IDH mutations as immune target and their effect on dendritic cell differentiation and metabolism

1 Generation of IDH2 R140Q specific T cells In the last decades, immunotherapy has become an important part of cancer treatment. For a cancer-specific immunotherapy, it is essential to find a target structure which is restricted to cancer cells but is not expressed on other cells. This is possible with “tumor-specific” antigens. In the first part of the project the goal was to create a T cell population expressing a T cell receptor specifically recognizing the mutated IDH2 R140Q enzyme. T cells were either stimulated with antigen presenting cells (APCs) loaded with a IDH2 R140Q protein specific peptide or with APCs transfected with the IDH2 R140Q protein. Suitable peptides were estimated by online tools (BIMAS, SYFPETHI and NET MHC Pan). The most promising binding properties were found for the HLA-B*15:01 restricted 9mer IQNILGGTV (IQN9) having the mutated amino acid at the second position (anchor position). Although we successfully generated IQN9-peptide specific T cells, it was not possible to find a T cell population specifically recognizing APCs expressing the IDH2 R140Q protein upon transfection. This could be explained by the fact that the mutated aminoacid was on an anchor position and not in the T cell receptor binding region. T cells stimulated with IQN9-peptide loaded APCs recognized a neuramidase protein (expressed by influenza A viruses) derived peptide which showed a similar amino acid sequence, in addition to the IQN9-peptide. Apparently, neuramidase-specific T cells were re-stimulated by the IDH2 R140Q derived peptide, a phenomenon known as molecular mimicry. 2 Effect of D-2-HG on metabolism and differentiation of monocyte-derived dendritic cells In the second part of the project the impact of the onco-metabolite D-2-Hydroxyglutarate (D 2 HG), produced by the mutated IDH, on myeloid cells was analyzed. D-2-HG is known to be released by different tumor entities as leukemia or glioma harbouring IDH mutations. The impact of D-2-HG on the differentiation of monocytes into DCs and on DC metabolism was examined. Monocytes are able to take up D-2-HG. Besides increased cell sizes, D-2-HG treated DCs had less and shorter dendrites than control cells after 7 days of culture. Cell yield and viability remained unaffected by D-2-HG treatment. CD1a, a differentiation marker was not expressed in D-2-HG treated DCs. DC-SIGN, HLA-DR and –DP, required for T cell priming and activation, were also significantly less expressed on D-2-HG treated DCs. Furthermore, D-2-HG treatment changed the cytokine profile of mature DCs. The pro-inflammatory cytokine IL-12 was significantly less secreted in the presence of D-2-HG. To analyze the functional consequences of the D-2-HG induced DC phenotype on T cell stimulation, we performed allogenic mixed lymphocyte reactions with CD4 T cells. D-2-HG treated DCs were characterized by a reduced stimulatory capacity, indicated by a reduced IFN production and diminished proliferation of CD4 T cells. In a next step, the metabolic profile of DCs was analyzed in the presence of D-2-HG. Respiration analyses revealed higher oxygen consumptions in D-2-HG treated DCs as well as in DCs expressing mutated IDH1 or IDH2 enzyme. Moreover, glycolytic activity analyzed by glucose consumption and lactate secretion was increased, in line with the earlier pH drop measured by the PreSens technology. The higher respiratory and glycolytic activity resulted in increased ATP levels. Furthermore, cytosolic reactive oxygen species (ROS) levels were significantly increased in the presence of D 2 HG. Since respiration displays an important source of ROS, we hypothesized that, antioxidants like vitamin C might reduce the increased ROS levels and thereby also the inhibitory effect of D-2-HG on DC differentiation. Vitamin C had a positive effect on HLA-DP expression, but only a minor effect on HLA-DR expression. DC-SIGN expression reached control levels in vitamin C and D-2-HG treated DCs. In another series of experiments, anti-diabetic drugs modulating the metabolism were applied with the aim to rescue inhibitory D-2-HG effects on DC differentiation, as metformin (an inhibitor of complex I of the electron transport chain) and pioglitazone (a ligand for peroxisome proliferator-activated receptor ). In contrast to metformin, pioglitazone treatment partially neutralized the effects induced by D-2-HG. As D 2 HG is a known modulator of DNA methylation, DCs were analyzed by MassARRAY in the presence of D 2 HG and vitamin C. Vitamin C acts as a co factor for TET2, an important demethylase in DC differentiation. We hypothesized that vitamin C could rescue marker expression on an epigenetic level. As expected, D 2 HG delayed the demethylation of sites, which are known to be important for DC differentiation. A single treatment with vitamin C did speed up the demethylation process. Vitamin C was able to partly compensate for the inhibitory effect of D-2-HG on demethylation in some regions, but levels of untreated DCs were not reached. Finally, primary AML blasts with IDH mutations were analyzed, regarding their HLA class expression. In line with the results obtained for D-2-HG treated monocyte-derived DCs, HLA class II expression was also decreased in primary IDH mutated AML blasts. Taken together, our results indicate that D-2-HG inhibits the differentiation of monocyte to DCs, which can have in turn an impact on T cell stimulation. Thereby D-2-HG is indirectly able to reduce the immunological anti-tumor response. D-2-HG can promote immune escape by reducing HLA class II expression in an autocrine and paracrine way. Moreover, D-2-HG diminishes the stimulatory capacity of DCs and decreases the immunogenicity of AML blasts

