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BACKGROUND: Overall survival of adult acute myeloid leukemia (AML) remains poor due to the lack of novel and effective therapies. The cancer cell death induced by some chemotherapeutic agents, especially anthracyclines, such as daunorubicin (DNR), named immunogenic cell death (ICD), is characterized by intra- and peri-cellular modifications, which favor the induction of anti-tumor T-cell immune response. Among them, the extracellular release of adenosine triphosphate (extracellular ATP, eATP) from dying tumor cells primes dendritic cells (DCs) by activating purinergic P2X7 receptors, thus eliciting the presentation of tumor antigens to T cell. DCs are key regulators of adaptive immunity, promoting or suppressing T-cell responses. One of the suppressive mechanisms involves the expression of indoleamine 2,3-dioxygenase 1 (IDO1), which plays a major role in the induction of T-cell tolerance through the expansion of regulatory T cells (Tregs). The present study aimed at evaluating the involvement of IDO-1 during ATP-driven ICD in AML. METHODS: AML patients were analyzed at diagnosis and after DNR-based chemotherapy. Ex vivo T cells were characterized by FACS and tested for their capacity to produce IFN-γ in response to autologous blasts. Then, CD8+IFN-γ-producing T cells were expanded and further characterized. ATP was used as an ICD representative model. In vitro, murine WEHI-3B and human HL-60 leukemic cell lines and primary blasts were tested for ATP release after DNR treatment. To in vivo investigate DNR-induced ICD, WEHI-3B cells stable transfected with luciferase PmeLUC were inoculated subcutaneously in BALB/c mice to measure ATP release directly from tumor mass. Tumor infiltrating DCs and T cells were characterized by FACS and immunohistochemistry after chemotherapy and plasma levels of cytokines were measured. In vitro DNR-treated AML cells were pulsed into immature DCs, previously generated from healthy donors. DCs maturation and IDO1 expression were examined (by FACS and western blot, respectively) and correlated with the presence of ATP in culture medium. IDO-driven Tregs induction was assessed. Finally, functional immunological tests were performed in vitro to test the ability of Tregs to inhibit leukemia antigen-specific IFN-γ production (FACS analysis) by ICD-activated T cells. RESULTS: After chemotherapy, 15/23 AML patients had an increase in leukemia-specific IFN-γ producing CD4+ and CD8+ T cells. Also an increase of Tregs was observed with a peak at day 21. CD8+ IFN-γ-producing T cells, which resulted in a skewing toward an effector memory phenotype, were activated and cytotoxic against autologous AML blasts but showed features of exhaustion and were defective in perforin production. In vitro and in vivo DNR induced ATP release from AML cells. In vivo the analysis of tumor-infiltrating T cells after treatment has shown an exhausted phenotype of cytotoxic CD8+ cells, increased IFN-γ+ Tregs and decreased TNF-α+ effector T cells. DNR treatment also increased in vivo plasma levels of cytokines IFN-γ, IL-1β, TNF-α, IL-12. Moreover, in DNR-treated mice we observed a significant increase of CD11c+ mature DCs which express IDO1 in tumor infiltrate. In vitro, loading of DNR-treated AML cells into DCs resulted in increased maturation, but also in IDO1 induction. Interestingly, extracellular ATP was directly involved in DCs maturation and IDO1 expression via purinergic receptor P2Y11. ICD-driven DCs were able to expand Tregs in an IDO-dependent manner. Finally, ICD both triggers a leukemia-specific IFN-γ production by CD8+T cells and induces Tregs, via IDO1-expressing DCs, which in turn inhibit leukemia-specific T cell. CONCLUSIONS: Overall, our data indicate that in AML chemotherapy-induced ICD has contrasting, and not fully elucidated, effects on T-cell immune response, resulting in the induction of leukemia-specific CTLs, albeit with defective features, and Tregs. In this scenario, the effects of ATP release from dying leukemia cells on DCs may be pivotal, as indicated by its capacity to concomitantly induce DC maturation and activation as well as tolerogenic function via IDO1. The combination of novel immunological drugs, such as IDO1 and/or checkpoint inhibitors, with conventional chemotherapy may represent an interesting approach to contrast tolerance induction and, then, fully exploit the immunogenic effect of chemotherapy. Disclosures Martinelli: Genentech: Consultancy; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Ariad: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; MSD: Consultancy; BMS: Speakers Bureau. Cavo:Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

