Adrian Schwarzer

Gene Therapy for Wiskott-Aldrich Syndrome—Long-Term Efficacy and Genotoxicity

Wiskott-Aldrich syndrome gene therapy is feasible, but γ-retroviral vectors contribute a substantial risk of leukemogenesis. Taking the Sting Out of Gene Therapy Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive disorder characterized by low platelet count, immune deficiency, autoimmunity, and high risk of cancer. WAS is primarily a disorder of blood cells, and hematopoietic stem cell transplantation (HSCT) has been the only hope of cure. However, HSCT is restricted to patients who can find matching donors. One way to overcome this limitation is through gene therapy that restores the function of the mutated protein in HSCs from the patient. Now, Braun et al. report correction of WAS protein (WASP) in 9 of 10 patients that underwent HSC gene therapy. The authors used a γ-retroviral vector to correct WASP expression in autologous HSCs. After transfer to patients, these cells engrafted and WASP was expressed in lymphoid and myeloid cells and platelets in 9 of 10 patients. What’s more, this therapy caused either partial or complete resolution of symptoms. However, seven patients developed acute leukemia, and further analysis revealed genetic alterations such as chromosomal translocations. These studies suggest that with improved vector design, gene therapy may be feasible and effective for patient with WAS. Wiskott-Aldrich syndrome (WAS) is characterized by microthrombocytopenia, immunodeficiency, autoimmunity, and susceptibility to malignancies. In our hematopoietic stem cell gene therapy (GT) trial using a γ-retroviral vector, 9 of 10 patients showed sustained engraftment and correction of WAS protein (WASP) expression in lymphoid and myeloid cells and platelets. GT resulted in partial or complete resolution of immunodeficiency, autoimmunity, and bleeding diathesis. Analysis of retroviral insertion sites revealed >140,000 unambiguous integration sites and a polyclonal pattern of hematopoiesis in all patients early after GT. Seven patients developed acute leukemia [one acute myeloid leukemia (AML), four T cell acute lymphoblastic leukemia (T-ALL), and two primary T-ALL with secondary AML associated with a dominant clone with vector integration at the LMO2 (six T-ALL), MDS1 (two AML), or MN1 (one AML) locus]. Cytogenetic analysis revealed additional genetic alterations such as chromosomal translocations. This study shows that hematopoietic stem cell GT for WAS is feasible and effective, but the use of γ-retroviral vectors is associated with a substantial risk of leukemogenesis.

Mutant IDH1 promotes leukemogenesis in vivo and can be specifically targeted in human AML

Mutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are frequently found in glioma, acute myeloid leukemia (AML), melanoma, thyroid cancer, and chondrosarcoma patients. Mutant IDH produces 2-hydroxyglutarate (2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators. We investigated the role of mutant IDH1 using the mouse transplantation assay. Mutant IDH1 alone did not transform hematopoietic cells during 5 months of observation. However, mutant IDH1 greatly accelerated onset of myeloproliferative disease-like myeloid leukemia in mice in cooperation with HoxA9 with a mean latency of 83 days compared with cells expressing HoxA9 and wild-type IDH1 or a control vector (167 and 210 days, respectively, P = .001). Mutant IDH1 accelerated cell-cycle transition through repression of cyclin-dependent kinase inhibitors Cdkn2a and Cdkn2b, and activated mitogen-activated protein kinase signaling. By computational screening, we identified an inhibitor of mutant IDH1, which inhibited mutant IDH1 cells and lowered 2HG levels in vitro, and efficiently blocked colony formation of AML cells from IDH1-mutated patients but not of normal CD34(+) bone marrow cells. These data demonstrate that mutant IDH1 has oncogenic activity in vivo and suggest that it is a promising therapeutic target in human AML cells.

Development of novel efficient SIN vectors with improved safety features for Wiskott-Aldrich syndrome stem cell based gene therapy.

Gene therapy is a promising therapeutic approach to treat primary immunodeficiencies. Indeed, the clinical trial for the Wiskott-Aldrich Syndrome (WAS) that is currently ongoing at the Hannover Medical School (Germany) has recently reported the correction of all affected cell lineages of the hematopoietic system in the first treated patients. However, an extensive study of the clonal inventory of those patients reveals that LMO2, CCND2 and MDS1/EVI1 were preferentially prevalent. Moreover, a first leukemia case was observed in this study, thus reinforcing the need of developing safer vectors for gene transfer into HSC in general. Here we present a novel self-inactivating (SIN) vector for the gene therapy of WAS that combines improved safety features. We used the elongation factor 1 alpha (EFS) promoter, which has been extensively evaluated in terms of safety profile, to drive a codon-optimized human WASP cDNA. To test vector performance in a more clinically relevant setting, we transduced murine HSPC as well as human CD34+ cells and also analyzed vector efficacy in their differentiated myeloid progeny. Our results show that our novel vector generates comparable WAS protein levels and is as effective as the clinically used LTR-driven vector. Therefore, the described SIN vectors appear to be good candidates for potential use in a safer new gene therapy protocol for WAS, with decreased risk of insertional mutagenesis.

