The chromatin remodeller Chd7 and the calcium ATPase Serca2 - adding new facets to the role of Notch in Hematopoiesis and T-ALL

Project I: Chd7 Deficiency does not affect N1-driven T-ALL Induction and Maintenance nor impair Hematopoiesis Notch1 has been shown to be a key driver in pediatric T-cell acute lymphobastic leukemia (T-ALL). Previous work in the lab identified the chromatin-remodeling enzyme Chd7 to be highly over-expressed in aggressive versus pre-malignant disease. The aim of this project was to study if Chd7 is required during T-ALL initiation and disease maintenance as well as to characterize its role in the hematopoietic system under physiological and stress conditions. Therefore, Chd7 cKO animals were crossed with the MxCre deleter to ablate Chd7 specifically in BM progenitors. Chd7 deficiency did not induce any overt phenotype in the hematopoietic system, when studied under âsteadyâ state or stress conditions. In addition, Chd7 proved to be dispensable in the initiation and maintenance of murine and human Notch1-driven T-ALL. Therefore, we can rule out a role of Chd7 during late-stage leukemogenesis in Notch1-induced murine and human T-ALL. Project II: Serca2, a putative drug target to fight Notch1-driven T cell acute lymphoblastic leukemia, and its role in Hematopoiesis The Notch signalling pathway represents a bona fide drug target. We and Roti et al. performed a chemical compound screen and identified Cyclopiazonic acid (CPA) as potent Notch inhibitor. Biological target of this compound is the Sarco/Endoplasmic Reticulum ATPase2 (Serca2) that is involved in calcium homeostasis within the cell. Recently, it was shown that Serca2 regulates Notch1 (N1) receptor maturation and inhibition of Serca ATPases, using thapsigargin, blocks T-ALL progression in xenografts (Roti et al., 2013). The putative global effects of Serca2 inhibition on hematopoiesis have, however, not been addressed in this study. Aim of this study was to characterize the function of Serca2 in the hematopoietic system. Therefore, we crossed Serca2 cKO with MxCre to ablate Serca2 function in BM progenitors. Serca2 haploinsufficiency did not lead to any overt phenotype under physiological or competitive conditions. In contrast, Serca2ÎMxCre chimeras showed (i) impaired long-term reconstitution capacity upon transplantation, (ii) a severe reduction of total BM, splenic and thymic cellularity and (iii) an enrichment of KLS cells, the cell pool that contains HSCs. Mature lineages were highly apoptotic, while KLS were not majorly affected by apoptosis, resided predominantly in G0 phase of the cell cycle, exhibited slower cycling kinetics and were functionally impaired as they formed less and smaller colony forming units in vitro. Deregulated calcium levels can lead to an accumulation of unfolded proteins in the ER. In order to resolve ER stress, the cell activates the unfolded protein response (UPR). In case of unresolved ER stress, however, the cell undergoes apoptosis. We demonstrate that mature hematopoietic lineages up-regulate UPR-associated and pro-apoptotic genes, while KLS cells, in contrast, show unperturbed expression of these genes. In addition, we performed in vitro experiments, inhibiting Serca function in Lin- BM cells via thapsigargin (TH) treatment. TH induced severe apoptosis in the overall cell population, while KLS accumulated and were blocked in the G0 phase, closely mimicking Serca2 LOF in vivo. We therefore question the therapeutic approach of targeting Serca2 in N1-driven T-ALL due to severe side effects induced by Serca2 inactivation in the BM.

Notch1-driven T-ALL chromatin landscape is regulated by Tcf1 in hematopoietic progenitors

Mateusz Waldemar Antoszewski

Notch1 receptor signaling is essential for T cell fate specification and physiological thymic T lymphocyte development. Its dysregulation and oncogenic activation are detected in almost 80% of pediatric patients suffering from T cell acute lymphoblastic leukemia (T-ALL). T-ALL is a hematological neoplasm arising from immature thymocytes. T cell factor 1 (Tcf1) is a crucial well-known transcription factor regulating the early stages of thymocyte maturation and Notch1 directly drives the expression of Tcf1 during normal T cell development. Here we assessed the role of Tcf1 in modulating chromatin topology which enables the initiation of T-ALL at the level of early hematopoietic progenitors. This allows for the discovery of stage-setting epigenetic regulators establishing a leukemia-prone chromatin landscape during Notch1-driven disease initiation. We found Tcf1 to be an essential mediator of Notch1 signaling during T-ALL induction. Inactivation of Tcf1 prevented leukemogenesis despite oncogenic Notch1 activation. Genomic analysis of hematopoietic progenitors revealed T cell lineage specification to be essential in T-ALL initiation prior to the appearance of phenotypic features of T cells. The expression of genes associated with the T cell lineage was dependent on Notch1 and Tcf1. Chromatin accessibility, chromosome conformation capture and ChIP-seq analysis revealed the co-regulatory epigenetic function of Notch1 and Tcf1 in imprinting a leukemic chromatin landscape. Epigenetic modulation of distal enhancers which regulate leukemic oncogenes has been well described in patient-derived T-ALL cells. The Notch1-Myc enhancer region was shown to be indispensable in the process of leukemogenesis. We have identified a novel regulatory locus, dependent on Tcf1 and Notch1, essential for the expression of Myc in pre-T-ALL cells. This genomic region termed Tcf1-regulated Myc enhancer (TMe) was highly conserved across species. TMe was essential for the transition of pre-leukemic cells to a transplantable full-blown T-ALL but was dispensable for the physiological development of T cells. After activation of the enhancer in hematopoietic progenitors, this regulatory site remained accessible and bound by Tcf1 in murine and human T-ALL cells. Tcf1 is therefore required for shaping the epigenetic landscape of hematopoietic progenitors overexpressing oncogenic Notch1, and thus regulates the expression of genes involved in leukemogenesis.

