Patient-derived Acute Myeloid Leukemia Blasts Research Articles

The CD33xCD123xCD70 Multispecific CD3-Engaging DARPin MP0533 Induces Selective T Cell-Mediated Killing of AML Leukemic Stem Cells.

The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging designed ankyrin repeat protein (DARPin) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell-mediated killing of AML cells coexpressing at least two of the antigens. In vitro, MP0533 induced selective T cell-mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen-targeting T-cell engagers. In xenograft AML mice models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity toward LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with a high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057).

Abstract 3604: NKX101, an allogeneic off-the-shelf NKG2D CAR-NK cell therapy, has potent in vitro cytotoxicity against patient-derived AML leukemic stem cells and non-leukemic stem cell blasts

Abstract Introduction: Acute myeloid leukemia (AML) is a malignancy of immature myeloid cells characterized by rapid proliferation of abnormal myeloblasts. Treatment options for patients with AML are limited, especially for relapsed/refractory (r/r) disease. Leukemic cells with stem cell features, known as leukemic stem cells (LSCs), have been implicated as the origin of relapse in minimal residual disease positive (MRD+) AML (Khaldoyanidi et al. 2022, Kamel et al. 2022). NKX101 is an allogeneic, off-the-shelf, healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D CAR and membrane bound IL-15. In a clinical trial, NKX101 has shown promise for the treatment of r/r AML, including generating complete responses (CR) with MRD negativity (MRD−). In this study, we utilize a flow cytometry-based approach to (i) evaluate the cell surface expression of NKG2D-Ls on normal cells vs. primary AML blasts, (ii) assess the expression pattern of NKG2D-Ls on AML blast subsets, and (iii) determine NKX101 cytotoxic killing of LSC and non-LSC AML blasts. Methods: NKX101 cells were generated from peripheral blood leukopaks from healthy donors. Cryopreserved bone marrow mononuclear cells (BMMCs) were obtained from AML patients (n=20) and healthy donors (n=10) in accordance with approved IRB protocols. AML somatic mutation status was determined using an Illumina TruSight® Myeloid Sequencing Panel. NKX101 cytotoxicity against patient-derived AML blasts was assessed using a flow cytometry-based assay that measured specific cytotoxicity and AML blast/LSC markers. NKG2D-L expression was determined using flow cytometry with antibodies specific to individual NKG2D-Ls. Results: Utilizing patient-derived primary samples, we demonstrate that NKG2D-Ls are expressed on AML blast cells and expressed at higher levels on AML blast cells compared to healthy normal hematopoietic cells (p = 0.002). Furthermore, we show that NKG2D-Ls are expressed at similar levels on LSCs compared to non-LSC AML blast populations. Target cell specific flow cytometry-based cytotoxicity assays revealed that NKX101 potently kills patient-derived AML blasts in a dose-dependent manner. Moreover, NKX101 had similar cytotoxic potency against both LSC and non-LSC AML blasts. Conclusions: In summary, we show that NKG2D-Ls are expressed on the surface of both LSC and non-LSC AML blasts and at levels above those observed in normal hematopoietic cells. Additionally, the ability of NKX101 to potently kill AML LSC blasts suggests that NKG2D-L targeting may be a viable mechanism for eliminating LSCs in the blood and bone marrow of patients with r/r AML. Taken together, these data support NKG2D-Ls as promising therapeutic targets for AML and support further investigation of NKX101 CAR-NK therapy for the treatment of r/r AML. Citation Format: Kyle Hansen, Cynthia Cho, Nishi Kothari, David Shook, James Trager. NKX101, an allogeneic off-the-shelf NKG2D CAR-NK cell therapy, has potent in vitro cytotoxicity against patient-derived AML leukemic stem cells and non-leukemic stem cell blasts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3604.

Efficient Combinatorial Adaptor-Mediated Targeting of Acute Myeloid Leukemia with CAR T-Cells

Introduction: Chimeric Antigen Receptor (CAR) T-cell therapies are remarkably efficient in treating B- and plasma-cell malignancies by targeting cell-of-origin antigens, eradicating both tumor cells as well as healthy B- and plasma-cell counterparts. Their loss can be compensated by immunoglobulin substitution until they regenerate from hematopoietic stem and progenitor cells (HSPCs). However, similar on-target, off-tumor cell-of-origin elimination would be detrimental in the context of HSPC-derived malignancies, such as acute myeloid leukemia (AML) or myelodysplastic neoplasia (MDS), as the CAR T-cell activity might lead to terminal ablation of hematopoiesis. In principle, CAR T-cells could be used to abrogate both, AML cells as well as HSPCs, followed by HSPC transplantation, ensuring hematopoietic recovery after termination of CAR T-cell activity and possibly without genotoxic preconditioning. This approach could be particularly beneficial for patients unable to tolerate radiation and high-dose chemotherapy. In order to address this challenge, we developed adaptor-mediated CAR T-cells displaying the single-chain variable fragment E2, AdFITC(E2), that binds to fluorescein conjugated to antigen-binding adaptors in diabody format (Db-FM). Such tagged adaptors, used as single linkers or in combination, would then act as safety switch, modulating CAR T-cell activation, proliferation, and lytic activity. Results: We generated second-generation AdFITC(E2)-CAR T-cells and multiple diabody-based adaptors against selected AML antigens. We then tested the diabodies targeting CD33 and CD117 with respect to their ability to mediate CAR T-cell biocidal activity as single adaptors or in combination. Combinatorial staining with CD117 Db-FM and CD33 Db-FM, each added at saturating concentration for the cognate antigen, resulted in higher fluorescein decoration of target cells, with more than two-fold increased fluorescence intensity detected by flow cytometry upon combined staining, thus enhancing AdFITC(E2)-CAR T-cell target density. In vitro cytotoxicity assays of healthy-donor-derived AdFITC(E2)-CAR T-cells, both against cell lines (MOLM14-CD117 +GFP +Luc +) as well as patient-derived AML blasts, revealed that dual adaptor use significantly improved tumor cell lysis compared to equimolar concentration of single adaptors. To further investigate the basis for increased killing by dual targeting, we performed live-cell imaging of effector-target cell interactions in microwells. The addition of both CD117 and CD33 Db-FM to the co-culture of MOLM14-CD117 +GFP +Luc + and AdFITC(E2)-CAR T-cells resulted in faster tumor cell lysis (measured by influx of PI) which correlated with a more rapid, durable engagement time of CAR T-cells with target cells. We next tested CAR T-cell biocidal activity in therapeutic xenogeneic mouse models engrafted with MOLM14-CD117 +GFP +Luc + where AdFITC(E2)-CAR T-cells in combination with CD117 or CD33 Db-FM administered as single adaptors were as efficient as direct CAR T-cells against the same antigens. Injection of AdFITC(E2)-CAR T-cells and both CD117 and CD33 Db-FM effectively inhibited tumor growth, outperforming monotherapies, and leading in some cases to full AML elimination in bone marrow and blood as shown by bioluminescence and terminal flow cytometry analysis (Figure 1). Conclusions: We here tested an adaptor CAR T-cell approach using multiple adaptors to on-off modulate and enhance CAR T-cell activity, focusing on AML as a relevant disease model with high clinical need for therapeutic improvement. The high heterogeneity in antigen expression on AML cells supports a combinatorial targeting strategy, individualized based on the respective AML immunophenotype. Due to their relatively small molecular weight (~55kDa), diabody-based adaptors are rapidly cleared from the body via the kidneys allowing for rapid control over AdFITC-CAR T-cell on-off activity prior to HSPC transplantation. We envision that this approach has the clinical potential to improve CAR T-cell safety profiles by enhancing the specificity towards target cells, while ameliorating the side-effects against healthy tissues expressing only one antigen of the combination.

