, 2020, 4 patients were dosed(1.5µg/kg:3 patients; 3µg/kg:1 patient). NKTR-255 was well tolerated. Most common treatment-related adverse events(AEs): flu-like symptoms, muscle stiffness, and myalgia. One Grade 3 event(pyrexia) was reported, resolving 24 hours with no accumulation following repeat dosing. NKTR-255 at 1.5µg/kg expanded NK and CD8+ T-cells in peripheral blood, with peak fold-changes in cell numbers of ~5-fold and ~3-fold respectively, maintained during the cycle. Bone marrow biopsy data indicate 7-fold(cycle 5) to 13-fold(cycle 9) increase from baseline in the CD56+ population in the MM patient. Proliferative capacity for NK and CD8+ T-cells was maintained across multiple treatment cycles. NKTR-255-dependent changes in inflammatory cytokines, including MCP-1 and IL-6, peaked by 6 hours and resolved to baseline levels by 24 hours, further supporting the safety of NKTR-255. Conclusions In heavily pretreated patients with hematologic malignancies, NKTR-255 is biologically active and demonstrated sustained increases in NK and CD8+ T-cells. NKTR-255 was well tolerated, with minimal treatment-related toxicities. Our preclinical and preliminary clinical data provide evidence of synergistic effects enhancing ADCC and support continued dose escalation of NKTR-255. Trial Registration NCT04136756 Ethics Approval The study was approved by the institutional review board of each participating site.

Background NKTR-262 is a small-molecule agonist of toll-like receptors (TLR) 7/8. Given by intratumoral (IT) injection, NKTR-262 is retained within the tumor microenvironment (TME) and promotes an immunostimulatory milieu and tumor antigen release. Bempegaldesleukin (BEMPEG) is a CD122-preferential IL-2 pathway agonist, which increases proliferation and tumor infiltration of CD8+ T cells and natural killer (NK) cells. Preclinically, NKTR-262 plus BEMPEG combined innate immune signaling and enhanced antigen presentation, with sustained T-cell activation, resulting in tumor growth inhibition of treated and abscopal lesions. Methods This phase 1 dose-escalation study enrolled patients with relapsed/refractory, advanced/metastatic solid tumors (REVEAL; NCT03435640). Patients received escalating doses of NKTR-262 (0.03 mg to 3.84 mg IT) followed 3 weeks‘ later by BEMPEG (0.006 mg/kg IV) q3wk utilizing a 3+3 design. The primary endpoint was safety and tolerability, including definition of the recommended phase 2 dose (RP2D). Other endpoints included antitumor activity, pharmacodynamics, and pharmacokinetics. Results As of June 15, 2020, 36 patients were enrolled. One dose-limiting toxicity, transient transaminase elevation, was observed at the highest NKTR-262 dose (3.84 mg). The most frequent treatment-related adverse events were flu-like symptoms, fatigue, nausea, and pruritus, consistent with the known profile of BEMPEG. Early evidence of clinical activity was observed in patients with metastatic melanoma, with a disease control rate (partial response [PR] + stable disease) of 41.2% (7/17 patients), including two patients with PRs after progression on two prior immunotherapy regimens. Preliminary analyses showed dose-dependent induction of CXCL10 and type 1 interferon genes, consistent with TLR7/8 engagement. CD11c+ target cells were significantly more abundant in baseline melanoma biopsies than other tumor types (p Conclusions NKTR-262 plus BEMPEG led to engagement of the entire immune activation cascade required for systemic tumor clearance. Robust TLR7/8 engagement supported the NKTR-262 mechanism of action, while the minimal toxicity profile underscored the benefit of local delivery of NKTR-262, and the BEMPEG combination induced systemic activation of T and NK cells. These data support the RP2D of NKTR-262 (3.84 mg IT) plus BEMPEG (0.006 mg IV) q3w, and the initiation of the phase 1b dose-expansion phase, which is exploring concurrent dosing, with or without nivolumab, in relapsed/refractory metastatic melanoma patients. Trial Registration NCT03435640 Ethics Approval The study was approved by the institutional review board of each participating site.

Background: Autologous T cells engineered to express a CD19 or BCMA-specific chimeric antigen receptor (CAR) have shown high overall response rates in treatment-refractory B-cell non-Hodgkin lymphoma (NHL) and BCMA + multiple myeloma (MM), respectively. However, most patients will eventually relapse, and thus strategies are needed to further improve the efficacy and durability of CAR-T cell products. NKTR-255 is an investigational polyethylene glycol-modified recombinant human IL-15 (rhIL-15) receptor agonist designed to engage the IL-15 pathway to stimulate and expand natural killer (NK) cells and promote the survival and expansion of memory CD8 + T cells. Preclinical data in B-cell lymphoma xenograft models have shown that NKTR-255 enhanced expansion, survival, and anti-tumor activity of human CD19 CAR-T cells. Furthermore, clinical studies have validated that IL-15 augments anti-tumor activity and promotes a stem-cell memory subset in tumor-specific T cells. Here we report pharmacodynamic (PD) analysis of CAR-T cells from relapsed/refractory (R/R) NHL or MM patients who had prior CAR-T therapy and were subsequently enrolled in an ongoing Phase 1 study of NKTR-255. Methods: Patients with R/R NHL or MM who progressed following prior CAR-T therapy were enrolled in a Phase 1 open-label trial evaluating safety, tolerability, and PD after treatment with NKTR-255 (NCT04136756). Patients received single-agent NKTR-255 IV administered every 3 weeks. Peripheral blood (PB) mononuclear cell samples were obtained before and at intervals following NKTR-255 infusion. CAR-T cells were identified by flow cytometry (FCM) using proprietary reagents to detect the CD19-CAR and BCMA-CAR in combination with antibodies to identify CD3 +, CD4 +, and CD8 + T cells. PD data were analyzed for patients with measurable CAR-T cells at baseline; fold change was calculated as treatment with NKTR-255 over baseline (baseline=1). Results: Patient characteristics are shown in Table 1. A total of 6 patients with prior CAR-T/CAR-NK treatment were evaluated for T/ CAR-T cell counts and Ki67 expression to assess proliferation before and after NKTR-255 administration. A total of 3 patients who received NKTR-255, at 1.5 µg/kg (n=1), 4.5 µg/kg (n=1), and 6.0 µg/kg (n=1), had detectable CAR-T cells at baseline. Following NKTR-255 administration, CD3+ CAR-T cell numbers in PB demonstrated a peak average increase of ~1.7-fold (~70% increase) compared to baseline (range 1.4 to 1.8-fold); Table 1. Three patients had low CAR-T/CAR-NK cell counts at baseline and/or post-NKTR-255 treatment. NKTR-255 treatment led to a reversal of the CD4 +:CD8 + CAR-T ratio in 1 patient, with a ~2-fold increase in CD8 + compared with CD4 + CAR-T cells. FCM in PBMC demonstrated an average of ~1.6-fold increase in total CD8 + T-cell with an average 9-fold increase in the percentage of Ki67 +CD8 + T cells in all CAR-T patients following 1 dose of NKTR-255. Among the 6 patients with prior CAR-T/CAR-NK therapy, NKTR-255 was generally well tolerated, with no treatment-related AEs leading to discontinuation, change of dose, or death. Conclusions: Following treatment with NKTR-255, there was an increase in CAR-T-cells as well as a reversal of the CD4 +:CD8 + ratio in 1 patient. Additionally, NKTR-255 induced expansion of total CD8 + cell fraction with an increase of proliferation index of Ki67 in all reported patients, supporting its role in enhancing the expansion and proliferative ability of cytotoxic T cells. Low baseline CAR-T/CAR-NK cell counts observed in patients may be due to longer time intervals between CAR-T infusion and the first dose of NKTR-255. Although preliminary, these data provide promising evidence of CAR-T cell rescue with NKTR-255 administration and represent a potentially novel means of CAR-T augmentation through enhancement of CD8 + T-cells and supports the further evaluation of NKTR-255 and CAR-T therapy as a potential strategy to enhancing the persistence of CAR-T therapy. Additional CAR-T cell patients are being evaluated following NKTR-255 treatment. Ethics approval The study was approved by the institutional review board of each participating site. Trial registration ClinicalTrials.gov NCT04136756 Figure 1 Figure 1. Disclosures Turtle: Century Therapeutics: Consultancy, Other: Scientific Advisory Board; Precision Biosciences: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Amgen: Consultancy; AstraZeneca: Consultancy, Research Funding; TCR2 Therapeutics: Research Funding; Allogene: Consultancy; PACT Pharma: Consultancy; Arsenal Bio: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Myeloid Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Asher Bio: Consultancy; Caribou Biosciences: Consultancy, Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Eureka Therapeutics: Current holder of stock options in a privately-held company, Other: Scientific Advisory Board; Juno Therapeutics/BMS: Patents & Royalties: Right to receive royalties from Fred Hutch for patents licensed to Juno Therapeutics, Research Funding; T-CURX: Other: Scientific Advisory Board; Nektar Therapeutics: Consultancy, Research Funding. Budde: Merck, Inc: Research Funding; Amgen: Research Funding; Astra Zeneca: Research Funding; Mustang Bio: Research Funding; Novartis: Consultancy; Gilead: Consultancy; Roche: Consultancy; Beigene: Consultancy. Patel: Pfizer: Consultancy; Janssen: Consultancy, Research Funding; BMS Celgene: Consultancy, Research Funding; Oncopeptides: Consultancy. Perales: Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Cidara: Honoraria; Equilium: Honoraria; Incyte: Honoraria, Other; Karyopharm: Honoraria; Kite/Gilead: Honoraria, Other; Medigene: Honoraria; Merck: Honoraria; Miltenyi Biotec: Honoraria, Other; MorphoSys: Honoraria; Nektar Therapeutics: Honoraria, Other; NexImmune: Honoraria; Novartis: Honoraria, Other; Omeros: Honoraria; Sellas Life Sciences: Honoraria; Servier: Honoraria; Takeda: Honoraria. Cowan: GSK: Consultancy; Bristol Myers Squibb: Research Funding; Nektar: Research Funding; Cellectar: Consultancy; Abbvie: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Harpoon: Research Funding; Sanofi Aventis: Consultancy, Research Funding; Secura Bio: Consultancy. Saeed: Bristol-Myers Squibb Company: Consultancy; sano-aventis U.S.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceutica Products, LP: Consultancy, Other: investigator; Celgene Corporation: Consultancy, Other: investigator; MEI Pharma Inc: Consultancy, Other: investigator; Kite Pharma: Consultancy, Other: investigator; Other-TG therapeutics: Consultancy, Other: investigator; Nektar Therapeutics: Consultancy, Other: research investigator; MorphoSys AG: Consultancy, Membership on an entity's Board of Directors or advisory committees; Other-Epizyme, Inc.: Consultancy; Other-Secura Bio, Inc.: Consultancy; Seattle Genetics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Chavez: Kite/Gilead: Consultancy; Abbvie: Consultancy; Epizyme: Speakers Bureau; Beigene: Speakers Bureau; ADC Therapeutics: Consultancy, Research Funding; Adaptive Biotech: Research Funding; Novartis: Consultancy; Astra Zeneca: Research Funding; Morphosys: Speakers Bureau; karyopharm: Consultancy; Merck: Research Funding. Hirayama: Bristol myers Squibb: Honoraria; Novartis: Honoraria. Janakiram: BMS: Honoraria; Fate: Research Funding; Nektar Therapeutics: Research Funding. Fong: Dendreon: Research Funding; Janssen: Research Funding; Merck: Research Funding; Roche genentech: Research Funding; BMS: Research Funding; Abbvie: Research Funding; Bavarian Nordic: Research Funding. Lee: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Dixit: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Wang: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Kai: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Marcondes: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Zalevsky: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Shah: Amgen: Consultancy; Janssen: Research Funding; Indapta Therapeutics: Consultancy; Precision Biosciences: Research Funding; Poseida: Research Funding; Nektar: Research Funding; Karyopharm: Consultancy; Sanofi: Consultancy; GSK: Consultancy; CSL Behring: Consultancy; CareDx: Consultancy; BMS/Celgene: Research Funding; Kite: Consultancy; Oncopeptides: Consultancy; Bluebird Bio: Research Funding; Sutro Biopharma: Research Funding; Teneobio: Research Funding.

