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.

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.

Disease-causing mutations in genes encoding transcription factors (TFs) can affect TF interactions with their cognate DNA-binding motifs. Whether and how TF mutations impact upon the binding to TF composite elements (CE) and the interaction with other TFs is unclear. Here, we report a distinct mechanism of TF alteration in human lymphomas with perturbed B cell identity, in particular classic Hodgkin lymphoma. It is caused by a recurrent somatic missense mutation c.295 T > C (p.Cys99Arg; p.C99R) targeting the center of the DNA-binding domain of Interferon Regulatory Factor 4 (IRF4), a key TF in immune cells. IRF4-C99R fundamentally alters IRF4 DNA-binding, with loss-of-binding to canonical IRF motifs and neomorphic gain-of-binding to canonical and non-canonical IRF CEs. IRF4-C99R thoroughly modifies IRF4 function by blocking IRF4-dependent plasma cell induction, and up-regulates disease-specific genes in a non-canonical Activator Protein-1 (AP-1)-IRF-CE (AICE)-dependent manner. Our data explain how a single mutation causes a complex switch of TF specificity and gene regulation and open the perspective to specifically block the neomorphic DNA-binding activities of a mutant TF.

Developmental control of gene expression critically depends on distal cis-regulatory elements including enhancers which interact with promoters to activate gene expression. To date no global experiments have been conducted that identify their cell type and cell stage-specific activity within one developmental pathway and in a chromatin context. Here, we describe a high-throughput method that identifies thousands of differentially active cis-elements able to stimulate a minimal promoter at five stages of hematopoietic progenitor development from embryonic stem (ES) cells, which can be adapted to any ES cell derived cell type. We show that blood cell-specific gene expression is controlled by the concerted action of thousands of differentiation stage-specific sets of cis-elements which respond to cytokine signals terminating at signalling responsive transcription factors. Our work provides an important resource for studies of hematopoietic specification and highlights the mechanisms of how and where extrinsic signals program a cell type-specific chromatin landscape driving hematopoietic differentiation.

C (PLC) signaling. We identi fi ed PLCgamma 1 (PLCG1) as a speci fi c 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 fi ndings are extended to and con fi rmed by pharmacologic perturbation of Ca 11 -signaling in AML1-ETO AML cells, indicating that the PLCG1 pathway poses an important therapeutic target for AML1-ETO 1 leukemic stem cells.

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.