Combined Metabolic Targeting With Metformin and the NSAIDs Diflunisal and Diclofenac Induces Apoptosis in Acute Myeloid Leukemia Cells

The accelerated metabolism of tumor cells, inevitable for maintaining high proliferation rates, is an emerging target for tumor therapy. Increased glucose and lipid metabolism as well as mitochondrial activity have been shown in solid tumors but also in leukemic cells. As tumor cells are able to escape the blockade of one metabolic pathway by a compensatory increase in other pathways, treatment strategies simultaneously targeting metabolism at different sites are currently developed. However, the number of clinically applicable anti-metabolic drugs is still limited. Here, we analyzed the impact of the anti-diabetic drug metformin alone or in combination with two non-steroidal anti-inflammatory drugs (NSAIDs) diclofenac and diflunisal on acute myeloid leukemia (AML) cell lines and primary patient blasts. Diclofenac but not diflunisal reduced lactate secretion in different AML cell lines (THP-1, U937, and KG-1) and both drugs increased respiration at low concentrations. Despite these metabolic effects, both NSAIDs showed a limited effect on tumor cell proliferation and viability up to a concentration of 0.2 mM. In higher concentrations of 0.4–0.8 mM diflunisal alone exerted a clear effect on proliferation of AML cell lines and blocked respiration. Single treatment with the anti-diabetic drug metformin blocked mitochondrial respiration, but proliferation and viability were not affected. However, combining all three drugs exerted a strong cytostatic and cytotoxic effect on THP-1 cells. Comparable to the results obtained with THP-1 cells, the combination of all three drugs significantly reduced proliferation of primary leukemic blasts and induced apoptosis. Furthermore, NSAIDs supported the effect of low dose chemotherapy with cytarabine and reduced proliferation of primary AML blasts. Taken together we show that low concentrations of metformin and the two NSAIDs diclofenac and diflunisal exert a synergistic inhibitory effect on AML proliferation and induce apoptosis most likely by blocking tumor cell metabolism. Our results underline the feasibility of applying anti-metabolic drugs for AML therapy

Anti-Thymocyte Globulin Treatment Augments 1,25-Dihydroxyvitamin D3 Serum Levels in Patients Undergoing Hematopoietic Stem Cell Transplantation

Application of anti-thymocyte globulin (ATG) is a widely used strategy for the prevention of graftversus-host disease (GvHD). As vitamin D3 serum levels are also discussed to affect hematopoietic stem cell transplantation (HSCT) outcome and GvHD development, we analysed a possible interplay between ATG treatment and serum levels of 25-hydroxyvitamin D3and 1,25-dihydroxyvitaminD3in 4HSCT cohorts withdifferent vitaminD3supplementation. ATG is significantly associated with higher serum level of 1,25 dihydroxyvitamin D3 around HSCT (day -2 to 7, peri-transplant), however only in patients with adequate levels of its precursor 25-hydroxyvitamin D3. ATG exposure had no impact on overall survival in patients supplemented with high dose vitamin D3, but was associated with higher risk of one-year treatment-related mortality (log rank test p=0.041) in patients with no/low vitamin D3 supplementation. However, the difference failed to reach significance applying a Cox-model regression without and with adjustment for baseline risk factors (unadjusted P=0,058, adjusted p=0,139). To shed some light on underlying mechanisms, we investigated the impact of ATG on 1,25-DihydroxyvitaminD3 production by human dendritic cells (DCs) in vitro.ATGincreased gene expression ofCYP27B1, the enzyme responsible for the conversion of 25-hydroxyvitamin D3 into 1,25-dihydroxyvitamin D3, which was accompanied by higher 1,25-dihydroxyvitamin D3levels in ATG-treatedDCculture supernatants.Our data demonstrate a cooperative effect of 25-hydroxyvitamin D3 and ATG in the regulation of 1,25-dihydroxyvitamin D3 production. This finding may be of importance in the context of HSCT, where early high levels of 1,25- dihydroxyvitamin D3 levels have been shown to be predictive for lower transplant related mortality and suggest that vitamin D3 supplementation may especially be important in patients receiving ATG for GvHD prophylaxis