Introduction: NKTR-255, an investigational novel polymer-conjugated recombinant human interleukin (IL)-15 agonist, maintains the full spectrum of IL-15 biology and provides sustained pharmacodynamic (PD) responses without the need for daily dosing. NKTR-255 engages IL-15Rα and IL-2/IL-15Rβγ leading to natural killer (NK) and CD8 + T-cell expansion, proliferation, and activation. In preclinical studies, NKTR-255 enhanced antibody-dependent cellular cytotoxicity (ADCC) of each of daratumumab, rituximab, trastuzumab, and cetuximab, resulting in synergistic anticancer activity. This ongoing Phase 1 trial (NCT04136756) evaluates the safety, tolerability, and pharmacokinetic (PK)/PD of NKTR-255 in patients with hematologic malignancies, including determination of the recommended Phase 2 dose (RP2D). Methods: Heavily pretreated patients with relapsed/refractory multiple myeloma (MM) or non-Hodgkin lymphoma (NHL) received escalating doses of NKTR-255 intravenously every 3 weeks. Patients were observed following the first NKTR-255 dose for dose-limiting toxicity. Preliminary PK and PD analyses were conducted. NKTR-255-mediated activation of the immune system was assessed by flow cytometry and plasma cytokine analysis. Fold change was calculated as treatment with NKTR-255 over baseline (baseline = 1). Results: As of June 8, 2021, 14 patients were enrolled, ranging in age from 49 to 80 years; 71% male. Eight (57%) had MM and 6 (43%) had NHL. Two (25%) of 8 patients with MM and 2 (33%) of 6 patients with NHL had received prior chimeric antigen receptor T-cell (CAR-T) therapy. The 14 patients were dosed at 4 levels: (1.5 µg/kg: 3 patients; 3.0 µg/kg: 4 patients; 4.5 µg/kg: 4 patients; 6.0 µg/kg: 3 patients). Among the 11 response-evaluable patients, 7 patients (64%) reported disease stabilization (4/5 [80%] MM patients; 3/6 [50%] NHL patients). Treatment-related adverse events occurring in ≥30% of patients were pyrexia (12/14; 86%), chills (10/14; 71%), nausea (8/14; 57%), headache (6/14; 43%), infusion-related reaction (5/14; 36%), and fatigue (5/14; 36%). Most adverse events were transient and resolved spontaneously, or by using standard treatment protocols. No dose-limiting toxicities were observed, and no patients discontinued NKTR-255 due to adverse events. NKTR-255- induced dose dependent, transient changes in inflammatory cytokines, including interferon-γ, monocyte chemoattractant protein-1 and IL-6 peaked at 3 to 6 hours post-infusion and resolved to baseline levels by 24 to 48 hours, supporting the safety profile of NKTR-255. Preliminary PK analyses showed target mediate disposition at the lowest dose level (1.5 µg/kg) and linear PK toward higher dose levels (>3.0 µg/kg). The average half-life of NKTR-255 was ~39 hours. No accumulation was observed following repeat dosing. There was a dose-dependent expansion in cell numbers of NK and CD8+ T cells in the peripheral blood of patients, with peak fold-changes of ~8-fold and ~2-fold respectively, within the first 2 cycles of NKTR-255 treatment at the 6.0 µg/kg dose. Consistent with NKTR-255 mechanism of action, minimal induction of Tregs was observed at all dose levels tested. Moreover, NK and CD8 + T cells demonstrated proliferative ability, which was maintained across multiple treatment cycles at all dose levels. Conclusions: In this heavily pretreated relapsed/refractory patient population with hematologic malignancies, NKTR-255 was biologically active, and demonstrated sustained increases in NK and CD8 + T cells. NKTR-255 was well tolerated with minimal treatment-related toxicities and transient upregulation and rapid decline of cytokines to baseline levels. RP2D for NKTR-255 monotherapy has not yet been reached; and dose escalation is ongoing at 9.0 µg/kg. NKTR-255 is currently being evaluated in multiple clinical studies in both hematologic malignancies and solid tumors as a monotherapy and in combination with agents that induce ADCC. Ethics approval: The study was approved by the institutional review board of each participating site. Trial registration: ClinicalTrials.gov NCT04136756 Disclosures Shah: Sanofi: Consultancy; Precision Biosciences: Research Funding; Nektar: Research Funding; Karyopharm: Consultancy; Indapta Therapeutics: Consultancy; CareDx: Consultancy; Kite: Consultancy; BMS/Celgene: Research Funding; Bluebird Bio: Research Funding; Amgen: Consultancy; CSL Behring: Consultancy; GSK: Consultancy; Janssen: Research Funding; Oncopeptides: Consultancy; Sutro Biopharma: Research Funding; Poseida: Research Funding; Teneobio: Research Funding. Budde: Merck, Inc: Research Funding; Amgen: Research Funding; Astra Zeneca: Research Funding; Mustang Bio: Research Funding; Novartis: Consultancy; Gilead: Consultancy; Roche: Consultancy; Beigene: Consultancy. Hofmeister: Sanofi: Other: National PI for CST; PI or co-PI IST; BMS/Celgene: Other: National PI for CST; PI or co-PI IST; Local PI of CST; Nektar Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Local PI of CST; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Local PI of CST; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Other: Local PI of CST; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Oncolytics: Other: National PI for CST; PI or co-PI IST; Takeda: Other: Local PI of CST; Genzyme: Membership on an entity's Board of Directors or advisory committees; Myeloma360: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; BioAscend: Other: CME speaker; Philips Gilmore: Other: CME speaker; Non-pharma speaker for education, research, marketing; BMS: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Non-CME speaker; BlueBird Bio: Other: Non-CME speaker; Aptitude Health: Other: Non-pharma speaker for education, research, marketing; Verascity: Other: Non-pharma speaker for education, research, marketing; TRM Oncology: Other: Non-pharma speaker for education, research, marketing; DAVA Oncology: Other: Non-pharma speaker for education, research, marketing; Medscape: Other: Non-pharma speaker for education, research, marketing; Ohio State University: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: IP rights, Patents & Royalties. Cowan: Bristol Myers Squibb: Research Funding; GSK: Consultancy; Abbvie: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Cellectar: Consultancy; Harpoon: Research Funding; Secura Bio: Consultancy; Nektar: Research Funding; Sanofi Aventis: Consultancy, Research Funding. Perales: Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Cidara: Honoraria; Equilium: Honoraria; Incyte: Honoraria, Other; Karyopharm: Honoraria; Kite/Gilead: Honoraria, Other; Medigene: Honoraria; Merck: Honoraria; Miltenyi Biotec: Honoraria, Other; MorphoSys: Honoraria; Nektar Therapeutics: Honoraria, Other; NexImmune: Honoraria; Novartis: Honoraria, Other; Omeros: Honoraria; Sellas Life Sciences: Honoraria; Servier: Honoraria; Takeda: Honoraria. Choi: Janssen Biotech: Speakers Bureau. Saeed: MorphoSys AG: Consultancy, Membership on an entity's Board of Directors or advisory committees; Nektar Therapeutics: Consultancy, Other: research investigator; Other-TG therapeutics: Consultancy, Other: investigator; Kite Pharma: Consultancy, Other: investigator; MEI Pharma Inc: Consultancy, Other: investigator; Celgene Corporation: Consultancy, Other: investigator; Janssen Pharmaceutica Products, LP: Consultancy, Other: investigator; sano-aventis U.S.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb Company: Consultancy; Other-Epizyme, Inc.: Consultancy; Other-Secura Bio, Inc.: Consultancy; Seattle Genetics, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Chavez: AbbVie: Consultancy; Adaptive: Research Funding; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Research Funding; BeiGene: Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Epizyme: Speakers Bureau; Karyopharm: Consultancy; Kite/Gilead: Consultancy; Merck: Research Funding; MorphoSys: Speakers Bureau; Novartis: Consultancy. Ye: Alexion, AstraZeneca Rare Disease: Other: Study investigator. Cairo: Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Speakers Bureau; Sanofi: Speakers Bureau; Servier: Speakers Bureau; Sobi: Speakers Bureau; Omeros: Membership on an entity's Board of Directors or advisory committees; Nektar: Membership on an entity's Board of Directors or advisory committees. Rizzieri: Celltron/Teva: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: presentation to FDA for biosimilar review ; Mustang: Consultancy, Honoraria; Bayer: Consultancy, Honoraria; Stemline Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Jazz: Other: personal fee; Gilead: Other: personal fee; Incyte: Other: personal fee; Amgen: Other: personal fee; Kite: Other: personal fee; AROG: Other; Pharmacyclics: Other. Lee: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Dixit: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Nieves: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Zhao: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Wang: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Kai: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Marcondes: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Tagliaferri: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Zalevsky: Nektar Therapeutics: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Patel: Pfizer: Consultancy; Janssen: Consultancy, Research Funding; BMS Celgene: Consultancy, Research Funding; Oncopeptides: Consultancy.