FLT3-ITD is a common activating receptor mutation in AML. We used genome-wide profiling to show that genes upregulated in FLT3-ITD+ AML had open chromatin regions (DHSs) enriched in binding sites for AP-1 and RUNX1 (1), within a gene regulatory network downstream of MAPK. We showed that MAPK inhibitors (i) suppress genes upregulated by FLT3-ITD, and (ii) inhibit growth of FLT3-ITD+ cells. Furthermore, concurrent RUNX1 mutations result in a suppression of genes normally activated by FLT3-ITD. We compared responses to two inhibitors targeting the FLT3-ITD gene regulatory network: (1)Gilteritinib (FLT3i) which targets FLT3-ITD, and (2)AI-14-91 (CBFbi) which blocks interactions between RUNX1 and CBFb (2). Overall, the responses to the 2 inhibitors were similar, suggesting that AP-1 and RUNX1 are in the same pathway. Treatment of FLT3-ITD+ AML cells with CBFbi or FLT3i led to a modest reduction in intensity of a subset of DHSs enriched in AP-1 and RUNX1 motifs. However, many pre-existing DHSs not normally bound by AP-1 acquired binding of FOS, at genes upregulated after either treatment. CBFbi suppressed FLT3-ITD AML cell cultures, but not healthy CD34+ blood stem cells, or AML cells with mutated RUNX1, suggesting that CBFbi plus FLT3i could form an AML therapy. To investigate CBFbi further, we performed cell culture assays on AML cells from a FLT3-ITD+ AML patient on AC220 FLT3 inhibitor therapy, who relapsed with a FLT3 D835H mutation. AML cells from this patient remained sensitive to culture in CBFbi, suggesting that CBFbi and MAPK inhibitors might form a second line of therapy in FLT3-ITD AML. (1)Assi et al. Subtype-specific regulatory network rewiring in acute myeloid leukemia. Nat Genet 51, 151-162 (2019). (2)Illendula et al. Small Molecule Inhibitor of CBFbeta-RUNX Binding for RUNX Transcription Factor Driven Cancers. EBioMedicine 8, 117-131 (2016). FLT3-ITD is a common activating receptor mutation in AML. We used genome-wide profiling to show that genes upregulated in FLT3-ITD+ AML had open chromatin regions (DHSs) enriched in binding sites for AP-1 and RUNX1 (1), within a gene regulatory network downstream of MAPK. We showed that MAPK inhibitors (i) suppress genes upregulated by FLT3-ITD, and (ii) inhibit growth of FLT3-ITD+ cells. Furthermore, concurrent RUNX1 mutations result in a suppression of genes normally activated by FLT3-ITD. We compared responses to two inhibitors targeting the FLT3-ITD gene regulatory network: (1)Gilteritinib (FLT3i) which targets FLT3-ITD, and (2)AI-14-91 (CBFbi) which blocks interactions between RUNX1 and CBFb (2). Overall, the responses to the 2 inhibitors were similar, suggesting that AP-1 and RUNX1 are in the same pathway. Treatment of FLT3-ITD+ AML cells with CBFbi or FLT3i led to a modest reduction in intensity of a subset of DHSs enriched in AP-1 and RUNX1 motifs. However, many pre-existing DHSs not normally bound by AP-1 acquired binding of FOS, at genes upregulated after either treatment. CBFbi suppressed FLT3-ITD AML cell cultures, but not healthy CD34+ blood stem cells, or AML cells with mutated RUNX1, suggesting that CBFbi plus FLT3i could form an AML therapy. To investigate CBFbi further, we performed cell culture assays on AML cells from a FLT3-ITD+ AML patient on AC220 FLT3 inhibitor therapy, who relapsed with a FLT3 D835H mutation. AML cells from this patient remained sensitive to culture in CBFbi, suggesting that CBFbi and MAPK inhibitors might form a second line of therapy in FLT3-ITD AML. (1)Assi et al. Subtype-specific regulatory network rewiring in acute myeloid leukemia. Nat Genet 51, 151-162 (2019). (2)Illendula et al. Small Molecule Inhibitor of CBFbeta-RUNX Binding for RUNX Transcription Factor Driven Cancers. EBioMedicine 8, 117-131 (2016).

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 ( CEBPA N/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.

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.

During open reduction of an irreducible anterior dislocation of a total hip replacement with an Oxinium femoral head, it was observed that the head had been significantly damaged. Gross and scanning electron microscopic examination revealed cracking, gouging, and delamination of the surface. Because of the risk which this poses for damaging the polyethylene acetabular liner, it is strongly recommended that patients with this type of prosthetic head be carefully monitored after a dislocation.

Acute myeloid leukemia (AML) is caused by recurrent mutations in members of the gene regulatory and signaling machinery that control hematopoietic progenitor cell growth and differentiation. Here, we show that the transcription factor WT1 forms a major node in the rewired mutation-specific gene regulatory networks of multiple AML subtypes. WT1 is frequently either mutated or upregulated in AML, and its expression is predictive for relapse. The WT1 protein exists as multiple isoforms. For two main AML subtypes, we demonstrate that these isoforms exhibit differential patterns of binding and support contrasting biological activities, including enhanced proliferation. We also show that WT1 responds to oncogenic signaling and is part of a signaling-responsive transcription factor hub that controls AML growth. WT1 therefore plays a central and widespread role in AML biology.

Acute myeloid leukemia development occurs in a stepwise fashion whereby an original driver mutation is followed by additional mutations. The first type of mutations tends to be in genes encoding members of the epigenetic/transcription regulatory machinery (i.e., RUNX1, DNMT3A, TET2), while the secondary mutations often involve genes encoding members of signaling pathways that cause uncontrolled growth of such cells such as the growth factor receptors c-KIT of FLT3. Patients usually present with both types of mutations, but it is currently unclear how both mutational events shape the epigenome in developing AML cells. To this end we generated an in vitro model of t(8;21) AML by expressing its driver oncoprotein RUNX1-ETO with or without a mutated (N822K) KIT protein. Expression of N822K-c-KIT strongly increases the self-renewal capacity of RUNX1-ETO-expressing cells. Global analysis of gene expression changes and alterations in the epigenome revealed that N822K-c-KIT expression profoundly influences the open chromatin landscape and transcription factor binding. However, our experiments also revealed that double mutant cells still differ from their patient-derived counterparts, highlighting the importance of studying patient cells to obtain a true picture of how gene regulatory networks have been reprogrammed during tumorigenesis.