Lentiviral Vector Induced Insertional Haploinsufficiency of Ebf1 Causes Murine Leukemia

Integrating vectors developed on the basis of various retroviruses have demonstrated therapeutic potential following genetic modification of long-lived hematopoietic stem and progenitor cells. Lentiviral vectors (LV) are assumed to circumvent genotoxic events previously observed with γ-retroviral vectors, due to their integration bias to transcription units in comparison to the γ-retroviral preference for promoter regions and CpG islands. However, recently several studies have revealed the potential for gene activation by LV insertions. Here, we report a murine acute B-lymphoblastic leukemia (B-ALL) triggered by insertional gene inactivation. LV integration occurred into the 8th intron of Ebf1, a major regulator of B-lymphopoiesis. Various aberrant splice variants could be detected that involved splice donor and acceptor sites of the lentiviral construct, inducing downregulation of Ebf1 full-length message. The transcriptome signature was compatible with loss of this major determinant of B-cell differentiation, with partial acquisition of myeloid markers, including Csf1r (macrophage colony-stimulating factor (M-CSF) receptor). This was accompanied by receptor phosphorylation and STAT5 activation, both most likely contributing to leukemic progression. Our results highlight the risk of intragenic vector integration to initiate leukemia by inducing haploinsufficiency of a tumor suppressor gene. We propose to address this risk in future vector design.

Activation of Evi1 inhibits cell cycle progression and differentiation of hematopoietic progenitor cells

The transcription factor Evi1 has an outstanding role in the formation and transformation of hematopoietic cells. Its activation by chromosomal rearrangement induces a myelodysplastic syndrome with progression to acute myeloid leukemia of poor prognosis. Similarly, retroviral insertion-mediated upregulation confers a competitive advantage to transplanted hematopoietic cells, triggering clonal dominance or even leukemia. To study the molecular and functional response of primary murine hematopoietic progenitor cells to the activation of Evi1, we established an inducible lentiviral expression system. EVI1 had a biphasic effect with initial growth inhibition and retarded myeloid differentiation linked to enhanced survival of myeloblasts in long-term cultures. Gene expression microarray analysis revealed that within 24 h EVI1 upregulated ‘stemness’ genes characteristic for long-term hematopoietic stem cells (Aldh1a1, Abca1, Cdkn1b, Cdkn1c, Epcam, among others) but downregulated genes involved in DNA replication (Cyclins and their kinases, among others) and DNA repair (including Brca1, Brca2, Rad51). Cell cycle analysis demonstrated EVI1’s anti-proliferative effect to be strictly dose-dependent with accumulation of cells in G0/G1, but preservation of a small fraction of long-term proliferating cells. Although confined to cultured cells, our study contributes to new hypotheses addressing the mechanisms and molecular targets involved in preleukemic clonal dominance or leukemic transformation by Evi1.

Impact of MLL5 expression on decitabine efficacy and DNA methylation in acute myeloid leukemia

Hypomethylating agents are widely used in patients with myelodysplastic syndromes and unfit patients with acute myeloid leukemia. However, it is not well understood why only some patients respond to hypomethylating agents. We found previously that the effect of decitabine on hematopoietic stem cell viability differed between Mll5 wild-type and null cells. We, therefore, investigated the role of MLL5 expression levels on outcome of acute myeloid leukemia patients who were treated with decitabine. MLL5 above the median expression level predicted longer overall survival independent of DNMT3A mutation status in bivariate analysis (median overall survival for high vs. low MLL5 expression 292 vs. 167 days; P=0.026). In patients who received three or more courses decitabine, high MLL5 expression and wild-type DNMT3A independently predicted improved overall survival (median overall survival for high vs. low MLL5 expression 468 vs. 243 days; P=0.012). In transformed murine cells, loss of Mll5 was associated with resistance to low-dose decitabine, less global DNA methylation in promoter regions, and reduced DNA demethylation upon decitabine treatment. Together, these data support our clinical observation of improved outcome in decitabine-treated patients who express MLL5 at high levels, and suggest a mechanistic role of MLL5 in the regulation of DNA methylation.

Enantiomer-specific and paracrine leukemogenicity of mutant IDH metabolite 2-hydroxyglutarate

Canonical mutations in IDH1 and IDH2 produce high levels of the R-enantiomer of 2-hydroxyglutarate (R-2HG), which is a competitive inhibitor of α-ketoglutarate (αKG)-dependent enzymes and a putative oncometabolite. Mutant IDH1 collaborates with HoxA9 to induce monocytic leukemia in vivo. We used two mouse models and a patient-derived acute myeloid leukemia xenotransplantation (PDX) model to evaluate the in vivo transforming potential of R-2HG, S-2HG and αKG independent of the mutant IDH1 protein. We show that R-2HG, but not S-2HG or αKG, is an oncometabolite in vivo that does not require the mutant IDH1 protein to induce hyperleukocytosis and to accelerate the onset of murine and human leukemia. Thus, circulating R-2HG acts in a paracrine manner and can drive the expansion of many different leukemic and preleukemic clones that may express wild-type IDH1, and therefore can be a driver of clonal evolution and diversity. In addition, we show that the mutant IDH1 protein is a stronger oncogene than R-2HG alone when comparable intracellular R-2HG levels are achieved. We therefore propose R-2HG-independent oncogenic functions of mutant IDH1 that may need to be targeted in addition to R-2HG production to exploit the full therapeutic potential of IDH1 inhibition.

