Isocitrate dehydrogenase 1 mutation drives leukemogenesis by PDGFRA activation due to insulator disruption in acute myeloid leukemia (AML).

Acute myeloid leukemia (AML) is characterized by complex molecular alterations and driver mutations. Elderly patients show increased frequencies of IDH mutations with high chemoresistance and relapse rates despite recent therapeutic advances. Besides being associated with global promoter hypermethylation, IDH1 mutation facilitated changes in 3D DNA-conformation by CTCF-anchor methylation and upregulated oncogene expression in glioma, correlating with poor prognosis. Here, we investigated the role of IDH1 p.R132H mutation in altering 3D DNA-architecture and subsequent oncogene activation in AML. Using public RNA-Seq data, we identified upregulation of tyrosine kinase PDGFRA in IDH1-mutant patients, correlating with poor prognosis. DNA methylation analysis identified CpG hypermethylation within a CTCF-anchor upstream of PDGFRA in IDH1-mutant patients. Increased PDGFRA expression, PDGFRA-CTCF methylation and decreased CTCF binding were confirmed in AML CRISPR cells with heterozygous IDH1 p.R132H mutation and upon exogenous 2-HG treatment. IDH1-mutant cells showed higher sensitivity to tyrosine kinase inhibitor dasatinib, which was supported by reduced blast count in a patient with refractory IDH1-mutant AML after dasatinib treatment. Our data illustrate that IDH1 p.R132H mutation leads to CTCF hypermethylation, disrupting DNA-looping and insulation of PDGFRA, resulting in PDGFRA upregulation in IDH1-mutant AML. Treatment with dasatinib may offer a novel treatment strategy for IDH1-mutant AML.

Dual Fc optimization to increase the cytotoxic activity of a CD19-targeting antibody.

Targeting CD19 represents a promising strategy for the therapy of B-cell malignancies. Although non-engineered CD19 antibodies are poorly effective in mediating complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), these effector functions can be enhanced by Fc-engineering. Here, we engineered a CD19 antibody with the aim to improve effector cell-mediated killing and CDC activity by exchanging selected amino acid residues in the Fc domain. Based on the clinically approved Fc-optimized antibody tafasitamab, which triggers enhanced ADCC and ADCP due to two amino acid exchanges in the Fc domain (S239D/I332E), we additionally added the E345K amino acid exchange to favor antibody hexamerization on the target cell surface resulting in improved CDC. The dual engineered CD19-DEK antibody bound CD19 and Fcγ receptors with similar characteristics as the parental CD19-DE antibody. Both antibodies were similarly efficient in mediating ADCC and ADCP but only the dual optimized antibody was able to trigger complement deposition on target cells and effective CDC. Our data provide evidence that from a technical perspective selected Fc-enhancing mutations can be combined (S239D/I332E and E345K) allowing the enhancement of ADCC, ADCP and CDC with isolated effector populations. Interestingly, under more physiological conditions when the complement system and FcR-positive effector cells are available as effector source, strong complement deposition negatively impacts FcR engagement. Both effector functions were simultaneously active only at selected antibody concentrations. Dual Fc-optimized antibodies may represent a strategy to further improve CD19-directed cancer immunotherapy. In general, our results can help in guiding optimal antibody engineering strategies to optimize antibodies' effector functions.

Engineering of CD19 Antibodies: A CD19-TRAIL Fusion Construct Specifically Induces Apoptosis in B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) Cells In Vivo.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent malignancy in children and also occurs in adulthood. Despite high cure rates, BCP-ALL chemotherapy can be highly toxic. This type of toxicity can most likely be reduced by antibody-based immunotherapy targeting the CD19 antigen which is commonly expressed on BCP-ALL cells. In this study, we generated a novel Fc-engineered CD19-targeting IgG1 antibody fused to a single chain tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) domain (CD19-TRAIL). As TRAIL induces apoptosis in tumor cells but not in healthy cells, we hypothesized that CD19-TRAIL would show efficient killing of BCP-ALL cells. CD19-TRAIL showed selective binding capacity and pronounced apoptosis induction in CD19-positive (CD19+) BCP-ALL cell lines in vitro and in vivo. Additionally, CD19-TRAIL significantly prolonged survival of mice transplanted with BCP-ALL patient-derived xenograft (PDX) cells of different cytogenetic backgrounds. Moreover, simultaneous treatment with CD19-TRAIL and Venetoclax (VTX), an inhibitor of the anti-apoptotic protein BCL-2, promoted synergistic apoptosis induction in CD19+ BCP-ALL cells in vitro and prolonged survival of NSG-mice bearing the BCP-ALL cell line REH. Therefore, IgG1-based CD19-TRAIL fusion proteins represent a new potential immunotherapeutic agent against BCP-ALL.

[COVID-19: Would compulsory vaccination be legally possible?] / COVID-19: Wäre eine Impfpflicht rechtlich möglich?

As the pandemic continues, there is increasing hope vaccinations are becoming available. As a solution for a nationwide immunization against the virus, a compulsory vaccination is repeatedly discussed. However, some opponents of vaccination are threatened by the idea of a possible mandatory vaccination. It is therefore necessary to discuss whether such a compulsory vaccination is theoretically legally enforceable. This article discusses the current legal situation in Germany. The introduction of a potential compulsory vaccination against the SARS-CoV-2 virus represents an encroachment on the fundamental right of physical integrity. According to §â€Š20 (6) IfSG, a protective vaccination for threatened parts of the population is permissible by statutory order, if a transmissible disease with clinically severe courses occurs and its epidemic spread can be expected.

Enhancing CDC and ADCC of CD19 Antibodies by Combining Fc Protein-Engineering with Fc Glyco-Engineering.

