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The CXXC domain is a reader of DNA methylation which preferentially binds to unmethylated CpG DNA motifs. Chromosomal translocations involving the

Histone H3 lysine 4 methylation (H3K4Me) is most often associated with chromatin activation, and removing H3K4 methyl groups has been shown to be coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a therapeutic target for multiple diseases, including for the potential treatment of β-globinopathies (sickle cell disease and β-thalassemia), because it is a component of γ-globin repressor complexes, and LSD1 inactivation leads to robust induction of the fetal globin genes. The effects of LSD1 inhibition in definitive erythropoiesis are not well characterized, so we examined the consequences of conditional inactivation of Lsd1 in adult red blood cells using a new Gata1creERT2 bacterial artificial chromosome transgene. Erythroid-specific loss of Lsd1 activity in mice led to a block in erythroid progenitor differentiation and to the expansion of granulocyte-monocyte progenitor–like cells, converting hematopoietic differentiation potential from an erythroid fate to a myeloid fate. The analogous phenotype was also observed in human hematopoietic stem and progenitor cells, coincident with the induction of myeloid transcription factors (eg, PU.1 and CEBPα). Finally, blocking the activity of the transcription factor PU.1 or RUNX1 at the same time as LSD1 inhibition rescued myeloid lineage conversion to an erythroid phenotype. These data show that LSD1 promotes erythropoiesis by repressing myeloid cell fate in adult erythroid progenitors and that inhibition of the myeloid-differentiation pathway reverses the lineage switch induced by LSD1 inactivation.

The formation of disulfide bonds in proteins is an essential process in both prokaryotes and eukaryotes. In gram-negative bacteria including Escherichia coli, the proteins DsbA and DsbB mediate the formation of disulfide bonds in the periplasm. DsbA acts as the periplasmic oxidant of periplasmic substrate proteins. DsbA is reoxidized by transfer of reducing equivalents to the 4 TM helix membrane protein DsbB, which transfers reducing equivalents to ubiquinone or menaquinone. Multiple structural studies of DsbB have provided detailed structural information on intermediates in the process of DsbB catalyzed oxidation of DsbA. These structures and the insights gained are described. In proteins with more than one pair of Cys residues, there is the potential for formation of non-native disulfide bonds, making it necessary for the cell to have a mechanism for the isomerization of such non-native disulfide bonds. In E. coli, this is mediated by the proteins DsbC and DsbD. DsbC reduces mis-formed disulfide bonds. The eight-TM-helix protein DsbD reduces DsbC and is itself reduced by cytoplasmic thioredoxin. DsbD also contributes reducing equivalents for the reduction of cytochrome c to facilitate heme attachment. The DsbD functional homolog CcdA is a six-TM-helix membrane protein that provides reducing equivalents for the reduction of cytochrome c. A recent structure determination of CcdA has provided critical insights into how reducing equivalents are transferred across the membrane that likely also provides understanding how this is achieved by DsbD as well. This structure and the insights gained are described.

RUNX1 has recently been shown to play an important role in determination of mammary epithelial cell identity. However, mechanisms by which loss of the RUNX1 transcription factor in mammary epithelial cells leads to epithelial-to-mesenchymal transition (EMT) are not known. Here, we report that interaction between RUNX1 and its heterodimeric partner CBFβ is essential for sustaining mammary epithelial cell identity. Disruption of RUNX1-CBFβ interaction, DNA binding, and association with mitotic chromosomes alters cell morphology, global protein synthesis, and phenotype-related gene expression. During interphase, RUNX1 is organized as punctate, predominantly nuclear, foci that are dynamically redistributed during mitosis, with a subset localized to mitotic chromosomes. Genome-wide RUNX1 occupancy profiles for asynchronous, mitotically enriched, and early G1 breast epithelial cells reveal RUNX1 associates with RNA Pol II-transcribed protein coding and long non-coding RNA genes and RNA Pol I-transcribed ribosomal genes critical for mammary epithelial proliferation, growth, and phenotype maintenance. A subset of these genes remains occupied by the protein during the mitosis to G1 transition. Together, these findings establish that the RUNX1-CBFβ complex is required for maintenance of the normal mammary epithelial phenotype and its disruption leads to EMT. Importantly, our results suggest, for the first time, that RUNX1 mitotic bookmarking of a subset of epithelial-related genes may be an important epigenetic mechanism that contributes to stabilization of the mammary epithelial cell identity.