Background CD19 CAR T immunotherapy has been successful in achieving durable remissions in some patients with relapsed/refractory B cell lymphomas, but disease progression and loss of CAR T cell persistence remains problematic. Interleukin 15 (IL-15) is known to support T cell proliferation and survival, and therefore may enhance CAR T cell efficacy, however, utilizing native IL-15 is challenging due to its short half-life and poor tolerability in the clinical setting. NKTR-255 is a polymer-conjugated IL-15 that retains binding affinity to IL15Rα and exhibits reduced clearance, providing sustained pharmacodynamic responses. We investigated the effects of NKTR-255 on human CD19 CAR T cells both in vitro and in an in vivo xenogeneic B cell lymphoma model and found improved survival of lymphoma bearing mice receiving NKTR-255 and CAR T cells compared to CAR T cells alone. Here, we extend upon these findings to further characterize CAR T cells in vivo and examine potential mechanisms underlying improved anti-tumor efficacy. Methods CD19 CAR T cells incorporating 4-1BB co-stimulation were generated from CD8 and CD4 T cells isolated from healthy donors. For in vitro studies, CAR T cells were incubated with NKTR-255 or native IL-15 with and without CD19 antigen. STAT5 phosphorylation, CAR T cell phenotype and CFSE dilution were assessed by flow cytometry and cytokine production by Luminex. For in vivo studies, NSG mice received 5x105 Raji lymphoma cells IV on day (D)-7 and a subtherapeutic dose (0.8x106) of CAR T cells (1:1 CD4:CD8) on D0. To determine optimal start date of NKTR-255, mice were treated weekly starting on D-1, 7, or 14 post CAR T cell infusion. Tumors were assessed by bioluminescence imaging. Tumor-free mice were re-challenged with Raji cells. For necropsy studies mice received NKTR-255 every 7 days following CAR T cell infusion and were euthanized at various timepoints post CAR T cell infusion. Results Treatment of CD8 and CD4 CAR T cells in vitro with NKTR-255 resulted in dose dependent STAT5 phosphorylation and antigen independent proliferation. Co-culture of CD8 CAR T cells with CD19 positive targets and NKTR-255 led to enhanced proliferation, expansion and TNFα and IFNγ production, particularly at lower effector to target ratios. Further studies showed that treatment of CD8 CAR T cells with NKTR-255 led to decreased expression of activated caspase 3 and increased expression of bcl-2. In Raji lymphoma bearing NSG mice, administration of NKTR-255 in combination with CAR T cells increased peak CAR T cell numbers, Ki-67 expression and persistence in the bone marrow compared to mice receiving CAR T cells alone. There was a higher percentage of EMRA like (CD45RA+CCR7-) CD4 and CD8 CAR T cells in NKTR-255 treated mice compared to mice treated with CAR T cells alone and persistent CAR T cells in mice treated with NKTR-255 were able to reject re-challenge of Raji tumor cells. Additionally, starting NKTR-255 on D7 post T cell infusion resulted in superior tumor control and survival compared to starting NKTR-255 on D-1 or D14. Conclusion Administration of NKTR-255 in combination with CD19 CAR T cells leads to improved anti-tumor efficacy making NKTR-255 an attractive candidate for enhancing CAR T cell therapy in the clinic. Disclosures Chou: Nektar Therapeutics: Other: Travel grant. Fraessle:Technical University of Munich: Patents & Royalties. Busch:Juno Therapeutics/Celgene: Consultancy, Equity Ownership, Research Funding; Kite Pharma: Equity Ownership; Technical University of Munich: Patents & Royalties. Miyazaki:Nektar Therapeutics: Employment, Equity Ownership. Marcondes:Nektar Therapeutics: Employment, Equity Ownership. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding; Adaptive Biotechnologies: Consultancy; Lyell Immunopharma: Equity Ownership, Patents & Royalties, Research Funding. Turtle:Allogene: Other: Ad hoc advisory board member; Novartis: Other: Ad hoc advisory board member; Humanigen: Other: Ad hoc advisory board member; Nektar Therapeutics: Other: Ad hoc advisory board member, Research Funding; Caribou Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; T-CURX: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Patents & Royalties: Co-inventor with staff from Juno Therapeutics; pending, Research Funding; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Other: Ad hoc advisory board member.

Background: IL-15-mediated responses have been shown to have a crucial role in the development, function, and survival of CD8+ T cells, natural killer (NK) cells, and NK T cells. However, exploiting native IL-15 is challenging due to its unfavorable pharmacokinetics and tolerability. NKTR-255 is a polymer-conjugated IL-15 that retains binding affinity to IL-15Ra, maintaining full spectrum of IL-15 biology. NKTR-255 also exhibits improved pharmacokinetics, thereby providing sustained pharmacodynamic responses without the need for daily dosing. Studies have indicated that NK cells from patients with multiple myeloma (MM) appear to be dysfunctional, and successful activity against MM cells requires highly active NK cells ideally activated from immunomodulatory agents or cytokine support. In recent years, several new agents have been introduced in the MM landscape to engage NK-mediated myeloma cell elimination, including the CD38-targeting monoclonal antibody daratumumab, and elotuzumab, further supporting the anti‐MM effect of NK cells in the post-autologous transplant setting. In non-Hodgkin lymphoma (NHL), studies have shown low peripheral blood NK cell counts are associated with poor clinical outcomes in diffuse large B-cell lymphoma patients receiving R-CHOP chemotherapy regimens. Recently published data indicate that NKTR-255 in combination with CAR T cells decreases tumor burden and increases survival compared to CAR T cells alone. NKTR-255 may address the need to boost NK cell quality and numbers in these patients with the purpose of aiding current approved therapies. Methods: In this phase 1, open-label, multi-center, dose escalation and dose expansion study of NKTR-255, patients with relapsed or refractory (r/r) MM or NHL with no available therapies will be eligible. In the dose escalation portion, approximately 46 patients will be enrolled. Successive cohorts of 3 patients each will receive single increasing doses of NKTR-255 until the maximum tolerated dose (MTD) is determined. All patients will receive NKTR-255 once every three weeks. Patients will be observed for a dose-limiting toxicity (DLT) window of three weeks following the first NKTR-255 dose. A two-parameter Bayesian logistic regression model (BLRM) will be used during the escalation part of the study for dose level selection and for determination of MTDs. The selected recommended phase 2 dose (RP2D) of NKTR-255 will be evaluated in two dose expansion cohorts. Cohort A will expand NKTR-255 in patients with r/r MM or NHL as a salvage regimen to further characterize safety and tolerability. Cohort B will combine NKTR-255 with daratumumab in patients with MM with progressive disease who have had at least 3 prior lines of therapy with no other regimens that would confer clinical benefit. Daratumumab will be administered IV at the standard approved regimen. The primary objectives of the study are to evaluate: safety, tolerability, MTD, and RP2D of NKTR-255 as a single agent, as well as safety and tolerability of NKTR-255 in combination with daratumumab in patients with r/r MM. Secondary objectives include measures biomarker and pharmacokinetic analyses. Figure Disclosures Shah: University of California, San Francisco: Employment; Nkarta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kite: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene, Janssen, Bluebird Bio, Sutro Biopharma: Research Funding; Poseida: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Teneobio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech, Seattle Genetics, Oncopeptides, Karoypharm, Surface Oncology, Precision biosciences GSK, Nektar, Amgen, Indapta Therapeutics, Sanofi: Membership on an entity's Board of Directors or advisory committees; Indapta Therapeutics: Equity Ownership. Turtle:Nektar Therapeutics: Other: Ad hoc advisory board member, Research Funding; T-CURX: Membership on an entity's Board of Directors or advisory committees; Allogene: Other: Ad hoc advisory board member; Humanigen: Other: Ad hoc advisory board member; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Patents & Royalties: Co-inventor with staff from Juno Therapeutics; pending, Research Funding; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Caribou Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Other: Ad hoc advisory board member; Novartis: Other: Ad hoc advisory board member. Cowan:Cellectar: Consultancy; Juno: Research Funding; Sanofi: Consultancy; Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Abbvie: Research Funding. Chavez:Kite: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Budde:F. Hoffmann-La Roche Ltd: Consultancy. Marcondes:Nektar Therapeutics: Employment, Equity Ownership. Lee:Nektar Therapeutics: Employment. Lin:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership. Tagliaferri:Nektar Therapeutics: Employment, Equity Ownership. Patel:Poseida Therapeutics, Cellectis, Abbvie: Research Funding; Oncopeptides, Nektar, Precision Biosciences, BMS: Consultancy; Takeda, Celgene, Janssen: Consultancy, Research Funding.

