Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood cancer and the leading cause of cancer-related mortality in children. T cell ALL (T-ALL) represents about 15% of pediatric ...ALL cases and is considered a high-risk disease. T-ALL is often associated with resistance to treatment, including steroids, which are currently the cornerstone for treating ALL; moreover, initial steroid response strongly predicts survival and cure. However, the cellular mechanisms underlying steroid resistance in T-ALL patients are poorly understood. In this study, we combined various genomic datasets in order to identify candidate genetic mechanisms underlying steroid resistance in children undergoing T-ALL treatment.
We performed whole genome sequencing on paired pre-treatment (diagnostic) and post-treatment (remission) samples from 13 patients, and targeted exome sequencing of pre-treatment samples from 69 additional T-ALL patients. We then integrated mutation data with copy number data for 151 mutated genes, and this integrated dataset was tested for associations of mutations with clinical outcomes and in vitro drug response. Our analysis revealed that mutations in JAK1 and KRAS, two genes encoding components of the interleukin 7 receptor (IL7R) signaling pathway, were associated with steroid resistance and poor outcome. We then sequenced JAK1, KRAS, and other genes in this pathway, including IL7R, JAK3, NF1, NRAS, and AKT, in these 69 T-ALL patients and a further 77 T-ALL patients. We identified mutations in 32% (47/146) of patients, the majority of whom had a specific T-ALL subtype (early thymic progenitor ALL or TLX). Based on the outcomes of these patients and their prednisolone responsiveness measured in vitro, we then confirmed that these mutations were associated with both steroid resistance and poor outcome. To explore how these mutations in IL7R signaling pathway genes cause steroid resistance and subsequent poor outcome, we expressed wild-type and mutant IL7R signaling molecules in two steroid-sensitive T-ALL cell lines (SUPT1 and P12 Ichikawa cells) using inducible lentiviral expression constructs. We found that expressing mutant IL7R, JAK1, or NRAS, or wild-type NRAS or AKT, specifically induced steroid resistance without affecting sensitivity to vincristine or L-asparaginase. In contrast, wild-type IL7R, JAK1, and JAK3, as well as mutant JAK3 and mutant AKT, had no effect. We then performed a functional study to examine the mechanisms underlying steroid resistance and found that, rather than changing the steroid receptor's ability to activate downstream targets, steroid resistance was associated with strong activation of MEK-ERK and AKT, downstream components of the IL7R signaling pathway, thereby inducing a robust antiapoptotic response by upregulating MCL1 and BCLXL expression. Both the MEK-ERK and AKT pathways also inactivate BIM, an essential molecule for steroid-induced cell death, and inhibit GSK3B, an important regulator of proapoptotic BIM. Importantly, treating our cell lines with IL7R signaling inhibitors restored steroid sensitivity. To address clinical relevance, we treated primary T-ALL cells obtained from 11 patients with steroids either alone or in combination with IL7R signaling inhibitors; we found that including a MEK, AKT, mTOR, or dual PI3K/mTOR inhibitor strongly increased steroid-induced cell death. Therefore, combining these inhibitors with steroid treatment may enhance steroid sensitivity in patients with ALL. The main limitation of our study was the modest cohort size, owing to the very low incidence of T-ALL.
Using an unbiased sequencing approach, we found that specific mutations in IL7R signaling molecules underlie steroid resistance in T-ALL. Future prospective clinical studies should test the ability of inhibitors of MEK, AKT, mTOR, or PI3K/mTOR to restore or enhance steroid sensitivity and improve clinical outcome.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Monoallelic inactivation of CCCTC-binding factor (CTCF) in human cancer drives altered methylated genomic states, altered CTCF occupancy at promoter and enhancer regions, and deregulated global gene ...expression. In patients with T cell acute lymphoblastic leukemia (T-ALL), we find that acquired monoallelic CTCF-inactivating events drive subtle and local genomic effects in nearly half of t(5; 14) (q35; q32.2) rearranged patients, especially when CTCF-binding sites are preserved in between the BCL11B enhancer and the TLX3 oncogene. These solitary intervening sites insulate TLX3 from the enhancer by inducing competitive looping to multiple binding sites near the TLX3 promoter. Reduced CTCF levels or deletion of the intervening CTCF site abrogates enhancer insulation by weakening competitive looping while favoring TLX3 promoter to BCL11B enhancer looping, which elevates oncogene expression levels and leukemia burden.