CKS1 inhibition depletes leukemic stem cells and protects healthy hematopoietic stem cells in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive hematological disorder comprising a hierarchy of quiescent leukemic stem cells (LSCs) and proliferating blasts with limited self-renewal ability. AML has a dismal prognosis, with extremely low two-year survival rates in the poorest cytogenetic risk patients, primarily due to the failure of intensive chemotherapy protocols to deplete LSCs, and the significant toxicity towards healthy hematopoietic cells. Whilst much work has been done to identify genetic and epigenetic vulnerabilities in AML LSCs, little is known about protein homeostasis in drug resistance and relapse. By targeting the proteostatic regulator CKS1, we demonstrate a dual role for CKS1-dependent protein degradation in reducing AML blasts in vivo, and importantly depleting LSCs, whilst inhibition of CKS1 has the opposite effect on normal hematopoiesis, protecting normal hematopoietic stem cells from chemotherapeutic toxicity. Together these findings demonstrate CKS1-dependent proteostasis is a key vulnerability in malignant stem cell biology.

Multi-Antigen Primed T Cells Promote Apoptosis of Acute Myeloid Leukemia (AML)

IntroductionAcute myeloid leukemia (AML), the most common acute leukemia in adults, is characterized by uncontrolled proliferation of immuature myeloid cells. Despite newly approved drugs, AML remains largely incurable due to the persistence of the leukemia stem cell (LSC) population which lie quiescent in the bone marrow niche. Immunotherapy has potential to eradicate LSCs, however, no unique LSC immunophenotype has been identified. Moreover, it is necessary to simultaneously target multiple antigens (Ags) to prevent immune escape and to overcome refractory disease. We present in vitro studies in support of a therapeutic platform capable of targeting multiple intracellular Ags which could meet this challenge. The adoptive transfer of activated T cells primed to engage diverse AML associated epitopes by ex vivo exposure to artificial Ag presenting cells (aAPC) has the potential to eliminate both primary leukemia blasts and LSCs.HypothesisEx vivo enrichment and expansion (E+E) of antigen-educated CD8+ T cells recognizing 5 peptides derived from 3 proteins, Cyclin A1, PRAME and WT1, can selectively identify, engage, and kill AML cell lines or patient-derived (PD) AML blasts in a HLA A*02:01 restricted manner in vitro.Materials and MethodsT cells from the peripheral blood mononuclear cell fraction of a healthy HLA A*02:01 donor were enriched for antigen-educated CD8+/CD4 -T cells. These cells were cultured with nanoparticles decorated with the 5 peptides and a costimulatory protein, resulting in the activation and expansion of those T cells expressing the cognate T cell receptors. These cells are composed of ~97% abT cells, 3% gdT cells and ~13% T scm, 41.5% T cm, 39.5%T em, 6%T emra and 1% T n.ResultsEx vivo expanded educated T cells exhibit target-specific anti-AML activity. T cell mediated cell apoptosis of HLA-matched THP1 cells is dose and time-dependent. At 10:1 effector to target (E:T) ratio, ~28% apoptosis occurred at 24 hrs, while apoptosis was at basal levels when antigen non-educated T cells were used (data not shown). Studies were extended to PD AML cells (Fig. 1A: 012; Fig. 1B: 415) where antigen educated T cells elicited rapid (<16 hrs) and extensive (~90%) apoptosis of target PD AML cells at all E:T ratios examined. Time lapse photography of T cell/PD AML incubations revealed antigen-educated T cells clustered around AML cells (Fig. 2A), a fraction of the latter disappearing over the course of 12 hrs while PD AML cells incubated with non-educated T cells (Fig. 2B) remained viable over 12 hrs. Furthermore, there is little or no T cell movement or clustering in the wells with unprimed, non-active T cells.Release of IFN-γ by educated T cells. T cells (Fig.3A: antigen-educated throughE+E) were incubated at E:T::5:1 for 24 to 48 hrs and IFN-γ in supernatants measured. The fold difference over non-educated T cells incubated with AML cells for the same time is shown and can reach over 5-fold. IFN-γ accumulation was time-dependent where antigen-educated T cells were combined with HLA-A2 matched THP1 or PD AML cells (012, 415, 470). Educated T cells were not active against target cells lacking HLA-A2 (K562) demonstrating HLA restricted killing (Fig. 3B). Additionally, antigen-educated T cells incubated without any target released slightly more IFN-γ than non-educated T cells under similar conditions but AML cells fail to stimulate IFN-γ release from non-educated T cells (data not shown).ConclusionsWe demonstrate HLA restricted cytotoxic activity of antigen-educated T cells against THP1 AML cells and PD AML blasts as shown by flow cytometry and microscopy. Consistent with target cell death, the supernatants from assays with antigen-educated T cells and HLA A*02:01 AML target cells exhibited over 5-fold more IFN-γ than media from assays of non-educated cells under identical conditions. Under these in vitro conditions, PD AML blasts were more readily killed than THP1 cells perhaps due to higher target antigen density (data not shown). These results support the use of multi-antigen-educated T cells for adoptive transfer to treat AML. To investigate the safety and establish the recommended phase II dose, a multi-center Phase I clinical study is underway in relapsed AML post-allo-HCT (NCT 04284228). Future studies will incorporate new antigens to enable broader targeting of a heterogeneous population of AML within and across patients [Display omitted] DisclosuresOelke: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Marcucci: Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings. Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Hansa Biopharma: Consultancy; Neximmune: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy.