Breast cancer stem cells (BCSCs) are competent to initiate tumor formation and growth and refractory to conventional therapies. Consequently BCSCs are implicated in tumor recurrence. Many signaling cascades associated with BCSCs are critical for epithelial-to-mesenchymal transition (EMT). We developed a model system to mechanistically examine BCSCs in basal-like breast cancer using MCF10AT1 FACS sorted for CD24 (negative/low in BCSCs) and CD44 (positive/high in BCSCs). Ingenuity Pathway Analysis comparing RNA-seq on the CD24-/low versus CD24+/high MCF10AT1 indicates that the top activated upstream regulators include TWIST1, TGFβ1, OCT4, and other factors known to be increased in BCSCs and during EMT. The top inhibited upstream regulators include ESR1, TP63, and FAS. Consistent with our results, many genes previously demonstrated to be regulated by RUNX factors are altered in BCSCs. The RUNX2 interaction network is the top significant pathway altered between CD24-/low and CD24+/high MCF10AT1. RUNX1 is higher in expression at the RNA level than RUNX2. RUNX3 is not expressed. While, human-specific quantitative polymerase chain reaction primers demonstrate that RUNX1 and CDH1 decrease in human MCF10CA1a cells that have grown tumors within the murine mammary fat pad microenvironment, RUNX2 and VIM increase. Treatment with an inhibitor of RUNX binding to CBFβ for 5 days followed by a 7-day recovery period results in EMT suggesting that loss of RUNX1, rather than increase in RUNX2, is a driver of EMT in early stage breast cancer. Increased understanding of RUNX regulation on BCSCs and EMT will provide novel insight into therapeutic strategies to prevent recurrence.

Mutated or dysregulated transcription factors represent a unique class of drug targets that mediate aberrant gene expression, including blockade of differentiation and cell death gene expression programmes, hallmark properties of cancers. Transcription factor activity is altered in numerous cancer types via various direct mechanisms including chromosomal translocations, gene amplification or deletion, point mutations and alteration of expression, as well as indirectly through non-coding DNA mutations that affect transcription factor binding. Multiple approaches to target transcription factor activity have been demonstrated, preclinically and, in some cases, clinically, including inhibition of transcription factor–cofactor protein–protein interactions, inhibition of transcription factor–DNA binding and modulation of levels of transcription factor activity by altering levels of ubiquitylation and subsequent proteasome degradation or by inhibition of regulators of transcription factor expression. In addition, several new approaches to targeting transcription factors have recently emerged including modulation of auto-inhibition, proteolysis targeting chimaeras (PROTACs), use of cysteine reactive inhibitors, targeting intrinsically disordered regions of transcription factors and combinations of transcription factor inhibitors with kinase inhibitors to block the development of resistance. These innovations in drug development hold great promise to yield agents with unique properties that are likely to impact future cancer treatment.

RUNX1 transcription factor regulates normal and malignant hematopoiesis. Somatic or germline mutant RUNX1 (mtRUNX1) is associated with poorer outcome in acute myeloid leukemia (AML). Knockdown or inhibition of RUNX1 induced more apoptosis of AML expressing mtRUNX1 versus wild-type RUNX1 and improved survival of mice engrafted with mtRUNX1-expressing AML. CRISPR/Cas9-mediated editing-out of RUNX1 enhancer (eR1) within its intragenic super-enhancer, or BET protein BRD4 depletion by short hairpin RNA, repressed RUNX1, inhibited cell growth, and induced cell lethality in AML cells expressing mtRUNX1. Moreover, treatment with BET protein inhibitor or degrader (BET–proteolysis targeting chimera) repressed RUNX1 and its targets, inducing apoptosis and improving survival of mice engrafted with AML expressing mtRUNX1. Library of Integrated Network–based Cellular Signatures 1000–connectivity mapping data sets queried with messenger RNA signature of RUNX1 knockdown identified novel expression-mimickers (EMs), which repressed RUNX1 and exerted in vitro and in vivo efficacy against AML cells expressing mtRUNX1. In addition, the EMs cinobufagin, anisomycin, and narciclasine induced more lethality in hematopoietic progenitor cells (HPCs) expressing germline mtRUNX1 from patients with AML compared with HPCs from patients with familial platelet disorder (FPD), or normal untransformed HPCs. These findings highlight novel therapeutic agents for AML expressing somatic or germline mtRUNX1.

AF9 (MLLT3) and ENL (MLLT1) are members of the YEATS family (named after the five proteins first shown to contain this domain: Yaf9, ENL, AF9, Taf14, Sas5) defined by the presence of a YEATS domain. The YEATS domain is an epigenetic reader that binds to acetylated and crotonylated lysines, unlike the bromodomain which can only bind to acetylated lysines. All members of this family have been shown to be components of various complexes with roles in chromatin remodeling, histone modification, histone variant deposition, and transcriptional regulation. MLLT3 is a critical regulator of hematopoiesis with a role in maintaining the hematopoietic stem or progenitor cell (HSPC) population. Approximately 10% of acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) patients harbor a translocation involving MLL (mixed lineage leukemia). In the context of MLL fusion patients with AML and ALL, MLL-AF9 and MLL-ENL fusions are observed in 34 and 31% of the patients, respectively. The intrinsically disordered C-terminal domain of MLLT3 (AHD, ANC1 homology domain) undergoes coupled binding and folding upon interaction with partner proteins AF4, DOT1L, BCOR, and CBX8. Backbone dynamics studies of the complexes suggest a role for dynamics in function. Inhibitors of the interaction of the intrinsically disordered AHD with partner proteins have been described, highlighting the feasibility of targeting intrinsically disordered regions. MLLT1 undergoes phase separation to enhance recruitment of the super elongation complex (SEC) and drive transcription. Mutations in MLLT1 observed in Wilms tumor patients enhance phase separation and transcription to drive an aberrant gene expression program.