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•Monoallelic CTCF aberrations are associated with patients with TLX3-BCL11B-rearranged T-ALL•CTCF aberrations coincide with intervening CTCF sites in BCL11B-TLX3 breakpoint regions•Intervening CTCF sites reduce oncogene levels by promoting alternative promoter loops•Aberrations that lower functional CTCF levels restore enhancer-promoter interactions
Smits et al. report that TLX3 chromosomal translocations in T cell acute lymphoblastic leukemia patients recurrently include intervening CTCF-binding sites in the breakpoint area. Pressure to acquire inactivating aberrations in CTCF abrogate consequential enhancer insulation that promotes TLX3 promoter to BCL11B enhancer looping boosts higher oncogene expression levels and leukemia burden.
Physiological and pathogenic interleukin-7-receptor (IL7R)-induced signaling provokes glucocorticoid resistance in a subset of patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL). ...Activation of downstream STAT5 has been suggested to cause steroid resistance through upregulation of anti-apoptotic BCL2, one of its downstream target genes. Here we demonstrate that isolated STAT5 signaling in various T-ALL cell models is insufficient to raise cellular steroid resistance despite upregulation of BCL2 and BCL-XL. Upregulation of anti-apoptotic BCL2 and BCLXL in STAT5-activated T-ALL cells requires steroid-induced activation of NR3C1. For the BCLXL locus, this is facilitated by a concerted action of NR3C1 and activated STAT5 molecules at two STAT5 regulatory sites, whereas for the BCL2 locus this is facilitated by binding of NR3C1 at a STAT5 binding motif. In contrast, STAT5 occupancy at glucocorticoid response elements does not affect the expression of NR3C1 target genes. Strong upregulation of BIM, a NR3C1 pro-apoptotic target gene, upon prednisolone treatment can counterbalance NR3C1/STAT5-induced BCL2 and BCL-XL expression downstream of IL7- induced or pathogenic IL7R signaling. This explains why isolated STAT5 activation does not directly impair the steroid response. Our study suggests that STAT5 activation only contributes to steroid resistance in combination with cellular defects or alternative signaling routes that disable the pro-apoptotic and steroid-induced BIM response.
Human T-cell development is less well understood than its murine counterpart. We previously described the transcriptional landscape of 11 immature human T-cell developmental stages developed from ...hematopoietic stem cells on the OP9-DL1 system that match those of ex vivo flow-sorted murine and human thymocyte subsets. We defined a gene signature comprising of 547 genes that distinguishes pre- from post- αβ T-cell commitment stages. Commitment is marked by loss of dim CD44 expression of early CD7+CD5+CD45dim cells, before acquiring CD1a surface expression. Unlike uncommitted CD44dimCD1a- thymocytes,CD44-CD1a- post-committed thymocytes undergo first TCR rearrangements while having lost myeloid-, B- or NK-lineage potential.1We used this gene signature to study whether genetic or transcription-based subtypes of human T-cell acute lymphoblastic leukemia are connected to pre- or post-commitment T-cell development.
Clustering of human T-ALL using our 547 pre- and post-commitment gene signature distinguished four T-ALL clusters that were highly similar to the four subgroups that we identified before using an unsupervised cluster analysis. This previous analysis distinguished immature/ETP ALL, TLX, Proliferative and TALLMO subtypes, strongly correlated with MEF2C or MEF2C -activating abnormalities, HOXA -activating or TLX3 rearrangements, NKX2-1 or TLX1 rearrangements, and aberrations affecting members of the TAL1 and/or LMO2 gene families, respectively.2 Further inspection of the original unsupervised gene expression signature revealed that nearly half of all genes overlapped with genes that were expressed at pre- or post-commitment. This implies strong dependency or collaboration of specific oncogene-driven pathogenic programs and pre- or post-commitment programs. In fact, Immature/ETP-ALL and TLX subtypes are each strongly characterized by expressing a pre-commitment program, whereas the Proliferative and TALLMO subtypes are associated with the post-commitment program. Interestingly, the TLX subgroup that has preferentially been associated with γδ-lineage arrest implies that normal γδ T-cell development does not transit through the αβ T-cell commitment point before branching off to the γδ-lineage. Consistent with this, rearrangements of the BCL11B locus that impact on critical levels of the haploinsufficient BCL11B transcription factor for T-cell commitment function are nearly exclusively associated with these ‘pre-commitment’ ETP-ALL and TLX subgroups. In contrast, TCR-driven chromosomal aberrations were almost exclusively found in the post-commitment Proliferative and TALLMO subtypes.