26 Background: NKTR-262 is a novel intratumoral prodrug designed to promote tumor antigen release, an immune stimulatory environment, and antigen presentation. NKTR-214 increases CD8+ T cells and NK cells in the tumor microenvironment. The combination of NKTR-262 with NKTR-214 accesses innate and adaptive immunity to provide an abscopal response and systemic tumor immunity. The Phase 1b/2 REVEAL trial is enrolling. Methods: In a 3+3 Phase 1 design, pts who are relapsed/refractory to ≥1 CPI receive escalating doses of intratumoral (IT) NKTR-262 followed by combined q3w treatment of IT NKTR-262 and fixed-dose intravenous (IV) NKTR-214 (0.006 mg/kg). Phase 1 will evaluate PK, safety, efficacy, and determine recommended Phase 2 dose. Scans are conducted every 9 wks (± 1 wk). Peripheral blood (PB) and tumor tissue are collected to measure biomarkers of immune activation. In Phase 2 expansion, NKTR-262 + NKTR-214 ± IV nivolumab (360 mg) will be assessed in specific tumor cohorts of IO-naïve and experienced pts. Results: As of 06 Nov 2018, 11 pts had received at least one cycle of NKTR-262 + NKTR-214 therapy. 7/11 pts were efficacy evaluable with at least one on-treatment scan (4 MEL, 2 SARC, 1 CRC). 2/4 R/R melanoma pts had uPRs with 100% (scan 2) and 40% (scan 1) reductions in target lesions per RECIST 1.1, respectively. Both patients remain on treatment. Two SARC pts had stable disease at scan 1. No pts experienced G3 or higher TRAEs or immune-related adverse events (AEs). Most common G1-2 AEs were transient flu-like symptoms. Gene expression analysis of tumor and PB show dose-dependent induction (4 to 16-fold) of TLR target genes. Consistent with the mechanism of action for NKTR-214, PB flow cytometry shows an average 13-fold increase from baseline in Ki67+ CD8+T cells in 6 pts. Conclusions: NKTR-262 + NKTR-214 engage the immune activation cascade from local tumor antigen production to anti-tumor T cell response. Initial dose levels of NKTR-262 with fixed-dose NKTR-214 were well-tolerated with early evidence of clinical activity. These data support continued evaluation of the combination +/- nivolumab. Clinical trial information: NCT03435640.

Introduction IL-15 is a common gamma chain cytokine that activates and provides a survival benefit to T-cells and NK cells and has long been recognized as having potential as an immunotherapeutic agent for the treatment of cancer. Therapeutic use of native IL-15 has been challenging due to, for example, its unfavorable pharmacokinetic and safety properties. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. Here we investigate the biological effects of NKTR-255 in naïve cynomolgus monkey. Methods In vitro monkey whole blood was treated with NKTR255 and the percentage of pSTAT5 positive populations in each NK, CD4 T and CD8 T cells was determined by flow cytometry. In an PK/PD study, monkeys received single IV doses of 0.001, 0.003, 0.01, 0.03, or 0.1 mg/kg NKTR-255. Blood samples were collected to determine the plasma concentrations of NKTR-255 and to assess the effects of NKTR-255 on NK and CD8 T cells at multiple time points; flow cytometry was used to measure STAT5 phosphorylation, Ki-67 expression and frequency of cell populations. Granzyme B expression was assessed in NK and CD8 T cells by flow cytometry. Results NKTR-255 induced dose-dependent phosphorylation of STAT5 in monkey whole blood (EC50 values NK cells: 6.9 ng/ml, CD8 T cells: 39 ng/ml, CD4 T cells: 53 ng/ml). The half-life and clearance of NKTR-255 were 26x longer and 38x lower, respectively, than IL-15. NKTR-255 engaged the IL-15 signaling pathway, in vivo, demonstrating both robust and sustained STAT5 phosphorylation in lymphocytes. NKTR-255 drove the proliferation of total CD8 T cells and NK cells in a dose-dependent manner, with dramatic and durable increases observed in Ki67 positive population and absolute cell numbers (NK cells: 6.1 fold; CD8 T cells: 7.8 fold from baseline on day 5 at 0.1 mg/kg). These effects were strongly biased towards CD8 T cells and NK cells, with substantially less induction of CD4 T cells. The Ki67 response analyses of the T cell subpopulation revealed a higher response of memory populations than for naive T cells. Among memory T cells, effector memory T cells showed the highest response over stem cell memory T cells and central memory T cells. Finally, NKTR-255 also increased the expression of Granzyme B in both NK and CD8 T cells, concomitant with an enhancement in target cell lysis. Conclusions Nektar has generated a novel and potent molecule in NKTR-255 that not only preserves the relevant biology of IL-15, but additionally provides enhanced PK and PD properties relative to the native IL-15 cytokine. NKTR-255 is being developed as an immune-stimulatory agent to target NK and CD8 T cell biology for the treatment of cancer. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Kuo:Nektar Therapeutics: Employment, Equity Ownership. Maiti:Nektar Therapeutics: Employment, Equity Ownership. Obalapur:Nektar Therapeutics: Employment, Equity Ownership. Addepalli:Nektar Therapeutics: Employment, Equity Ownership. Rubas:Nektar Therapeutics: Employment, Equity Ownership. Sims:Nektar Therapeutics: Employment, Equity Ownership. Zhang:Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership.

The transcription factor TWIST-1 is up-regulated in CD34(+) cells in myelodysplastic syndrome and is involved in resistance to apoptosis. There is evidence that TWIST-1 affects apoptosis via microRNAs (miRs). Expression of miRs was determined in myeloid cell lines and primary CD34(+) marrow cells from patients with myelodysplastic syndrome and healthy donors using NanoString/array and validated by real-time-polymerase chain reaction. Expression levels of miR10a and miR10b were significantly higher in CD34(+) marrow cells from 28 patients with myelodysplastic syndrome than in CD34(+) cells from healthy donors (P=0.05 and P=0.012, respectively). Levels of miR10a/b correlated with TWIST-1 miR levels in CD34(+) myelodysplastic marrow cells (miR10a, R=+0.69, P

Patients with low-grade myelodysplastic syndromes (MDS) show high levels of tumor necrosis factor α (TNFα) and up-regulation of apoptosis in the marrow. In contrast, marrow cells in advanced MDS are typically resistant to TNFα-induced apoptosis but are rendered apoptosis-sensitive on coculture with stroma. The present studies show that CD34+ marrow cells in advanced MDS express high levels of TWIST, a basic helix-loop-helix transcription factor that opposes p53 function. TWIST levels correlated with disease stage (advanced > low grade; P = .01). Coculture with HS5 stroma resulted in down-regulation of TWIST and increased apoptosis in response to TNFα in CD34+ cells from advanced MDS; the same effect was achieved by TWIST-specific RNA interference in CD34+ cells. In primary MDS marrow stroma TWIST expression was lower than in healthy controls; suppression of TWIST in stroma interfered with induction of apoptosis sensitivity in cocultured CD34+ cells. Stroma cells so modified expressed reduced levels of intercellular adhesion molecule-1 (ICAM1; CD54); blockade of ICAM1 in unmodified stroma was associated with reduced apoptosis in cocultured CD34+ MDS marrow cells. These data suggest role for dysregulation of TWIST in the pathophysiology of MDS. Conceivably, TWIST or components in the signaling pathway could serve as therapeutic targets for patients with MDS.

There is growing evidence for a role of the hemopoietic microenvironment in the pathophysiology of myelodysplastic syndromes (MDS). Effects of various cytokines on the marrow microenvironment of patients with MDS have been studied. Autoimmunity, i.e. a reaction of autologous T lymphocytes against components of the marrow is also operative in a proportion of patients with MDS. The negative feed-back loop that controls tumor necrosis factor (TNF)α levels in healthy individuals is apparently disrupted in MDS due to auto-amplification signals involving TNFα and interleukin (IL-32). IL-32 mRNA levels in primary adherent cells from patients with MDS are 14- to 17-fold higher than in controls. In contrast, cells from patients with chronic myelomonocytic leukemia (CMML), a myeloproliferative disorder with low TNFα levels, express IL-32 at only 1/10 the level observed in controls. Damage in the microenvironment may occur secondary to oxidative stress, which may also lead to accelerated shortening of telomeres. This is, indeed, true for hematopoietic cells in MDS marrow, but telomere length in marrow stroma does not differ from that in age-matched healthy individuals. Nevertheless, the stroma shows functional aberrancies. Stroma-derived signals facilitate apoptosis in clonal hematopoietic cells but not in normal CD34+ cells. Thus while stroma dysfunction is likely due to signals derived from the hematopoietic clone rather than being intrinsic, it does affect clonal death or survival, respectively. Therefore, signals exchanged between both compartments could serve as targets for therapeutic interventions.

TNFα levels are elevated in the marrows of patients with myelodysplastic syndrome (MDS) and are associated with high rates of apoptosis, which contributes to hematopoietic failure. We observed that exposure of human marrow stroma cell lines HS5 and HS27a to TNFα increases levels of IL-32 mRNA. IL-32, in turn, induces TNFα. Marrow stroma from patients with MDS expressed 14- to 17-fold higher levels of IL-32 mRNA than healthy controls. In contrast, cells from patients with chronic myelomonocytic leukemia (CMML) expressed only one tenth the level of IL-32 measured in healthy controls. Human KG1a leukemia cells underwent apoptosis when cocultured with HS5 stromal cells, but knockdown of IL-32 in the stromal cells by using siRNA abrogated apoptosis in the leukemia cells. IL-32 knockdown cells also showed dysregulation of VEGF and other cytokines. Furthermore, CD56+natural killer cells from patients with MDS and CMML expressed IL-32 at lower levels than controls and exhibited reduced cytotoxic activity, which was unaffected by IL-2 treatment. We propose that IL-32 is a marrow stromal marker that distinguishes patients with MDS and CMML. Furthermore, IL-32 appears to contribute to the pathophysiology of MDS and may be a therapeutic target.