The ETV6-RUNX1 onco-fusion arises in utero, initiating a clinically silent pre-leukemic state associated with the development of pediatric B-acute lymphoblastic leukemia (B-ALL). We characterize the ETV6-RUNX1 regulome by integrating chromatin immunoprecipitation- and RNA-sequencing and show that ETV6-RUNX1 functions primarily through competition for RUNX1 binding sites and transcriptional repression. In pre-leukemia, this results in ETV6-RUNX1 antagonization of cell cycle regulation by RUNX1 as evidenced by mass cytometry analysis of B-lineage cells derived from ETV6-RUNX1 knock-in human pluripotent stem cells. In frank leukemia, knockdown of RUNX1 or its co-factor CBFβ results in cell death suggesting sustained requirement for RUNX1 activity which is recapitulated by chemical perturbation using an allosteric CBFβ-inhibitor. Strikingly, we show that RUNX1 addiction extends to other genetic subtypes of pediatric B-ALL and also adult disease. Importantly, inhibition of RUNX1 activity spares normal hematopoiesis. Our results suggest that chemical intervention in the RUNX1 program may provide a therapeutic opportunity in ALL.

Pulmonary fibrosis (PF) can arise from unknown causes, as in idiopathic PF, or as a consequence of infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current treatments for PF slow, but do not stop, disease progression. We report that treatment with a runt-related transcription factor 1 (RUNX1) inhibitor (Ro24-7429), previously found to be safe, although ineffective, as a Tat inhibitor in patients with HIV, robustly ameliorates lung fibrosis and inflammation in the bleomycin-induced PF mouse model. RUNX1 inhibition blunted fundamental mechanisms downstream pathologic mediators of fibrosis and inflammation, including transforming growth factor-β1 and tumor necrosis factor-α, in cultured lung epithelial cells, fibroblasts, and vascular endothelial cells, indicating pleiotropic effects. RUNX1 inhibition also reduced the expression of angiotensin-converting enzyme 2 and FES Upstream Region (FURIN), host proteins critical for SARS-CoV-2 infection, in mice and in vitro. A subset of human lungs with SARS-CoV-2 infection overexpress RUNX1. These data suggest that RUNX1 inhibition via repurposing of Ro24-7429 may be beneficial for PF and to battle SARS-CoV-2, by reducing expression of viral mediators and by preventing respiratory complications.

Histone H3 lysine 4 methylation (H3K4Me) is proximally associated with chromatin activation, and therefore removing H3K4 methyl groups is normally coincident with gene repression. H3K4Me demethylase KDM1a/LSD1 is a potential therapeutic target for multiple diseases, including for the treatment of the {beta}-globinopathies (sickle cell disease and {beta}-thalassemia) since it is a component of multiple{gamma} -globin repressor complexes, and its inactivation leads to robust induction of the fetal globin genes. However, the effects of LSD1 inhibition in definitive erythroid cells are not well characterized. Here we examined the consequences of erythroid-specific conditional inactivation of Lsd1 in vivo using a new Gata1creERT2 bacterial artificial chromosome (BAC) transgene. Conditional loss of Lsd1 in adult mice led to a differentiation block in erythroid progenitor cells and the surprising expansion of a GMP-like cell pool, apparently converting hematopoietic differentiation potential from an erythroid to a myeloid fate. The analogous phenotype was also observed in human cells: inactivation of LSD1 in hematopoietic stem and progenitor cells (HSPC) also blocked erythroid differentiation, coincident with robust induction of myeloid transcription factor genes (e.g. Pu.1 and Cebpa). Remarkably, blocking the activity of PU.1 or RUNX1 (a transcriptional activator of Pu.1) at the same time as blocking LSD1 activity reverted the myeloid lineage conversion back to an erythroid phenotype. Taken together, the data show that LSD1 maintains erythropoiesis by reversibly repressing a myeloid cell fate in adult erythroid cell precursors, and that inhibition of the myeloid differentiation pathway can reverse the negative effects of LSD1 inactivation on erythroid differentiation.

SummaryThe ETV6-RUNX1 onco-fusion arises in utero, initiating a clinically silent pre-leukemic state associated with the development of pediatric B-acute lymphoblastic leukemia (B-ALL). We characterize the ETV6-RUNX1 regulome by integrating chromatin immunoprecipitation- and RNA-sequencing and show that ETV6-RUNX1 functions primarily through competition for RUNX1 binding sites and transcriptional repression. In pre-leukemia, this results in ETV6-RUNX1 antagonization of cell cycle regulation by RUNX1 as evidenced by mass cytometry analysis of B-lineage cells derived from ETV6-RUNX1 knock-in human pluripotent stem cells. In frank leukemia, knockdown of RUNX1 or its co-factor CBFβ results in cell death suggesting sustained requirement for RUNX1 activity which is recapitulated by chemical perturbation using an allosteric CBFβ-inhibitor. Strikingly, we show that RUNX1 addiction extends to other genetic subtypes of pediatric B-ALL and also adult disease. Importantly, inhibition of RUNX1 activity spares normal hematopoiesis. Our results implicate chemical intervention in the RUNX1 program as an exciting therapeutic opportunity in ALL.