BACKGROUND: Native cluster of differentiation (CD) 19 targeting antibodies are poorly effective in triggering antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which are crucial effector functions of therapeutic antibodies in cancer immunotherapy. Both functions can be enhanced by engineering the antibody's Fc region by altering the amino acid sequence (Fc protein-engineering) or the Fc-linked glycan (Fc glyco-engineering). We hypothesized that combining Fc glyco-engineering with Fc protein-engineering will rescue ADCC and CDC in CD19 antibodies. RESULTS: Four versions of a CD19 antibody based on tafasitamab's V-regions were generated: a native IgG1, an Fc protein-engineered version with amino acid exchanges S267E/H268F/S324T/G236A/I332E (EFTAE modification) to enhance CDC, and afucosylated, Fc glyco-engineered versions of both to promote ADCC. Irrespective of fucosylation, antibodies carrying the EFTAE modification had enhanced C1q binding and were superior in inducing CDC. In contrast, afucosylated versions exerted an enhanced affinity to Fcγ receptor IIIA and had increased ADCC activity. Of note, the double-engineered antibody harboring the EFTAE modification and lacking fucose triggered both CDC and ADCC more efficiently. CONCLUSIONS: Fc glyco-engineering and protein-engineering could be combined to enhance ADCC and CDC in CD19 antibodies and may allow the generation of antibodies with higher therapeutic efficacy by promoting two key functions simultaneously.

Combination analysis of activator protein-1 family members, Sp1 and an activator protein-2alpha-related factor binding to different regions of the urokinase receptor gene in resected colorectal cancers.

PURPOSE: Studies on the transactivation of genes via promoter elements have mostly been done on cell lines rather than resected tissues. This, however, is essential to address an in vivo or clinical relevance. We have previously shown tumor-specific binding of Sp1 and an activator protein (AP)-2-related factor to promoter region -152/-135 of the metastasis-related u-PAR gene in 60% of in vivo-resected cancer tissues. Cell lines have implicated an additional role, and potential synergism, of an AP-1 region (-190/-171) in u-PAR regulation. This study was done to (a) analyze AP-1 binding to this region in resected tumor and normal tissues, and define subgroups in which it is tumor-specific, and (b) to analyze transcription factor-binding patterns to both promoter motifs in resected tissues, supporting synergism, and draw first prognostic conclusions. EXPERIMENTAL DESIGN: In 103 patients with colorectal cancer, electrophoretic mobility shift assay/supershift analysis for u-PAR promoter region -190/-171 was done in tumors and normal tissues. In 71 patients, region -152/-135 was also analyzed. U-PAR protein was measured by ELISA. RESULTS: Tumor-specific AP-1 binding to region -190/-171 of the u-PAR promoter was found in 40% of patients. Subgroup analysis showed tumor-specific binding for c-Fos in 58%, for c-Jun in 50%, for JunD in 39%, and for Fra-1 in 4% of cases. AP-1 binding correlated significantly with u-PAR protein amounts in both normal and tumor tissues (P<0.001), in contrast to a tumor-specific correlation with u-PAR of the AP-2/Sp1 region. In analyses for both promoter regions, 62% of cancers showed simultaneous binding for AP-1, AP-2, and Sp1, 11% for AP-1 and AP-2, 16% for AP-2 and Sp1, 4% for AP-2 only, 3% for AP-1 only, and 0% for Sp1 only. The binding of AP-1, AP-2, and Sp1 correlated significantly with each other (P<0.001), the combination of AP-1 and AP-2 showing the highest correlation with u-PAR (P=0.008). Preliminary survival analysis indicated a trend for poorer prognosis for binding of all three transcription factors. CONCLUSION: This is the first study differentiating transcription factor-binding to two important u-PAR promoter regions in a large series of resected tumors and normal tissues. The AP-1 site seems to be a less tumor-specific regulator than the Sp1/AP-2 motif. Nevertheless, data corroborate the hypothesis of synergism between both elements in resected tumors.

Tumor-specific transcription factor binding to an activator protein-2/Sp1 element of the urokinase-type plasminogen activator receptor promoter in a first large series of resected gastrointestinal cancers.

PURPOSE: Evidence for transactivation of genes via specific promoter elements has been derived from studies on tumor cell lines but rarely on resected tumors. However, the proof of an in vivo relevance and the identification of patients with a potential tumor-specific gene expression is essential to transfer molecular-targeting strategies into clinical applications. This study gives the first clinical evidence that urokinase-type plasminogen activator receptor (u-PAR) gene expression is tumor-specifically regulated via an activator protein (AP)-2/Sp1 promoter element in a large patient subpopulation. EXPERIMENTAL DESIGN: In 145 gastrointestinal cancer patients, electrophoretic mobility shift analysis and supershift assays for u-PAR-promoter region -152/-135 were performed in tumors and corresponding normal tissues. u-PAR protein levels were measured by ELISA. RESULTS: Binding of Sp1 to region -152/-135 in tumors in contrast to corresponding normal mucosae was observed in 55% of colorectal and in 52% of gastric cancer patients. Tumor-specific binding of an AP-2-related factor was seen in 59% of colorectal and in 63% of gastric cancer patients. A significant correlation between AP-2 (P < 0.0001) and Sp1 (P = 0.0003) binding with a high u-PAR expression was observed in tumors but not in normal mucosae. Tissues of five nontumor patients did not show transcription factor binding to this region. CONCLUSIONS: This is the first study to show the tumor-specific binding of trans-activators to the u-PAR promoter region (-152/-135) biochemically in a large series of resected tumors. For the subpopulation of approximately 60% of patients with tumor-restricted u-PAR-transactivation, a molecular targeting of this region or its activating pathways should be pursued as a new antimetastasis therapy approach.