Aberrant regulation of the tumour necrosis factor alpha gene (TNF) and stroma-derived signals are involved in the pathophysiology of myelodysplasia. Therefore, KG1a, a myeloid leukaemia cell line, was exposed to Tnf in the absence or presence of either HS-5 or HS-27a cells, two human stroma cell lines. While KG1a cells were resistant to Tnf-induced apoptosis in the absence of stroma cells, Tnf-promoted apoptosis of KG1a cells in co-culture experiments with stroma cells. To investigate the Tnf-induced signals from the stroma cells, we examined expression changes in HS-5 and HS-27a cells after Tnf exposure. DNA microarray studies found both discordant and concordant Tnf-induced expression responses in the two stroma cell lines. Tnf promoted an increased mRNA expression of pro-inflammatory cytokines [e.g. interleukin (IL)6, IL8 and IL32]. At the same time, Tnf decreased the mRNA expression of anti-apoptotic genes (e.g. BCL2L1) and increased the mRNA expression of pro-apoptotic genes (e.g. BID). Overall, the results suggested that Tnf induced a complex set of pro-inflammatory and pro-apoptotic signals in stroma cells that promote apoptosis in malignant myeloid clones. Additional studies will be required to determine which of these signals are critical for the induction of apoptosis in the malignant clones. Those insights, in turn, may point the way to novel therapeutic approaches.

α1-Antitrypsin (A1AT) purified from human plasma upregulates expression and release of angiopoietin-like protein 4 (Angptl4) in adherent human blood monocytes and in human lung microvascular endothelial cells, providing a mechanism for the broad immune-regulatory properties of A1AT independent of its antiprotease activity. In this study, we demonstrate that A1AT (Prolastin), a potent inducer of Angptl4, contains significant quantities of the fatty acids (FA) linoleic acid (C18:2) and oleic acid (C18:1). However, only trace amounts of FAs were present in preparations that failed to increase Angplt4 expression, for example, A1AT (Zemaira) or M-type A1AT purified by affinity chromatography. FA pull-down assays with Western blot analysis revealed a FA-binding ability of A1AT. In human blood-adherent monocytes, A1AT-FA conjugates upregulated expression of Angptl4 (54.9-fold, p < 0.001), FA-binding protein 4 (FABP4) (11.4-fold, p < 0.001), and, to a lesser degree, FA translocase (CD36) (3.1-fold, p < 0.001) relative to A1AT devoid of FA (A1AT-0). These latter effects of A1AT-FA were blocked by inhibitors of peroxisome proliferator-activated receptor (PPAR) β/δ (ST247) and PPARγ (GW9662). When compared with controls, cell pretreatment with ST247 diminished the effect of A1AT-LA on Angptl4 mRNA (11.6- versus 4.1-fold, p < 0.001) and FABP4 mRNA (5.4- versus 2.8-fold, p < 0.001). Similarly, preincubation of cells with GW9662 inhibited inducing effect of A1AT-LA on Angptl4 mRNA (by 2-fold, p < 0.001) and FABP4 mRNA (by 3-fold, p < 0.001). Thus, A1AT binds to FA, and it is this form of A1AT that induces Angptl4 and FABP4 expression via a PPAR-dependent pathway. These findings provide a mechanism for the unexplored area of A1AT biology independent of its antiprotease properties.

Left ventricular hypertrophy is common in renal transplant patients but the factors influencing its development remain to be determined. The present investigation was conducted to study the effect of blood pressure load on the left ventricular mass of recently transplanted patients using 24-h ambulatory blood pressure monitoring (ABPM).We studied 30 renal transplant (RT) patients (36.1+/-13.7 years old, 11 males, 26 Whites, 4 diabetics, 15 under antihypertensive medication, 21 recipients of cadaver donors, and all treated with steroids, cyclosporin and azathioprine and with adequate (serum creatinine1.8 mg/100 ml) renal function). The median duration of dialysis treatment before transplant was 37 months, and the studies were performed during the first 40 days post-transplantation. Blood pressure was measured after a 15-min rest (casual blood pressure) and during a 24-h period with a SpaceLabs apparatus. Echocardiograms were obtained from all patients.Mean left ventricular mass index (LVMI) was 153+/-44 g/m2; casual systolic and diastolic BP (mmHg) was 152+/-25 and 92+/-13, whereas systolic and diastolic 24-h BP was 133+/-12 and 85+/-8, respectively. The systolic sleeping BP/awake systolic BP (SSBP/ASBP) ratio was 0.94+/-0.07, and 73% of the patients did not show a significant (10%) fall of systolic blood pressure during sleep. Multivariate analysis showed that awake systolic blood pressure was the only variable that independently influenced LVMI after adjusting for confounding factors (regression coefficient = 0.49, p = 0.01). Casual systolic and diastolic BP, sleeping systolic and diastolic blood pressure, 24-h heart rate, age, race, gender, smoking, body mass index, duration of dialysis, diabetes, antihypertensive and immunosuppressive drugs and levels of hematocrit, creatinine and serum lipids did not correlate with LVMI.The data indicate that left ventricular hypertrophy during the early post-transplant period is mainly influenced by awake blood pressure load. They also suggest that ABPM may be more useful in the diagnosis and management of post-transplant hypertension than casual BP. The findings emphasize the importance of rigid blood pressure control in renal transplant recipients.

Introduction: Idelalisib (IDELA) is a targeted PI3Kd inhibitor approved for the treatment of patients (pts) with relapsed CLL/FL. The established toxicity profile of IDELA includes diarrhea/colitis with a grade ≥3 incidence of ~15%. Here, we present the histopathological-as well as immune profiling and viral testing-results of gastrointestinal tissue specimens from pts who received diagnostic biopsies for abdominal pain or diarrhea during treatment with IDELA. Methods: Intestinal biopsy specimens obtained from 31 pts during treatment with IDELA across 10 trials (monotherapy n = 3 or in combination with: rituximab n = 9; ofatumumab n = 14; and bendamustine/rituximab n = 5) were reviewed separately by 2 independent pathologists with expertise in the area of gastrointestinal and transplant pathology. If more than 1 biopsy time point per pt was available (n = 3), all time points would be evaluated and, if discordant, would be reported separately. Evaluation included hematoxylin and eosin stain; and immunohistochemistry (IHC) for CD3, CD8, FOXP3, CD79a, CMV, and adenovirus (as adequate tissue allowed). Additionally, droplet digital PCR (ddPCR) was performed for cytomegalovirus (CMV), adenovirus, and human herpesvirus 6 (HHV-6). Colon/small bowel tissues from 18 normal individuals who underwent biopsy during routine screening colonoscopy and had normal pathology results were included as controls. Results: Baseline characteristics of pts included CLL n = 24 (77.4%) and iNHL n = 7 (22.6%), age range 50 to 82 years with median 65.6 years, male n = 19 (61.3%). IDELA median dose = 150 mg BID. Diarrhea/colitis (Grade 1-3) was reported in 24 cases (75%) including 1 case of hemorrhagic colitis. One patient each had concurrent laboratory-confirmed Mycobacterium avium-intracellulare and norovirus and 2 patients were excluded from histomorphological analysis, 1 due to celiac disease and 1 due to total loss of glands secondary to CMV. Four distinct morphological patterns were identified: 1) predominantly normal, 2) apoptotic, 3) inflammatory/ischemic, and 4) mixed apoptotic/inflammatory/ischemic. CMV IHC was performed in all 31 cases; 2 showed strong/moderate positivity, and 2 were weakly positive. Adenovirus IHC was performed on 29 specimens, and 1 was weakly positive. ddPCR performed on 27 formalin-fixed paraffin-embedded tissues identified 6 positive CMV cases (including all 4 IHC+ cases), 0 adenovirus cases, and 1 HHV-6-positive case. Among the CMV cases, the most common pattern was mixed, seen in 4 of 6 cases, whereas the most common morphologic feature was apoptosis, seen in 5 of 6 cases. A summary of findings is shown in Table 1. Conclusion: In this subset of pts who underwent intestinal biopsy during IDELA clinical trials, most biopsies (87%) were performed in the context of ongoing diarrhea/colitis and the majority of tissue samples (90%) revealed some component of inflammation. Immune profiling by IHC showed increased Tregs in patterns associated with glandular destruction. Immunohistochemistry and ddPCR for virus in conjunction with microbiology studies revealed that in 28% of these cases, a pathogen was identified. This dataset suggests that in a significant number of cases, idelalisib-associated diarrhea/colitis may be due to an infectious etiology secondary to immune dysfunction that results in either an inflammatory/ischemic or a mixed histologic pattern; however, the majority of these cases have no identifiable infectious etiology. Based on these findings, we recommend that a workup of pts with idelalisib-associated diarrhea/colitis should include a complete evaluation for infectious causes including intestinal biopsy when a pathogen is elusive with standard testing. Disclosures Yeung: Gilead Sciences: Research Funding. Hockenbery:Gilead Sciences: Research Funding. Coutre:Gilead Sciences: Consultancy, Research Funding. Dubowy:Gilead Sciences: Employment, Equity Ownership. Marcondes:Gilead Sciences: Employment, Equity Ownership. Munugalavadla:Gilead Sciences: Employment, Equity Ownership. Bosch:Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Research Funding.