Runt‐related transcription factor 1 (RUNX1) acts as a mediator of aberrant retinal angiogenesis and has been implicated in the progression of proliferative diabetic retinopathy (PDR). Patients with PDR, retinopathy of prematurity (ROP), and wet age‐related macular degeneration (wet AMD) have been found to have elevated levels of Tumor Necrosis Factor‐alpha (TNF‐α) in the eye. In fibrovascular membranes (FVMs) taken from patients with PDR RUNX1 expression was increased in the vasculature, while in human retinal microvascular endothelial cells (HRMECs), TNF‐α stimulation causes increased RUNX1 expression, which can be modulated by RUNX1 inhibitors. Using TNF‐α pathway inhibitors, we determined that in HRMECs, TNF‐α‐induced RUNX1 expression occurs via JNK activation, while NF‐κB and p38/MAPK inhibition did not affect RUNX1 expression. JNK inhibitors were also effective at stopping high d‐glucose‐stimulated RUNX1 expression. We further linked JNK to RUNX1 through Activator Protein 1 (AP‐1) and investigated the JNK‐AP‐1‐RUNX1 regulatory feedback loop, which can be modulated by VEGF. Additionally, stimulation with TNF‐α and d‐glucose had an additive effect on RUNX1 expression, which was downregulated by VEGF modulation. These data suggest that the downregulation of RUNX1 in conjunction with anti‐VEGF agents may be important in future treatments for the management of diseases of pathologic ocular angiogenesis.

MLL is a target of chromosomal translocations in acute leukemias with poor prognosis. The common MLL fusion partner AF9 (MLLT3) can directly bind to AF4, DOT1L, BCOR, and CBX8. To delineate the relevance of BCOR and CBX8 binding to MLL-AF9 for leukemogenesis, here we determine protein structures of AF9 complexes with CBX8 and BCOR, and show that binding of all four partners to AF9 is mutually exclusive. Using the structural analyses, we identify point mutations that selectively disrupt AF9 interactions with BCOR and CBX8. In bone marrow stem/progenitor cells expressing point mutant CBX8 or point mutant MLL-AF9, we show that disruption of direct CBX8/MLL-AF9 binding does not impact in vitro cell proliferation, whereas loss of direct BCOR/MLL-AF9 binding causes partial differentiation and increased proliferation. Strikingly, loss of MLL-AF9/BCOR binding abrogated its leukemogenic potential in a mouse model. The MLL-AF9 mutant deficient for BCOR binding reduces the expression of the EYA1 phosphatase and the protein level of c-Myc. Reduction in BCOR binding to MLL-AF9 alters a MYC-driven gene expression program, as well as altering expression of SIX-regulated genes, likely contributing to the observed reduction in the leukemia-initiating cell population. Significance: Direct recruitment of BCOR to MLL-AF9 is essential for leukemia via EYA1 phosphatase regulation, altering MYC and SIX gene expression programs. Specific partner binding (AF4, DOT1L, and BCOR) contributes in distinct ways to MLL leukemia. This may provide a rationale for combination DOT1L and EYA1 inhibition for MLL fusion leukemia treatment. This article is highlighted in the In This Issue feature, p. 127

Protein tyrosine phosphatase (PTP) 4A3 is frequently overexpressed in human solid tumors and hematologic malignancies and is associated with tumor cell invasion, metastasis, and a poor patient prognosis. Several potent, selective, and allosteric small molecule inhibitors of PTP4A3 were recently identified. A lead compound in the series, JMS-053 (7-imino-2-phenylthieno[3,2-c]pyridine-4,6(5H,7H)-dione), has a long plasma half-life (∼ 24 hours) in mice, suggesting possible binding to serum components. We confirmed by isothermal titration calorimetry that JMS-053 binds to human serum albumin. A single JMS-053 binding site was identified by X-ray crystallography in human serum albumin at drug site 3, which is also known as subdomain IB. The binding of JMS-053 to human serum albumin, however, did not markedly alter the overall albumin structure. In the presence of serum albumin, the potency of JMS-053 as an in vitro inhibitor of PTP4A3 and human A2780 ovarian cancer cell growth was reduced. The reversible binding of JMS-053 to serum albumin may serve to increase JMS-053’s plasma half-life and thus extend the delivery of the compound to tumors. SIGNIFICANCE STATEMENT X-ray crystallography revealed that a potent, reversible, first-in-class small molecule inhibitor of the oncogenic phosphatase protein tyrosine phosphatase 4A3 binds to at least one site on human serum albumin, which is likely to extend the compound’s plasma half-life and thus assist in drug delivery into tumors.

Osteosarcoma remains the most common primary bone tumour in dogs with half of affected dogs unable to survive 1 year beyond diagnosis. New therapeutic options are needed to improve outcomes for this disease. Recent investigations into potential therapeutic targets have focused on cell surface molecules with little clear therapeutic benefit. Transcription factors and protein interactions represent underdeveloped areas of therapeutic drug development. We have utilized allosteric inhibitors of the core binding factor transcriptional complex, comprised of core binding factor beta (CBFβ) and RUNX2, in four canine osteosarcoma cell lines Active inhibitor compounds demonstrate anti-tumour activities with concentrations demonstrated to be achievable in vivo while an inactive, structural analogue has no activity. We show that CBFβ inhibitors are capable of inducing apoptosis, inhibiting clonogenic cell growth, altering cell cycle progression and impeding migration and invasion in a cell line-dependent manner. These effects coincide with a reduced interaction between RUNX2 and CBFβ and alterations in expression of RUNX2 target genes. We also show that addition of CBFβ inhibitors to the commonly used cytotoxic chemotherapeutic drugs doxorubicin and carboplatin leads to additive and/or synergistic anti-proliferative effects in canine osteosarcoma cell lines. Taken together, we have identified the interaction between components of the core binding factor transcriptional complex, RUNX2 and CBFβ, as a potential novel therapeutic target in canine osteosarcoma and provide justification for further investigations into the anti-tumour activities we describe here.

