Descriptive sequencing of human leukemia samples has revealed the diversity and combinatorial complexity of oncogenic effector genes which can occur within all or just a subset of tumor cells. With ...the aim of deconvoluting the individual contributions of T-cell acute lymphoblastic leukemia (T-ALL) oncogenes, we employed our recently developed synthetic model of human T-ALL (Kusakabe et al, 2019) to perform a systematic analysis of transcriptional signatures associated with each of the four oncogenes used in our model. We identified synthetic leukemias to cluster based on two gene signatures which resembled distinct stages in T-cell development and had marked variation in MYCN expression, with its highest expression found on the more “immature” cluster. All synthetic leukemias carried activated NOTCH1, which is best known for upregulating MYC, but not MYCN. By analyzing publicly available RNA sequencing datasets, we found MYCN to be highly expressed in early T-cell progenitors (ETPs) and T-ALLs of more immature phenotype.
To assess oncogene “addiction” to MYCN, cell lines and primary synthetic leukemias were transduced with shRNAs or sgRNAs to knock-down (KD) or knock-out (KO) MYCN, respectively. By both approaches, bulk cell growth and/or clonogenic activity were reduced in KD/KO cells as compared to controls, supporting their functional dependence on MYCN. MYCN overexpression resulted in increased clonogenic activity and differentiation delay of NOTCH1-transduced cells in vitro but was still insufficient to generate leukemia in vivo. We are currently exploring other genes with similar expression patterns to MYCN to identify upstream/downstream mediators of MYCN dependence in this context. Our results emphasize stage-specific oncogene dependencies and identify alternate therapeutic targets in T-ALLs with high MYCN expression.
The Wnt signaling pathway has been shown to play important roles in normal hematopoietic stem cell biology and in the development of both acute and chronic myelogenous leukemia. Its role in ...maintaining established leukemia stem cells, which are more directly relevant to patients with disease, however, is less clear. To address what role Wnt signaling may play in T-cell acute lymphoblastic leukemia (T-ALL), we used a stably integrated fluorescent Wnt reporter construct to interrogate endogenous Wnt signaling activity in vivo. In this study, we report that active Wnt signaling is restricted to minor subpopulations within bulk tumors, that these Wnt-active subsets are highly enriched for leukemia-initiating cells (LICs), and that genetic inactivation of β-catenin severely reduces LIC frequency. We show further that β-catenin transcription is upregulated by hypoxia through hypoxia-inducible factor 1α (Hif1α) stabilization, and that deletion of Hif1α also severely reduces LIC frequency. Of note, the deletion of β-catenin or Hif1α did not impair the growth or viability of bulk tumor cells, suggesting that elements of the Wnt and Hif pathways specifically support leukemia stem cells. We also confirm the relevance of these findings to human disease using cell lines and patient-derived xenografts, suggesting that targeting these pathways could benefit patients with T-ALL.
•A real-time, integrated fluorescent Wnt reporter marks rare leukemia stem cells in T-ALL.•Deletion of β-catenin or Hif1α reduces LIC frequency in established tumors, but does not affect the growth of bulk cells.
•Noncanonical interactions of β-catenin with FOXO3 transcription factor promote LIC activity in early T-cell precursor ALL.•β-Catenin– and FOXO3-dependent gene signature identifies LIC-enriched ...CD82+CD117+ cell subsets, highly selected in MRD.
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T-cell acute lymphoblastic leukemia (T-ALL) is a T-cell malignancy characterized by cell subsets and enriched with leukemia-initiating cells (LICs). β-Catenin modulates LIC activity in T-ALL. However, its role in maintaining established leukemia stem cells remains largely unknown. To identify functionally relevant protein interactions of β-catenin in T-ALL, we performed coimmunoprecipitation followed by liquid chromatography–mass spectrometry. Here, we report that a noncanonical functional interaction of β-catenin with the Forkhead box O3 (FOXO3) transcription factor positively regulates LIC-related genes, including the cyclin-dependent kinase 4, which is a crucial modulator of cell cycle and tumor maintenance. We also confirm the relevance of these findings using stably integrated fluorescent reporters of β-catenin and FOXO3 activity in patient-derived xenografts, which identify minor subpopulations with enriched LIC activity. In addition, gene expression data at the single-cell level of leukemic cells of primary patients at the time of diagnosis and minimal residual disease (MRD) up to 30 days after the standard treatments reveal that the expression of β-catenin– and FOXO3-dependent genes is present in the CD82+CD117+ cell fraction, which is substantially enriched with LICs in MRD as well as in early T-cell precursor ALL. These findings highlight key functional roles for β-catenin and FOXO3 and suggest novel therapeutic strategies to eradicate aggressive cell subsets in T-ALL.