Background:Spleen tyrosine kinase (SYK) is a nonreceptor cytoplasmic tyrosine kinase primarily expressed in cells of hematopoietic lineage. Constitutive activation of SYK in acute myeloid leukemia (AML) has been reported and targeted inhibition of SYK induced differentiation in vitro and demonstrated anti-leukemia activity in AML mouse models. SYK has also been shown to directly phosphorylate the FLT3 receptor, modulating its activation and possibly promoting its role in leukemogenesis. Entospletinib is an orally bioavailable, selective inhibitor of SYK shown to be clinically active in B-cell malignancies. Here we evaluate the combination of entospletinib in patients with untreated AML using a 14-day window phase to assess single-agent activity, then adding standard intensive chemotherapy. Methods: In this phase 1b/2 study (NCT02343939), patients age 18 to 70 years with previously untreated AML, preserved organ function, and ECOG ≤ 2 were eligible to receive dose escalated entospletinib for 14 days as monotherapy (days -14 to 0) followed by combination with daunorubicin 60 mg/m2/d, cycle 1 day 1 to 3, and cytarabine 100 mg/m2/d, cycle 1 day 1 to 7. All patients received entospletinib monotherapy for up to 14 days prior to starting induction. Chemotherapy could be initiated after 5 days of monotherapy (and entospletinib continued for 4+ weeks) in patients with leukemia-related complications necessitating chemotherapy. Patients enrolled to dose level (DL) 0 and DL 1 received entospletinib 200 mg po BID and 400 mg po BID, respectively. Patients with residual disease two weeks after chemotherapy received a second induction cycle identical to the first. Entospletinib was continued without interruption until remission was assessed at count recovery. Results:Twelve patients enrolled with a median age of 54 (range, 18-69) years. Patients were in the following European LeukemiaNet genetic risk groups: favorable (n=1), intermediate I (n=3), intermediate II (n=2), and adverse (n=4), respectively. Three patients were not evaluable for dose limiting toxicity (DLT) assessment and were replaced (due to detection of CNS disease requiring non-study therapy (n=1), and withdrawal of consent unrelated to drug toxicity (n=2)). Single-agent entospletinib during the window period was well tolerated; toxicities after combination with intensive chemotherapy were common and typical. Among 3 patients treated at 200mg BID, no DLT was observed. Of 3 patients treated at 400mg BID, a patient with documented fungal pneumonia developed grade 3 pneumonitis that was possibly related to entospletinib. Although this did not meet DLT criteria, DL 1 was expanded with 3 additional patients, none of whom experienced DLT. Overall, the most common non hematologic adverse events (inclusive of intensive chemotherapy periods) were febrile neutropenia, nausea, and diarrhea. Based on this clinical experience and compiled pharmacokinetic data demonstrating lack of benefit to further dose escalation, 400 mg BID was selected as the recommended phase 2 dose. Responses were seen at both levels. Among the 3 patients treated at 200 mg BID, two required a second induction but all achieved a complete remission (CR) (3/3; 100%). Of the 6 patients treated at 400mg BID, none required a second induction and the CR rate was also 100%. Remarkably, an 18 year old male with 11q23-rearranged AML achieved morphologic and cytogenetic CR after only the 14 day entospletinib monotherapy window (prior to chemotherapy). Another patient with 11q23-rearranged AML had significant platelet response during the window period (this patient refused disease evaluation by marrow aspiration prior to chemotherapy). Conclusions: Entospletinib appears to have significant clinical activity in AML, and its combination at doses up to 400mg BID with intensive chemotherapy is well tolerated. An extended phase 2 program is now underway. Patients with 11q23-rearranged AML may be uniquely sensitive to SYK inhibition by entospletinib. Detailed molecular analysis of these patients is ongoing and will be presented. Disclosures Walker: Gilead Sciences: Research Funding. Bhatnagar:Karyopharm: Research Funding. Marcondes:Gilead Sciences: Employment, Equity Ownership. DiPaolo:Gilead Sciences: Employment, Equity Ownership. Abella-Dominicis:Gilead Sciences: Employment, Equity Ownership.

Hepatic iron overload is common in patients undergoing hematopoietic cell transplantation. We showed previously in a murine model that transplantation of allogeneic T cells induced iron deposition and down-regulation of hepcidin (Hamp) in hepatocytes. We hypothesized that hepatic injury was related to disrupted iron homeostasis triggered by the interaction of Fas-ligand, expressed on activated T cells, with Fas on hepatocytes. In the current study, we determined the effects of modified expression of the Flice inhibitory protein (FLIP long [FLIPL]), which interferes with Fas signaling, on the impact of Fas-initiated signals on the expression of IL-6 and Stat3 and their downstream target, Hamp. To exclude a possible contribution by other pathways, we used agonistic anti-Fas antibodies (rather than allogeneic T cells) to trigger Fas signaling. Inhibition of FLIPL by RNA interference resulted, as expected, not only in enhanced hepatocyte apoptosis in response to Fas signals, but also in decreased levels of IL-6, Stat3, and Hamp. In contrast, overexpression of FLIPL protected hepatocytes against agonistic anti-Fas antibody-mediated apoptosis and increased the levels of IL-6 and Stat3, thereby maintaining the expression of Hamp in an NF-κB–dependent fashion. In vivo overexpression of FLIPL in the liver via hydrodynamic transfection, similarly, interfered with Fas-initiated apoptosis and prevented down-regulation of IL-6, Stat3, and Hamp. These data indicate that Fas-dependent signals alter the regulation of iron homeostasis and suggest that signals initiated by Fas may contribute to peritransplantation iron accumulation.

The role of aberrant DNA methylation in Ewing sarcoma is not completely understood. The methylation status of 503 genes in 52 formalin-fixed paraffin-embedded EWS tumors and 3 EWS cell lines was compared to human mesenchymal stem cell primary cultures (hMSCs) using bead chip methylation analysis. Relative expression of methylated genes was assessed in 5-Aza-2-deoxycytidine-(5-AZA)-treated EWS cell lines and in a cohort of primary EWS samples and hMSCs by gene expression and quantitative RT-PCR. 129 genes demonstrated statistically significant hypermethylation in EWS tumors compared to hMSCs. Thirty-six genes were profoundly methylated in EWS and unmethylated in hMSCs. 5-AZA treatment of EWS cell lines resulted in upregulation of expression of hundreds of genes including 162 that were increased by at least 2-fold. The expression of 19 of 36 candidate hypermethylated genes was increased following 5-AZA. Analysis of gene expression from an independent cohort of tumors confirmed decreased expression of six of nineteen hypermethylated genes (AXL, COL1A1, CYP1B1, LYN, SERPINE1,) andVCAN. Comparing gene expression and DNA methylation analyses proved to be an effective way to identify genes epigenetically regulated in EWS. Further investigation is ongoing to elucidate the role of these epigenetic alterations in EWS pathogenesis.

The HCT-comorbidity index (CI) was developed as a measure of health-status that could stratify risks of mortality after HCT. Information are lacking on the possible biologic tissue-specific process that underlies the association between pre-transplant comorbidities and post-transplant mortality. Further, there is a continuously increasing need for novel biomarkers that could increase the magnitude of objectivity in diagnosing pre-HCT comorbidities and increase the predictive power for post-HCT NRM. MiRs are a class of small non-coding RNAs (~22 nt) that negatively regulate gene expression. Studies have uncovered the functional role of miRs in diverse pathophysiological processes. Moreover, a single miR could be implicated in different pathological processes. To this end, we analyzed miRs as diagnostic for relevant comorbidities before and as prognostic for mortality after HCT. Peripheral blood mononuclear cell samples were previously collected from 36 pts within 30 days prior to HCT as a part of research repository. All samples were collected in EDTA tubes and processed and frozen at -80 Celsius degrees within 8 hours of draw. All pts were in CR before HCT. Patients were divided into two risk groups: low-risk included those with HCTCI score of 0 before HCT, who survived allogeneic HCT per last-follow up; while the high-risk group included those with HCT-CI scores of ≥4, who deceased after allogeneic HCT (Table). RNA was isolated from PBMC using previously described methods (Xie LN et al, Clinical Transplant. 2014; 28:314). For discovery of relevant miRs, we used NanoString nCounter miR assay as previously described (Knouf EC et al, 2013. PLoS ONE 8: e69630) comprising 654 endogenous miRs. Analysis of miR raw data was done using nSolverTM 2.0 Software (NanoString Technologies, Inc.) applying standard quality control tests. All samples contributed to the discovery analysis. MiRs were filtered to include only those expressed with at least 50 counts for the NanoString abundance analyses. MiR raw data was normalized using the geometric mean of top 100 miRs (probes with highest 100 counts) as recommended by the manufacturer. Fold change was calculated with partitioning by the low vs high-risk groups computing two-tailed t-test on the log transformed normalized data that assumes unequal variance. We used p-value cut-off of Among 654 tested miRs, 19 met the significance level of p We identified a group of MiRs as diagnostic biomarkers for relevant pre-transplant comorbidities and as prognostic biomarkers for post-transplant mortality. Comparing the predictive power of a clinical comorbidity index to that of the combined model of 7 miRs in a large dataset is underway. We will also investigate gene associations with these 7 miRs and how they predict specific post-HCT morbidities. Our results promise objectively accurate assessment of transplant risks and could also pave the way to potential interventions to lessen risks of mortality after allogeneic HCT. Table. Pt characteristics Characteristics Low risk (n=18) High-risk (n=18) Age (median, range), years 44 (22-70) 54 (24-68) % AL Myeloid 78 67 Lymphoid 22 33 CR First 67 61 Second 33 39 Conditioning High-dose 50 39 Reduced-intensity 50 61 Donor Related 17 28 Unrelated 83 72 Gender Male 50 50 Female 50 50 Race White 88 83 Others 12 17 Figure 1. Figure 1. Disclosures Maclean: NanoString Technologies: Employment. Roy:3NanoString Technologies: Employment.

Hematopoietic cell transplantation (HCT) offers potentially curative therapy for patients with myelodysplastic syndromes (MDS). However, who should and can be transplanted, with which approach, and when is a matter of debate. Various classification schemes and prognostic scoring systems have helped in the decision-making process. Offering HCT to patients who were not considered transplant candidates in the past is now possible with the development of new transplant strategies. In addition to disease stage, patient age, comorbid conditions, preHCT chemotherapy, type of donor, source of stem cells, and possibly other factors, need to be considered prior to transplant. Patients without substantial comorbid conditions up to 60 years with the availability of unrelated donor grafts or 65 years with related donor grafts can be transplanted with more conventional (higher dose) regimens (e.g. targeted BU/CY; Flu/BU). Older patients and patients with comorbid conditions should be enrolled in trials aimed at optimizing RIC/nonmyeloablative HCT (e.g. Flu/melphalan; Flu/low dose TBI). Patients who present with ‘advanced’ MDS or are transfusion dependent, and do not have a del(5q), should probably be transplanted early in their course. GVHD (in all patients) and post-HCT relapse (in patients with high risk disease) remain major problems. Modifications of transplant conditioning regimens have reduced transplant-related mortality and allow successful HCT even in older patients. However, prospective randomized trials are needed to determine the role of pre-HCT chemotherapy and the type of transplant conditioning regimen best suited for a given patient.

