Validating genetic variants in innate immunity linked to infectious events in acute myeloid leukemia post-induction chemotherapy.

Infectious events, such as sepsis and invasive fungal disease (IFD), pose significant risks in patients with acute myeloid leukemia (AML). Previous studies, including our own, have suggested a potential role of single nucleotide polymorphisms (SNPs) within the innate immune system in influencing individual infection susceptibility. However, many of these associations lack validation in independent cohorts. This study sought to validate the impact of 11 candidate SNPs across 6 genes (TLR2, TLR4, Dectin-1, DC-SIGN, PTX3, L-Ficolin) in an independent cohort of patients. Two cohorts with newly diagnosed AML patients receiving intensive induction chemotherapy were analyzed: a stratification cohort comprising 186 patients and a validation cohort consisting of 138 patients. Multiple SNPs in each cohort were found to be associated to infectious complications, notably the DC-SIGN SNP rs4804800 demonstrated a significant association with sepsis in both cohorts. SNPs within the PTX3 and Dectin-1 genes were linked to IFD development in one cohort each. This study represents the first validation study of candidate genes associated with infectious events in AML patients after intensive induction chemotherapy. Identifying genetic predispositions to infections could significantly impact the management of antimicrobial prophylaxis and treatment in AML patients.

Synergistic Effects of the RARalpha Agonist Tamibarotene and the Menin Inhibitor Revumenib in Acute Myeloid Leukemia Cells with KMT2A Rearrangement or NPM1 Mutation.

Inhibition of menin in acute myeloid leukemia (AML) harboring histone-lysine-N-methyltransferase 2A rearrangement (KMT2Ar) or the mutated Nucleophosmin gene (NPM1c) is considered a novel and effective treatment approach in these patients. However, rapid acquisition of resistance mutations can impair treatment success. In patients with elevated retinoic acid receptor alpha (RARA) expression levels, promising effects are demonstrated by the next-generation RARalpha agonist tamibarotene, which restores differentiation or induces apoptosis. In this study, the combination of revumenib and tamibarotene was investigated in various KMT2Ar or NPM1c AML cell lines and patient-derived blasts, focusing on the potential synergistic induction of differentiation or apoptosis. Both effects were analyzed by flow cytometry and validated by Western blot analysis. Synergy calculations were performed using viability assays. Regulation of the relevant key mediators for the MLL complex were quantified by RT-qPCR. In MV4:11 cells characterized by the highest relative mRNA levels of RARA, highly synergistic induction of apoptosis is demonstrated upon combination treatment. Induction of apoptosis by combined treatment of MV4:11 cells is accompanied by pronounced induction of the pro-apoptotic protein BAX and a synergistic reduction in CDK6 mRNA levels. In MOLM13 and OCI-AML3 cells, an increase in differentiation markers like PU.1 or a decreased ratio of phosphorylated to total CEBPA is demonstrated. In parts, corresponding effects were observed in patient-derived AML cells carrying either KMT2Ar or NPM1c. The impact of revumenib on KMT2Ar or NPM1c AML cells was significantly enhanced when combined with tamibarotene, demonstrating synergistic differentiation or apoptosis initiation. These findings propose promising strategies for relapsed/refractory AML patients with defined molecular characteristics.

Modulation of FLT3-ITD Localization and Targeting of Distinct Downstream Signaling Pathways as Potential Strategies to Overcome FLT3-Inhibitor Resistance.

