Acute myeloid leukemia remains difficult to treat due to strong genetic heterogeneity between and within individual patients. Here, we show that Pyruvate dehydrogenase kinase 1 (PDK1) acts as a ...targetable determinant of different metabolic states in acute myeloid leukemia (AML). PDK1
AMLs are OXPHOS-driven, are enriched for leukemic granulocyte-monocyte progenitor (L-GMP) signatures, and are associated with FLT3-ITD and NPM1cyt mutations. PDK1
AMLs however are OXPHOS
, wild type for FLT3 and NPM1, and are enriched for stemness signatures. Metabolic states can even differ between genetically distinct subclones within individual patients. Loss of PDK1 activity releases glycolytic cells into an OXPHOS state associated with increased ROS levels resulting in enhanced apoptosis in leukemic but not in healthy stem/progenitor cells. This coincides with an enhanced dependency on glutamine uptake and reduced proliferation in vitro and in vivo in humanized xenograft mouse models. We show that human leukemias display distinct metabolic states and adaptation mechanisms that can serve as targets for treatment.
Maintenance of epigenetic modifiers is of utmost importance to preserve the epigenome and consequently appropriate cellular functioning. Here, we analyzed Polycomb group protein (PcG) complex ...integrity in response to heat shock (HS). Upon HS, various Polycomb Repressive Complex (PRC)1 and PRC2 subunits, including CBX proteins, but also other chromatin regulators, are found to accumulate in the nucleolus. In parallel, binding of PRC1/2 to target genes is strongly reduced, coinciding with a dramatic loss of H2AK119ub and H3K27me3 marks. Nucleolar-accumulated CBX proteins are immobile, but remarkably both CBX protein accumulation and loss of PRC1/2 epigenetic marks are reversible. This post-heat shock recovery of pan-nuclear CBX protein localization and reinstallation of epigenetic marks is HSP70 dependent. Our findings demonstrate that the nucleolus is an essential protein quality control center, which is indispensable for recovery of epigenetic regulators and maintenance of the epigenome after heat shock.
Gene therapy in the central nervous system (CNS) is hindered by the presence of the blood-brain barrier, which restricts access of serum constituents and peripheral cells to the brain parenchyma. ...Expression of exogenously administered genes in the CNS has been achieved in vivo using highly invasive routes, or ex vivo relying on the direct implantation of genetically modified cells into the brain. Here we provide evidence for a novel, noninvasive approach for targeting potential therapeutic factors to the CNS. Genetically-modified hematopoietic cells enter the CNS and differentiate into microglia after bone-marrow transplantation. Up to a quarter of the regional microglial population is donor-derived by four months after transplantation. Microglial engraftment is enhanced by neuropathology, and gene-modified myeloid cells are specifically attracted to the sites of neuronal damage. Thus, microglia may serve as vehicles for gene delivery to the nervous system.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Upregulation of the plasma membrane receptor IL1RAP in Acute Myeloid Leukemia (AML) has been reported but its role in the context of the leukemic bone marrow niche is unclear. Here, we studied the ...signaling events downstream of IL1RAP in relation to leukemogenesis and normal hematopoiesis. High IL1RAP expression was associated with a leukemic GMP-like state, and knockdown of IL1RAP in AML reduced colony-forming capacity. Stimulation with IL1β resulted in the induction of multiple chemokines and an inflammatory secretome via the p38 MAPK and NFκB signaling pathways in IL1RAP-expressing AML cells, but IL1β-induced signaling was dispensable for AML cell proliferation and NFκB-driven survival. IL1RAP was also expressed in stromal cells where IL1β induced expression of inflammatory chemokines and cytokines as well. Intriguingly, the IL1β-induced inflammatory secretome of IL1RAPexpressing AML cells grown on a stromal layer of mesenchymal stem cells affected normal hematopoiesis including hematopoietic stem/progenitor cells while AML cell proliferation was not affected. The addition of Anakinra, an FDA-approved IL1 receptor antagonist, could reverse this effect. Therefore, blocking the IL1-IL1RAP signaling axis might be a good therapeutic approach to reduce inflammation in the bone marrow niche and thereby promote normal hematopoietic recovery over AML proliferation after chemotherapy.