The marrow microenvironment contributes to the pathogenesis of ineffective hematopoiesis in Myelodysplastic Syndromes (MDS). Since mutations and cytogenetic alterations seen in the hematopoietic compartment are generally not present in marrow stromal cells, we hypothesized that epigenetic alterations may be responsible for altered stroma function in MDS. To elucidate the epigenome of MDS microenvironment, we used the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay to study cytosine methylation patterns of 50,000 CpGs loci covering 14,000 promoters in primary stromal cells. Global DNA methylation of 6 MDS marrow-derived stroma was analyzed by HELP assay and compared to 3 healthy controls. MDS stroma showed aberrant hypermethylation compared with controls (3626 hypermethylated vs 306 hypomethylated loci in untreated MDS stromal cells) that preferentially occurred outside of CpG islands (Test of Proportions, P value After demonstrating aberrant hypermethylation in MDS stroma, we next examined the functional role of these alterations by treatment with DNMT inhibitors. 5-Aza treatment of MDS stroma enhanced hematopoietic activity and erythroid differentiation from co-cultured healthy CD34+ cells. Significantly increased erythroid differentiation was seen in CD34+ cells cocultured with aza pretreated MDS stroma as shown by their glycophorin A positivity. Conclusions: These results reveal widespread aberrant epigenetic changes in the MDS marrow microenvironmentthat affect many important pathways. A recent report had shown that activation of beta-catenin in the bone marrow niche can trigger MDS/AML in vivo. Our results demonstrate that epigenetic silencing of WNT antagonists can potentially lead to activation of the WNT pathway in MDS stroma. Finally, we demonstrate that DNA methyl transferase inhibitors alter the epigenomic profiles of stromal cells and it raises the possibility that epigenetic modulation of the stroma by these drugs contributing to their therapeutic efficacy. Disclosures No relevant conflicts of interest to declare.

Background and Hypothesis DNA and histone methylation as well as microRNA (miR) expression are dysregulated in MDS. The histone demethylase KDM2B suppresses let7b, which post-transcriptionally controls EZH2, a histone-lysine N-methyl transferase that methylates lysine 27 on histone 3. We hypothesized that epigenetic dysregulation in MDS involves the KDM2B/ let7b/EZH2 axis (Figure). DZNep (cyclopentanyl analog of 3-deazaadenosine) is a novel compound, which results in loss of lysine27 methylation, re-expression of epigenetically silenced genes, and decreased cell proliferation.We propose that DZNep will bypass cellular dysregulation via the KDM2B/let7b/EZH2 axis and thereby enhance therapeutic efficacy when combined with azanucleosides. Methods CD34+ MDS marrow cells were obtained by density separation and enriched by magnetic-activated cell sorting (MACS; Milteny Biotec, Auburn, CA); purity 95% to 98% by flow cytometry. For NanoString nCounter miR array total cell derived RNA or synthetic miR pools (IDT; 30 pmol per oligonucleotide) were used as input. Nano-string data were validated by real-time polymerase chain reaction (PCR). RNA was extracted using the RNeasy Mini kit (QIAGEN, Valencia, CA). Primers for let7b, U6, KDM2b and EZH2 were synthesized by Applied Biosystems. Total RNA was reverse transcribed with Taqman MiR Reverse Transcriptase kit (Applied Biosystems, Carlsbad, CA). Potential functional roles of KDM2B, let7b and EZH2 were determined in myeloid cell lines MDS-L, PL-21, KG1a,OCI-AML3 and ML1. Conditional knock-down of KDM2B was achieved with 2 short hairpins (open-biosystems). Expression of KDM2B was forced using pCMV-Myc-KDM2B (gift of Dr. Tzatsos) in cell lines with low/absent constitutive levels of KDM2B and high levels of let7b. miRZip plasmids (miRZip anti-sense) were used to knock-down let7b; pre-BAB-let7b (origene) served to overexpress let7b. Gene expression, protein levels and cell proliferation were analyzed by RQ-PCR, western blot and BrdU uptake. To determine the impact of modified let7b and KDM2B expression on treatment responses, MDS-L cells (high constitutive levels of EZH2) were treated with DZNep and methyltransferase inhibitor, 5-azacytidine, and subjected to the same analyses. Results Let7b levels were significantly higher in primary CD34+ MDS marrow cells (n=44) than in healthy controls (p= 0.0304), while KDM2B and EZH2 expression was reduced (n=21, p Summary and Conclusion The KDM2B/ let7b/EZH2 axis is involved in epigenetic regulation in MDS. KDM2B, via let-7b/EZH2, promotes transcriptional repression in myeloid cell lines and primary MDS cells; KDM2B controls epigenetic modifications and cell proliferation. DZNep bypasses the “inhibitory” KDM2B/let7b/EZH2 axis by preventing H3K27 methylation and reducing cell proliferation, independent of EZH2 expression. Thus, the use of DZNep would add histone demethylation to the effect of azanucleosides on DNA methylation, thereby enhancing the therapeutic success in MDS. Disclosures: No relevant conflicts of interest to declare.

The bone marrow microenvironment plays an important role in the pathogenesis and perpetuation of stem cell defects in Myelodysplastic Syndrome (MDS). However, while distinct cytogenetic alterations have been described in the stem cell compartment in MDS, the bone marrow (BM) stroma has never been shown to be part of the clone. Thus, aberrant epigenetic alterations may be responsible for altered function of BM stroma in MDS. DNA methyl transferase (DNMT) inhibitors, which are therapeutically effective in MDS, can potentially affect both hematopoietic cells and the stroma, providing further rationale for studying DNA methylation profiles of BM stroma in MDS. To accomplish this aim, adherent non-hematopoietic cells were isolated from MDS patients and controls by immunomagnetic CD45 negative selection and then used for whole genome methylation studies using the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR) . Global epigenetic profiling revealed that MDS stroma (n=7) was epigenetically distinct from normal BM stroma (n=3) (ANOVA, P In subsequent studies, we profiled stroma from another set of MDS patients treated with the DNMT inhibitor, 5-Azacytidine (5-Aza) (n=4). In contrast to untreated MDS patients, the 5-Aza treated MDS patients and healthy controls (p = NS) were epigenetically similar. These 5-Aza exposed stroma cells did not demonstrate increased cytosine methylation thus suggesting that these cells were also targeted by DNMT depletion by the drug. Finally, the ability of MDS and control stromal cells to support the growth of healthy CD34+ cells was tested by in-vitro colony formation assay. MDS stromal cell co-culture led to significant reduction in erythroid and myeloid colony formation, demonstrating the MDS stroma contributes to ineffective hematopoiesis seen in this disease. Strikingly, pretreatment of MDS stromal cells with 5-aza increased the colony formation seen in co-cultures, demonstrating again that DNMT inhibitors exert effects on the microenvironment. Thus our results reveal for the first time that MDS is characterized by widespread aberrant epigenetic changes in the BM microenvironment. Our results also demonstrate that DNMT inhibitors can alter the epigenomic profiles of stromal cells, contributing in part to their clinical efficacy. Overall, these studies underscore the importance of studying the entire BM, including the microenvironment to improve our understanding of the pathophysiology of MDS and therapy. Citation Format: Tushar D. Bhagat, Matthias Bartenstein, Yiting Yu, A. Mario Marcondes, Davendra Sohal, John Greally, H Joachim Deeg, Amit Verma. Myelodysplasia bone marrow stromal cells have widespread aberrant hypermethylation that is targeted by treatment with DNMT inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1503. doi:10.1158/1538-7445.AM2013-1503

Abstract 4142 Background: Injury to healthy (non-target) tissues still is a major limitation of radiation therapy, at least in part related to release of cytokines, including TNFα and IL-1β, which amplify tissue damage. Cytokine release is particularly prominent in patients who receive total body irradiation (TBI) before hematopoietic cell transplantation (HCT), as interactions of allogeneic donor cells with patient tissue contribute to the resulting “cytokine storm” and the development of graft versus-host-disease (GVHD). In murine models, administration of alpha-1 anti-trypsin (AAT) in the peri-transplant period decreased GVHD incidence and mortality. AAT, a member of the serine protease inhibitor (serpin) family, is a major protective protein in the circulation and has been used successfully in the clinic for other indications. Methods: We were interested in determining the potential benefits of AAT in preventing toxicity related to the transplant conditioning regimen, specifically TBI. AAT inhibits proteinase-3 (PR3) which, among other targets, cleaves IL-32 thereby leading to activation of TNFa and enhancing the cytokine storm. Since others have also suggested that AAT, via enhanced expression of heme oxygenase-1 (HMOX-1), leads to activation of Nrf2, thereby enhancing transcription of anti-inflammatory cytokines such as IL10 and the IL-1 receptor antagonist (IL1Ra), we determined the overall shift in the cytokine milieu and cell death. Thus, we irradiated male C3H/HeN mice (n = 5 mice per group; 6–8 weeks old) with sub-lethal doses (500, 600 and 700 cGy) of TBI from a 137Cs source. AAT (300 μg/animal) was administered intra-peritoneally 1 hour before and every 48 hours after TBI for a total of 6 doses. The effect of TBI/AAT on hematopoiesis was analyzed by growing granulocyte-macrophage colony forming units (GM-CFU) from bone marrow cells, on days 3, 7 and 14 after TBI. Results: Results were compared to those with cells from albumin-treated controls. Marrows (days 3 and 7) from AAT-treated mice generated higher GM-CFU counts than those from controls (mean = 25 vs. 5 colonies per 25 × 103 cells plated after 500cGy, and 15 vs. 3 colonies per 25 × 103 cells after 600 cGy). Peripheral blood and unsorted bone marrow from AAT-treated mice showed up-regulation of HMOX-1 and Nrf2 (30 and 50 log2 increase, respectively), and enhanced transcription of IL-10 and IL-1Ra (50 and 10 log2, respectively) compared to albumin treated donors. PR3 and TNFα, in contrast, were down-regulated (5 log2 and 7 log2 decrease in comparison to albumin treated controls). The cytokine mitigating effects of AAT were accompanied by attenuation of ATM-p53-dependent DNA damage responses in bone marrow cells (determined on days 3, 7, and 14), showing a 3-fold decrease in p53 protein levels, and a 6-fold decrease in phospho-ATM in comparison to albumin treated controls (peak day 7). In addition, protein levels of caspase 3 and caspase 9 were decreased (2-fold and 5-fold, respectively; peak on day 7) in unsorted marrow cells and spleen lysates of AAT treated mice in comparison to albumin treated controls. Bone marrow histopathology revealed normo-cellularity and a decrease in cleaved caspase 3 staining in AAT-treated mice compared to albumin treated animals. Summary and conclusions: AAT treatment significantly mitigated the hematopoietic toxicity induced by sub-lethal TBI. The mechanism involves cytokine suppression, associated with attenuation of ATM-p53 mediated DNA damage response. Taken together, these data suggest that AAT treatment pre-TBI provides protection against radiation injury. Disclosures: No relevant conflicts of interest to declare.