T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy characterized by an uncontrolled proliferation of immature T-cells. While current therapies cure ~80% of pediatric patients, adults fare ...more poorly with ~40% overall survival. Refractory cases and relapses are presumably due to the ineffective targeting of leukemia stem cells (LSC), previously described in human and in mouse models of T-ALL, and thought to be resistant to standard treatments.
Recently, we have reported that active signaling through the Wnt/β-catenin pathway is a defining feature of LSC in T-ALL, and that interruption of this signaling pathway abrogates disease propagation in vivo. Using an integrated, real-time reporter of Wnt/β-catenin signaling (7TGC; composed of 7 Tcf/Lef-binding sites upstream of a minimal promoter and GFP marker), we identified leukemia-initiating cell (LIC) activity to reside asymmetrically within the minor proportion of Wnt-active, GFP+ cells in primary mouse NOTCH1-induced T-cell leukemias. Moreover, inducible deletion of β-catenin in this context eliminated LIC activity.
Here, we report that LIC activity in this Wnt-active subpopulation is dependent on Lef1. Using Lef1loxP/loxP animals, we show that inducible Cre-mediated deletion of Lef1 in established leukemias extinguishes both Wnt/GFP reporter expression and LIC activity. To explore mechanisms underlying asymmetry of LIC activity within the tumor population, we have also investigated the differential expression of various Lef1 protein isoforms in Wnt-active (GFP+) vs. Wnt-inactive (GFP-) leukemic subsets and assessed their function in supporting LIC activity. These results suggest that β-catenin acts via Lef1 to support asymmetric LIC activity within the Wnt-active subset of leukemia cells.
No relevant conflicts of interest to declare.
Reactive oxygen species (ROS), a byproduct of cellular metabolism, damage intracellular macromolecules and, when present in excess, can promote normal hematopoietic stem cell differentiation and ...exhaustion. However, mechanisms that regulate the amount of ROS in leukemia-initiating cells (LICs) and the biological role of ROS in these cells are largely unknown. We show here that the ROS(low) subset of CD44(+) cells in T cell acute lymphoblastic leukemia (T-ALL), a malignancy of immature T cell progenitors, is highly enriched in the most aggressive LICs and that ROS accumulation is restrained by downregulation of protein kinase C θ (PKC-θ). Notably, primary mouse T-ALLs lacking PKC-θ show improved LIC activity, whereas enforced PKC-θ expression in both mouse and human primary T-ALLs compromised LIC activity. We also show that PKC-θ is regulated by a new pathway in which NOTCH1 induces runt-related transcription factor 3 (RUNX3), RUNX3 represses RUNX1 and RUNX1 induces PKC-θ. NOTCH1, which is frequently activated by mutation in T-ALL and required for LIC activity in both mouse and human models, thus acts to repress PKC-θ. These results reveal key functional roles for PKC-θ and ROS in T-ALL and suggest that aggressive biological behavior in vivo could be limited by therapeutic strategies that promote PKC-θ expression or activity, or the accumulation of ROS.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy with diverse oncogenic drivers, the relative contributions of which remain obscure. Enforced expression of select oncogenes is ...sufficient to generate T-ALL in mice, however, mouse models are limited in their value for understanding human disease. We recently developed the first “synthetic” model of human T-ALL by lentiviral transduction of normal human CD34+ cord blood progenitors with a combination of four known T-ALL oncogenes: NOTCH1, LMO2, TAL1, and BMI1 (NLTB). These cells expand robustly in culture and produce aggressive, serially transplantable T-ALL in immunodeficient mice. Additionally, we found that LTB alone fails to perform in vitro or in vivo, highlighting an essential role of NOTCH1. To determine the minimal complement of oncogenes required to generate de novo human T-ALL, we executed a “leave-one-out” and “leave-two-out” strategy. We scored the following oncogene combinations in vitro and in vivo: NLB, NLT, NTB, NL, NT, and NB. We found that the various oncogene combinations yielded a spectrum of aberrant phenotypes affecting cell differentiation and proliferation, and a subset produced aggressive leukemias in mice. We also performed RNA-seq on non-leukemogenic and preleukemic cell populations, and fully transformed leukemic cells to define gene expression programs necessary for cellular transformation. Our approach allows us to attribute specific gene programs and cellular phenotypes to each oncogene individually and in combination. Our synthetic approach is flexible, reproducible, and experimentally tractable, allowing functional testing of individual genetic variants and can also serve as a customizable platform for testing of targeted therapeutics.
T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T-cell progenitors, characterized by activating NOTCH1 mutations in over 50% of children and adult cases. Although intensive ...multiagent chemotherapy achieves cure in most pediatric patients, the majority of adults succumb quickly to their disease. The basis for this divergence is likely multifactorial, but we sought in this study to investigate whether cell intrinsic features might contribute to the disparate biologies in pediatric and adult patients.