The Impact of Hypomethylating Agents and BCL-2 Inhibitor on HLA Expression on THP-1 Cells

Note: BP, MV and LG, KG contributed equallyBackgroundRelapsed acute myeloid leukemia (AML) remains the most common reason for allogeneic hematopoietic cell transplant (HCT) failure. Thus, understanding AML immune escape mechanism is important for improving the odds of curing HCT patients with AML. Downregulation of HLA Class I and II expression by AML is one of the potential immune escape mechanisms. Therefore, treatment to restore HLA surface expression is crucial to prevent and treat relapse. Endogenous cytokines, such as IFN-γ, have been shown to stimulate HLA expression but are poorly tolerated by patients. However, two hypomethylating agents (HMA), decitabine (Dec) and azacitadine (Aza), that are routinely used in AML treatment are known to augment HLA expression. For AML, HMAs are often combined with venetoclax (Ven), a drug that blocks the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein. Thus, while HMAs have been reported to increase HLA expression, what is unknown is whether these agents impact individual HLA loci differently and whether Ven has any impact on HLA expression. To address these questions, we treated the THP-1 cell line with Dec, Aza or Ven and measured changes in cell-surface expression of HLA proteins by flow cytometry using locus-specific HLA mAbs.MethodsTHP-1 cells were incubated with IFN-γ (500 U/mL), Aza (2µM), Dec (5µM), or Ven (30nM) for 48 hours (drug concentrations were determined by earlier titration experiments). THP-1 cells are a monocytic cell line, derived from the peripheral blood of a childhood case of acute monocytic leukemia (M5 subtype), that express HLA Class I and HLA-DR but not HLA-DQ or -DP under basal conditions, although they are inducible by IFN-γ. Thus, the induction of HLA Class II expression by IFN-γ serves as a positive control. Isotype controls were included to measure background. Data is presented as the difference in MFI (delta MFI) between cells treated with a drug and those treated with diluent only.ResultsTreatment of THP-1 cells with either IFN-γ or Dec led to increases in Class I HLA-A, -B & -C (Figure 1) compared to untreated cells (a mean fold increase of 1.4 and 1.2, respectively). Notably, Aza did not stimulate additional HLA-C expression and induced less of an increase in HLA-A & -B expression (an increase of 1.1-fold) than IFN-γ or Dec. Treatment of THP-1 cells by Ven did not induce a change in HLA Class I expression.For Class II, IFN-γ or Dec increased HLA-DR, -DQ and -DP expression in comparison to untreated cells (Figure 1). IFN-γ induced greater HLA-DR expression compared to Dec (an increase of 2.3-fold and 1.5-fold, respectively), and both stimulated similar increases in HLA-DQ (increases of 1.5-fold and 1.4-fold, respectively) & -DP (increases of 1.9-fold and 1.5-fold, respectively). However, treatment of cells with either Aza or Ven did not lead to changes in HLA Class II expression.DiscussionPrevious studies have illustrated the ability of IFN-γ to induce HLA Class II expression in THP-1 cells, however, data for Dec to induce HLA Class II expression was unconfirmed. We report differences in the degree to which IFN-γ and Dec are capable of stimulating HLA-DR with IFN-γ being more potent. The inability of Aza to induce HLA Class II expression in THP-1 cells may be related to the differing drug activating pathways of the two HMAs. Indeed, there are conflicting reports as to whether Aza can stimulate HLA Class II expression. Though Ven treatment of THP-1 cells did not impact HLA expression, because it is given with HMAs, it remains to be seen what effect these drugs may have on HLA expression when administered together. Additional studies to confirm these observations in patient-derived AML blasts are ongoing.ConclusionWe report that HMAs increased expression of HLA-A, -B, & -C loci and Dec but not Aza stimulated HLA-DR, -DQ, and -DP expression in THP-1 cells. Given these data, Dec may be superior in increasing HLA Class II expression post-HCT. [Display omitted] DisclosuresMarcucci: Abbvie: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings. Al Malki: Neximmune: Consultancy; CareDx: Consultancy; Jazz Pharmaceuticals, Inc.: Consultancy; Rigel Pharma: Consultancy; Hansa Biopharma: Consultancy.

Targeting Acute Myeloid Leukemia Using the RevCAR Platform: A Programmable, Switchable and Combinatorial Strategy.

Simple SummaryAcute myeloid leukemia (AML) is a type of blood malignancy particularly affecting the myeloid lineage and one of the most common types of leukemia in adults. It is characterized by high heterogeneity among patients leading to immune escape and disease relapse, which challenges the development of immunotherapies such as chimeric antigen receptor (CAR) T-cells. In this way, the aim of our work was to establish the modular RevCAR platform as a combinatorial tumor targeting approach for the treatment of AML. Herein, we demonstrate the preclinical flexibility and efficiency of RevCAR T-cells in targeting patient-derived AML cells expressing CD33 and CD123. Furthermore, AND gate logic targeting these antigens was successfully established using the RevCAR platform. These accomplishments pave the way towards the future clinical translation of such an improved and personalized immunotherapy for AML patients aiming long-lasting anticarcinogenic responses.Clinical translation of novel immunotherapeutic strategies such as chimeric antigen receptor (CAR) T-cells in acute myeloid leukemia (AML) is still at an early stage. Major challenges include immune escape and disease relapse demanding for further improvements in CAR design. To overcome such hurdles, we have invented the switchable, flexible and programmable adaptor Reverse (Rev) CAR platform. This consists of T-cells engineered with RevCARs that are primarily inactive as they express an extracellular short peptide epitope incapable of recognizing surface antigens. RevCAR T-cells can be redirected to tumor antigens and controlled by bispecific antibodies cross-linking RevCAR T- and tumor cells resulting in tumor lysis. Remarkably, the RevCAR platform enables combinatorial tumor targeting following Boolean logic gates. We herein show for the first time the applicability of the RevCAR platform to target myeloid malignancies like AML. Applying in vitro and in vivo models, we have proven that AML cell lines as well as patient-derived AML blasts were efficiently killed by redirected RevCAR T-cells targeting CD33 and CD123 in a flexible manner. Furthermore, by targeting both antigens, a Boolean AND gate logic targeting could be achieved using the RevCAR platform. These accomplishments pave the way towards an improved and personalized immunotherapy for AML patients.