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

D-2-Hydroxyglutarate and L-2-Hydroxyglutarate Inhibit IL-12 Secretion by Human Monocyte-Derived Dendritic Cells

Mutations in isocitrate dehydrogenase (IDH) or a reduced expression of L-2-hydroxyglutarate (HG)-dehydrogenase result in accumulation of D-2-HG or L-2-HG, respectively, in tumor tissues. D-2-HG and L-2-HG have been shown to affect T-cell differentiation and activation; however, effects on human myeloid cells have not been investigated so far. In this study we analyzed the impact of D-2-HG and L-2-HG on activation and maturation of human monocyte-derived dendritic cells (DCs). 2-HG was taken up by DCs and had no impact on cell viability but diminished CD83 expression after Lipopolysaccharides (LPS) stimulation. Furthermore, D-2-HG and L-2-HG significantly reduced IL-12 secretion but had no impact on other cytokines such as IL-6, IL-10 or TNF. Gene expression analyses of the IL-12 subunits p35/IL-12A and p40/IL-12B in DCs revealed decreased expression of both subunits. Signaling pathways involved in LPS-induced cytokine expression (NFkB, Akt, p38) were not altered by D-2-HG. However, 2-HG reprogrammed LPS-induced metabolic changes in DCs and increased oxygen consumption. Addition of the ATP synthase inhibitor oligomycin to DC cultures increased IL-12 secretion and was able to partially revert the effect of 2-HG. Our data show that both enantiomers of 2-HG can limit activation of DCs in the tumor environment

HLA-DPB1 Reactive T Cell Receptors for Adoptive Immunotherapy in Allogeneic Stem Cell Transplantation

HLA-DPB1 antigens are mismatched in about 80% of allogeneic hematopoietic stem cell transplantations from HLA 10/10 matched unrelated donors and were shown to be associated with a decreased risk of leukemia relapse. We recently developed a reliable in vitro method to generate HLA-DPB1 mismatch-reactive CD4 T-cell clones from allogeneic donors. Here, we isolated HLA-DPB1 specific T cell receptors (TCR DP) and used them either as wild-type or genetically optimized receptors to analyze in detail the reactivity of transduced CD4 and CD8 T cells toward primary AML blasts. While both CD4 and CD8 T cells showed strong AML reactivity in vitro, only CD4 T cells were able to effectively eliminate leukemia blasts in AML engrafted NOD/SCID/IL2Rγc−/− (NSG) mice. Further analysis showed that optimized TCR DP and under some conditions wild-type TCR DP also mediated reactivity to non-hematopoietic cells like fibroblasts or tumor cell lines after HLA-DP upregulation. In conclusion, T cells engineered with selected allo-HLA-DPB1 specific TCRs might be powerful off-the-shelf reagents in allogeneic T-cell therapy of leukemia. However, because of frequent (common) cross-reactivity to non-hematopoietic cells with optimized TCR DP T cells, safety mechanisms are mandatory

D-2-hydroxyglutarate supports a tolerogenic phenotype with lowered major histocompatibility class II expression in non-malignant dendritic cells and acute myeloid leukemia cells

D-2-hydroxyglutarate (D-2-HG) accumulates in primary acute myeloid leukemia (AML) patients with mutated isocitrate dehydrogenase (IDH) and other malignancies. D-2-HG suppresses antitumor T cell immunity but little is known about potential effects on non-malignant myeloid cells. Here we show that D-2-HG impairs human but not murine dendritic cell (DC) differentiation, resulting in a tolerogenic phenotype with low major histocompatibility (MHC) class II expression. In line, IDH-mutated AML blasts exhibited lower expression of HLA-DP and were less susceptible to lysis by HLA-DP-specific T cells. Interestingly, D-2-HG reprogrammed metabolism towards increased lactate production in DCs and AML besides its expected impact on DNA demethylation. Vitamin C accelerated DNA demethylation, but only the combination of vitamin C and glycolytic inhibition lowered lactate levels and supported MHC class II expression. Our results indicate an unexpected link between the immunosuppressive metabolites 2-HG and lactic acid and suggest a potentially novel therapeutic strategy with combinations of anti-glycolytic drugs and epigenetic modulators (hypomethylating agents) or other therapeutics for the treatment of AML