Translocation t(12;21), resulting in the ETV6-RUNX1 (or TEL-AML1) fusion protein, is present in 25% of pediatric patients with B-cell precursor acute lymphoblastic leukemia and is considered a first ...hit in leukemogenesis. A targeted therapy approach is not available for children with this subtype of leukemia. To identify the molecular mechanisms underlying ETV6-RUNX1-driven leukemia, we performed gene expression profiling of healthy hematopoietic progenitors in which we ectopically expressed ETV6-RUNX1. We reveal an ETV6-RUNX1-driven transcriptional network that induces proliferation, survival and cellular homeostasis. In addition, Vps34, an important regulator of autophagy, was found to be induced by ETV6-RUNX1 and up-regulated in ETV6-RUNX1-positive leukemic patient cells. We show that induction of Vps34 was transcriptionally regulated by ETV6-RUNX1 and correlated with high levels of autophagy. Knockdown of Vps34 in ETV6-RUNX1-positive cell lines severely reduced proliferation and survival. Inhibition of autophagy by hydroxychloroquine, a well-tolerated autophagy inhibitor, reduced cell viability in both ETV6-RUNX1-positive cell lines and primary acute lymphoblastic leukemia samples, and selectively sensitized primary ETV6-RUNX1-positive leukemia samples to L asparaginase. These findings reveal a causal relationship between ETV6-RUNX1 and autophagy, and provide pre-clinical evidence for the efficacy of autophagy inhibitors in ETV6-RUNX1-driven leukemia.
Pompe disease is an autosomal recessive lysosomal storage disorder characterized by progressive muscle weakness. The disease is caused by mutations in the acid α-glucosidase (GAA) gene. Despite the ...currently available enzyme replacement therapy (ERT), roughly half of the infants with Pompe disease die before the age of 3 years. Limitations of ERT are immune responses to the recombinant enzyme, incomplete correction of the disease phenotype, lifelong administration, and inability of the enzyme to cross the blood-brain barrier. We previously reported normalization of glycogen in heart tissue and partial correction of the skeletal muscle phenotype by ex vivo hematopoietic stem cell gene therapy. In the present study, using a codon-optimized GAA (GAAco), the enzyme levels resulted in close to normalization of glycogen in heart, muscles, and brain, and in complete normalization of motor function. A large proportion of microglia in the brain was shown to be GAA positive. All astrocytes contained the enzyme, which is in line with mannose-6-phosphate receptor expression and the key role in glycogen storage and glucose metabolism. The lentiviral vector insertion site analysis confirmed no preference for integration near proto-oncogenes. This correction of murine Pompe disease warrants further development toward a cure of the human condition.
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This publication reports that stem cell gene therapy using a codon-optimized gene encoding acid α-glucosidase (GAA) cures the mouse model of Pompe disease, a lysosomal storage disorder.
Severe congenital neutropenia (SCN) patients treated with CSF3/G-CSF to alleviate neutropenia frequently develop acute myeloid leukemia (AML). A common pattern of leukemic transformation involves the ...appearance of hematopoietic clones with CSF3 receptor (CSF3R) mutations in the neutropenic phase, followed by mutations in RUNX1 before AML becomes overt. To investigate how the combination of CSF3 therapy and CSF3R and RUNX1 mutations contributes to AML development, we make use of mouse models, SCN-derived induced pluripotent stem cells (iPSCs), and SCN and SCN-AML patient samples. CSF3 provokes a hyper-proliferative state in CSF3R/RUNX1 mutant hematopoietic progenitors but does not cause overt AML. Intriguingly, an additional acquired driver mutation in Cxxc4 causes elevated CXXC4 and reduced TET2 protein levels in murine AML samples. Expression of multiple pro-inflammatory pathways is elevated in mouse AML and human SCN-AML, suggesting that inflammation driven by downregulation of TET2 activity is a critical step in the malignant transformation of SCN.
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Combinatorial CSF3R and RUNX1 mutations seen in SCN-AML do not result in AML in miceAn additional mutation in Cxxc4 causes AML development in CSF3R/RUNX1 mutant miceMutant CXXC4 protein is more stable than wild-type and reduces TET2 protein levelsCXXC4 mutations are also found in de novo AML patients
Olofsen et al. show that acquisition of a mutation in Cxxc4 results in increased CXXC4 protein levels, reduced TET2 protein, increased inflammatory signaling, and leukemic progression of a CSF3R/RUNX1 mutant mouse model of severe congenital neutropenia (SCN).