Abstract 4287 Purpose: To evaluate safety, feasibility and anti-leukemia potential of haploidentical KIR-L mismatched natural killer (NK) cell infusion in elderly high risk acute myeloid leukemia (AML) patients. Patients and Methods: Thirteen patients (5 active disease, 2 molecular relapse and 6 complete remissions) with median age 62 years (range 53–73) received NK cell infusion after immunosuppressive chemotherapy (fludarabine/cyclophosphamide), followed by interleukin-2. Highly purified CD56+CD3- NK cells from haploidentical KIR-L mismatched donors were used. The median number of infused NK cells was 2.74 × 106/Kg. T cells were less than 105/Kg. NK cell chimerism, phenotyping, and functional assays were performed. Results: No significant toxicity, including graft versus host disease, related to NK cell infusion was observed. Among patients with active disease, 1/5 obtained transient complete remission (CR), whereas 4/5 patients had no clinical benefit. Both patients in molecular relapse obtained CR, which lasted 9 and 4 months. Three/6 patients in morphologic CR are disease-free after 34, 32 and 18 months. Donor NK cells were demonstrated in the peripheral blood (PB) of all evaluable patients with a peak at day 10 after infusion and, in some cases, also in the bone marrow (BM). NK alloreactivity was demonstrated in vivo by the detection of donor-derived postinfusion NK clones capable of killing recipient targets. Conclusion: Infusion of purified CD56+CD3- NK cells is feasible and safe in elderly high risk AML patients. Adoptively transferred NK cells, which can be detected in PB and BM after infusion, are alloreactive against recipient cells and may induce an anti-leukemic activity. Disclosures: No relevant conflicts of interest to declare.

Alkylphosphocholines are promising new drugs for cancer treatment. They kill cancer cells by evoking multiple signaling pathways: Interference with phospholipid turnover and lipid signaling, Induction of stress signaling (SAPK/JNK) and apoptosis, Inhibition of survival and proliferation pathways (PI3K/AKT, MEK/ERK). We have investigated the therapeutic potential of the novel alkylphosphocholine, erucylphosphohomocholine (ErPC3), on human acute myelogenous leukemia (AML) cells. At variance with other alkylphosphocholines, such as perifosine, ErPC3 can be administered i.v. as it does not cause hemolysis. ErPC3 was tested on THP-1 cells (which display activation of both PI3K/AKT and MEK/ERK pathways), and on HL60 and NB4 cell lines (which only have MEK/ERK activation), as well as primary AML cells. THP-1, HL60, and NB4 cells have a non-functional p53 pathway. At short (6 h) incubation times, the drug blocked AML cells in G2/M phase of the cell cycle, whereas at longer incubation times (24 hr), it decreased survival and induced cell death by apoptosis. The IC50 for ErPC3 with THP-1 cells was 5.0 μM, whereas with HL60 and NB4 cells it was 10.0 μM. In THP-1 cells, ErPC3 caused Akt dephosphorylation on Ser 473 and Thr 308, however it also downregulated total Akt expression levels. Downregulation of total Akt levels was blocked by a caspase-3 inhibitor. At 3 μM ErPC3, induced a complete dephosphorylation of ERK 1/2, whereas a sizable MEK1 dephosphorylation was seen only at 10 μM. The protein phosphatase inhibitor okadaic acid blocked ERK 1/2 dephosphorylation induced by ErPC3, suggesting dephosphorylation was due to increased phosphatase activity. ErPC3 activated JNK 2 (54- kDa), and a JNK 1/2 peptide inhibitor markedly reduced ErPC3-elicited apoptosis in a dose-dependent manner. ErPC3 did not significantly affect the expression of proteins which are involved in mitochondrial control of apoptosis, including