EPFL2021

Assessing the role of Hes1 and identification of target genes in human and murine T-ALL

Silvia Wirth

The Notch signalling pathway is an ancient cell signalling mechanism that enables short-range communications between cells and controls a broad spectrum of cell fates and developmental processes. In the haematopoietic system Notch signalling has been linked to physiological, but also to pathological phenotypes of T cell development. Identification of numerous activating mutations within the NOTCH1 receptor in human T cell acute lymphoblastic leukaemia (T-ALL) patient samples demonstrated that aberrant activation of this signalling pathway is a frequent event in T-ALL. Active Notch signalling leads to the expression of a plethora of genes that might be drivers or passengers in tumorigenesis including Hes1, which encodes a transcriptional repressor. We have therefore studied Hes1 in the context of T-ALL in mouse models that mimic human disease and in human T-ALL cell lines with the aim to evaluate whether Hes1 affects disease development and disease maintenance and to characterize its function on a molecular level. The first part of this study is dedicated to the functional characterisation of Hes1 in vivo in T-ALL mouse models and in vitro in human T-ALL cell lines. Using a retroviral bone marrow transplant model of T-ALL in which progenitor cells are transduced with a retrovirus expressing constitutive active Notch1 intracellular domain (NICD) we could show that Hes1 is not required in established, late stage T-ALL cells, but that it promotes tumour progression in the early stages of the disease. We have also investigated the function of HES1 in human T-ALL and can show that the overexpression of dominant negative versions of Hes1 in two patient derived cell lines (T-ALL1, CUTLL-1) does not affect proliferation or apoptosis, emphasising that HES1 does not affect cell viability once cells are fully transformed to the leukemic state. In the second part of the study we set out to systematically delineate the gene regulatory networks down-stream of Hes1 by combining RNA-seq and ChIP-seq analysis. Comparing the gene expression of murine premalignant, early stage NICD induced CD4+CD8+ DP cells in the presence and absence of Hes1 by RNA-seq, we unexpectedly found a predominant upregulation of genes in cells expressing Hes1 indicating that Hes1 might rather act as a positive regulator of transcription than as a transcriptional repressor of distinct target genes. Hes1 might influence transcription through secondary or indirect effects as combinatorial analysis of RNA-seq and ChIP-seq led to the identification of only a small number of potential direct target genes that are transcriptionally regulated by Hes1. In summary, the role of Hes1 in promoting the early stages of the disease appears to be highly complex and is likely to involve both direct and indirect control of gene regulatory networks.

EPFL2014

The Cell Differentiation Status Influences the Outcome of Notch-Induced Malignancy

Caroline Poisson

The Notch signaling cascade is a well conserved pathway that regulates many aspects of embryonic development and plays a crucial role in the differentiation process and tissue homeostasis in multiple organs of adult species. Several human pathological disorders, including cancer, arise as a consequence of the deregulation of the Notch signaling pathway. Notch deregulation, up to date, has been best characterized in acute T-cell lymphoblastic leukemia (T-ALL). Indeed, in T-ALL pediatric patients activating Notch1 mutations have been identified in more than 50% of the cases. Increased knowledge of the mechanisms underlying T-ALL development and maintenance is clearly needed as one out of 5 patients with T-ALL has a very poor prognosis. Moreover, the high toxicity of chemotherapeutic regimens has been associated with elevated risks of developing severe secondary health problems. In the present study, we characterized the role of Notch in T-ALL induction and maintenance. Using transgenic mouse models, we could demonstrate that the oncogenic potential of Notch is dependent on RBP-Jκ transcriptional activity. Indeed, the canonical Notch signaling pathway, mediated by the RBP-Jκ transcription factor, is essential for both Notch-mediated T-ALL induction and maintenance, as well as for the induction of a Notch-mediated lymphoproliferative disorder. Moreover, constitutive overexpression of Notch1 at discrete developmental stages within the hematopoietic system, led us to identify a correlation between the differentiation status of a cell, within the hierarchy of the hematopoietic tree, and its sensitivity to Notch1-induced transformation. We demonstrated that forced Notch1 expression in hematopoietic stem cells was not sufficient to induce T-ALL. Nevertheless, aberrant Notch1 expression in the bone marrow resulted in the development of a lethal lymphoproliferative disorder. Overexpression of Notch1 in thmus at the DN2 to DN3 stage of T-cell delvelopment generated an aggressive transplantable T-ALL with important metastatic potential. This suggested that progenitors already committed to the T-cell lineage are highly susceptible to Notch-mediated transformation, which is relevant to the human disease. Nevertheless, forced Notch1 expression after the β-selection checkpoint in the thymys, was no longer sufficient to induce transformation of late DN3 to DN4 thymocytes. We therefore established a murine genetic system that allowed us to dissect the mechanisms of aberrant Notch1 signaling in a lymphoproliferative disorder and a true T-ALL model. We were able to assess the genetic signature that is involved in the transplantability and metastatic potential of leukemic cells and generated a list of candidate genes implicated in these processes. In addition, we correlated genes identified in our murine models with genes associated as well in human T-ALL profiles to identify genes implicated in T-ALL pathogenesis in both mouse and human.

EPFL2011