OBJECTIVES: Internal tandem duplications (ITDs) of the Fms-like tyrosine kinase 3 (FLT3) represent the most frequent molecular aberrations in acute myeloid leukemia (AML) and are associated with an inferior prognosis. The pattern of downstream activation by this constitutively activated receptor tyrosine kinase is influenced by the localization of FLT3-ITD depending on its glycosylation status. Different pharmacological approaches can affect FLT3-ITD-driven oncogenic pathways by the modulation of FLT3-ITD localization. AIMS: The objective of this study was to investigate the effects of N-glycosylation inhibitors (tunicamycin or 2-deoxy-D-glucose) or the histone deacetylase inhibitor valproic acid (VPA) on FLT3-ITD localization and downstream activity. We sought to determine the potential differences between the distinct FLT3-ITD variants, particularly concerning their susceptibility towards combined treatment by addressing either N-glycosylation and the heat shock protein 90 (HSP90) by 17-AAG, or by targeting the PI3K/AKT/mTOR pathway by rapamycin after treatment with VPA. METHODS: Murine Ba/F3 leukemia cell lines were stably transfected with distinct FLT3-ITD variants resulting in IL3-independent growth. These Ba/F3 FLT3-ITD cell lines or FLT3-ITD-expressing human MOLM13 cells were exposed to tunicamycin, 2-deoxy-D-glucose or VPA, and 17-AAG or rapamycin, and characterized in terms of downstream signaling by immunoblotting. FLT3 surface expression, apoptosis, and metabolic activity were analyzed by flow cytometry or an MTS assay. Proteome analysis by liquid chromatography-tandem mass spectrometry was performed to assess differential protein expression. RESULTS: The susceptibility of FLT3-ITD-expressing cells to 17-AAG after pre-treatment with tunicamycin or 2-deoxy-D-glucose was demonstrated. Importantly, in Ba/F3 cells that were stably expressing distinct FLT3-ITD variants that were located either in the juxtamembrane domain (JMD) or in the tyrosine kinase 1 domain (TKD1), response to the sequential treatments with tunicamycin and 17-AAG varied between individual FLT3-ITD motifs without dependence on the localization of the ITD. In all of the FLT3-ITD cell lines that were investigated, incubation with tunicamycin was accompanied by intracellular retention of FLT3-ITD due to the inhibition of glycosylation. In contrast, treatment of Ba/F3-FLT3-ITD cells with VPA was associated with a significant increase of FLT3-ITD surface expression depending on FLT3 protein synthesis. The allocation of FLT3 to different cellular compartments that was induced by tunicamycin, 2-deoxy-D-glucose, or VPA resulted in the activation of distinct downstream signaling pathways. Whole proteome analyses of Ba/F3 FLT3-ITD cells revealed up-regulation of the relevant chaperone proteins (e.g., calreticulin, calnexin, HSP90beta1) that are directly involved in the stabilization of FLT3-ITD or in its retention in the ER compartment. CONCLUSION: The allocation of FLT3-ITD to different cellular compartments and targeting distinct downstream signaling pathways by combined treatment with N-glycosylation and HSP90 inhibitors or VPA and rapamycin might represent new therapeutic strategies to overcome resistance towards tyrosine kinase inhibitors in FLT3-ITD-positive AML. The treatment approaches addressing N-glycosylation of FLT3-ITD appear to depend on patient-specific FLT3-ITD sequences, potentially affecting the efficacy of such pharmacological strategies.

Polymethine Dye-Functionalized Nanoparticles for Targeting CML Stem Cells.

In chronic myelogenous leukemia (CML), treatment with tyrosine kinase inhibitors (TKI) is unable to eradicate leukemic stem cells (LSC). Polymethine dye-functionalized nanoparticles can be internalized by specific cell types using transmembrane carrier proteins. In this study we investigated the uptake behavior of various polymethine dyes on leukemia cell lines and searched for carrier proteins that guide dye transport using RNA interference. The results show that the uptake of DY-635 is dependent on organic anion transport protein 1B3 (OATP1B3) in CML cells and immature myeloid precursor cells of CML patients. In contrast to nonspecific poly(lactide-co-glycolic acid) (PLGA) nanoparticle constructs, DY-635-functionalization of nanoparticles led to an uptake in CML cells. Investigation of these nanoparticles on bone marrow of CML patients showed a preferred uptake in LSC. The transcription of OATP1B3 is known to be induced under hypoxic conditions via the hypoxia-inducing factor 1 alpha (HIF1α), thus also in the stem cells niche. Since these cells have the potential to repopulate the bone marrow after CML treatment discontinuation, eliminating them by means of drug-loaded DY-635-functionalized PLGA nanoparticles deployed as a selective delivery system to LSC is highly relevant to the ongoing search for curative treatment options for CML patients.

Cell cycle-dependent and -independent telomere shortening accompanies murine brain aging.

Replication-based telomere shortening during lifetime is species- and tissue-specific, however, its impact on healthy aging is unclear. In particular, the contribution of telomere truncation to the aging process of the CNS, where replicative senescence alone fails to explain organ aging due to low to absent mitotic activity of intrinsic populations, is undefined. Here, we assessed changes in relative telomere length in non-replicative and replicative neural brain populations and telomerase activity as a function of aging in C57BL/6 mice. Telomeres in neural cells and sub-selected neurons shortened with aging in a cell cycle-dependent and -independent manner, with preponderance in replicative moieties, implying that proliferation accelerates, but is not prerequisite for telomere shortening. Consistent with this telomere erosion, telomerase activity and nuclear TERT protein were not induced with aging. Knockdown of the Rela subunit of NF-κB, which controls both telomerase enzyme and subcellular TERT protein allocation, did also not influence telomerase activity or telomere length, in spite of its naive up-regulation selectively under aging conditions. We conclude that telomere instability is intrinsic to physiological brain aging beyond cell replication, and appears to occur independently of a functional interplay with NF-κB, but rather as a failure to induce or relocate telomerase.