In our prior abstract, we modeled pediatric and adult leukemias by transduction of hematopoietic stem/progenitor cells (HSPC) derived from mouse fetal liver (FL) and adult bone marrow (ABM) with activated NOTCH1 virus followed by transplantation into histocompatible recipient animals. We observed that whereas FL- and ABM-derived HSPC generate similar primary acute T-cell leukemias in terms of penetrance, latency, disease burden/distribution, and immunophenotype, FL leukemias exhibit much greater cycling activity than ABM leukemias, yet are dramatically impaired in their ability to propagate disease in secondary and tertiary recipients compared to ABM leukemias. Using a combination of gene expression profiling and in vitro culture assays, we attributed this differential behavior to NOTCH1-induced autocrine IGF signaling that is operative in FL, but not ABM-derived HSPC.
Here we report that NOTCH1 mediates its effects on IGF1 in FL-derived HSPC directly by physical occupancy over the IGF1 promoter in a dimerization-dependent fashion. As well, increased NOTCH1 occupancy at the IGF1 promoter region in FL tissues is associated with reduced histone H3K27 trimethylation (a mark of transcriptionally silent chromatin), yet there is equivalent histone H3K4 trimethylation (a mark identifying transcriptionally active promoters) in both FL and ABM tissues, suggesting that NOTCH1 may be responsible for interconverting the IGF1 locus between active and inactive, but poised chromatin states. NOTCH1 occupancy is also associated with enhanced physical interactions between the IGF1 promoter region and distant genomic loci as revealed by circularized chromosome conformation capture (4C) assay and confirmed by chromosome conformation capture (3C) assay, including sites with H3K4 monomethylation (a mark of transcriptional enhancers) suggesting that NOTCH1 promotes "looping in" of distant enhancer elements that drive IGF1 expression in FL tissues. We conclude from these studies that NOTCH1 enacts differential, developmental stage-specific transcriptional programs by a combination of local epigenetic patterning and long-range genomic interactions. These findings support the notion that pediatric and adult T-ALL may potentially be regarded as related, but biologically distinct diseases, and that novel, age-specific therapies that exploit these differences may improve clinical outcomes.
No relevant conflicts of interest to declare.
Reactive oxygen species (ROS), a byproduct of cellular metabolism, damage intracellular macromolecules and, when present in excess, can promote normal hematopoietic stem cell differentiation and ...exhaustion. However, mechanisms that regulate the amount of ROS in leukemia-initiating cells (LICs) and the biological role of ROS in these cells are largely unknown. We show here that the ROS(low) subset of CD44(+) cells in T cell acute lymphoblastic leukemia (T-ALL), a malignancy of immature T cell progenitors, is highly enriched in the most aggressive LICs and that ROS accumulation is restrained by downregulation of protein kinase C theta (PKC-theta). Notably, primary mouse T-ALLs lacking PKC-theta show improved LIC activity, whereas enforced PKC-theta expression in both mouse and human primary T-ALLs compromised LIC activity. We also show that PKC-theta is regulated by a new pathway in which NOTCH1 induces runt-related transcription factor 3 (RUNX3), RUNX3 represses RUNX1 and RUNX1 induces PKC-theta. NOTCH1, which is frequently activated by mutation in T-ALL and required for LIC activity in both mouse and human models, thus acts to repress PKC-theta. These results reveal key functional roles for PKC-theta and ROS in T-ALL and suggest that aggressive biological behavior in vivo could be limited by therapeutic strategies that promote PKC-theta expression or activity, or the accumulation of ROS.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Reactive oxygen species (ROS), a by-product of cellular metabolism, damage intracellular macromolecules and, in excess, can promote normal hematopoietic stem cell differentiation and exhaustion
1
–
3
.... However, mechanisms that regulate ROS levels in leukemia-initiating cells (LICs) and the biological role of ROS in these cells remain largely unknown. We show here the ROS
low
subset of CD44
+
cells in T-cell acute lymphoblastic leukemia (T-ALL), a malignancy of immature T-cell progenitors, to be highly enriched in the most aggressive LICs, and that ROS are maintained at low levels by downregulation of protein kinase C theta (PKCθ). Strikingly, primary mouse T-ALLs lacking PKCθ show improved LIC activity whereas enforced PKCθ expression in both mouse and human primary T-ALLs compromised LIC activity. We also demonstrate that PKCθ is positively regulated by RUNX1, and that NOTCH1, which is frequently activated by mutation in T-ALL
4
–
6
and required for LIC activity in both mouse and human models
7
,
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, downregulates PKCθ and ROS via a novel pathway involving induction of RUNX3 and subsequent repression of RUNX1. These results reveal key functional roles for PKCθ and ROS in T-ALL and suggest that aggressive biological behavior in vivo could be limited by therapeutic strategies that promote PKCθ expression/activity or ROS accumulation.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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