Constitutive IRF8 expression inhibits AML by activation of repressed immune response signaling

Myeloid differentiation is blocked in acute myeloid leukemia (AML), but the molecular mechanisms are not well characterized. Meningioma 1 (MN1) is overexpressed in AML patients and confers resistance to all-trans retinoic acid-induced differentiation. To understand the role of MN1 as a transcriptional regulator in myeloid differentiation, we fused transcriptional activation (VP16) or repression (M33) domains with MN1 and characterized these cells in vivo. Transcriptional activation of MN1 target genes induced myeloproliferative disease with long latency and differentiation potential to mature neutrophils. A large proportion of differentially expressed genes between leukemic MN1 and differentiation-permissive MN1VP16 cells belonged to the immune response pathway like interferon-response factor (Irf) 8 and Ccl9. As MN1 is a cofactor of MEIS1 and retinoic acid receptor alpha (RARA), we compared chromatin occupancy between these genes. Immune response genes that were upregulated in MN1VP16 cells were co-targeted by MN1 and MEIS1, but not RARA, suggesting that myeloid differentiation is blocked through transcriptional repression of shared target genes of MN1 and MEIS1. Constitutive expression of Irf8 or its target gene Ccl9 identified these genes as potent inhibitors of murine and human leukemias in vivo. Our data show that MN1 prevents activation of the immune response pathway, and suggest restoration of IRF8 signaling as therapeutic target in AML.

Hyperactivation of mTORC1 and mTORC2 by multiple oncogenic events causes addiction to eIF4E-dependent mRNA translation in T-cell leukemia

High activation of the PI3K–AKT–mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL). The activity of the master regulator of this pathway, PTEN, is often impaired in T-ALL. However, experimental evidence suggests that input from receptor tyrosine kinases (RTKs) is required for sustained mTOR activation, even in the absence of PTEN. We previously reported the expression of Neurotrophin receptor tyrosine kinases (TRKs) and their respective ligands in primary human leukemia samples. In the present study we aimed to dissect the downstream signaling cascades of TRK-induced T-ALL in a murine model and show that T-ALLs induced by deregulated receptor tyrosine kinase signaling acquire activating mutations in Notch1 and lose PTEN during clonal evolution. Some clones additionally lost one allele of the homeodomain transcription factor Cux1. All events independently led to a gradual hyperactivation of both mTORC1 and mTORC2 signaling. We dissected the role of the individual mTOR complexes by shRNA knockdown and found that the separate depletion of mTORC1 or mTORC2 reduced the growth of T-ALL blasts, but was not sufficient to induce apoptosis. In contrast, knockdown of the mTOR downstream effector eIF4E caused a striking cytotoxic effect, demonstrating a critical addiction to cap-dependent mRNA-translation. Although high mTORC2–AKT activation is commonly associated with drug-resistance, we demonstrate that T-ALL displaying a strong mTORC2–AKT activation were specifically susceptible to 4EGI-1, an inhibitor of the eIF4E–eIF4G interaction. To decipher the mechanism of 4EGI-1, we performed a genome-wide analysis of mRNAs that are translationally regulated by 4EGI-1 in T-ALL. 4EGI-1 effectively reduced the ribosomal occupancy of mRNAs that were strongly upregulated in T-ALL blasts compared with normal thymocytes including transcripts important for translation, mitochondria and cell cycle progression, such as cyclins and ribosomal proteins. These data suggest that disrupting the eIF4E–eIF4G interaction constitutes a promising therapy strategy in mTOR-deregulated T-cell leukemia.

The non-coding RNA landscape of human hematopoiesis and leukemia

Non-coding RNAs have emerged as crucial regulators of gene expression and cell fate decisions. However, their expression patterns and regulatory functions during normal and malignant human hematopoiesis are incompletely understood. Here we present a comprehensive resource defining the non-coding RNA landscape of the human hematopoietic system. Based on highly specific non-coding RNA expression portraits per blood cell population, we identify unique fingerprint non-coding RNAs—such as LINC00173 in granulocytes—and assign these to critical regulatory circuits involved in blood homeostasis. Following the incorporation of acute myeloid leukemia samples into the landscape, we further uncover prognostically relevant non-coding RNA stem cell signatures shared between acute myeloid leukemia blasts and healthy hematopoietic stem cells. Our findings highlight the importance of the non-coding transcriptome in the formation and maintenance of the human blood hierarchy.While micro-RNAs are known regulators of haematopoiesis and leukemogenesis, the role of long non-coding RNAs is less clear. Here the authors provide a non-coding RNA expression landscape of the human hematopoietic system, highlighting their role in the formation and maintenance of the human blood hierarchy.