Isoform localization of Dectin-1 regulates the signaling quality of anti-fungal immunity.

Dectin-1 is recognized as a major receptor for fungal ß-glucans and contributes to anti-fungal immunity. Human monocyte populations express Dectin-1 isoforms A and B, which differ by the presence of a stalk region and its N-linked glycosylation site. Here, we analyzed the expression of both isoforms in human monocyte-derived cells. The cellular localization on cell lines stably expressing either Dectin-1 isoform A or B was studied by flow cytometry and confocal laser scanning microscopy. Intracellular protein signaling and cytokine production were analyzed by immunoblotting and cytometric bead array, respectively. Monocyte-derived cells showed cell type-specific expression of the two isoforms. Glycosylated Dectin-1 isoform A was predominantly localized at the cell surface, non-glycosylated isoform B was retained intracellularly. Inhibition of glycosylation resulted in efficient abrogation of cell surface expression of isoform A. Signaling quality following Dectin-1 stimulation was reduced in isoform B cells. Differential isoform specific cytokine secretion was observed by cytometric bead array. We show here that n-glycosylation of Dectin-1 is crucial for its cell surface expression and consequently signal transduction. Taken together, unique cytokine secretion and varying expression levels of human Dectin-1 isoforms on monocyte-derived cells may indicate distinct isoform usage as a cell type-specific mechanism of regulating anti-fungal immunity.

Polymorphisms of Dectin-1 and TLR2 Predispose to Invasive Fungal Disease in Patients with Acute Myeloid Leukemia.

BACKGROUND: Patients with acute myeloid leukemia (AML) who undergo induction chemotherapy are at high risk for invasive fungal disease (IFD). Dectin-1, a C-type lectin family member represents one of the most important pattern recognition receptors of the innate immune system and single nucleotide polymorphisms (SNPs) in the Dectin-1 gene have been associated with an increased risk of infectious complications. We sought to investigate the impact of three different Dectin-1 SNPs and one TLR2 SNP on developing IFD in 186 adult patients with newly diagnosed AML following anthracycline-based induction chemotherapy. PATIENTS AND METHODS: Genotyping of Dectin-1 SNPs (rs16910526, rs3901533 and rs7309123) and TLR2 SNP (rs5743708) was performed by TaqMan method and pyrosequencing. IFD was defined according to the EORTC/MSG consensus guidelines. Multiple logistic regression analyses were applied to evaluate the association between the polymorphisms and the occurrence of pulmonary infections. Dectin-1 expression studies with SNP genotyped human monocytes were performed to elucidate susceptibility to IFD following chemotherapy. RESULTS: We could demonstrate that patients carrying the Dectin-1 SNP rs7309123 G/G (n = 47) or G/G and C/G (n = 133) genotype revealed a significant higher risk for developing both pneumonia in general (adjusted odds ratio (OR): 2.5; p = 0.014 and OR: 3.0, p = 0.004) and pulmonary IFD (OR: 2.6; p = 0.012 and OR: 2.4, p = 0.041, respectively). Patients carrying the TLR2 SNP rs5743708 (R753Q, GA/AA genotype, n = 12) also revealed a significantly higher susceptibility to pneumonia including IFD. Furthermore, Dectin-1 mRNA expression in human monocytes was lower following chemotherapy. CONCLUSION: To our best knowledge, this study represents the first analysis demonstrating that harbouring polymorphisms of Dectin-1 (rs7309123) or TLR2 (rs5743708) represents an independent risk factor of developing IFD in patients with AML undergoing induction chemotherapy.

Paradoxical MAPK-activation in response to treatment with tyrosine kinase inhibitors in CML: flow cytometry loses track.

BACKGROUND: Paradoxical activation of the MAP-kinases, ERK1, and ERK2 (ERK1/2) is observed in CML cell lines and primary CML patient cells treated with tyrosine kinase inhibitors (TKI) in vitro. The commonly accepted assumption is that activated ERK1/2 is key regulators of survival of leukemic cells treated with kinase inhibitors. Hence, paradoxical ERK1/2-activation may trigger resistance in vivo, which yet has to be shown. We therefore sought to establish a flow cytometric assay that enables us to measure paradoxical TKI-induced ERK1/2-activation on a single cell basis in primary CML cells. METHODS: Side-by-side Western blot and intracellular flow cytometry (FCM) after in vitro exposure of cell lines and primary cells to nilotinib were performed. Detailed analysis of pre-analytical factors and the issue of compartmentalization of phosphorylated ERK1/2 by confocal laser scanning microscopy were performed. RESULT: Results were conflicting in that pERK-activation was robustly detected in Western blot assays, but not when cells were analyzed by FCM despite well functioning positive and negative controls. This is in contrast to experiments on other targets such as phospho-CrkL, where also in our hands TKI-dependent inhibition of phosphorylation is trackable by both Western blot and FCM assays. CONCLUSIONS: To our knowledge this is the first report of discordant results in phospho-protein analysis in TKI-treated cells analyzed by Western blot vs. FCM. We speculate that a substance specific interaction interferes with fluorescence dependent methods seeking to track phosphorylated ERK1/2 in TKI-treated cells.

