Pharmacological inhibition of RAS overcomes FLT3 inhibitor resistance in FLT3-ITD+ AML through AP-1 and RUNX1.

AML is characterized by mutations in genes associated with growth regulation such as internal tandem duplications (ITD) in the receptor kinase FLT3. Inhibitors targeting FLT3 (FLT3i) are being used to treat patients with FLT3-ITD+ but most relapse and become resistant. To elucidate the resistance mechanism, we compared the gene regulatory networks (GRNs) of leukemic cells from patients before and after relapse, which revealed that the GRNs of drug-responsive patients were altered by rewiring their AP-1-RUNX1 axis. Moreover, FLT3i induces the upregulation of signaling genes, and we show that multiple cytokines, including interleukin-3 (IL-3), can overcome FLT3 inhibition and send cells back into cycle. FLT3i leads to loss of AP-1 and RUNX1 chromatin binding, which is counteracted by IL-3. However, cytokine-mediated drug resistance can be overcome by a pan-RAS inhibitor. We show that cytokines instruct AML growth via the transcriptional regulators AP-1 and RUNX1 and that pan-RAS drugs bypass this barrier.

Leukemic stem cells activate lineage inappropriate signalling pathways to promote their growth.

Acute Myeloid Leukemia (AML) is caused by multiple mutations which dysregulate growth and differentiation of myeloid cells. Cells adopt different gene regulatory networks specific to individual mutations, maintaining a rapidly proliferating blast cell population with fatal consequences for the patient if not treated. The most common treatment option is still chemotherapy which targets such cells. However, patients harbour a population of quiescent leukemic stem cells (LSCs) which can emerge from quiescence to trigger relapse after therapy. The processes that allow such cells to re-grow remain unknown. Here, we examine the well characterised t(8;21) AML sub-type as a model to address this question. Using four primary AML samples and a novel t(8;21) patient-derived xenograft model, we show that t(8;21) LSCs aberrantly activate the VEGF and IL-5 signalling pathways. Both pathways operate within a regulatory circuit consisting of the driver oncoprotein RUNX1::ETO and an AP-1/GATA2 axis allowing LSCs to re-enter the cell cycle while preserving self-renewal capacity.

Chromatin priming elements direct tissue-specific gene activity before hematopoietic specification.

Tissue-specific gene regulation during development involves the interplay between transcription factors and epigenetic regulators binding to enhancer and promoter elements. The pattern of active enhancers defines the cellular differentiation state. However, developmental gene activation involves a previous step called chromatin priming which is not fully understood. We recently developed a genome-wide functional assay that allowed us to functionally identify enhancer elements integrated in chromatin regulating five stages spanning the in vitro differentiation of embryonic stem cells to blood. We also measured global chromatin accessibility, histone modifications, and transcription factor binding. The integration of these data identified and characterised cis-regulatory elements which become activated before the onset of gene expression, some of which are primed in a signalling-dependent fashion. Deletion of such a priming element leads to a delay in the up-regulation of its associated gene in development. Our work uncovers the details of a complex network of regulatory interactions with the dynamics of early chromatin opening being at the heart of dynamic tissue-specific gene expression control.

Gene regulatory network analysis predicts cooperating transcription factor regulons required for FLT3-ITD+ AML growth.

Acute myeloid leukemia (AML) is a heterogeneous disease caused by different mutations. Previously, we showed that each mutational subtype develops its specific gene regulatory network (GRN) with transcription factors interacting within multiple gene modules, many of which are transcription factor genes themselves. Here, we hypothesize that highly connected nodes within such networks comprise crucial regulators of AML maintenance. We test this hypothesis using FLT3-ITD-mutated AML as a model and conduct an shRNA drop-out screen informed by this analysis. We show that AML-specific GRNs predict crucial regulatory modules required for AML growth. Furthermore, our work shows that all modules are highly connected and regulate each other. The careful multi-omic analysis of the role of one (RUNX1) module by shRNA and chemical inhibition shows that this transcription factor and its target genes stabilize the GRN of FLT3-ITD+ AML and that its removal leads to GRN collapse and cell death.

Gene regulatory network analysis predicts cooperating transcription factor regulons required for FLT3-ITD+ AML growth.

