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Repression of GSK3 restores NK cell cytotoxicity in AML patients.

ABSTRACT: Natural killer cells from acute myeloid leukaemia patients (AML-NK) show a dramatic impairment in cytotoxic activity. The exact reasons for this dysfunction are not fully understood. Here we show that the glycogen synthase kinase beta (GSK3?) expression is elevated in AML-NK cells. Interestingly, GSK3 overexpression in normal NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK3 inactivation enhances their cytotoxic activity. Mechanistic studies reveal that the increased cytotoxic activity correlates with an increase in AML-NK cell conjugates. GSK3 inhibition promotes the conjugate formation by upregulating LFA expression on NK cells and by inducing ICAM-1 expression on AML cells. The latter is mediated by increased NF-?B activation in response to TNF-? production by NK cells. Finally, GSK3-inhibited NK cells show significant efficacy in human AML mouse models. Overall, our work provides mechanistic insights into the AML-NK dysfunction and a potential NK cell therapy strategy.

SUBMITTER: Parameswaran R

PROVIDER: S-EPMC4822012 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Repression of GSK3 restores NK cell cytotoxicity in AML patients.

Parameswaran Reshmi R Ramakrishnan Parameswaran P Moreton Stephen A SA Xia Zhiqiang Z Hou Yongchun Y Lee Dean A DA Gupta Kalpana K deLima Marcos M Beck Rose C RC Wald David N DN

Nature communications 20160404

Natural killer cells from acute myeloid leukaemia patients (AML-NK) show a dramatic impairment in cytotoxic activity. The exact reasons for this dysfunction are not fully understood. Here we show that the glycogen synthase kinase beta (GSK3β) expression is elevated in AML-NK cells. Interestingly, GSK3 overexpression in normal NK cells impairs their ability to kill AML cells, while genetic or pharmacological GSK3 inactivation enhances their cytotoxic activity. Mechanistic studies reveal that the ...[more]

PMID: 27040177

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Project description:Natural killer (NK) cell-based adoptive immunotherapy in leukemia patients is an emerging field of interest based on clinical evidence of efficacy and safety. Elderly acute myeloid leukemia (AML) patients have been successfully treated with NK cells from HLA-haploidentical donors, especially when high amounts of alloreactive NK cells were infused. The aim of this study was comparing two approaches to define the size of alloreactive NK cells in haploidentical donors for AML patients recruited in two clinical trials with the acronym "NK-AML" (NCT03955848), and "MRD-NK". The standard methodology was based on the frequency of NK cell clones capable of lysing the related patient-derived cells. The alternative approach consisted of the phenotypic identification of freshly derived NK cells expressing, as inhibitory receptors, only the inhibitory KIR(s) specific for the mismatched KIR-Ligand(s) (HLA-C1, HLA-C2, HLA-Bw4). However, in KIR2DS2+ donors and HLA-C1+ patients, the unavailability of reagents staining only the inhibitory counterpart (KIR2DL2/L3) may lead to an underestimated identification of the alloreactive NK cell subset. Conversely, in the case of HLA-C1 mismatch, the alloreactive NK cell subset could be overestimated due to the ability of KIR2DL2/L3 to recognize with low-affinity also HLA-C2. Especially in this context, the additional exclusion of LIR1-expressing cells might be relevant to refine the size of the alloreactive NK cell subset. We could also associate degranulation assays, using as effector cells IL-2 activated donor peripheral blood mononuclear cells (PBMC) or NK cells upon co-culture with the related patient target cells. The donor alloreactive NK cell subset always displayed the highest functional activity, confirming its identification accuracy by flow cytometry. Despite the phenotypic limitations and considering the proposed corrective actions, a good correlation was shown by the comparison of the two investigated approaches. In addition, the characterization of receptor expression on a fraction of NK cell clones revealed expected but also few unexpected patterns. Thus, in most instances, the quantification of phenotypically defined alloreactive NK cells from PBMC can provide data similar to the analysis of lytic clones, with several advantages, such as a shorter time to achieve the results and, perhaps, higher reproducibility/feasibility in many laboratories.