Objective Ovarian cancer survival and treatment have improved minimally in the past 20years. Novel treatment strategies are needed to combat this disease. This study investigates the effects of chemical inhibition of the CBFβ/RUNX protein-protein interaction on ovarian cancer cell lines. Methods Ovarian cancer cell lines were treated with CBFβ/RUNX inhibitors, and the effects on proliferation, DNA replication, wound healing, and anchorage-independent growth were measured. RNA-Seq was performed on compound-treated cells to identify differentially expressed genes. Genes altered by compound treatment were targeted with siRNAs, and effects on DNA replication and wound healing were measured. Results Chemical inhibition of the CBFβ/RUNX interaction decreases ovarian cancer cell proliferation. Inhibitor treatment leads to an S-phase cell cycle delay, as indicated by an increased percentage of cells in S-phase, and a decreased DNA replication rate. Inhibitor treatment also reduces wound healing and anchorage-independent growth. RNA-Seq on compound-treated cells revealed changes in a small number of genes related to proliferation and epithelial-to-mesenchymal transition. siRNA-mediated knockdown of INHBA and MMP1 – two genes whose expression decreases with compound treatment – slowed DNA replication and impaired wound healing. Conclusions Chemical inhibition of the CBFβ/RUNX interaction is a viable strategy for the treatment of ovarian cancer.

Membrane oxidoreductase CcdA plays a central role in supplying reducing equivalents from the bacterial cytoplasm to the envelope. It transports electrons across the membrane using a single pair of cysteines by a mechanism that has not yet been elucidated. Here we report an NMR structure of the Thermus thermophilus CcdA (TtCcdA) in an oxidized and outward-facing state. CcdA consists of two inverted structural repeats of three transmembrane helices (2 × 3-TM). We computationally modeled and experimentally validated an inward-facing state, which suggests that CcdA uses an elevator-type movement to shuttle the reactive cysteines across the membrane. CcdA belongs to the LysE superfamily, and thus its structure may be relevant to other LysE clan transporters. Structure comparisons of CcdA, semiSWEET, Pnu, and major facilitator superfamily (MFS) transporters provide insights into membrane transporter architecture and mechanism.

Signal transduction pathways regulate the proliferation and viability of acute myeloid leukemia (AML) blasts. The regulation in the expression of cytokine receptors in AML is not well understood. In this study, we investigated how the CBFβ-SMMHC fusion protein regulates expression of cytokine receptors in inv(16) AML, with focus on the co-receptor Neuropilin-1 (NRP1). Knock-down of CBFβ-SMMHC expression, utilizing shRNA transduction, induced G1 phase of cell cycle arrest and reduced the viability of inv(16) ME-1 cells in culture. Expression profile analysis of CBFβ-SMMHC knock-down cells revealed a significant repression of genes associated with transmembrane receptor protein kinase pathways, including NRP1 (-5 fold), FGFR1 (-4.2 fold) , FLT3 (-2 fold) and TGFBR2 (-1.2 fold). The expression of NRP1 was significantly upregulated in inv(16) AML cases when compared to other AML sub-types and to hematopoietic stem and progenitor cells. Functionally, NRP1 knock-down reduced the viability of ME-1 cells with a similar dynamics as when using CBFβ-SMMHC shRNAs. In addition, the proliferation of inv(16) AML cells was reduced 4.1-fold when treated with a function-blocking antibody for the FV/VIII extracellular NRP1 domain, while having no effect in non-inv(16) AML cells or when using blocking antibody for the CUB extracellular domain. Furthermore, deletion of Nrp1 by gene editing reduced the colony-forming unit capacity of primary mouse Cbfb +/MYH11 leukemic cells and extended the median leukemia latency in vivo. To identify the genes regulated by NRP1 in inv(16) AML, we analysed the transcription profile of NRP1 knock-down in ME-1 cells. Gene Set Enrichment and Pathway Analysis revealed a repression in STAT5 pathway, and in signalling receptor activity, including FLT3 (-1.8 fold) and TGFBR2 (-1.8 fold) expression, indicating that NRP1 mediates transcriptional regulation of FLT3 and TGFBR2 expression in inv(16) AML. Furthermore, the regulation of FLT3 and TGFB2 expression by CBFβ-SMMHC and by NRP1 was validated by gene editing in inv(16) AML blasts. Accordingly, NRP1 knock-down in AML cells reduced SMAD2/3 phosphorylation. The repression of RUNX1/CBFβ function, using small molecule inhibitors, in inv(16) AML cells with CBFβ-SMMHC knockdown restored NRP1 expression, suggesting that RUNX1 may repress NRP1 expression in AML cells. To evaluate if RUNX1 directly regulates NRP1 expression, we tested RUNX1 binding in the NRP1 locus of AML cells with CBFβ-SMMHC knockdown. RUNX1 binding at one of six sites with RUNX1 occupancy identified by chromatin immunoprecipitation followed by sequencing (RE5, regulatory element 5) was increased in the CBFβ-SMMHC knock-down cells. The RE5 is located 178kb upstream of the NRP1 transcription start site and it is evolutionarily conserved in vertebrates. The deletion of RE5 by gene editing (~50% editing efficiency) increased NRP1 expression 1.8-fold, suggesting that RUNX1 may repress NRP1 expression at by binding to the RE5 enhancer. Taken together, these studies demonstrate that CBFβ-SMMHC regulates expression of cytokine receptors in inv(16) AML. Specifically, it directly regulates expression of the co-receptor NRP1, which is essential for AML survival, acting (at least in part) by regulating FLT3 and TGFB pathways. Disclosures Guzman: SeqRx: Consultancy; BridgeMedicines: Consultancy; Cellectis: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.