Intra-tumor heterogeneity caused by clonal evolution is a major problem in cancer treatment. To address this problem, we performed label-free quantitative proteomics on primary acute myeloid leukemia ...(AML) samples. We identified 50 leukemia-enriched plasma membrane proteins enabling the prospective isolation of genetically distinct subclones from individual AML patients. Subclones differed in their regulatory phenotype, drug sensitivity, growth, and engraftment behavior, as determined by RNA sequencing, DNase I hypersensitive site mapping, transcription factor occupancy analysis, in vitro culture, and xenograft transplantation. Finally, we show that these markers can be used to identify and longitudinally track distinct leukemic clones in patients in routine diagnostics. Our study describes a strategy for a major improvement in stratifying cancer diagnosis and treatment.
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•Identification of clinically relevant leukemia-enriched plasma membrane proteins•Proteomics-informed prospective isolation of genetically distinct AML subclones•Subclones differ in transcription factor occupancy and transcriptional regulation•AML subclones display functional differences in vitro and in vivo
de Boer et al. identify plasma membrane proteins enriched on acute myeloid leukemia (AML) that enable prospective isolation of genetically distinct subclones with different functions from individual AML patients and can be used to longitudinally track distinct leukemic clones in patients in routine diagnostics.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Upregulation of the plasma membrane receptor IL1RAP in Acute Myeloid Leukemia (AML) has been reported but its role in the context of the leukemic bone marrow niche is unclear. We studied the ...signaling events downstream of IL1RAP in relation to leukemogenesis and healthy hematopoiesis. IL1RAP was significantly upregulated across various AML subtypes and was expressed on the outer membrane, whereby high IL1RAP expression was associated with a leukemic GMP-like state. Stimulation with IL1-beta resulted in the induction of multiple chemokines and an inflammatory secretome via the p38 MAPK and NF-kB signaling pathways. IL1-beta-induced signaling through IL1RAP was dispensable for AML cell proliferation and NF-kB-driven survival. IL1RAP was also expressed in stromal cells where IL1-beta-induced expression of inflammatory chemokines and cytokines as well. Intriguingly, the IL1-beta-induced inflammatory secretome of AML cells grown on stroma affected the proliferation of healthy hematopoietic cells while AML cell proliferation was much less affected. The addition of Anakinra, an FDA-approved IL1 receptor antagonist, could partially reverse this effect. Blocking the IL1-IL1RAP signaling axis might be a good therapeutic approach to reduce inflammation in the bone marrow niche and thereby promote normal hematopoietic recovery over AML proliferation after chemotherapy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The development of acute myeloid leukemia (AML) is a stepwise process with early founder mutations that give rise to hematopoietic stem cells (HSCs) with a clonal advantage, followed by subsequent ...driver mutations that result in transformation and full-blown leukemia. To study the development and progression of AML through time requires consecutive samples of (pre-)leukemic cells, however, this also comes with many practical challenges. Therefore, we implemented a recently published CRISPR methodology that allows modeling of AML mutations including DNMT3A, TET2, ASXL1, RUNX1, GATA2, and CEBPA p30 in primary HSCs. In short, bone marrow (BM) derived CD34+ cells are expanded, electroporated with ribonucleoprotein complexes, and in case of homologous directed repair, directly followed by HDR-template delivery using adeno-associated viruses. The HDR template consists of a SFFV-GFP, which is integrated in an early exon resulting in a loss of function (LOF) that can be traced through GFP expression. In general, we reach editing efficiencies ranging from 60-90% and HDR efficiencies of around 30-50%. Modeling CEBPA p30 in BM CD34+ cells resulted in a block in differentiation and increased colony forming potential in multiple replates, and uncontrolled cell growth. Proteome analysis revealed both known and unknown CEBPA p30 targets being differentially expressed. Injecting CEBPA p30 cells in NSGS mice resulted in a (pre-)leukemic phenotype with increased myeloid output, aberrant plasma membrane marker expression, and an immature phenotype. Currently, we are further evaluating the additional effect of recurrently co-mutated genes TET2 and GATA2. In addition, we also study the consequences of LOF mutations in DNMT3A, ASXL1, TET2, and RUNX1 in NSG and NSGS mice potentially followed by secondary hits, stresses and treatments in order to study the leukemic evolution through time.