We then further explored whether particular networks or signaling routes as captured by specific Hallmark gene sets are activated in pre- or post commitment programs and associated with particular T-ALL subtypes. We identified hallmark gene sets connected to interferon gamma and inflammatory responses that are activated in ETP-ALL, as well as signaling of the KRAS, the IL2-STAT5 and the IL6-JAK-STAT3 pathways including CD44 . For the TLX subgroup, hallmark gene sets included NOTCH signaling, coagulation, angiogenesis and myogenesis. These data are in agreement with activating mutations in IL7R-JAK-STAT5B and RAS pathways that are most prevalent in ETP ALL and TLX subtypes. The TLX subtype is particularly associated with strongly-activating NOTCH mutations in line with the requirement of strong NOTCH signaling for γδ T-cell development. Hallmark gene sets for the Proliferative group include E2F and MYC targets, G2M checkpoint and glycolysis. Surprisingly, we did not identify specific Hallmark gene sets for the TALLMO subtype, implying that it may include patients that have arrested at several distinct stages of αβ development.
In conclusion, our data demonstrate that T-ALL oncogenes can exert their pathogenic effects only in the context of particular T-cell developmental programs.
1Canté-Barrett, K. et al. Front Immunol 2017; 8:32. doi: 10.3389/fimmu.2017.00032
2Homminga, I. et al. Cancer Cell 2011; 19(4). doi: 10.1016/j.ccr.2011.02.008
No relevant conflicts of interest to declare.
Physiological and pathogenic interleukin-7-receptor (IL7R)-induced signaling provokes glucocorticoid resistance in a subset of patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL). ...Activation of downstream STAT5 has been suggested to cause steroid resistance through upregulation of anti-apoptotic
BCL2
, one of its downstream target genes. Here we demonstrate that isolated STAT5 signaling in various T-ALL cell models is insufficient to raise cellular steroid resistance despite upregulation of BCL2 and BCL-XL. Upregulation of anti-apoptotic
BCL2
and
BCLXL
in STAT5-activated T-ALL cells requires steroid-induced activation of NR3C1. For the
BCLXL
locus, this is facilitated by a concerted action of NR3C1 and activated STAT5 molecules at two STAT5 regulatory sites, whereas for the
BCL2
locus this is facilitated by binding of NR3C1 at a STAT5 binding motif. In contrast, STAT5 occupancy at glucocorticoid response elements does not affect the expression of NR3C1 target genes. Strong upregulation of BIM, a NR3C1 pro-apoptotic target gene, upon prednisolone treatment can counterbalance NR3C1/STAT5-induced BCL2 and BCL-XL expression downstream of IL7-induced or pathogenic IL7R signaling. This explains why isolated STAT5 activation does not directly impair the steroid response. Our study suggests that STAT5 activation only contributes to steroid resistance in combination with cellular defects or alternative signaling routes that disable the pro-apoptotic and steroid-induced BIM response.
Abstract
The thymus has a remarkable ability to sustain T cell development and T cell output from thymuses with quantitative and qualitative deficiencies in T cell progenitors, suggesting a role for ...homeostatic pathways in controlling T cell development. However, these homeostatic pathways are currently uncharacterized. We find that Delta-like 4 (Dll4), which is expressed on thymic epithelial cells (TECs) and is essential for driving T cell lineage commitment, is dynamically regulated in response to thymocyte deficiencies, and plays a major role in thymocyte homeostasis. We observe that TECs express elevated Dll4 levels in multiple lines of mice that have defective T cell development, as well as in mice subjected to antibody or irradiation-induced thymocyte depletion. Recovery of thymus cellularity after thymic injury is accompanied by re-establishment of normal levels of Dll4. In vitro proliferation of thymocytes increases proportionally in response to elevated levels of Dll4 expressed on stromal cells. Furthermore, Dll4 levels on stromal cells diminish in response to interactions with thymocytes themselves. These findings implicate dynamic control of Dll4 levels as a major homeostatic mechanism that regulates thymocyte proliferation.
The thymus is the central site of T-cell development and thus is of fundamental importance to the immune system, but little information exists regarding molecular regulation of thymus development in ...humans. Here we demonstrate, via spatial and temporal expression analyses, that the genetic mechanisms known to regulate mouse thymus organogenesis are conserved in humans. In addition, we provide molecular evidence that the human thymic epithelium derives solely from the third pharyngeal pouch, as in the mouse, in contrast to previous suggestions. Finally, we define the timing of onset of hematopoietic cell colonization and epithelial cell differentiation in the human thymic primordium, showing, unexpectedly, that the first colonizing hematopoietic cells are CD45(+)CD34(int/-). Collectively, our data provide essential information for translation of principles established in the mouse to the human, and are of particular relevance to development of improved strategies for enhancing immune reconstitution in patients.