Ubiquitination of cytokine receptors controls intracellular receptor routing and signal duration, but the underlying molecular determinants are unclear. The suppressor of cytokine signaling protein ...SOCS3 drives lysosomal degradation of the granulocyte colony-stimulating factor receptor (G-CSFR), depending on SOCS3-mediated ubiquitination of a specific lysine located in a conserved juxtamembrane motif. Here, we show that, despite ubiquitination of other lysines, positioning of a lysine within the membrane-proximal region is indispensable for this process. Neither reallocation of the motif nor fusion of ubiquitin to the C-terminus of the G-CSFR could drive lysosomal routing. However, within this region, the lysine could be shifted 12 amino acids toward the C-terminus without losing its function, arguing against the existence of a linear sorting motif and demonstrating that positioning of the lysine relative to the SOCS3 docking site is flexible. G-CSFR ubiquitination peaked after endocytosis, was inhibited by methyl-β-cyclodextrin as well as hyperosmotic sucrose and severely reduced in internalization-defective G-CSFR mutants, indicating that ubiquitination mainly occurs at endosomes. Apart from elucidating structural and spatio-temporal aspects of SOCS3-mediated ubiquitination, these findings have implications for the abnormal signaling function of G-CSFR mutants found in severe congenital neutropenia, a hematopoietic disorder with a high leukemia risk.
We have studied the intracellular distribution and internalization kinetics of the granulocyte colony-stimulating factor receptor (G-CSF-R) in living cells using fusion constructs of wild-type or ...mutant G-CSF-R and enhanced green fluorescent protein (EGFP). Under steady-state conditions the G-CSF-R localized predominantly to the Golgi apparatus, late endosomes, and lysosomes, with only low expression on the plasma membrane, resulting from spontaneous internalization. Internalization of the G-CSF-R was significantly accelerated by addition of G-CSF. This ligand-induced switch from slow to rapid internalization required the presence of G-CSF-R residue Trp650, previously shown to be essential for its signaling ability. Both spontaneous and ligand-induced internalization depended on 2 distinct amino acid stretches in the G-CSF-R COOH-terminus: 749-755, containing a dileucine internalization motif, and 756-769. Mutation of Ser749 at position –4 of the dileucine motif to Ala significantly reduced the rate of ligand-induced internalization. In contrast, mutation of Ser749 did not affect spontaneous G-CSF-R internalization, suggesting the involvement of a serine-threonine kinase specifically in ligand-accelerated internalization of the G-CSF-R. COOH-terminal truncation mutants of G-CSF-R, found in severe congenital neutropenia, lack the internalization motifs and were completely defective in both spontaneous and ligand-induced internalization. As a result, these mutants showed constitutively high cell-surface expression.
Introduction: Severe congenital neutropenia (SCN) is a genetically heterogeneous disease characterized by recurrent infections and a predisposition for malignant transformation. A wide variety of ...autosomal dominant or sporadic mutations in ELANE encoding neutrophil elastase (NE) are the most frequent cause of SCN, whereas recessive mutations in HAX1 are responsible for the autosomal recessive form of SCN known as Kostmann syndrome. How ELANE and HAX1 mutations cause SCN is still unclear. A prevailing hypothesis is that cellular stresses either caused by protein misfolding or malfunction in the case of ELANE-SCN, or by mitochondrial dysfunction in the case of HAX1-SCN, are drivers of the neutropenia. We focused on the role of the promyelocytic leukemia protein (PML) because PML is implicated in controlling cellular stress responses caused by reactive oxygen species (ROS) and protein misfolding and may exert both oncogenic and tumor-suppressive functions.
Aims: (1) To elucidate which cellular stress mechanisms are involved in different genetic subtypes of SCN. (2) To assess the role of PML in SCN with a predicted ELANE misfolding mutation.
Methods: We generated induced pluripotent stem cells (iPSCs) from healthy control and SCN patients with non-overlapping mutations: ELANE-I60F, ELANE-R103L and HAX1-W44X. CD34+CD45+ Hematopoietic Stem and Progenitor cells (HSPCs) were derived from iPSCs using the STEMdiff™ Hematopoietic Kit (STEMCELL Technologies). PML-/- iPSCs were created by introducing a stop codon in exon 3, shared by all PML isoforms, using CRISPR/Cas9 mediated genome editing.