Abstract 1889 Background: Graft-versus-host disease (GVHD) is a frequent complication of allogeneic hematopoietic cell transplantation (HCT). While cytolytic effector function of donor-derived CD8+ cytotoxic T lymphocytes (CTLs) is critical for the elimination of the underlying malignant disease, the goal is to achieve this graft-versus-leukemia (GVL) effect without inducing GVHD. We and others showed in murine models that administration of AAT in the peri-transplant period decreased GVHD incidence and mortality. Concurrently, levels of pro-inflammatory cytokines were suppressed compared to albumin-treated recipients. Since both GVHD and GVL effects are initiated by donor cells, we asked whether donor treatment with AAT would modify GVHD or GVL effects in recipients. Of concern was the observation that AAT enhanced expression of heme oxygenase-1 (HMOX-1) which, via generation of carbon monoxide, enables the transcription factor Nrf2 to bind to the promoters of IL10 and IL1Ra, which might interfere with GVL activity. Methods: We determined the effect of donor AAT treatment on GVHD and GVL in two murine transplant models: C3H/SW(H-2Kbc) into C57Bl/6(H-2Kb) (minor mismatch), and C57Bl/6(H-2Kb) into BALB/C (H-2Kd) (major mismatch). Four regimens of donor and recipient treatment were studied: 1) Donor albumin /recipient albumin; 2) donor albumin/recipient AAT; 3) donor AAT/recipient albumin; 4) donor AAT /recipient AAT. AAT and albumin were given to donors at 3 μg/ dose every 72 hours for two weeks before cell harvesting. Recipients received 3 μ/dose every 72 hours following total body irradiation with 800 cGy. On day 4, recipients were injected with 5 × 106 T cell-depleted bone marrow (TCD-BM) plus 5 × 105 CD4+/CD8+ splenic T cells to induce acute GVHD. To test our hypothesis that donor AAT treatment would interfere with the GVL effect, BALB/C (H-2Kd) recipients were injected i.v. with 20 × 103 A-20 lymphoma cells containing a luciferase reporter construct (A20-luc/yfp; H-2Kd), following TBI. The infusion of TCD-BM and T cells from C57Bl/6(H-2Kb) donors was delayed until day 4 to allow the tumor to home and get established. Results: RT-PCR analysis of donor BM and T cells prior to transplantation showed up-regulation of HMOX-1 and Nrf2 (10 and 15 log2 increase, respectively), and enhanced transcription of IL-10 and IL-1Ra (50 and 10 log2, respectively, compared to albumin treated donors). In contrast, TNFa, was down-regulated (7 log2 decrease in comparison to albumin-treated donors) in both unsorted bone marrow and in CD8+/CD4+spleen cells from AAT pre-treated donors. FACS analysis revealed a 4-fold increase in frequency of CD56+NK cells, and a 5-fold increase in CD4+/CD24+FoxP3+ Treg cells, associated with a 3 fold increase in CD8+/CD205+ APCs/ DC in comparison to albumin treated donors. Mice transplanted with cells from AAT-treated donors showed lower clinical GVHD scores and decreased mortality (Figure 1) in comparison to mice transplanted from albumin-treated donors. BALB/C (H-2Kd) animals transplanted from AAT-treated donors showed significantly lower A-20 tumor signal (luciferase) intensity (over the first 3 weeks post-HCT) than did mice transplanted from albumin-treated donors (n=6, mean 4.8 × 106 (AAT) versus,7 × 106 (albumin) photons/sec/mouse, P =.03). Summary and conclusions: These data suggest that pre-transplant in vivo AAT exposure of donor cells results in protection against acute GVHD. Tumor growth was not increased in mice transplanted from AAT treated donors and, in fact, appeared to be suppressed, presumably related to a significant increase in NK cells. Further studies on donor pre-treatment or direct administration of AAT to the transplant recipients are warranted. Disclosures: No relevant conflicts of interest to declare.

Abstract 522 Graft-versus-host disease (GVHD) is an important complication of allogeneic hematopoietic cell transplantation (HCT). The role of various cell populations, cytokines and chemokines, present pre and post-transplantation, in the development of GVHD has been studied extensively.We investigated the potential role of Interleukin (IL)-32 in alloreactivity and GVHD. IL-32 is the protein product of the NK4 transcript first reported in IL-2 activated T lymphocytes and natural killer cells. IL-32 has pro-inflammatory and pro-apoptotic properties and induces expression of TNF-α in several cell targets. We used one-way mixed lymphocyte cultures (MLC) as a simple in vitro model of GVHD to determine IL-32 expression upon alloactivation. The α and γ isforms of (IL)-32 protein were 2-fold upregulated in allogeneic MLC compared to autologous controls (n=4, p=0.037). Concurrently, the concentrations of TNF-α, IL-6 and IL-8 in the allogeneic MLC supernatants were, as expected, upregulated significantly. This finding led us to evaluate IL-32 expression as a potential marker for GVHD after allogeneic HCT in 45 patients with either active acute GVHD or chronic GVHD, and 16 patients who did not show clinical evidence of GVHD. IL-32 mRNA levels in peripheral blood unsorted white blood cells, correlated with acute GVHD (RT-PCR values expressed as mean +/− SEM of the ratio of expression of IL-32/GUS-B = 1.01, p=0.011, vs control values IL-32/GUS-B = 0.23) but not with chronic GVHD, (IL-32/GUS-B = 0.26, p=0.16) (Figure 1). As the serine protease neutrophil proteinase 3 (PR3) has been shown to serve as activator of IL-32, and to process several inflammatory cytokines, including IL-32, TNF-α and IL-8, we postulated that the addition of the serine protease inhibitor α-1 anti-trypsin (AAT), would interfere with the processing of IL-32 by PR3, and as a result would lead to decreased proliferation of cells in MLC, and reduced cytokine production. To test this hypothesis, MLCs were treated with AAT at concentrations of 1–20 ug/ml and expression of IL-32 and PR3 were determined. In MLCs treated with AAT at the optimal concentration of 5 ug/ml, added to cultures on alternate days for a period of 7 days T lymphocyte proliferation was suppressed when compared to vehicle-treated MLC, (mean CPM=33.000 versus CPM=67.000; p=0.012). Concurrently there was a 2.5 fold decrease in IL-32 and PR3 protein levels (n=4, p=0.023). CONCLUSION: IL-32 is upregulated in patients with acute GVHD. Determination of IL-32 in patients with suspected GVHD may support the diagnosis and the decision to treat.AAT interferred with T cell activation and the release of cytokines, including IL-32, suggesting that administration of AAT may have therapeutic potential in patients with acute GVHD. Disclosures: No relevant conflicts of interest to declare.

The bone marrow microenvironment plays an important role in the pathogenesis and perpetuation of stem cell defects in Myelodysplastic Syndrome (MDS). However, while distinct cytogenetic alterations have been described in the stem cell compartment in MDS, the bone marrow stroma has never been shown to be part of the clone. Thus, aberrant epigenetic alterations may be responsible for altered function of bone marrow stroma in MDS. DNA methyl transferase (DNMT) inhibitors, which are therapeutically effective in MDS, affect both hematopoietic cells and the stroma, providing further rationale for studying DNA methylation profiles of bone marrow stroma in this disease. To accomplish this aim, bone marrow mononuclear cells from MDS patients and controls were grown to form adherent cell layers and then depleted for hematopoietic elements by immunomagnetic CD45 negative selection. CD45 negative adherent cells were subsequently expanded and then used for whole genome methylation studies using a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) which uses differential methylation-specific digestion by HpaII and MspI followed by amplification, two color labeling and hybridization to quantify individual promoter CpG island methylation. A custom whole genome human promoter array (Roche-Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Global epigenetic profiling revealed that MDS stroma (n=6) was epigenetically distinct from normal bone marrow stroma (n=4) (ANOVA, P In subsequent studies, we profiled stroma from another set of MDS patients who had been treated with the DNMT inhibitor, 5-Azacytidine (n=4). In contrast to untreated MDS patients, there were no significant epigenetic differences between these 5-Azacytidine treated MDS patients and healthy controls (p = NS). These 5-Azacytidine exposed stroma cells did not demonstrate global hypomethylation (as hypothesized after DNMT inhibitor treatment) and were characterized by both hyper- and hypo-methylated loci similar to healthy controls. Thus our results reveal that MDS is characterized by widespread aberrant epigenetic changes in the bone marrow microenvironment. Our results also demonstrate that DNMT inhibitors can alter the epigenomic profiles of stromal cells, and we hypothesize that those stroma effects contribute in part to their clinical efficacy. Overall, these studies underscore the importance of studying the entire bone marrow, including the microenvironment, if we are to improve our understanding of the pathophysiology of MDS and further improve therapy.