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Epigenetic regulators are frequently mutated in hematological malignancies including acute myeloid leukemia (AML). Thus, the identification and characterization of novel epigenetic drivers affecting ...AML biology holds potential to improve our basic understanding of AML and to uncover novel options for therapeutic intervention. To identify novel tumor suppressive epigenetic regulators in AML, we performed an in vivo short hairpin RNA (shRNA) screen in the context of CEBPA mutant AML. This identified the Histone 3 Lysine 4 (H3K4) demethylase KDM5C as a tumor suppressor, and we show that reduced Kdm5c/KDM5C expression results in accelerated growth both in human and murine AML cell lines, as well as in vivo in Cebpa mutant and inv(16) AML mouse models. Mechanistically, we show that KDM5C act as a transcriptional repressor through its demethylase activity at promoters. Specifically, KDM5C knockdown results in globally increased H3K4me3 levels associated with up-regulation of bivalently marked immature genes. This is accompanied by a de-differentiation phenotype that could be reversed by modulating levels of several direct and indirect downstream mediators. Finally, the association of KDM5C levels with long-term disease-free survival of female AML patients emphasizes the clinical relevance of our findings and identifies KDM5C as a novel female-biased tumor suppressor in AML.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Although there is growing evidence that N-terminal pro-brain natriuretic peptide (NT-proBNP) can be used as a powerful tool in risk prediction in patients with non–ST-elevation acute coronary ...syndrome (NSTEACS), the dynamic variation of serum concentrations in time is poorly understood. To gain insight into the dynamics of NT-proBNP, a study was performed using serial serum samples in patients admitted with NSTEACS.
A total of 24 patients admitted with NSTEACS was included in this study. Serial samples were taken at baseline, 8 hours, 16 hours, 24 hours, and 36 hours after admittance.
A highly dynamic pattern in serial measurements of NT-proBNP was observed. Although an increase in NT-proBNP serum levels already existed 8 hours after admittance, it did not reach significance as compared with baseline. The samples obtained 16, 24, and 36 hours after admission were all significantly increased as compared with the values at admission (
P < .01), generally leading to a >2-fold increase with peak values at 16 to 24 hours after admittance. Furthermore, considerable differences in NT-proBNP concentrations between patients were observed.
It was shown that NT-proBNP is a highly dynamic cardiac peptide. Strategic sampling at 16 to 24 hours after admittance could prove representative regarding the assessment of risk prediction and subsequent clinical decision making.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
To begin to understand the mechanisms that regulate self-renewal, differentiation, and transformation of human hematopoietic stem cells or to evaluate the efficacy of novel treatment modalities, stem ...cells need to be studied in their own species-specific microenvironment. By implanting ceramic scaffolds coated with human mesenchymal stromal cells into immune-deficient mice, we were able to mimic the human bone marrow niche. Thus, we have established a human leukemia xenograft mouse model in which a large cohort of patient samples successfully engrafted, which covered all of the important genetic and risk subgroups. We found that by providing a humanized environment, stem cell self-renewal properties were better maintained as determined by serial transplantation assays and genome-wide transcriptome studies, and less clonal drift was observed as determined by exome sequencing. The human leukemia xenograft mouse models that we have established here will serve as an excellent resource for future studies aimed at exploring novel therapeutic approaches.
•Humanized niche xenograft mouse models were generated that enabled engraftment of patients' leukemia cells covering all risk groups.•Self-renewal was better maintained in the humanized niches as determined by serial transplantation and genome-wide transcriptome studies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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