Results: HSPCs derived from the SCN-iPSCs showed increased ROS levels as measured with CellROX Deep Red. Consequently, nuclear translocation of the antioxidant regulatory factor NRF2 was significantly elevated in both ELANE- and HAX1-mutant SCN HSPCs relative to controls. Mutation prediction analysis (Venselaar, BMC Bioinformatics 2010) showed that ELANE-I60F likely causes NE protein misfolding, whereas the ELANE-R103L mutation predictably causes NE malfunction by disrupting interactions with other proteins. The mutation in HAX1 was predicted to result in nonsense-mediated mRNA decay. Transcriptome analysis using Gene Set Enrichment Analysis (GSEA) confirmed upregulation of the nonsense mediated decay pathway in HAX1 mutant HSPCs and in line with previous studies (Klein et al, 2008), FACS analysis using TMRM and Mitotracker Red showed that loss of HAX1 protein reduced mitochondrial membrane integrity. Surprisingly, and in apparent conflict with the mutation prediction analysis, GSEA on ELANE-I60F HSPCs did not show increased expression of the classical unfolded protein response (UPR) pathway. Because PML has been implicated as an alternative player involved in degrading misfolded proteins (Guo, Mol Cell 2014), we investigated a possible link between ELANE-I60F and PML. Immunofluorescent staining showed increased numbers of PML nuclear bodies (PML-NBs) in ELANE-I60F derived HPSCs, but not in ELANE-R103L or HAX1-W44X HSPCs. Furthermore, GSEA showed upregulation of transcripts associated with PML chromatin binding in ELANE-I60F, but not in ELANE-R103L or HAX1-W44X cells. Deletion of PML by CRISPR-Cas9 revealed that PML enhanced MYC and mTORC1-induced transcription and cell cycle signatures in HSPCs from ELANE-I60F, suggestive of an oncogenic role of PML by inducing proliferation and metabolism in ELANE-I60F. In contrast, PML inhibited these pathways in HSPCs derived from healthy control iPSCs, indicative of its tumor-suppressive function in normal HSPCs. Finally, and perhaps most intriguingly, transcriptome analysis revealed that ELANE-I60F HSPCs expressed 5-fold higher levels of (mutant) ELANE transcripts than control HSPCs, which were reduced to basal levels after deletion of PML.
Conclusion: HAX1 and ELANE mutations cause oxidative stress in SCN-HSPCs by distinct mechanisms. We provide evidence for a dual role of PML in the pathogenesis of SCN caused by an ELANE mutation (I60F) associated with NE misfolding: (1) NE misfolding and increased oxidative stress cause elevated formation of PML-NBs, leading to increased expression of proliferation, cell cycle and metabolism associated transcripts, (2) PML strongly enhances the levels of ELANE transcripts, thus driving the expression of the disease causative ELANE mutant through a feed-forward mechanism.
No relevant conflicts of interest to declare.
Introduction: Severe congenital neutropenia (SCN) patients receive life-long treatment with CSF3/G-CSF to alleviate neutropenia and have a high risk to develop MDS or AML. The appearance of ...hematopoietic clones with CSF3 receptor (CSF3R) mutations represents a first step in MDS/AML progression, which is followed by mutations in RUNX1 shortly before MDS/AML becomes clinically overt. How intracellular signaling pathways are affected by the combination of CSF3 therapy, CSF3R and RUNX1 mutations, and how deregulation of these pathways contributes to MDS/AML development is unknown. Also, it has remained unclear how defects in epigenetic regulators such as ASXL1 or SUZ12, which are recurrently but less frequently mutated in SCN/AML, contribute to full malignant transformation.
Aims: (i) To elucidate how CSF3R and RUNX1 mutations in conjunction with CSF3 treatment contribute to leukemic progression in an in vivo serial transplantation mouse model (ii) To determine whether additional mutations, specifically in epigenetic regulators, are needed for full malignant transformation and (iii) To validate the relevance of the findings for clinical SCN and AML.
Mouse model and patient samples: Bone marrow (BM) cells from Csf3r-d715 mice, copying the most frequent CSF3R truncation mutant in SCN patients, were transduced with RUNX1 mutant D171N or empty vector control lentivirus and serially transplanted. Recipient mice were treated 3x a week with CSF3 or PBS (solvent control). Transcriptome analysis (RNA-sequencing + GSEA) and whole exome sequencing (WES) on FACS purified Lineage- cKit+ (LK) populations were done to identify molecular pathways associated with leukemic progression. Sequential CD34+ cell samples from a SCN/AML patient with identical CSF3R and RUNX1 mutations (Beekman, Blood 2012) and whole genome sequencing data from diagnostic AML samples were used for clinical comparisons.