Therapy‐Induced Senescence Is a Predictor of Treatment Outcome in Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease, for which biologically grounded predictors of outcome remain an urgent clinical need. In addition to tumor cell death, antineoplastic drugs have been shown to mediate a long-lasting growth arrest of vital, metabolically active tumor cells termed therapy-induced senescence (TIS) in various cancer entities and model systems, but predictive investigations on TIS are lacking. We hypothesized here that an ex vivo assessment of TIS in blast samples from AML patients at diagnosis might serve as a biomarker of outcome.Methods: We established assays to quantitatively detect TIS in a primary culture setting of purified primary human AML specimens (n > 30) assessed through cytochemical and fluorescence-based analysis of senescence-associated β-galactosidase (SA-β-gal) activity and other senescence markers (such as p16INK4a, H3K9me3) in concert with downregulation of proliferation markers (Ki67, EdU). AML samples were subjected to standard karyotype and molecular genetics analyses, defining risk groups according to the European Leukemia Net (ELN) genetic risk stratification.Results: We found the individual fractions of senescent AML blasts in response to the standard anti-leukemic agents Daunorubicin and Cytarabine samples to be positively correlated with better disease-free (p= 0.042) and overall survival (p= 0.004) of the patients. Furthermore, a more favorable molecular risk group (p= 0.005), normal karyotype (p= 0.007), and NPM1 mutations (p= 0.018) as well as DNMT3A mutations (p < 0.001) were associated with higher TIS levels in our ex vivo senescence assay.Discussion: Our study is the first report providing direct evidence that cellular senescence induced in patient-derived AML blasts by chemotherapeutic drugs ex vivo serves as a predictor of patient long-term responsiveness to standard induction therapy. TIS might explain the underlying biology of current para-clinical risk indicators, and also seems to identify subgroups of superior outcome in pre-defined risk groups including patients with DNMT3A-mutant disease. Additional investigations exploring senescence in response to other AML-relevant agents and functional analyses expanding to patient-derived xenograft (PDX) AML models will be presented at the meeting. DisclosuresNo relevant conflicts of interest to declare.

A trispecific killer engager molecule against CLEC12A effectively induces NK-cell mediated killing of AML cells

The low five-year survival rate for patients with acute myeloid leukemia (AML), primarily caused due to disease relapse, emphasizes the need for better therapeutic strategies. Disease relapse is facilitated by leukemic stem cells (LSCs) that are resistant to standard chemotherapy and promote tumor growth. To target AML blasts and LSCs using Natural Killer (NK) cells, we have developed a trispecific killer engager (TriKE™) molecule containing a humanized anti-CD16 heavy chain camelid single domain antibody (sdAb) that activates NK cells, an IL-15 molecule that drives NK cell priming, expansion and survival, and a single-chain variable fragment (scFv) against human CLEC12A (CLEC12A TriKE). CLEC12A is a myeloid lineage antigen that is highly expressed by AML cells and LSCs, but not expressed by normal hematopoietic stem cells (HSCs), thus minimizing off-target toxicity. The CLEC12A TriKE induced robust NK cell specific proliferation, enhanced NK cell activation and killing of both AML cell lines and primary patient derived AML blasts in vitro while sparing healthy HSCs. Additionally, the CLEC12A TriKE was able to reduce tumor burden in pre-clinical mouse models. These findings highlight the clinical potential of the CLEC12A TriKE for the effective treatment of AML.

Mechanistic basis and efficacy of targeting the β-catenin–TCF7L2–JMJD6–c-Myc axis to overcome resistance to BET inhibitors

AbstractThe promising activity of BET protein inhibitors (BETi's) is compromised by adaptive or innate resistance in acute myeloid leukemia (AML). Here, modeling of BETi-persister/resistance (BETi-P/R) in human postmyeloproliferative neoplasm (post-MPN) secondary AML (sAML) cells demonstrated accessible and active chromatin in specific superenhancers/enhancers, which was associated with increased levels of nuclear β-catenin, TCF7L2, JMJD6, and c-Myc in BETi-P/R sAML cells. Following BETi treatment, c-Myc levels were rapidly restored in BETi-P/R sAML cells. CRISPR/Cas9-mediated knockout of TCF7L2 or JMJD6 reversed BETi-P/R, whereas ectopic overexpression conferred BETi-P/R in sAML cells, confirming the mechanistic role of the β-catenin–TCF7L2–JMJD6–c-Myc axis in BETi resistance. Patient-derived, post-MPN, CD34+ sAML blasts exhibiting relative resistance to BETi, as compared with sensitive sAML blasts, displayed higher messenger RNA and protein expression of TCF7L2, JMJD6, and c-Myc and following BETi washout exhibited rapid restoration of c-Myc and JMJD6. CRISPR/Cas9 knockout of TCF7L2 and JMJD6 depleted their levels, inducing loss of viability of the sAML blasts. Disruption of colocalization of nuclear β-catenin with TBL1 and TCF7L2 by the small-molecule inhibitor BC2059 combined with depletion of BRD4 by BET proteolysis-targeting chimera reduced c-Myc levels and exerted synergistic lethality in BETi-P/R sAML cells. This combination also reduced leukemia burden and improved survival of mice engrafted with BETi-P/R sAML cells or patient-derived AML blasts innately resistant to BETi. Therefore, multitargeted disruption of the β-catenin–TCF7L2–JMJD6–c-Myc axis overcomes adaptive and innate BETi resistance, exhibiting preclinical efficacy against human post-MPN sAML cells.