AML is a heterogenous disease caused by different mutations. We have previously shown that each mutational sub-type develops its specific gene regulatory network (GRN) with transcription factors interacting with multiple gene modules, many of which are transcription factor genes themselves. Here we hypothesized that highly connected nodes within such networks comprise crucial regulators of AML maintenance. We tested this hypothesis using FLT3-ITD mutated AML as a model and conducted an shRNA drop-out screen informed by this analysis. We show that AML-specific GRNs predict identifying crucial regulatory modules required for AML but not normal cellular growth. Furthermore, our work shows that all modules are highly connected and regulate each other. The careful multi-omic analysis of the role of one (RUNX1) module by shRNA and chemical inhibition shows that this transcription factor and its target genes stabilize the GRN of FLT3-ITD AML and that its removal leads to GRN collapse and cell death.

Identification and interrogation of the gene regulatory network of CEBPA-double mutant acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy caused by mutations in genes encoding transcriptional and epigenetic regulators together with signaling genes. It is characterized by a disturbance of differentiation and abnormal proliferation of hematopoietic progenitors. We have previously shown that each AML subtype establishes its own core gene regulatory network (GRN), consisting of transcription factors binding to their target genes and imposing a specific gene expression pattern that is required for AML maintenance. In this study, we integrate gene expression, open chromatin and ChIP data with promoter-capture Hi-C data to define a refined core GRN common to all patients with CEBPA-double mutant (CEBPAN/C) AML. These mutations disrupt the structure of a major regulator of myelopoiesis. We identify the binding sites of mutated C/EBPα proteins in primary cells, we show that C/EBPα, AP-1 factors and RUNX1 colocalize and are required for AML maintenance, and we employ single cell experiments to link important network nodes to the specific differentiation trajectory from leukemic stem to blast cells. Taken together, our study provides an important resource which predicts the specific therapeutic vulnerabilities of this AML subtype in human cells.

Epigenetic regulator genes direct lineage switching in MLL/AF4 leukemia.

The fusion gene MLL/AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukemia. Relapse can be associated with a lineage switch from acute lymphoblastic to acute myeloid leukemia, resulting in poor clinical outcomes caused by resistance to chemotherapies and immunotherapies. In this study, the myeloid relapses shared oncogene fusion breakpoints with their matched lymphoid presentations and originated from various differentiation stages from immature progenitors through to committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programs, including alternative splicing. These findings indicate that the execution and maintenance of lymphoid lineage differentiation is impaired. The relapsed myeloid phenotype is recurrently associated with the altered expression, splicing, or mutation of chromatin modifiers, including CHD4 coding for the ATPase/helicase of the nucleosome remodelling and deacetylation complex. Perturbation of CHD4 alone or in combination with other mutated epigenetic modifiers induces myeloid gene expression in MLL/AF4+ cell models, indicating that lineage switching in MLL/AF4 leukemia is driven and maintained by disrupted epigenetic regulation.

Stable Epigenetic Programming of Effector and Central Memory CD4 T Cells Occurs Within 7 Days of Antigen Exposure In Vivo.

T cell immunological memory is established within days of an infection, but little is known about the in vivo changes in gene regulatory networks accounting for their ability to respond more efficiently to secondary infections. To decipher the timing and nature of immunological memory we performed genome-wide analyses of epigenetic and transcriptional changes in a mouse model generating antigen-specific T cells. Epigenetic reprogramming for Th differentiation and memory T cell formation was already established by the peak of the T cell response after 7 days. The Th memory T cell program was associated with a gain of open chromatin regions, enriched for RUNX, ETS and T-bet motifs, which remained stable for 56 days. The epigenetic programs for both effector memory, associated with T-bet, and central memory, associated with TCF-1, were established in parallel. Memory T cell-specific regulatory elements were associated with greatly enhanced inducible Th1-biased responses during secondary exposures to antigen. Furthermore, memory T cells responded in vivo to re-exposure to antigen by rapidly reprograming the entire ETS factor gene regulatory network, by suppressing Ets1 and activating Etv6 expression. These data show that gene regulatory networks are epigenetically reprogrammed towards memory during infection, and undergo substantial changes upon re-stimulation.

Different mutant RUNX1 oncoproteins program alternate haematopoietic differentiation trajectories.