Project description:SOT101 is a superagonist fusion protein of interleukin (IL)-15 and the IL-15 receptor α (IL-15Rα) sushi+ domain, representing a promising clinical candidate for the treatment of cancer. SOT101 among other immune cells specifically stimulates natural killer (NK) cells and memory CD8+ T cells with no significant expansion or activation of the regulatory T cell compartment. In this study, we showed that SOT101 induced expression of cytotoxic receptors NKp30, DNAM-1 and NKG2D on human NK cells. SOT101 stimulated dose-dependent proliferation and the relative expansion of both major subsets of human NK cells, CD56brightCD16- and CD56dimCD16+, and these displayed an enhanced cytotoxicity in vitro. Using human PBMCs and isolated NK cells, we showed that SOT101 added concomitantly or used for immune cell pre-stimulation potentiated clinically approved monoclonal antibodies Cetuximab, Daratumumab and Obinutuzumab in killing of tumor cells in vitro. The anti-tumor efficacy of SOT101 in combination with Daratumumab was assessed in a solid multiple myeloma xenograft in CB17 SCID mouse model testing several combination schedules of administration in the early and late therapeutic setting of established tumors in vivo. SOT101 and Daratumumab monotherapies decreased with various efficacy tumor growth in vivo in dependence on the advancement of the tumor development. The combination of both drugs showed the strongest anti-tumor efficacy. Specifically, the sequencing of both drugs did not matter in the early therapeutic setting where a complete tumor regression was observed in all animals. In the late therapeutic treatment of established tumors Daratumumab followed by SOT101 administration or a concomitant administration of both drugs showed a significant anti-tumor efficacy over the respective monotherapies. These results suggest that SOT101 might significantly augment the anti-tumor activity of therapeutic antibodies by increasing NK cell-mediated activity in patients. These results support the evaluation of SOT101 in combination with Daratumumab in clinical studies and present a rationale for an optimal clinical dosing schedule selection.

Project description:BACKGROUND:Previous evidence demonstrated that restoration of wild type VHL in human renal cancer cells decreased in vitro NK susceptibility. To investigate on the role of tumoral VHL status versus NK capability in renal cancer patients, 51 RCC patients were characterized for VHL mutational status and NK function. METHODS:VHL mutational status was determined by direct DNA sequencing on tumor tissue. NK cytotoxicity was measured against specific target cells K562, VHL-wild type (CAKI-1) and VHL-mutated (A498) human renal cancer cells through externalization of CD107a and IFN-? production. Activating NK receptors, NKp30, NKp44, NKp46, NKG2D, DNAM-1, NCAM-1 and Fc?RIIIa were evaluated through quantitative RT-PCR. RCC tumoral Tregs were characterized as CD4+CD25+CD127lowFoxp3+ and Treg function was evaluated as inhibition of T-effector proliferation. RESULTS:VHL mutations were detected in 26/55 (47%) RCC patients. IL-2 activated whole-blood samples (28 VHL-WT-RCC and 23 VHL-MUT-RCC) were evaluated for NK cytotoxicity toward human renal cancer cells A498, VHL-MUT and CAKI-1, VHL-WT. Efficient NK degranulation and increase in IFN-? production was detected when IL-2 activated whole-blood from VHL-MUT-RCC patients were tested toward A498 as compared to CAKI-1 cells (CD107a+NK: 7?±?2% vs 1?±?0.41%, p?=?0.015; IFN-?+NK: 6.26?±?3.4% vs 1.78?±?0.9% respectively). In addition, IL-2 activated NKs induced higher CD107a exposure in the presence of RCC autologous tumor cells or A498 as compared to SN12C (average CD107a+NK: 4.7 and 2.7% vs 0.3% respectively at 10E:1?T ratio). VHL-MUT-RCC tumors were NKp46+ cells infiltrated and expressed high NKp30 and NKp46 receptors as compared to VHL-WT-RCC tumors. A significant lower number of Tregs was detected in the tumor microenvironment of 13 VHL-MUT-RCC as compared to 13 VHL-WT-RCC tumors (1.84?±?0.36% vs 3.79?±?0.74% respectively, p?=?0.04). Tregs isolated from VHL-MUT-RCC patients were less suppressive of patients T effector proliferation compared to Tregs from VHL-WT-RCC patients (Teff proliferation: 6.7?±?3.9% vs 2.8?±?1.1%). CONCLUSIONS:VHL tumoral mutations improve NKs effectiveness in RCC patients and need to be considered in the evaluation of immune response. Moreover therapeutic strategies designed to target NK cells could be beneficial in VHL-mutated-RCCs alone or in association with immune checkpoints inhibitors.

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Repression of GSK3 restores NK cell cytotoxicity in AML patients.

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Repression of GSK3 restores NK cell cytotoxicity in AML patients.

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