Introduction. The CCCTC-binding factor (CTCF) regulates the 3D chromatin architecture by facilitating chromosomal loops and forming the boundaries of structural domains. In addition, CTCF is an ...important transcription factor and regulator of antigen receptor and T cell receptor recombination events. CTCF inactivating events have been found in various human cancers. Loss-of-heterozygosity (LOH) or inactivating missense mutations in specific zinc- fingers have been identified in many human cancers including sporadic breast cancer, prostate cancer, Wilms-tumors and acute lymphoblastic leukemia (ALL). Heterozygous deletions or point mutations have been identified in over half of the patients with breast cancer or uterine endometrial cancers, deregulating global gene expression by altering methylated genomic states and poor survival. Here, we investigated the functional significance and molecular-cytogenetic associations of CTCF aberrations in T-cell acute lymphoblastic leukemia patients.
Methods. Biopsies from a cohort of 181 pediatric T-ALL patients who enrolled on DCOG or COALL protocols and/or their derivative patient-derived xenograft models were screened for alterations in global DNA copy number, methylation status, topologically associating domain organization and CTCF and cohesion binding patterns and changes in local TLX3 and BCL11B promoter enhancer loops using array-comparative genomic hybridization, single molecule Molecular Inversion Probe sequencing, targeted locus amplification, gene expression and DNA methylation microarrays, Hi-C sequencing, Chromatin Immunoprecipitation and/or real-time quantitative PCR. Ctcf f/fl mice 1 were crossed on a the Lck-cre transgenic background 2 to study the impact of Ctcf loss during early T-cell development.
Results. We here describe that inactivation of CTCF can drive subtle and local genomic effects that elevate oncogene expression levels from driver chromosomal rearrangements. We find that for T cell acute lymphoblastic leukemia (T-ALL), heterozygous CTCF deletions or inactivating mutations are present in nearly 50 percent of t(5;14)(q35;q32.2) rearranged patients that positions the TLX3 oncogene in the vicinity of the BCL11B enhancer. Functional CTCF loss results in diminished expression of the αβ-lineage commitment factor BCL11B from the non-rearranged allele and γδ-lineage development. Unexpectedly, it also drives higher levels of the TLX3 oncogene from the translocated allele. We demonstrate that heterozygous CTCF aberrations specifically occur in TLX3-rearranged patients with distal breakpoints that preserve CTCF bindings sites in the translocation breakpoint areas in between the BCL11B enhancer and the TLX3 oncogene. We show that these intervening CTCF sites insulate TLX3 from the enhancer by forming competitive loops with TLX3. Upon loss of CTCF, or the deletion of the intervening CTCF sites, these competitive loops are weakened and loops with the BCL11B enhancer are stimulated, boosting TLX3 oncogene expression levels and leukemia burden in these T-ALL patients.
Conclusions. CTCF aberrations are especially associated with t(5;14)(q35;q32.2) rearranged T-ALL patients who maintain TLX3-proximal CTCF sites reflects a necessity to neutralize these sites in order to topologically enable the distal BCL11B enhancer to interact with the TLX3 oncogene and to boost its expression. Collectively, this provides direct demonstration of a mechanism in which loss of CTCF result in removal of enhancer insulation that facilitates elevated levels of an oncogene in leukemia.
References.
1. Heath H, Ribeiro de Almeida C, Sleutels F, et al. CTCF regulates cell cycle progression of alphabeta T cells in the thymus. EMBO J. 2008;27(21):2839-2850.
2. Lee PP, Fitzpatrick DR, Beard C, et al. A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival. Immunity. 2001;15(5):763-774.
Splinter: Cergentis BV: Current Employment. Van Eyndhoven: Agilent Technologies Netherland: Current Employment. Van Min: Cergentis BV: Current Employment. Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company.
Leucine‐rich repeat‐containing G protein‐coupled receptor (Lgr)5 is a marker for epithelial stem cells in the adult intestine of mice. Lgr5 transcripts have also been detected in the developing ...murine thymus, leading to speculation that Lgr5 is a marker for the long‐sought stem cell of the thymus. To address the nature of the Lgr5‐expressing thymic epithelial cells (TECs), we used Lgr5‐GFP reporter mice. We show that epithelial cells expressing Lgr5 protein are present in the fetal thymus during a specific developmental window yet are no longer detectable at birth. To analyze the function of the Lgr5 protein during thymus development, we generated Lgr5−/− mice. These experiments unequivocally show that thymus development is not perturbed in the absence of Lgr5, that all TEC subsets develop in Lgr5−/− mice and that T cells are produced in the expected ratios. Finally, by using an inducible lineage tracing system to track the progeny of Lgr5+ fetal TECs in vivo, we demonstrated that Lgr5+ fetal TECs have no detectable progeny in the later fetal thymus. In sum, we show that presence of the Lgr5 protein is not a prerequisite for proper thymus organogenesis.
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