White matter lesions (WMLs) are a common manifestation of small vessel disease (SVD) in the elderly population. They are associated with an enhanced risk of developing gait abnormalities, poor executive function, dementia, and stroke with high mortality. Hypoperfusion and the resulting endothelial damage are thought to contribute to the development of WMLs. The focus of the present study was the analysis of the microvascular bed in SVD patients with deep WMLs (DWMLs) by using double- and triple-label immunohistochemistry and immunofluorescence. Simultaneous visualization of collagen IV (COLL4)-positive membranes and the endothelial glycocalyx in thick sections allowed us to identify endothelial recession in different types of string vessels, and two new forms of small vessel/capillary pathology, which we called vascular bagging and ghost string vessels. Vascular bags were pouches and tubes that were attached to vessel walls and were formed by multiple layers of COLL4-positive membranes. Vascular bagging was most severe in the DWMLs of cases with pure SVD (no additional vascular brain injury, VBI). Quantification of vascular bagging, string vessels, and the density/size of CD68-positive cells further showed widespread pathological changes in the frontoparietal and/or temporal white matter in SVD, including pure SVD and SVD with VBI, as well as a significant effect of the covariate age. Plasma protein leakage into vascular bags and the white matter parenchyma pointed to endothelial damage and basement membrane permeability. Hypertrophic IBA1-positive microglial cells and CD68-positive macrophages were found in white matter areas covered with networks of ghost vessels in SVD, suggesting phagocytosis of remnants of string vessels. However, the overall vessel density was not altered in our SVD cohort, which might result from continuous replacement of vessels. Our findings support the view that SVD is a progressive and generalized disease process, in which endothelial damage and vascular bagging drive remodeling of the microvasculature.Electronic supplementary materialThe online version of this article (10.1186/s40478-018-0632-z) contains supplementary material, which is available to authorized users.
AMPK (adenosine monophosphate‐activated protein kinase) is phosphorylated (AMPK‐P) in response to low energy through allosteric activation by Adenosine mono‐ or diphosphate (AMP/ADP). Folliculin (FLCN) and the FLCN‐interacting proteins 1 and 2 (FNIP1, 2) modulate AMPK. FNIP1 deficiency patients have a AMPK‐P gain of function phenotype with hypertrophic cardiomyopathy, Wolff‐Parkinson‐White pre‐excitation syndrome, myopathy of skeletal muscles and combined immunodeficiency.
We previously identified an association of rapid engraftment of patient-derived leukemia cells transplanted into NOD/SCID mice with early relapse in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In search for the cellular and molecular profiles associated with this phenotype, we investigated the expression of microRNAs (miRNAs) in different engraftment phenotypes and patient outcomes and found high miR-497/195 expression in patient-derived xenograft samples with slow engraftment, derived from patients with favorable outcome. In contrast, epigenetic repression and low expression of these miRNAs was observed in rapidly engrafting samples associated with early relapse. Overexpression of miR-497/195 in patient-derived leukemia cells suppressed in vivo growth of leukemia and prolonged recipient survival. Conversely, inhibition of miR-497/195 led to increased leukemia cell growth. Key cell cycle regulators were downregulated upon miR-497/195 overexpression and we identified CDK4/CCND3-mediated control of G1/S transition as a principal mechanism for the suppression of BCP-ALL progression by miR-497/195. The critical role for miR-497/195-mediated cell cycle regulation was underscored by the finding in an additional independent series of patient samples, showing that high miR-497/195 expression together with a full sequence of CDKN2A/B was associated with excellent outcome, while deletion of CDKN2A/B together with low expression of miR-497/195 was associated with clearly inferior relapse-free survival. These findings point to the cooperative loss of cell cycle regulators as new prognostic factor indicating possible therapeutic targets for pediatric BCP-ALL.
Targeting BCL-2, a key regulator of survival in B-cell malignancies including precursor B-cell acute lymphoblastic leukemia, has become a promising treatment strategy. However, given the redundancy of anti-apoptotic BCL-2 family proteins (BCL-2, BCL-XL, MCL-1), single targeting may not be sufficient. When analyzing the effects of BH3-mimetics selectively targeting BCL-XL and MCL-1 alone or in combination with the BCL-2 inhibitor venetoclax, heterogeneous sensitivity to either of these inhibitors was found in ALL cell lines and in patient-derived xenografts. Interestingly, some venetoclax-resistant leukemias were sensitive to the MCL-1-selective antagonist S63845 and/or BCL-XL-selective A-1331852 suggesting functional mutual substitution. Consequently, co-inhibition of BCL-2 and MCL-1 or BCL-XL resulted in synergistic apoptosis induction. Functional analysis by BH3-profiling and analysis of protein complexes revealed that venetoclax-treated ALL cells are dependent on MCL-1 and BCL-XL, indicating that MCL-1 or BCL-XL provide an Achilles heel in BCL-2-inhibited cells. The effect of combining BCL-2 and MCL-1 inhibition by venetoclax and S63845 was evaluated in vivo and strongly enhanced anti-leukemia activity was found in a pre-clinical patient-derived xenograft model. Our study offers in-depth molecular analysis of mutual substitution of BCL-2 family proteins in acute lymphoblastic leukemia and provides targets for combination treatment in vivo and in ongoing clinical studies.