Anticancer Activity of a Highly Potent Small Molecule Tubulin Polymerization Inhibitor, AB8939

We have identified the small chemical molecule AB8939 as being a structurally novel, synthesized tubulin inhibitor that can circumvent resistance mechanisms known to limit the effectiveness of existing tubulin inhibitors; e.g., P-glycoprotein (Pgp) and myeloperoxidase (MPO) mediated resistance. A series of in vitro preclinical studies provide proof-of-concept that AB8939 has broad applicability as a potent anticancer drug, particularly in tumors of hematopoietic and lymphoid tissues, including acute myeloid leukemia (AML). Regarding mechanism of action, x-ray crystallography demonstrated that AB8939 binds to the colchicine-binding site on the beta-subunit of tubulin. Cell cycle arrest in the G2/M phase was evaluated using HCT116 cells (a human colorectal tumor), treated at various concentrations of AB8939 for 24 hours. It was seen that AB8939 produced a strong mitotic arrest at the sub-micromolar concentration range (90% of cells in G2/M phase at 10 nM), which was of comparable strength to that of established microtubule targeting agents, each at a concentration of 100 nM. Additional assays using cytarabine (Ara-C) resistant MOLM14 AML cells confirmed this activity, also demonstrating dose dependent (2 to 20 nM) G2/M phase cell cycle arrest in patient-derived AML blasts and that G2/M cell cycle arrest lead to cellular death by apoptosis at nanomolar concentrations. The effect of AB8939 (100 nM) on the integrity of the microtubule and actin networks was tested in 3T3NIH cells (murine embryonic fibroblast cell line). AB8939 induced a rapid (within 1 hour) and radical destabilization of the microtubule network but did not affect the actin network. Similarly, destabilization of the microtubule network was observed in human primary cardiomyocytes and primary human lung fibroblast cells treated for 24 hours at 10 to 1000 nM AB8939. Further in vitro analysis showed that AB8939 produces a direct and potent, dose-dependent depolymerization effect (50% inhibition of in vitro microtubule polymerization at around 1 µM, with 100% inhibition at &gt;5 µM). The potential of AB8939 to overcome resistance to chemotherapeutic agents in Pgp-dependent multidrug-resistant cell lines was assessed using the drug-sensitive human sarcoma cell line MES-SA (parental) and its multidrug-resistant counterparts MES-SA/MX2 and MES-SA/Dx5 in a 6-day proliferation/survival assay. AB8939 efficiently inhibited each of these cells with an IC50 ≤10 nM. By comparison, the MES-SA/MX2 and MES-SA/Dx5 cell lines were highly resistant to the chemotherapeutic agents of doxorubicin and vincristine, as compared with the effect on parental cells (IC50 &lt;1.5 - 2.0 µM versus 20 nM, respectively). Additional tests showed that AB8939 is a very poor substrate of Pgp efflux pump, comparable with combretastatin-4, and therefore has the potential to overcome multidrug resistance in cancer patients. The anti-proliferative activity of AB8939 in various hematopoietic tumors and solid tumors was evaluated using a colorimetric cell proliferation and viability assay. AB8939 produced good anti-tumor activity after 72 hours (IC50 of ≤50 nM) in 19 hematopoietic tumor cell lines tested, including AML (3 cell lines), B cell lymphoma (8 cell lines), T cell lymphoma (6 cell lines), and multiple myeloma (2 cell lines). AB8939 also showed good anti-tumor activity after 6 days (IC50 of ≤10 nM) in several solid tumor cell lines, including breast, colon, glioblastoma, head and neck, lung, kidney, melanoma neuroblastoma, ovary, pancreas and prostate cell lines. The therapeutic potential of AB8939 in refractory/resistant AML was investigated further on doxorubicin-resistant AML cell lines (HL60 and U937), doxorubicin being a commonly used AML induction drug and Pgp substrate. AB8939 produced a strong anti-proliferative effect in both cell lines whereas both were resistant to doxorubicin, thus demonstrating AB8939's potential to overcome refractory/resistant AML. Notably, HL60 and U937 are respectively MPO-positive and MPO-negative, indicating that unlike vinca alkaloids (e.g. vincristine or vinblastine) AB8939 it is not deactivated by this myeloid enzyme. These data show that AB8939 is a prolific and highly potent (nanomolar concentrations) Pgp-independent, next-generation microtubule-destabilizer drug for cancer therapy; in particular, difficult to treat hematopoietic tumors such as relapsed/refractory AML. Disclosures Humbert: AB Science: Employment. Goubard:AB Science: Employment. Mansfield:AB Science: Employment, Patents &amp; Royalties. Hermine:AB Science: Membership on an entity's Board of Directors or advisory committees. Dubreuil:AB Science: Employment, Membership on an entity's Board of Directors or advisory committees, Research Funding. AB8939 Study Group:AB Science: Consultancy, Employment.

A Chemo-enzymatically Linked Bispecific Antibody Retargets T Cells to a Sialylated Epitope on CD43 in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a high-risk disease with a poor prognosis, particularly in elderly patients. Because current AML treatment relies primarily on untargeted therapies with severe side effects that limit patient eligibility, identification of novel therapeutic AML targets is highly desired. We recently described AT1413, an antibody produced by donor B cells of a patient with AML cured after allogeneic hematopoietic stem cell transplantation. AT1413 binds CD43s, a unique sialylated epitope on CD43, which is weakly expressed on normal myeloid cells and overexpressed on AML cells. Because of its selectivity for AML cells, we considered CD43s as a target for a bispecific T-cell-engaging antibody (bTCE) and generated a bTCE by coupling AT1413 to two T-cell-targeting fragments using chemo-enzymatic linkage. In vitro, AT1413 bTCE efficiently induced T-cell-mediated cytotoxicity toward different AML cell lines and patient-derived AML blasts, whereas endothelial cells with low binding capacity for AT1413 remained unaffected. In the presence of AML cells, AT1413 bTCE induced upregulation of T-cell activation markers, cytokine release, and T-cell proliferation. AT1413 bTCE was also effective in vivo. Mice either coinjected with human peripheral blood mononuclear cells or engrafted with human hematopoietic stem cells [human immune system (HIS) mice] were inoculated with an AML cell line or patient-derived primary AML blasts. AT1413 bTCE treatment strongly inhibited tumor growth and, in HIS mice, had minimal effects on normal human hematopoietic cells. Taken together, our results indicate that CD43s is a promising target for T-cell-engaging antibodies and that AT1413 holds therapeutic potential in a bTCE-format. SIGNIFICANCE: These findings offer preclinical evidence for the therapeutic potential of a bTCE antibody that targets a sialylated epitope on CD43 in AML.