The core binding factor (CBF) gene RUNX1 is a target of chromosomal translocations in leukemia, including t(8;21) in acute myeloid leukemia (AML). Normal CBF function is essential for activity of AML1-ETO, product of the t(8;21), and for survival of several leukemias lacking RUNX1 mutations. Using virtual screening and optimization, we developed Runt domain inhibitors which bind to the Runt domain and disrupt its interaction with CBFβ. On-target activity was demonstrated by the Runt domain inhibitors' ability to depress hematopoietic cell formation in zebrafish embryos, reduce growth and induce apoptosis of t(8;21) AML cell lines, and reduce progenitor activity of mouse and human leukemia cells harboring the t(8;21), but not normal bone marrow cells. Runt domain inhibitors had similar effects on murine and human T cell acute lymphocytic leukemia (T-ALL) cell lines. Our results confirmed that Runt domain inhibitors might prove efficacious in various AMLs and in T-ALL.

Transcription factors have traditionally been viewed with skepticism as viable drug targets, but they offer the potential for completely novel mechanisms of action that could more effectively address the stem cell like properties, such as self-renewal and chemo-resistance, that lead to the failure of traditional chemotherapy approaches. Core binding factor is a heterodimeric transcription factor comprised of one of 3 RUNX proteins (RUNX1-3) and a CBFβ binding partner. CBFβ enhances DNA binding of RUNX subunits by relieving auto-inhibition. Both RUNX1 and CBFβ are frequently mutated in human leukemia. More recently, RUNX proteins have been shown to be key players in epithelial cancers, suggesting the targeting of this pathway could have broad utility. In order to test this, we developed small molecules which bind to CBFβ and inhibit its binding to RUNX. Treatment with these inhibitors reduces binding of RUNX1 to target genes, alters the expression of RUNX1 target genes, and impacts cell survival and differentiation. These inhibitors show efficacy against leukemia cells as well as basal-like (triple-negative) breast cancer cells. These inhibitors provide effective tools to probe the utility of targeting RUNX transcription factor function in other cancers.

Aberrant ERG (v-ets avian erythroblastosis virus E26 oncogene homolog) expression drives leukemic transformation in mice and high expression is associated with poor patient outcomes in acute myeloid leukemia (AML) and T-acute lymphoblastic leukemia (T-ALL). Protein phosphorylation regulates the activity of many ETS factors but little is known about ERG in leukemic cells. To characterize ERG phosphorylation in leukemic cells, we applied liquid chromatography coupled tandem mass spectrometry and identified five phosphorylated serines on endogenous ERG in T-ALL and AML cells. S283 was distinct as it was abundantly phosphorylated in leukemic cells but not in healthy hematopoietic stem and progenitor cells (HSPCs). Overexpression of a phosphoactive mutant (S283D) increased expansion and clonogenicity of primary HSPCs over and above wild-type ERG. Using a custom antibody, we screened a panel of primary leukemic xenografts and showed that ERG S283 phosphorylation was mediated by mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling and in turn regulated expression of components of this pathway. S283 phosphorylation facilitates ERG enrichment and transactivation at the ERG +85 HSPC enhancer that is active in AML and T-ALL with poor prognosis. Taken together, we have identified a specific post-translational modification in leukemic cells that promotes progenitor proliferation and is a potential target to modulate ERG-driven transcriptional programs in leukemia.