Results: CSF3 treatment of primary recipients of Csf3r-RUNX1 mutant BM cells resulted in sustained (30+ weeks) presence of LK cells (16.5% ± 7), which we morphologically and functionally identified as myeloblasts, in the peripheral blood (PB). None of the mice succumbed to symptoms of AML, suggesting that the elevated PB myeloblast counts reflected a pre-leukemic state. Upon transplantation in secondary and tertiary recipients, mice developed a Csf3r-RUNX1 mutant AML that no longer depended on CSF3 administration. Transcriptome profiles of purified progenitor cells at the sequential steps of transformation in the mouse model and an SCN/AML patient with identical mutations shared striking similarities in deregulation of signaling mechanisms, particularly showing progressive upregulation of TNFα-, interferon- and interleukin-6-driven inflammatory responses during leukemic progression.
WES on the myeloblasts from these stages revealed a single additional clonal mutation in Cxxc4, an Internal tandem duplication (ITD) resulting in the in-frame insertion of two glycines at a.a. position 157 of the protein. This heterozygous mutation appeared in a subclone in the primary recipient (VAF: 0.27) and expanded in secondary and tertiary recipients, in which all AML cells harbored the Csf3r, RUNX1 and Cxxc4 mutations (VAF 0.52±0.06). The mutation resulted in a 10.8 fold (± 2.3) higher expression of a CXXC4 isoform. CXXC4 was shown to inhibit TET2 protein levels (Ko, Nature 2013). In agreement with this, TET2 levels were strongly (6.03 fold ± 0.78) reduced in the CXXC4 overexpressing leukemic samples.
Intriguingly, CXXC4 mutations have also been detected in human AML cases (n=13), including the homologous ITD mutations identified in our mouse model. We are currently investigating the frequencies and co-occurrence of CXXC4 mutations in RUNX1 mutant and other molecular subtypes of AML. Also, studies are in progress to determine how the CXXC4-ITD alters CXXC4 and TET2 protein expression.
Conclusion: By studying the leukemic progression of SCN, driven by CSF3 treatment and mutations in CSF3R and RUNX1, we have identified a mechanism of AML development that involves activation of multiple inflammatory pathways, mutation of CXXC4 and reduction of TET2 expression. These findings corroborate a recent study showing that inflammatory responses drive pre-leukemic myeloproliferation in Tet2 deficient mice (Meisel, Nature 2018).
Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
Summary
Severe congenital neutropenia (SCN) patients are prone to develop myelodysplastic syndrome (MDS) or acute myeloid leukaemia (AML). Leukaemic progression of SCN is associated with the early ...acquisition of CSF3R mutations in haematopoietic progenitor cells (HPCs), which truncate the colony‐stimulating factor 3 receptor (CSF3R). These mutant clones may arise years before MDS/AML becomes overt. Introduction and activation of CSF3R truncation mutants in normal HPCs causes a clonally dominant myeloproliferative state in mice treated with CSF3. Paradoxically, in SCN patients receiving CSF3 therapy, clonal dominance of CSF3R mutant clones usually occurs only after the acquisition of additional mutations shortly before frank MDS or AML is diagnosed. To seek an explanation for this discrepancy, we introduced a patient‐derived CSF3R‐truncating mutation in ELANE‐SCN and HAX1‐SCN derived and control induced pluripotent stem cells and compared the CSF3 responses of HPCs generated from these lines. In contrast to CSF3R‐mutant control HPCs, CSF3R‐mutant HPCs from SCN patients do not show increased proliferation but display elevated levels of inflammatory signalling. Thus, activation of the truncated CSF3R in SCN‐HPCs does not evoke clonal outgrowth but causes a sustained pro‐inflammatory state, which has ramifications for how these CSF3R mutants contribute to the leukaemic transformation of SCN.
Reactive oxygen species (ROS) regulate growth factor receptor signalling at least in part by inhibiting oxidation-sensitive phosphatases. An emerging concept is that ROS act locally to affect signal ...transduction in different subcellular compartments and that ROS levels are regulated by antioxidant proteins at the same local level. Here, we show that the ER-resident antioxidant peroxiredoxin 4 (Prdx4) interacts with the cytoplasmic domain of the granulocyte colony-stimulating factor receptor (G-CSFR). This interaction occurs when the activated G-CSFR resides in early endosomes. Prdx4 inhibits G-CSF-induced signalling and proliferation in myeloid progenitors, depending on its redox-active cysteine core. Protein tyrosine phosphatase 1b (Ptp1b) appears to be a major downstream effector controlling these responses. Conversely, Ptp1b might keep Prdx4 active by reducing its phosphorylation. These findings unveil a new signal transduction regulatory circuitry involving redox-controlled processes in the ER and activated cytokine receptors in endosomes.
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