Participation of vitamin D-upregulated protein 1 (TXNIP)-ASK1-JNK1 signalosome in the enhancement of AML cell death by a post-cytotoxic differentiation regimen

Standard therapy for Acute Myeloid Leukemia (AML) is rarely curative, and several suggested improvements have had little success so far. We have reported that in an in vitro model of a potential therapeutic regimen for AML, the activity of cytarabine (AraC) is enhanced by a sequential treatment with a combination of the vitamin D2 analog Doxercalciferol (D2) and the plant-derived antioxidant carnosic acid (CA). Importantly, the enhancement occurred selectively in patient-derived AML blasts, but not in the normal bone marrow cells. We now demonstrate that TXNIP, previously known as Vitamin D up-regulated protein 1 (VDUP1) [PMID 808674] plays a part in signaling cell death (CD) in this regimen. This is shown by the reduced CD when TXNIP protein levels are decreased by the CRISPR/CAS9 or RNAi technology. Further, we show that direct activation of ASK1 kinase by TXNIP is required for the optimal transmission of the CD signal to apoptotic machinery, regulated by JNK and BIM. These studies provide a rationale for a projected clinical trial of this vitamin D-based new therapeutic regimen for AML.

Human Acute Myeloid Leukemia Inhibits Normal Erythroid Differentiation through the Paracrine Effects of IL-6

Acute Myeloid Leukemia (AML) is an aggressive cancer resulting in severe cytopenias related to bone marrow (BM) failure. The common assumption for AML-induced BM failure is overcrowding due to clonal expansion of immature myeloid blasts, leading to failure of normal hematopoiesis. However, in a cohort of 293 AML patients, we found that disease burden (% of blasts determined on diagnostic BM aspirate) did not predict severity of cytopenia (hemoglobin rs=-0.053; p=0.49; WBC rs=-0.030, p=0.70; platelet rs=0.091, p=0.026), strongly arguing against simple crowding as the main mechanism underlying AML-induced BM failure. Thus, the goal of our study is to identify novel mechanism(s) associated with AML-induced BM failure, potentially enabling development of new therapies to improve AML management and reverse morbidity.Conventional xenograft models of human AML do not typically exhibit cytopenias, making them unsuitable to study AML-induced BM failure. We speculated that in the mouse, increased splenic extramedullary hematopoiesis compensated for failed intramedullary hematopoiesis due to AML. To test this hypothesis, we performed surgical splenectomy on NSG mice prior to their transplantation with human AML. Strikingly, splenectomized NSG mice engrafted with primary human AML at a 30-70% disease burden (n=8 for primary AML samples, n=5-10 for each group of splenectomized NSG mice) developed leukopenia and severe anemia compared to sham-operated AML-engrafted controls. AML-engrafted splenectomized NSG mice showed early mortality compared to AML-engrafted NSG mice with intact spleens (10.1 weeks vs. 34.2 weeks p<0.0001). Thus, our splenectomized murine xenograft model uniquely recapitulates human disease and allows for novel characterization of AML-induced BM failure.Utilizing our model, splenectomized NSG mice engrafted with human AML demonstrated depletion of normal hematopoietic progenitors (HSPCs) including HSCs (11.1 fold p<0.0001), MPPs (8.90 fold, p<0.001)), CMPs (11.4 fold p<0.0001), MEPs (1.76 fold p=0.0005) and GMPs (5.07 fold p<0.0001). Focusing on anemia, AML also depleted erythroid progenitors including proerythroblasts (1.39 fold p=0.04), normoblasts (4.96 fold p<0.0001), late normoblasts (10.0 fold p<0.0001), and reticulocytes (4.34 fold p=0.007). The relative preservation of proerythroblasts and depletion of normoblasts indicated that AML blasts impart a specific in vivo differentiation blockade in erythroid differentiation.To explore mechanisms by which AML blasts inhibit normal HSPCs, we generated conditioned media (CM) from patient-derived AML blasts, and found that AML-CM suppressed BFU-E colony formation from normal HSPCs (3.1-5.1 fold). Furthermore, in a direct co-culture system with AML blasts and CD34+ HSPCs, AML blasts inhibited erythroid differentiation from the CFU-E to normoblast stage by 81%. Removing CD34+ cells from the AML co-culture allowed cells to resume differentiation to the proerythroblast stage. These experiments demonstrate that AML imparts a differentiation blockade along the MEP-proerythroblast axis in a cell non-autonomous, reversible fashion.Using cytokine array analysis, we identified elevated IL-6 levels in AML sample-derived CM (n=10, 2841±766.4 pg/ml, 7.80 fold increase, p=0.03) compared to CD34+-derived CM (n=5, 364±36.0 pg/ml). Increased IL-6 was also found in BM aspirates from human AML engrafted splenectomized NSG mice (715±125pg/ml, p=0.001) compared to mice engrafted with normal CD34+ HSPCs (undetectable). Thus, we hypothesized that IL-6 produced by AML blasts acts as a paracrine factor to suppress erythropoiesis. Consistent with this hypothesis, an IL-6 neutralizing antibody reversed the inhibition of BFU-E formation imparted by AML-CM. Furthermore, the addition of recombinant IL-6 to liquid cultures of erythroid differentiation resulted in a 23% reduction in proerythroblasts.Together, our data suggests that (1) overcrowding is not the primary mechanism resulting in BM failure in AML; (2) AML blasts play a previously unrecognized role in imparting a differentiation blockade along the MEP-proerythroblast axis, resulting in progressive anemia; and (3) this differentiation blockade is at least partially attributable to IL-6 secreted from AML blasts as a paracrine factor. DisclosuresNo relevant conflicts of interest to declare.

Abstract 20: Oncolytic virotherapy enhances Smac mimetic treatment of acute myeloid leukemia