The E3 ubiquitin ligase TRAF2 can contribute to TNF-α resistance in FLT3-ITD-positive AML cells.

TNF-α has pleiotropic effects on cell survival and apoptosis. The E3 ubiquitin ligase TRAF2 plays a crucial role for TNF-α mediated signaling since NF-κB activation by TNF-α is at least partially mediated by TRAF2. The objective of this study was to investigate whether TNF-α can induce apoptosis in FLT3-ITD-positive AML cells and to elucidate the influence of TRAF2. Stable lentiviral mediated down-regulation of TRAF2 resulted in a decrease of phosphorylation of the anti-apoptotic protein AKT and its downstream target GSK-3ß. Induction of apoptosis and impaired proliferation after TNF-α exposure were observed. Co-treatment of FLT3-ITD-positive cells with the specific FLT3 inhibitor AC220 was able to overcome TNF-α resistance. Taken together, we conclude that TRAF2 plays an important role in signal transduction and survival of AML cells.

The E3 ubiquitin ligase TRAF6 inhibits LPS-induced AKT activation in FLT3-ITD-positive MV4-11 AML cells.

PURPOSE: The aim of the study was to investigate the activation of the PI3K/AKT (phosphatidylinositol-3-kinase) pathway after stimulation of TLR-4 (Toll-like receptor 4) with LPS (lipopolysaccharide) in FLT3-ITD (internal tandem duplication)-positive AML (acute myeloid leukemia) cells. TRAF6 (tumor necrosis factor receptor-associated factor 6), an E3 ubiquitin ligase, is critically involved in TLR-signaling. Based on the observation that TRAF6 might play a role in AKT phosphorylation, we hypothesized that TRAF6 can enhance the constitutive FLT3-ITD-driven activation of AKT after LPS stimulation. MATERIALS AND METHODS: Human MV4-11 FLT3-ITD cells were silenced for TRAF6 by stable shRNA expression. Western blotting was used to analyze signal transduction by detection of phosphorylated proteins. LPS-induced activation of the NF-κB pathway was ensured by the induction of IκBα expression. To evaluate a potential functional role of TRAF6, we also performed chemosensitivity assays. RESULTS: In MV4-11 cells, AKT was activated in response to LPS treatment. Surprisingly, shRNA-mediated knockdown of TRAF6 resulted in a significant increase in basal AKT phosphorylation. By LPS stimulation, the gain of AKT phosphorylation was more pronounced in the TRAF6 knockdown cell line than in the control. In addition, the concentration-dependent induction of apoptosis in response to treatment with the cytostatic drugs cytarabine or daunorubicin was significantly reduced in TRAF6-depleted MV4-11 cells. CONCLUSION: Our data strongly suggest that the E3 ubiquitin ligase TRAF6 plays an important functional role in signal transduction and survival of AML cells. We hypothesize that LPS-mediated stimulation of TLR-4 leads to the induction of NF-κB-mediated signaling. However, TRAF6 might prevent a synergistic activation of the PI3K/AKT pathway after activation of TLR-4 signaling in FLT3-ITD-positive cells.

Poly(I:C)-induced tumour cell death leads to DC maturation and Th1 activation.

Dendritic cells (DCs) have the ability to generate peptide epitopes for MHC class I molecules derived from apoptotic tumour cells for direct recognition by cytotoxic T cells. This function has lead to DCs being used in vaccine strategies. In this study, we investigate the effect of inducing apoptosis in tumour cell lines using IFN-γ and poly(I:C), the subsequent maturation of the endocytosing DC and its ability to direct the resulting T cell response. We show that uptake of poly(I:C)-induced apoptotic tumour cells leads to DC maturation and activation with a Th1 cell polarising capacity. In contrast, these effects are not seen by DCs loaded with γ-irradiated apoptotic tumour cells. We propose that the manner in which tumour cells are induced to die can have a profound effect on the endocytosing DC and the resulting T cell response.