Deregulation of cell death pathways is a hallmark of many cancers and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Different pro- and anti-apoptotic molecules control apoptosis signaling. While pro-apoptotic BCL-2 homology domain 3 (BH3) proteins induce cellular death, apoptosis induction is counter-regulated by anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2). Therefore, inhibition of anti-apoptotic molecules has been developed as therapeutic strategy. Venetoclax (VEN) selectively binds to BCL-2, which leads to the release of pro-apoptotic molecules such as BIM resulting in apoptosis induction. In BCP-ALL, we and others showed preclinical activity of VEN and first clinical trials have started to evaluate VEN in ALL. Despite high activity, resistance can be acquired over time and acquisition of BCL2 mutations has been reported in CLL patients. In this study, we modeled VEN resistance in BCP-ALL and investigated underlying mechanisms in order to identify potential strategies to overcome VEN insensitivity. Starting from the BCP-ALL cell line RS4;11, five parallel VEN insensitive lines were generated by exposure to increasing concentrations of VEN over time (49 passages, over 8 months of continuous treatment). Simultaneously, five control lines were exposed to corresponding concentrations of solvent (DMSO). Measuring half maximal effective concentrations (EC50) over time showed increasing EC50 values from 4 nM to 26.2 µM in all VEN treated lines, reflecting acquired resistance in our model. Importantly, no mutations of the BCL2 gene were identified by sequencing at high coverage (Illumina AmpliSeq, 650- to 5951-fold), excluding acquisition of BCL2 mutations as mechanism of resistance. Next, we assessed expression levels of the mitochondrial apoptosis regulators BCL-2, MCL-1 and BCL-XL by western blot analysis. Comparing VEN insensitive to control cell lines, no differences in expression of the target molecule BCL-2 and similar levels of BCL-XL were observed. Most interestingly, all five VEN insensitive lines showed significant up-regulation of MCL-1 compared to all control lines. We next investigated the dependence of apoptosis signaling on different BCL-2 family members by exposing ALL cells to synthetic BH3-only peptides, which specifically bind to different regulators of mitochondrial apoptosis signaling (BH3-profiling), followed by analysis of apoptosis induction. Interestingly, dependence on BCL-2 was clearly reduced in all VEN insensitive lines indicated by almost lost mitochondrial priming. On the other hand, VEN insensitive ALL cells showed increased dependence on MCL-1. In order to analyze whether our modeled VEN resistance can be overcome by targeting MCL-1, we investigated sensitivity of these cells to the MCL-1 inhibitor S63845. High EC50 values for S63845 were found in all VEN insensitive and corresponding control lines, indicating low anti-MCL-1 activity. Importantly, the combination of S63845 with VEN synergistically induced cell death, showing that acquired VEN insensitivity in BCP-ALL can be overcome by co-targeting BCL-2 and MCL-1. To get insight into the molecular mechanisms underlying the synergism, we treated VEN insensitive ALL cells with VEN, S63845 or both and investigated binding of BIM to either BCL-2 or MCL-1 by immunoprecipitation. In the presence of VEN, clearly lower co-precipitation of BIM with BCL-2 but increased co-precipitation with MCL-1 was observed, indicating that pro-apoptotic BIM displaced from BCL-2 by VEN is sequestered by MCL-1, thereby counter-regulating VEN activity. Inversely, the MCL-1 inhibitor S63845 reduced binding of BIM to MCL-1 but increased BIM binding to BCL-2. Most interestingly, upon combination of both inhibitors, lower BIM binding to both BCL-2 and MCL-1 was found. This indicates that co-targeting MCL-1 can block the sequestration of pro-apoptotic BIM from BCL-2 to MCL-1, overcoming VEN resistance. Taken together, we show that acquired VEN resistance in BCP-ALL is characterized by up-regulated expression of counter-regulatory MCL-1 and can be overcome by simultaneous BCL-2 and MCL-1 inhibition, which prevents sequestration of BIM by MCL-1 after release from BCL-2. Disclosures Tausch: Roche: Consultancy, Honoraria, Speakers Bureau; AbbVie: Consultancy, Honoraria, Other: travel support, Speakers Bureau. Stilgenbauer:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau.
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