Abstract This study demonstrates that the combination of Smac mimetic and oncolytic virotherapy (OVT) induces cell death in acute myeloid leukemia (AML) cell lines and primary patient samples; this is the first time the efficacy of this combination approach has been investigated in a human cancer model and for the treatment of AML. Methods: The Smac mimetic LCL161 and several oncolytic viruses (OVs) were tested for in vitro cytotoxicity against a panel of diverse AML cell lines. Bystander killing of LCL161-treated AML cells was explored using conditioned media from OV-treated PBMCs. Multiplex immunoassays were used to detect potential mediators of bystander cytotoxicity produced following OV treatment. Several upregulated cytokines were tested for their ability to enhance LCL161 toxicity in AML cell lines by MTS assay. The activation of healthy donor peripheral blood mononuclear cells (PBMC) was investigated by flow cytometry and immune cell-mediated death of AML cells was measured using chromium release assays. In vitro direct and bystander toxicity was further verified using fresh blood samples from AML patients. Results: A rhabdovirus, MG1, showed the greatest cytotoxicity across a panel of AML cell lines compared to several other OVs. In resistant cell lines the addition of LCL161 treatment led to increased cell death, indicating the potential merits of a combination strategy. Primary AML samples exposed to both LCL161 and MG1 showed greater levels of cell death than either treatment alone, confirming combinational efficacy. Treating healthy donor PBMCs with virus induced the activation of innate immune effector cells, as indicated by an increase in CD69 on NK cells as well as stimulating CD14+ monocytes to produce membrane-bound TNFα-related apoptosis-inducing ligand (TRAIL). Immune cell-mediated death was increased following activation by MG1, and LCL161 sensitized AML cell lines to death by the membrane mimicking KillerTRAIL. Conditioned media from MG1-treated healthy and patient-derived PBMCs displayed an inflammatory milieu that was toxic to AML cell lines. Further analysis identified several cytokines relevant to AML therapy, including the anti-viral interferon alpha (IFNα), soluble TRAIL, and tumor necrosis factor alpha (TNFα); LCL161 was able to increase the sensitivity of AML cells to MG1-conditioned media and recombinant versions of IFNα and TNFα. Discussion: OVT has shown positive clinical efficacy in many solid tumors; however, it is underexplored in the context of hematologic malignancies. Many cases of spontaneous remission following viral infection, the correlation of cytomegalovirus reactivation and positive prognosis following hematologic stem cell transplant, along with the identification of OVs which target leukemic cells without harming healthy hematologic stem cells, suggest that AML patients may be suitable for OVT. Indeed, data from our lab suggest that PBMCs from AML patients are able to recognize and respond to OV, to induce antitumor cytotoxicity. MG1 is cytotoxic to AML cells and acts as an immunogenic agent to induce the release of soluble inflammatory mediators by PBMCs that lead to the death of bystander cells without the need for direct infection. Recent studies into the use of Smac mimetics in hematologic malignancies have demonstrated the synergistic action of combining treatment with inflammatory cytokines, suggesting that combination with an immunogenic agent may be beneficial. Indeed, LCL161 is able to sensitize both AML cell lines and patient-derived AML blasts to MG1 treatment. The release of inflammatory mediators following MG1 treatment also stimulates the activation of innate immune effector cells to induce AML cell death, indicating the ability to initiate a cell-mediated innate immune attack on AML cells using this regime. In conclusion, these data suggest that MG1 should be considered in combination with the Smac mimetic, LCL161, for the treatment of AML. Citation Format: Joanne L. Hopper, Louise ME Müller, Victoria A. Jennings, Gina B. Scott, Stewart McConnell, Richard Kelly, Fiona Errington-Mais. Oncolytic virotherapy enhances Smac mimetic treatment of acute myeloid leukemia [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 20.

Development of A Chimeric Antigen Receptor Targeting C-Type Lectin-Like Molecule-1 for Human Acute Myeloid Leukemia

The treatment of patients with acute myeloid leukemia (AML) with targeted immunotherapy is challenged by the heterogeneity of the disease and a lack of tumor-exclusive antigens. Conventional immunotherapy targets for AML such as CD33 and CD123 have been proposed as targets for chimeric antigen receptor (CAR)-engineered T-cells (CAR-T-cells), a therapy that has been highly successful in the treatment of B-cell leukemia and lymphoma. However, CD33 and CD123 are present on hematopoietic stem cells, and targeting with CAR-T-cells has the potential to elicit long-term myelosuppression. C-type lectin-like molecule-1 (CLL1 or CLEC12A) is a myeloid lineage antigen that is expressed by malignant cells in more than 90% of AML patients. CLL1 is not expressed by healthy Hematopoietic Stem Cells (HSCs), and is therefore a promising target for CAR-T-cell therapy. Here, we describe the development and optimization of an anti-CLL1 CAR-T-cell with potent activity on both AML cell lines and primary patient-derived AML blasts in vitro while sparing healthy HSCs. Furthermore, in a disseminated mouse xenograft model using the CLL1-positive HL60 cell line, these CAR-T-cells completely eradicated tumor, thus supporting CLL1 as a promising target for CAR-T-cells to treat AML while limiting myelosuppressive toxicity.

Therapeutic targeting of necroptosis by Smac mimetic bypasses apoptosis resistance in acute myeloid leukemia cells.

Resistance to apoptosis, for example due to overexpression of Inhibitor of Apoptosis (IAP) proteins, is associated with poor prognosis in acute myeloid leukemia (AML). Here, we identify that Smac mimetics such as BV6, which antagonizes IAP proteins, elicit necroptosis in AML cells, in which apoptosis is inhibited pharmacologically by caspase inhibitors or genetically by caspase-8 knockdown. Importantly, BV6 triggers necroptosis also in apoptosis-resistant patient-derived AML blasts, underlining the clinical relevance of our findings. Mechanistically, we show that BV6-induced cell death depends on key components of necroptosis signaling such as RIP1, RIP3 and MLKL, since pharmacological or genetic inhibition of these proteins significantly protects AML cells from BV6-mediated cell death, whereas PGAM5 is dispensable. Interestingly, we identify constitutive tumor necrosis factor-alpha (TNFα) secretion and an autocrine/paracrine TNFα loop as critical mediators of BV6-induced necroptosis in AML cell lines and patient-derived blasts, as the TNFα-blocking antibody Enbrel or tumor necrosis factor-alpha receptor 1 (TNFR1) knockdown significantly rescue cell death. Notably, AML cells exhibit high basal levels of TNFα compared to non-malignant CD34+ cells, which is further increased by BV6. In conclusion, this is the first report showing that Smac mimetics circumvent apoptosis resistance in AML cells by inducing necroptosis in a TNFα-dependent manner, which has important implications for the development of new strategies to overcome treatment resistance in AML.

RALB provides critical survival signals downstream of Ras in acute myeloid leukemia.

