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.

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.

Molecular Basis of Hematological Disease Caused by Inherited or Acquired RUNX1 Mutations.

The transcription factor RUNX1 is essential for correct hematopoietic development; in its absence in the germ line, blood stem cells are not formed. RUNX1 orchestrates dramatic changes in the chromatin landscape at the onset of stem cell formation, which set the stage for both stem self-renewal and further differentiation. However, once blood stem cells are formed, the mutation of the RUNX1 gene is not lethal but can lead to various hematopoietic defects and a predisposition to cancer. Here we summarize the current literature on inherited and acquired RUNX1 mutations, with a particular emphasis on mutations that alter the structure of the RUNX1 protein itself, and place these changes in the context of what is known about RUNX1 function. We also summarize which mutant RUNX1 proteins are actually expressed in cells and discuss the molecular mechanism underlying how such variants reprogram the epigenome setting stem cells on the path to malignancy.

PLCG1 is required for AML1-ETO leukemia stem cell self-renewal.

In an effort to identify novel drugs targeting fusion-oncogene-induced acute myeloid leukemia (AML), we performed high-resolution proteomic analysis. In AML1-ETO (AE)-driven AML, we uncovered a deregulation of phospholipase C (PLC) signaling. We identified PLCgamma 1 (PLCG1) as a specific target of the AE fusion protein that is induced after AE binding to intergenic regulatory DNA elements. Genetic inactivation of PLCG1 in murine and human AML inhibited AML1-ETO dependent self-renewal programs, leukemic proliferation, and leukemia maintenance in vivo. In contrast, PLCG1 was dispensable for normal hematopoietic stem and progenitor cell function. These findings are extended to and confirmed by pharmacologic perturbation of Ca++-signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO+ leukemic stem cells.

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.

t(8;21) Acute Myeloid Leukemia as a Paradigm for the Understanding of Leukemogenesis at the Level of Gene Regulation and Chromatin Programming.

Acute myeloid leukemia (AML) is a heterogenous disease with multiple sub-types which are defined by different somatic mutations that cause blood cell differentiation to go astray. Mutations occur in genes encoding members of the cellular machinery controlling transcription and chromatin structure, including transcription factors, chromatin modifiers, DNA-methyltransferases, but also signaling molecules that activate inducible transcription factors controlling gene expression and cell growth. Mutant cells in AML patients are unable to differentiate and adopt new identities that are shaped by the original driver mutation and by rewiring their gene regulatory networks into regulatory phenotypes with enhanced fitness. One of the best-studied AML-subtypes is the t(8;21) AML which carries a translocation fusing the DNA-binding domain of the hematopoietic master regulator RUNX1 to the ETO gene. The resulting oncoprotein, RUNX1/ETO has been studied for decades, both at the biochemical but also at the systems biology level. It functions as a dominant-negative version of RUNX1 and interferes with multiple cellular processes associated with myeloid differentiation, growth regulation and genome stability. In this review, we summarize our current knowledge of how this protein reprograms normal into malignant cells and how our current knowledge could be harnessed to treat the disease.

Correction: Increased proliferation and altered cell cycle regulation in pancreatic stem cells derived from patients with congenital hyperinsulinism.

[This corrects the article DOI: 10.1371/journal.pone.0222350.].

Increased proliferation and altered cell cycle regulation in pancreatic stem cells derived from patients with congenital hyperinsulinism.

Congenital hyperinsulinism (CHI) is characterised by inappropriate insulin secretion causing profound hypoglycaemia and brain damage if inadequately controlled. Pancreatic tissue isolated from patients with diffuse CHI shows abnormal proliferation rates, the mechanisms of which are not fully resolved. Understanding cell proliferation in CHI may lead to new therapeutic options, alongside opportunities to manipulate ß-cell mass in patients with diabetes. We aimed to generate cell-lines from CHI pancreatic tissue to provide in vitro model systems for research. Three pancreatic mesenchymal stem cell-lines (CHIpMSC1-3) were derived from patients with CHI disease variants: focal, atypical and diffuse. All CHIpMSC lines demonstrated increased proliferation compared with control adult-derived pMSCs. Cell cycle alterations including increased CDK1 levels and decreased p27Kip1 nuclear localisation were observed in CHIpMSCs when compared to control pMSCs. In conclusion, CHIpMSCs are a useful in vitro model to further understand the cell cycle alterations leading to increased islet cell proliferation in CHI.