Mutations of the haematopoietic master regulator RUNX1 are associated with acute myeloid leukaemia, familial platelet disorder and other haematological malignancies whose phenotypes and prognoses depend upon the class of the RUNX1 mutation. The biochemical behaviour of these oncoproteins and their ability to cause unique diseases has been well studied, but the genomic basis of their differential action is unknown. To address this question we compared integrated phenotypic, transcriptomic, and genomic data from cells expressing four types of RUNX1 oncoproteins in an inducible fashion during blood development from embryonic stem cells. We show that each class of mutant RUNX1 deregulates endogenous RUNX1 function by a different mechanism, leading to specific alterations in developmentally controlled transcription factor binding and chromatin programming. The result is distinct perturbations in the trajectories of gene regulatory network changes underlying blood cell development which are consistent with the nature of the final disease phenotype. The development of novel treatments for RUNX1-driven diseases will therefore require individual consideration.

RUNX1-EVI1 disrupts lineage determination and the cell cycle by interfering with RUNX1 and EVI1 driven gene regulatory networks.

Hematological malignancies are characterised by a block in differentiation, which in many cases is caused by recurrent mutations affecting the activity of hematopoietic transcription factors. RUNX1-EVI1 is a fusion protein formed by the t(3;21) translocation linking two transcription factors required for normal hematopoiesis. RUNX1-EVI1 expression is found in myelodysplastic syndrome, secondary acute myeloid leukemia, and blast crisis of chronic myeloid leukemia; with clinical outcomes being worse than in patients with RUNX1-ETO, RUNX1 or EVI1 mutations alone. RUNX1-EVI1 is usually found as a secondary mutation, therefore the molecular mechanisms underlying how RUNX1-EVI1 alone contributes to poor prognosis are unknown. To address this question, we induced expression of RUNX1-EVI1 in hematopoietic cells derived from an embryonic stem cell differentiation model. Induction resulted in disruption of the RUNX1-dependent endothelial-hematopoietic transition, blocked the cell cycle and undermined cell fate decisions in multipotent hematopoietic progenitor cells. Integrative analyses of gene expression with chromatin and transcription factor binding data demonstrated that RUNX1-EVI1 binding caused the re-distribution of endogenous RUNX1 within the genome and interfered with both RUNX1 and EVI1 regulated gene expression programs. In summary, RUNX1-EVI1 expression alone leads to extensive epigenetic reprogramming which is incompatible with healthy blood production.

IL-2/IL-7-inducible factors pioneer the path to T cell differentiation in advance of lineage-defining factors.

When dormant naïve T cells first become activated by antigen-presenting cells, they express the autocrine growth factor IL-2 which transforms them into rapidly dividing effector T cells. During this process, hundreds of genes undergo epigenetic reprogramming for efficient activation, and also for potential reactivation after they return to quiescence as memory T cells. However, the relative contributions of IL-2 and T cell receptor signaling to this process are unknown. Here, we show that IL-2 signaling is required to maintain open chromatin at hundreds of gene regulatory elements, many of which control subsequent stimulus-dependent alternative pathways of T cell differentiation. We demonstrate that IL-2 activates binding of AP-1 and STAT5 at sites that can subsequently bind lineage-determining transcription factors, depending upon what other external factors exist in the local T cell environment. Once established, priming can also be maintained by the stroma-derived homeostatic cytokine IL-7, and priming diminishes if Il7r is subsequently deleted in vivo. Hence, IL-2 is not just a growth factor; it lays the foundation for T cell differentiation and immunological memory.

Chromatin Priming Renders T Cell Tolerance-Associated Genes Sensitive to Activation below the Signaling Threshold for Immune Response Genes.

Immunological homeostasis in T cells is maintained by a tightly regulated signaling and transcriptional network. Full engagement of effector T cells occurs only when signaling exceeds a critical threshold that enables induction of immune response genes carrying an epigenetic memory of prior activation. Here we investigate the underlying mechanisms causing the suppression of normal immune responses when T cells are rendered anergic by tolerance induction. By performing an integrated analysis of signaling, epigenetic modifications, and gene expression, we demonstrate that immunological tolerance is established when both signaling to and chromatin priming of immune response genes are weakened. In parallel, chromatin priming of immune-repressive genes becomes boosted, rendering them sensitive to low levels of signaling below the threshold needed to activate immune response genes. Our study reveals how repeated exposure to antigens causes an altered epigenetic state leading to T cell anergy and tolerance, representing a basis for treating auto-immune diseases.

