Natural Killer Cell Function, an Important Target for Infection and Tumor Protection, Is Impaired in Type 2 Diabetes

<div><p></p><p>Patients with Type 2 diabetes (T2D) are highly susceptible to infection and have an increased incidence of some tumors, possibly due to immune system dysfunction. In the innate cellular immune system, Natural Killer (NK) lymphocytes are important effectors responsible for controlling infections and combating tumor development. We analyzed NK cell subsets in 51 patients with long-standing T2D. Compared with healthy blood donors, diabetic patients showed a profound decrease in both NKG2D-positive NK cells (44% <i>vs.</i> 55.5%, P<0.01) and NKp46-positive cells (26% <i>vs.</i> 50%, P<0.01). Decreased expression of these receptors was associated with functional defects, such as reduced NK degranulation capacity when challenged with the tumor target cell line K562 (10.3 <i>vs.</i> 15.8%, P<0.05). This defect could be restored <i>in vitro</i> by stimulating NK cells from T2D patients with IL-15 (P<0.05). NKG2D expression was found to be negatively correlated with HBA1c level (r = −0.50; P = 0.009), suggesting that sustained hyperglycemia could directly influence NK cell defects. We demonstrated that endoplasmic reticulum (ER) stress, an important mediator in diabetes-associated complications, was inducible <i>in vitro</i> in normal NK cells and that tunicamycin treatment resulted in a significant decrease in NKG2D expression (P<0.05). Furthermore, markers of the Unfolded Protein Response (UPR) BiP, PDI and sXBP1 mRNAs were significantly increased in NK cells from T2D patients (P<0.05, P<0.01, P<0.05, respectively), indicating that ER stress is activated in vivo through both PERK and IRE1 sensors. These results demonstate for the first time defects in NK cell-activating receptors NKG2D and NKp46 in T2D patients, and implicate the UPR pathway as a potential mechanism. These defects may contribute to susceptibility to infections and malignancies and could be targetted therapeutically.</p> </div

Tunicamycin induces ER stress and UPR activation, and decreases NKG2D expression in normal PBMCs <b><i>in vitro</i></b><b>.</b>

<p>PBMCs from healthy donors were incubated in the absence (Ctrl) or in the presence (Tm) of 1.25 µg/mL tunicamycin for 6 hours. (A) ER stress markers <i>BiP</i>, <i>HERP</i>, <i>GRP94</i> and <i>PDI</i>, (B) IRE1α pathway marker <i>spliced XBP1</i> (s<i>XBP1</i>) and (C) PERK pathway markers <i>ATF4</i>, <i>GADD34</i> and <i>CHOP</i> mRNA amounts were determined by quantitative RT-PCR in normal PBMCs. Transcript levels are presented as mean±SEM (n = 6). *:P<0.05. (D) NKG2D, NKp46 and NKG2C expressions were quantified by flow cytometry (n = 12). *:P<0.05.</p

Demographic characteristics of the 51 diabetic patients.

<p>Demographic characteristics of the 51 diabetic patients.</p

Assessing ER stress and UPR activation in NK cells from diabetic patients.

<p>mRNA levels for <i>BiP</i>, <i>PDI</i> and s<i>XBP1</i> were quantified by quantitative RT-PCR in NK cells from healthy donors (n = 17) and type 2 diabetic patients (n = 18). Transcript levels are presented as mean±SEM. *:P<0.05; **: P<0.01.</p

Decreased NKp46 mRNA expression in NK cells from type 2 diabetic patients.

<p>Quantitative RT-PCR was used to assess expression of NKp46 and NKG2D mRNAs in NK cells from healthy donors (n = 7) and diabetic patients (n = 7). Transcript levels are presented as mean±SEM. Only NKp46 mRNA is down-regulated. *:P<0.05.</p

Decreased functional properties of diabetic NK cells and effects of IL-15.