We have recently identified R-spondin3 (RSPO3) as a novel key modulator of breast cancer development that may become a potential target for treatment of the basal subtype, which lacks efficient therapeutic options to date. RSPO3 is expressed in the basal stem cell-enriched compartment of mouse mammary glands whereas it is absent in the mature luminal cells. RSPO3 knockdown in mouse mammary tumor cells reduces canonical Wnt signaling pathway, epithelial-to-mesenchymal transition (EMT)-like features, migration capacity, and tumor formation in vivo. To evaluate the relevance of RSPO3 in human breast cancer, we analyzed samples from 75 patients with ductal infiltrating breast carcinomas, where we found a high prevalence of positive immunoreactivity for RSPO3 (70%). Expression data of The Cancer Genome Atlas Network from 1985 human breast carcinoma samples showed that RSPO3 is primarily expressed in Claudin-low breast cancer subtype, which is characterized for having stem cells features. In culture assays revealed that knockdown of RSPO3 expression reduced SOX2 expression and impaired mammary tumor cell ability to form mammospheres. Previous reports suggested the potential involvement of RUNX1-CBFβ on RSPO3 transcription in mammary tumor cells. Therefore, we evaluated if pharmacologic inhibition of RUNX1 activity would reduce RSPO3 expression and activity in the cancer stem cell compartment. Treatment of breast cancer cells with AI-10-104, a small molecule that inhibits CBFβ-RUNX interaction, reduced RSPO3 mRNA and protein levels, and reduced the migration ability of MDA-MB231 breast cancer cells, ability that was recovered upon treatment with recombinant RSPO3. In addition, AI-10-104 inhibited the expression of canonical Wnt pathway targets, EMT and stem-cell markers. Therefore, our results demonstrate that inhibition of RUNX-CBFβ transcriptional activity leads to a down-regulation of RSPO3 expression, constituting a new molecular target for therapeutic modulation in breast cancer treatment. Citation Format: Carla M. Felcher, Johanna M. Tocci, Martin E. Garcia Sola, John H. Bushweller, Lucio H. Castilla, Edith C. Kordon. Inhibition of RUNX-CBFβ binding reduces RSPO3 expression and EMT features in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5041.

RUNX1 has recently been shown to play an important role in determination of mammary epithelial cell identity. However, mechanisms by which loss of the RUNX1 transcription factor in mammary epithelial cells leads to epithelial-to-mesenchymal transition (EMT) are not known. Here, we report mitotic bookmarking of genes by RUNX1 as a potential mechanism to convey regulatory information through successive cell divisions for coordinate control of mammary cell proliferation, growth, and identity. Genome-wide RUNX1 occupancy profiles for asynchronous, mitotically enriched, and early G1 breast epithelial cells reveal RUNX1 is retained during the mitosis to G1 transition on protein coding and long non-coding RNA genes critical for mammary epithelial proliferation, growth, and phenotype maintenance. Disruption of RUNX1 DNA binding and association with mitotic chromosomes alters cell morphology, global protein synthesis, and phenotype-related gene expression. Together, these findings show for the first time that RUNX1 bookmarks a subset of epithelial-related genes during mitosis that remain occupied as cells enter the next cell cycle. Compromising RUNX1 DNA binding initiates EMT, an essential first step in the onset of breast cancer.SignificanceThis study elucidates mitotic gene bookmarking as a potential epigenetic mechanism that impacts breast epithelial cell growth and phenotype and has potential implications in breast cancer onset.

Ikaros family zinc finger protein 1 and 3 (IKZF1 and IKZF3) are transcription factors that promote multiple myeloma (MM) proliferation. The immunomodulatory imide drug (IMiD) lenalidomide promotes myeloma cell death via Cereblon (CRBN)-dependent ubiquitylation and proteasome-dependent degradation of IKZF1 and IKZF3. Although IMiDs have been used as first-line drugs for MM, the overall survival of refractory MM patients remains poor and demands the identification of novel agents to potentiate the therapeutic effect of IMiDs. Using an unbiased screen based on mass spectrometry, we identified the Runt-related transcription factor 1 and 3 (RUNX1 and RUNX3) as interactors of IKZF1 and IKZF3. Interaction with RUNX1 and RUNX3 inhibits CRBN-dependent binding, ubiquitylation and degradation of IKZF1 and IKZF3 upon lenalidomide treatment. Inhibition of RUNXs, via genetic ablation or a small molecule (AI-10-104), results in sensitization of myeloma cell lines and primary tumors to lenalidomide. Thus, RUNX inhibition represents a valuable therapeutic opportunity to potentiate IMiDs therapy for the treatment of multiple myeloma.

The fusion oncoprotein CBFβ-SMMHC, expressed in leukemia cases with chromosome 16 inversion, drives leukemia development and maintenance by altering the activity of the transcription factor RUNX1. Here, we demonstrate that CBFβ-SMMHC maintains cell viability by neutralizing RUNX1-mediated repression of MYC expression. Upon pharmacologic inhibition of the CBFβ-SMMHC/RUNX1 interaction, RUNX1 shows increased binding at three MYC distal enhancers, where it represses MYC expression by mediating the replacement of the SWI/SNF complex component BRG1 with the polycomb-repressive complex component RING1B, leading to apoptosis. Combining the CBFβ-SMMHC inhibitor with the BET inhibitor JQ1 eliminates inv(16) leukemia in human cells and a mouse model. Enhancer-interaction analysis indicated that the three enhancers are physically connected with the MYC promoter, and genome-editing analysis demonstrated that they are functionally implicated in deregulation of MYC expression. This study reveals a mechanism whereby CBFβ-SMMHC drives leukemia maintenance and suggests that inhibitors targeting chromatin activity may prove effective in inv(16) leukemia therapy.