Bax, Bcl-XL, Mcl-1, AIF, Bcl2, p-Bcl2, and survivin. Only Puma expression was downregulated. ErPC3 activated apical caspases −2, −8, −9, and −10, as well as the executioner caspase−3. Pharmacological inhibitors of either caspase−3 or −9 completely blocked ErPC3-induced apoptotic cell death. ErPC3 synergized with etoposide (CI:0.16), doxorubicin (CI: 0.48), and mitoxantrone (CI: 0.33) when the drugs were administered together. In contrast, when the chemotherapeutic drugs were administered prior to ErPC3, synergism was detected with mitoxantrone (CI: 0.67) and etoposide (CI: 0.15), whereas combinations with doxorubicin resulted in antagonism (CI:1.71). If ErPC3 was administered prior to the chemotherapeutic drugs, synergism was detected with doxorubicin (CI: 0.79) and etoposide (CI: 0.40), but not with mitoxantrone (CI: 1.41). Moreover, ErPC3 was cytotoxic for AML blasts with activated PI3K/Akt and MEK/ERK pathways (IC50: 10 μM at 72 h) and for AML primary cells with only activation of MEK/ERK signaling (IC50: 13 μM at 72 h). Remarkably, ErPC3 induced a significant apoptosis (30–40%) of primary blast cells, especially in the compartment (CD34+, CD38Low/Neg, CD123+) enriched in putative leukemic stem cells. This observation was also supported by the cytotoxic effects of ErPC3 on AML blasts displaying high aldehyde dehydrogenase activity. ErPC3 also reduced the clonogenic activity of CD34+ cells from AML patients displaying constitutive PI3K/Akt and/or MEK/ERK upregulation, but not CD34+ cells from healthy donors. Our findings indicate that ErPC3, either alone or in combination with existing drugs, is a promising therapeutic agent for the treatment of those AML cases characterized by upregulation of the PI3K/Akt and/or MEK/ERK survival pathways, even in the absence of a functional p53.

The Tumor Necrosis Factor (TNF) family member TNF-Related Apoptosis Inducing Ligand (TRAIL) was originally reported to induce apoptosis in many tumor cells but not in normal cells in vivo and thus represents a promising anticancer cytokine. The in vitro cytotoxic response of acute myelogenous leukemia (AML) cell lines to recombinant TRAIL varies from good to moderate, however, a number of in vitro studies have convincingly demonstrated that AML primary cells are resistant to the proapoptotic activity of TRAIL used as a single agent. To potentiate the response of AML cells to TRAIL cytotoxicity, we have adopted a strategy of combining perifosine, a novel Akt inhibitor, with recombinant TRAIL. The rationale for using such a combination is that perifosine was recently described to increase TRAIL-R2 receptor expression and decrease cellular FLICEinhibitory (c-FLIP, an inhibitor of caspase-8 activation) protein in human lung cancer cells. Both perifosine and TRAIL, when used alone, induced cell death by apoptosis in THP- 1 AML cells, which normally express constitutively active Akt and nonfunctional p53. Perifosine treatment, at concentrations well below the IC50 (0.5 μM), dephosphorylated Akt on Ser473 and increased TRAIL-R2 levels, as demonstrated by flow cytometry, western blot, and RT-PCR. Perifosine also downmodulated cFLIP-L and XIAP levels. However, perifosine did not affect expression of TRAIL-R1 and TRAIL-R4 receptors, or of other proteins which are critical for TRAIL-mediated proapoptotic signaling, including FADD and Mcl-1. Perifosine and TRAIL strongly synergized to induce cytotoxicity as suggested by calculation of the combination index (CI range: 0.15–0.37). The combined treatment resulted in upregulation of caspase-8 activity and apoptosis which was markedly reduced by a selective caspase-8 inhibitor (Z-IETD-FMK). While cFLIP-L and XIAP downregulation was dependent on inhibition of NF-κB activity caused by perifosine, upregulation of TRAIL-R2 expression was dependent on generation of reactive oxygen species (ROS) by perifosine which in turn sequentially activated protein kinase C (PKC)α, JNK2, and c-Jun. A ROS scavenger (N-acetylcysteine), siRNA downregulation of either PKCα or c-Jun, or a JNK1/2 selective pharmacological inhibitor (SP600125), all markedly impaired perifosine-dependent TRAIL-R2 upregulation. Perifosine synergized with TRAIL by inducing