Expression of RUNX1-ETO Rapidly Alters the Chromatin Landscape and Growth of Early Human Myeloid Precursor Cells.

Acute myeloid leukemia (AML) is a hematopoietic malignancy caused by recurrent mutations in genes encoding transcriptional, chromatin, and/or signaling regulators. The t(8;21) translocation generates the aberrant transcription factor RUNX1-ETO (RUNX1-RUNX1T1), which by itself is insufficient to cause disease. t(8;21) AML patients show extensive chromatin reprogramming and have acquired additional mutations. Therefore, the genomic and developmental effects directly and solely attributable to RUNX1-ETO expression are unclear. To address this, we employ a human embryonic stem cell differentiation system capable of forming definitive myeloid progenitor cells to express RUNX1-ETO in an inducible fashion. Induction of RUNX1-ETO causes extensive chromatin reprogramming by interfering with RUNX1 binding, blocks differentiation, and arrests cellular growth, whereby growth arrest is reversible following RUNX1-ETO removal. Single-cell gene expression analyses show that RUNX1-ETO induction alters the differentiation of early myeloid progenitors, but not of other progenitor types, indicating that oncoprotein-mediated transcriptional reprogramming is highly target cell specific.

Robust hematopoietic specification requires the ubiquitous Sp1 and Sp3 transcription factors.

BACKGROUND: Both tissue-specific and ubiquitously expressed transcription factors, such as Sp-family members, are required for correct development. However, the molecular details of how ubiquitous factors are involved in programming tissue-specific chromatin and thus participate in developmental processes are still unclear. We previously showed that embryonic stem cells lacking Sp1 DNA-binding activity (Sp1ΔDBD/ΔDBD cells) are able to differentiate into early blood progenitors despite the inability of Sp1 to bind chromatin without its DNA-binding domain. However, gene expression during differentiation becomes progressively deregulated, and terminal differentiation is severely compromised. RESULTS: Here, we studied the cooperation of Sp1 with its closest paralogue Sp3 in hematopoietic development and demonstrate that Sp1 and Sp3 binding sites largely overlap. The complete absence of either Sp1 or Sp3 or the presence of the Sp1 DNA-binding mutant has only a minor effect on the pattern of distal accessible chromatin sites and their transcription factor binding motif content, suggesting that these mutations do not affect tissue-specific chromatin programming. Sp3 cooperates with Sp1ΔDBD/ΔDBD to enable hematopoiesis, but is unable to do so in the complete absence of Sp1. Using single-cell gene expression analysis, we show that the lack of Sp1 DNA binding leads to a distortion of cell fate decision timing, indicating that stable chromatin binding of Sp1 is required to maintain robust differentiation trajectories. CONCLUSIONS: Our findings highlight the essential contribution of ubiquitous factors such as Sp1 to blood cell development. In contrast to tissue-specific transcription factors which are required to direct specific cell fates, loss of Sp1 leads to a widespread deregulation in timing and coordination of differentiation trajectories during hematopoietic specification.

Prospective Isolation and Characterization of Genetically and Functionally Distinct AML Subclones.

Intra-tumor heterogeneity caused by clonal evolution is a major problem in cancer treatment. To address this problem, we performed label-free quantitative proteomics on primary acute myeloid leukemia (AML) samples. We identified 50 leukemia-enriched plasma membrane proteins enabling the prospective isolation of genetically distinct subclones from individual AML patients. Subclones differed in their regulatory phenotype, drug sensitivity, growth, and engraftment behavior, as determined by RNA sequencing, DNase I hypersensitive site mapping, transcription factor occupancy analysis, in vitro culture, and xenograft transplantation. Finally, we show that these markers can be used to identify and longitudinally track distinct leukemic clones in patients in routine diagnostics. Our study describes a strategy for a major improvement in stratifying cancer diagnosis and treatment.