<p>(A) CD107a degranulation assay using PBMCs from diabetic (n = 18) or control subjects (n = 16) challenged with K562 cells. Representative flow cytometry experiments are shown. (B) Overnight incubation in the presence of IL-15 (10 ng/mL) significantly increases NKG2D expression on NK cells from T2D patients (n = 7), as assessed by flow cytometry. A representative flow cytometry experiment is shown. (C) Stimulation with IL-15 (10 ng/mL) restores CD107a degranulation for PBMCs from diabetic patients (n = 7) challenged with K562 target cells. A representative flow cytometry experiment is shown *: P<0.05.</p

BET inhibitor OTX015 targets BRD2 and BRD4 and decreases c-MYC in acute leukemia cells

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The bromodomain (BRD) and extraterminal (BET) proteins including BRD2, BRD3 and BRD4 have been identified as key targets for leukemia maintenance. A novel oral inhibitor of BRD2/3/4, the thienotriazolodiazepine compound OTX015, suitable for human use, is available. Here we report its biological effects in AML and ALL cell lines and leukemic samples. Exposure to OTX015 lead to cell growth inhibition, cell cycle arrest and apoptosis at submicromolar concentrations in acute leukemia cell lines and patient-derived leukemic cells, as described with the canonical JQ1 BET inhibitor. Treatment with JQ1 and OTX15 induces similar gene expression profile

Abstract 803: Bromodomain and extra-terminal BET inhibitors induce TP53 independent apoptosis, maturation and oncoprotein degradation in NPM1 mutated acute myeloid leukemia

Abstract Background: Differentiation based therapy by all trans retinoic acid (ATRA) and arsenic trioxide (ATO) results in cure of &gt;90% of patients with acute promyelocytic leukemia (APL). ATRA+ATO is highly biologically active in NPM1c AML, accounting for 30-40% of AML patients. ATO/ATRA induces proteasomal degradation of NPM1c, differentiation, growth arrest and TP53 dependent apoptosis in NPM1c cells. Furthermore, ATRA/ATO exposure restores nuclear localization of NPM1wt and significantly reduces blasts in NPMc AML patients. It was shown that the BET inhibitors OTX015/MK-8628 and JQ1 yield antileukemic activity and here we demonstrate their effects in NPM1c leukemia cells compared to ATRA/ATO. Methods : NPMc OCI-AML3 cell line or patient bone marrow (BM) blast cells obtained after informed consent were exposed to ATRA/ATO or OTX015/MK-8628 and JQ1. Apoptosis was assessed by annexin V/PI and caspase 3/PARP cleavage by WB. TP53 expression was detected by WB. Knock down of TP53 was performed with siRNA. Differentiation of OCI-AML3 cells was studied by CD11b surface expression and morphologic studies after MGG stain. Gene expression profiling was performed with GeneChip Array (Affymetrix®). NPMc expression was assessed by WB (+/- bortezomib) and cellular localization of NPMc/NPMwt was studied by immunofluorescence. Results : Exposure of OCI-AML3 cells to OTX015/MK-8628 and JQ1 was more potent to induce apoptosis as compared to ATRA/ATO. All treatments lead to caspase 3 and PARP cleavage. In OCI-AML3 cells, ATO-ATRA induced strong upregulation of genes of the TP53 dependent pathway (BAX/GADD45) while the anti apoptotic gene BCL2 was downregulated. In contrast, treatment with BET inhibitors lead to strong down regulation of the TP53 dependent pathway. In line, ATRA/ATO induced TP53 protein expression and TP53 knock down by siRNA decreased significantly ATRA/ATO induced apoptosis suggesting that apoptosis induced by BET inhibitors is TP53 independent. As compared to ATRA/ATO, OTX015/MK-8628 and JQ1 were more potent to induce differentiation as detected by CD11b surface expression and by morphologic analysis of OCI-AML3 cells. Interestingly, gene expression profiling of human leukocyte differentiation pathways in OCI-AML3 cells revealed different expression profiles for exposure to BET inhibitors compared to ATRA/ATO. Treatment of OCI-AML3 cells either by OTX015/MK8628, JQ1 or ATRA/ATO lead to proteosomal degradation of the NPMc protein. Exposure of OCI-AML3 cells and primary BM blasts of patients either by OTX015/MK8628, JQ1 or ATRA/ATO led to nuclear relocalization of NPMwt protein to the nucleus. Conclusion : BET inhibitors induce TP53 independent apoptosis, differentiation, proteasomal degradation and NPMwt relocalization in NPMc cells. Thus, clinical testing of bromodomain inhibitors in NPMc AML is indicated. Citation Format: Thorsten Braun, Marie-Magdelaine Coudé, Jeannig Berrou, Hanene Djamai, Mélanie Dupont, Anna Kaci, Marc Delord, Raphael Itzykson, Emmanuel Raffoux, Caroline Berthier, Hugues de Thé, André Baruchel, Claude Gardin, Hervé Dombret. Bromodomain and extra-terminal BET inhibitors induce TP53 independent apoptosis, maturation and oncoprotein degradation in NPM1 mutated acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 803