apoptosis exclusively in primary AML cells displaying constitutive activation of the Akt pathway. Also in primary AML blasts, perifosine upregulated TRAIL-R2 levels, downmodulated the expression of both c-FLIP and XIAP, and increased Ser 63 p-c-Jun levels, without affecting the expression of FADD. Remarkably, perifosine increased p-JNK2 levels and TRAIL-R2 expression in primary AML patient blasts (CD34+, CD38Low/Neg, CD123+) enriched in putative leukemic stem cells. Perifosine and TRAIL combined treatment was effective in inducing apoptosis (55–60%) in this immature blast population, as documented by a quadruple staining flow cytometric technique for CD34+, CD38Low/Neg, CD123+, Annexin V+ blast cells. The combined treatment negatively affected the clonogenic activity of CD34+ cells from AML patients with Akt activation. In contrast, CD34+ cells from healthy donors and AML patients without Akt activation were resistant to perifosine plus recombinant TRAIL treatment. Our findings suggest that a combination consisting of perifosine plus recombinant TRAIL might offer a novel therapeutic strategy for AML displaying enhanced Akt signaling by overcoming critical mechanisms of apoptosis resistance. Moreover, this kind of combination therapy could be effective also in AML cases exhibiting nonfunctional p53 or low levels of p53.

Natural killer (NK) cells play a critical role in innate immunity and are relevant for the immune control of tumors. Their effector function is regulated by a number of activating and inhibitory receptors, termed killer immunoglobulin receptors (KIRs). In haploidentical T-cell depleted transplantation the donor/recipient KIR mismatch significantly impacts on tumor cell killing, particularly in acute myeloid leukemia (AML). Therefore, haploidentical KIR-mismatched NK cells may be used as adoptive immunotherapy for high risk AML patients in non-transplantation settings. Thirty-one high risk AML patients entered a phase I-II study of NK-cell based immunotherapy and were screened for the avalilability of one haploidentical KIR ligand mismatched donor. Ten of them resulted as having one suitable donor. NK cells were enriched from steady-state leukaphereses by using a double-step immunomagnetic separation system (Miltenyi Biotec, Germany), consisting in depletion of CD3+ T cells followed by positive selection of CD56+ NK cells, and then cryopreserved. CD56+ NK cells were enriched from 8.28±3.7% to 85.9±3.2% (recovery 67.9±15%, viability >92%). So far, 8 patients (1 partial remission, 4 progressions and 3 complete remissions) received NK cell infusion which was preceeded by immunosuppressive chemotherapy, including fludarabine 125 mg/m2 and cyclophosphamide 4g/m2. AML patients received 6 subcutaneous injections of interleukin 2 (IL-2) after adoptive immunotherapy. The median number of reinfused NK cells was 3.6 × 106/Kg and contaminating CD3+ T cells were always less than 1×105/Kg. NK cells were capable to kill NK-sensitive K562 cells in a flow-cytometry-based cytotoxicity assay. The procedure was well-tolerated and no significant toxicity, including GVHD, related to NK cell infusion was observed. The patient in partial remission obtained a complete remission, which lasted for 6 months; among the 4 patients with active/progressive disease 2 patients showed the persistence of disease and one patient died during the aplastic phase. The 3 patients in complete remission are stable after 5, 3 and 4 months of follow up. By using a RT-PCR-based chimerism assay, we demonstrated the presence of adoptively transferred NK cells in 2/4 patients with a peak in circulating NK cell number at day 12 after infusion. Functional biological studies demonstrated the presence of alloreactive NK cell clones in all the donors (at the frequencies of 11 ± 4%) and in 3/4 patients (at the frequences of 16 ± 5%) only at day 3 after infusion of NK cells. In summary, a two-step enrichment of CD56+ NK cells allows the collection of a suitable number of target cells to be used as adoptive immunotherapy in AML patients. Infusion of cryopreserved NK cells is feasible and safe and adoptively transferred NK cells can be detected in the peripheral blood after infusion.