Mutations that activate RAS proto-oncogenes and their effectors are common in acute myeloid leukemia (AML); however, efforts to therapeutically target Ras or its effectors have been unsuccessful, and have been hampered by an incomplete understanding of which effectors are required for AML proliferation and survival. We investigated the role of Ras effector pathways in AML using murine and human AML models. Whereas genetic disruption of NRAS(V12) expression in an NRAS(V12) and Mll-AF9-driven murine AML induced apoptosis of leukemic cells, inhibition of phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) signaling did not reproduce this effect. Conversely, genetic disruption of RALB signaling induced AML cell death and phenocopied the effects of suppressing oncogenic Ras directly – uncovering a novel role for RALB signaling in AML survival. Knockdown of RALB led to decreased phosphorylation of TBK1 and reduced BCL2 expression, providing mechanistic insight into RALB survival signaling in AML. Notably, we found that patient-derived AML blasts have higher levels of RALB-TBK1 signaling compared to normal blood leukocytes, supporting a pathophysiologic role for RALB signaling for AML patients. Overall, our work provides new insight into the specific roles of Ras effector pathways in AML and has identified RALB signaling as a key survival pathway.

Redirected EBV-Specific Stem Cell-Memory and Central-Memory CD8+ T Lymphocytes Exhibit Effective and Sustained Antileukemic Immunity to Acute Myeloid Leukemia in a Patient-Tailored Humanized NSG Mouse Model

Introduction: Adoptive cellular therapy (ACT) of donor-derived cytolytic T lymphocytes (CTL) directed to leukemia or herpesvirus has proven promising to improve antiviral and antileukemic immunity in patients following allogeneic hematopoietic stem cell transplantation (AHSCT). However, durable clinical responses are often hampered by detrimental graft-versus host (GvH) reactivity and limited persistence of transferred, fully differentiated antileukemic effector T cells (TEFF). We thus explored memory and tumoricidal features of in vitro generated EBV-specific stem cell-memory T cells (TSCM) and central-memory T (TCM) cells, T cell-receptor (TCR) redirected to primary acute myeloid leukemia (AML) blasts, for improved immunotherapy using a patient-tailored minimal residual leukemia NOD/scidIL2Rcg-null (NSG) mouse model.Methods: Purifiednaive CD8+CD45RA+ T cells from 2 EBV-negative healthy donors were stimulated with EBV-peptide loaded autologous DCs or EBV-transformed B cells (B-LCL) derived from a HLA-class I-matched AML patient in the presence of IL-12, -15, -21 and inhibitors targeting the glycogen synthase kinase-3β thereby blocking the canonical Wnt-signaling pathway for 3-4 weeks. EBV-specific CTL generated from unselected CD8+ T cells served as controls. Phenotype and function as well as metabolic and migratory properties of EBV-specific TSCM/TCM was analyzed by standard assays, glucose uptake and transmigration analyses. αβ-TCR genes isolated from AML-reactive CTL-clones (Albrecht et al. Cancer Immunol. Immunther. 2011), retroviral (RV) transduction and AML-reactivity of eGFP+ TCRVβ7.1+ and TCRVβß21.3+ RV-transduced T(SCM) and T(CM) was tested in vitro according to standard procedures. For in vivo studies redirected T cells were adoptively transferred into AML- and B-LCL-engrafted NSG mice, respectively, and monitored for engraftment, persistence and antileukemic reactivity. All experimental procedures were performed with human specimens obtained with informed consent and were approved by the local ethics committee in compliance of the Helsinki Declaration. Animal studies were approved by institutional supervisory boards and federal law.Results: Upon 3 to 4 weekly stimulations TSCM and TCM expressing a CD8+CD45RA+CD45RO-CD62L+CCR7+ and CD8+CD45RA-CD45RO+CD62L+CCR7+ phenotype, respectively, elicited strong and to EBV-specific CD8+ TEFF comparable reactivity to HLA class I-matched B-LCL. Additionally, reduced glucose uptake but enhanced CCL-21 driven migration properties confirmed their TSCM and TCM status as described previously. Moreover, TCR-redirected EBV-specific CTL elicited comparable reactivity to AML blasts derived from the same patient as the B-LCL when compared with the original AML-reactive CTL. Finally, adoptive transfer of redirected TSCM and TCM into NSG mice engrafted with ≤ 5% of HLA-class I matched patient-derived AML blasts resembling a minimal residual disease situation upon odoptive transfer resulted in strong reduction of AML-burden and long term persistence (>90d) in NSG mice. Additional studies on boosting antileukemic immunity by additional transfer of irradiated autologous B-LCL into treated mice or supporting T cell homeostasis by weekly i.p. injection of a novel IL-15 superagonist are currently in progress.Conclusion: These results show that EBV-specific TSCM and TCM redirected to AML can induce sustained antileukemic immunity and thus might represent a promising tool to improve ACT in AHSCT. DisclosuresNo relevant conflicts of interest to declare.

Upregulation of SOCS-1 by Nutlin-3 in acute myeloid leukemia cells but not in primary normal cells

OBJECTIVE:It has been shown that SOCS-1 plays an important role in the proper control of cytokine/growth factor responses and acts as a tumor suppressor in acute myeloid leukemias. Therefore, the objective of the present study was to evaluate the in vitro effect of treatment with Nutlin-3, a small molecule inhibitor of the MDM2/p53 interaction, on the expression of the suppressor of cytokine signaling 1 in primary acute myeloid leukemia cells and in myeloid cell lines with differential p53 status.METHOD:The expression of the suppressor of cytokine signaling 1 was quantitatively analyzed by real-time PCR in myeloid p53wild-type (OCI and MOLM) and p53null HL-60, leukemic cell lines, in patient-derived acute myeloid leukemia blasts, and in primary normal cell types, such as macrophages, endothelial cells, and bone marrow mesenchymal stem cells. The p53-dependence of the suppressor of cytokine signaling 1 upregulation that is induced by Nutlin-3 was analyzed in experiments performed using siRNA for p53, while the functional upregulation of the suppressor of cytokine signaling 1 was analyzed by assessing the levels of phosphorylated STAT-3.RESULTS:Nutlin-3 significantly upregulated the transcription of the suppressor of cytokine signaling 1 in p53wild-type OCI and MOLM but not in p53deleted p53null HL60, myeloid leukemic cell lines, as well as in primary acute myeloid leukemia blasts. Conversely, and somewhat unexpectedly, Nutlin-3 did not modulate the suppressor of cytokine signaling 1 expression in primary normal macrophages, endothelial cells, and bone marrow mesenchymal stem cells. The p53-dependent upregulation of the suppressor of cytokine signaling 1 by Nutlin-3 was associated with the downregulation of phosphorylated STAT-3, a major molecular target of the suppressor of cytokine signaling 1.CONCLUSION:Overall, our data suggest a potential role for the suppressor of cytokine signaling 1 as a therapeutic target of Nutlin-3 in p53 wild-type acute myeloid leukemias.