C/EBPα overrides epigenetic reprogramming by oncogenic transcription factors in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease caused by recurrent mutations in the transcription regulatory machinery, resulting in abnormal growth and a block in differentiation. One type of recurrent mutations affects RUNX1, which is subject to mutations and translocations, the latter giving rise to fusion proteins with aberrant transcriptional activities. We recently compared the mechanism by which the products of the t(8;21) and the t(3;21) translocation RUNX1-ETO and RUNX1-EVI1 reprogram the epigenome. We demonstrated that a main component of the block in differentiation in both types of AML is direct repression of the gene encoding the myeloid regulator C/EBPα by both fusion proteins. Here, we examined at the global level whether C/EBPα is able to reverse aberrant chromatin programming in t(8;21) and t(3;21) AML. C/EBPα overexpression does not change oncoprotein expression or globally displace these proteins from their binding sites. Instead, it upregulates a core set of common target genes important for myeloid differentiation and represses genes regulating leukemia maintenance. This study, therefore, identifies common CEBPA-regulated pathways as targets for therapeutic intervention.

Intron Length Coevolution across Mammalian Genomes.

Although they do not contribute directly to the proteome, introns frequently contain regulatory elements and can extend the protein coding potential of the genome through alternative splicing. For some genes, the contribution of introns to the time required for transcription can also be functionally significant. We have previously shown that intron length in genes associated with developmental patterning is often highly conserved. In general, sets of genes that require precise coordination in the timing of their expression may be sensitive to changes in transcript length. A prediction of this hypothesis is that evolutionary changes in intron length, when they occur, may be correlated between sets of coordinately expressed genes. To test this hypothesis, we analyzed intron length coevolution in alignments from nine eutherian mammals. Overall, genes that belong to the same protein complex or that are coexpressed were significantly more likely to show evidence of intron length coevolution than matched, randomly sampled genes. Individually, protein complexes involved in the cell cycle showed the strongest evidence of coevolution of intron lengths and clusters of coexpressed genes enriched for cell cycle genes also showed significant evidence of intron length coevolution. Our results reveal a novel aspect of gene coevolution and provide a means to identify genes, protein complexes and biological processes that may be particularly sensitive to changes in transcriptional dynamics.

Promiscuous mRNA splicing under the control of AIRE in medullary thymic epithelial cells.

MOTIVATION: The expression of tissue-restricted antigens (TRAs) in the thymus is required to ensure efficient negative selection of potentially auto-reactive T lymphocytes and avoid autoimmune disease. This promiscuous expression is under the control of the autoimmune regulator (AIRE), a transcription factor expressed in medullary thymic epithelial cells (mTECs). Tissue-specific alternative splicing may also produce TRAs but the extent to which splice isoforms that are restricted to specific tissues are expressed in mTECs is yet to be investigated. RESULTS: We reanalyzed microarray and RNA-Seq datasets from mouse mTECs and other epithelial and non-epithelial cell types and found that the diversity of splice isoforms in mTECs was greater than in any of the other cell types or tissues studied. We identified tissue-specific isoforms from a panel of mouse tissues and found several examples of such isoforms that are expressed in mTECs. The number of isoforms with restricted expression found in mTECs was significantly higher than for comparable cell types. Furthermore, we found evidence that AIRE influences the increased splicing diversity observed in mTECs as the genes for which tissue restricted isoforms are produced in mTECs were significantly more likely than other genes to be differentially spliced between AIRE knock-out and wild-type samples. Our results suggest that developing T lymphocytes are exposed to diverse tissue-restricted splice isoforms in the thymus and that AIRE has a direct or indirect role in this process, representing a novel aspect of its role in the maintenance of immune self-tolerance. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Implication of beta3-adrenoceptors in the antidepressant-like effects of amibegron using Adrb3 knockout mice in the chronic mild stress.

The involvement of the beta3-adrenoceptor in the antidepressant-like effects of the beta3-adrenoceptor-agonist amibegron is addressed in the chronic mild stress, using adrenergic beta3-receptor knockout (Adrb3tm1Lowl) mice. Amibegron (3 mg/kg/day, i.p., 33 days), attenuated the physical alteration due to the application of repeated stress in wild-type littermates, but not in knockout mice. This result suggests that the beta3-adrenoceptor plays a predominant role in mediating antidepressant-like actions of amibegron.