SummaryEarly endosomes represent the first sorting station for vesicular ubiquitylated cargo. Tollip, through its C2 domain, associates with endosomal phosphatidylinositol 3-phosphate (PtdIns(3)P) and binds ubiquitylated cargo in these compartments via its C2 and CUE domains. Tom1, through its GAT domain, is recruited to endosomes by binding to the Tollip Tom1-binding domain (TBD) through an unknown mechanism. Nuclear magnetic resonance data revealed that Tollip TBD is a natively unfolded domain that partially folds at its N terminus when bound to Tom1 GAT through high-affinity hydrophobic contacts. Furthermore, this association abrogates binding of Tollip to PtdIns(3)P by additionally targeting its C2 domain. Tom1 GAT is also able to bind ubiquitin and PtdIns(3)P at overlapping sites, albeit with modest affinity. We propose that association with Tom1 favors the release of Tollip from endosomal membranes, allowing Tollip to commit to cargo trafficking.

SummaryThe MLL gene is a common target of chromosomal translocations found in human leukemia. MLL-fusion leukemia has a consistently poor outcome. One of the most common translocation partners is AF9 (MLLT3). MLL-AF9 recruits DOT1L, a histone 3 lysine 79 methyltransferase (H3K79me1/me2/me3), leading to aberrant gene transcription. We show that DOT1L has three AF9 binding sites and present the nuclear magnetic resonance (NMR) solution structure of a DOT1L-AF9 complex. We generate structure-guided point mutations and find that they have graded effects on recruitment of DOT1L to MLL-AF9. Chromatin immunoprecipitation sequencing (ChIP-seq) analyses of H3K79me2 and H3K79me3 show that graded reduction of the DOT1L interaction with MLL-AF9 results in differential loss of H3K79me2 and me3 at MLL-AF9 target genes. Furthermore, the degree of DOT1L recruitment is linked to the level of MLL-AF9 hematopoietic transformation.

Toward drugging the undruggable in cancer Many human cancers are characterized by inappropriate activity of transcription factors. These proteins are attractive drug targets in principle, but normalizing their function requires drugs that modulate specific protein-protein interactions, a goal that has been challenging. In acute myeloid leukemia, a chromosomal translocation creates an aberrant form of the transcription factor CBF-beta, which outcompetes “normal” CBF-beta for binding to another transcription factor called RUNX1, thereby deregulating its activity. Illendula et al. identified and chemically optimized a small molecule that selectively disrupts the interaction between the aberrant CBF-beta and RUNX1 (see the Perspective by Koehler and Chen). This molecule restored normal gene expression patterns and delayed leukemia progression in mice. Thus, transcription factors may not be as undruggable as once thought. Science , this issue p. 779 ; see also p. 713

The gene encoding the RUNX1 transcription factor is mutated in a subset of T-cell acute lymphoblastic leukemia (T-ALL) patients, and RUNX1 mutations are associated with a poor prognosis. These mutations cluster in the DNA-binding Runt domain and are thought to represent loss-of-function mutations, indicating that RUNX1 suppresses T-cell transformation. RUNX1 has been proposed to have tumor suppressor roles in T-cell leukemia homeobox 1/3-transformed human T-ALL cell lines and NOTCH1 T-ALL mouse models. Yet, retroviral insertional mutagenesis screens identify RUNX genes as collaborating oncogenes in MYC-driven leukemia mouse models. To elucidate RUNX1 function(s) in leukemogenesis, we generated Tal1/Lmo2/Rosa26-CreERT2Runx1f/f mice and examined leukemia progression in the presence of vehicle or tamoxifen. We found that Runx1 deletion inhibits mouse leukemic growth in vivo and that RUNX silencing in human T-ALL cells triggers apoptosis. We demonstrate that a small molecule inhibitor, designed to interfere with CBFβ binding to RUNX proteins, impairs the growth of human T-ALL cell lines and primary patient samples. We demonstrate that a RUNX1 deficiency alters the expression of a crucial subset of TAL1- and NOTCH1-regulated genes, including the MYB and MYC oncogenes, respectively. These studies provide genetic and pharmacologic evidence that RUNX1 has oncogenic roles and reveal RUNX1 as a novel therapeutic target in T-ALL.

Acute myeloid leukemia (AML) is characterized by recurrent chromosomal rearrangements that encode for fusion proteins which drive leukemia initiation and maintenance. The inv(16) (p13q22) rearrangement is a founding mutation and the associated CBFβ-SMMHC fusion protein is essential for the survival of inv(16) AML cells. This Chapter will discuss our understanding of the function of this fusion protein in disrupting hematopoietic homeostasis and creating pre-leukemic blasts, in its cooperation with other co-occurring mutations during leukemia initiation, and in leukemia maintenance. In addition, this chapter will discuss the current approaches used for the treatment of inv(16) AML and the recent development of AI-10-49, a selective targeted inhibitor of CBFβ-SMMHC/RUNX1 binding, the first candidate targeted therapy for inv(16) AML.

Leukaemia is the most common childhood cancer and acute lymphoblastic leukaemia (ALL) is responsible for a third of all childhood cancer deaths. Despite the overall good prognosis, failure to eradicate the disease leads to relapse in ∼20% of patients and survival rates for infants are less than 50%. There is therefore a clear unmet need for less toxic and more effective targeted treatments. ETV6-RUNX1 translocation is the first-hit event in ∼25% of B-ALLs, initiating a clinically silent pre-leukaemia in utero. The translocation fuses almost the entire RUNX1 protein to the repressor domain of ETV6 and the chimeric product is proposed to mediate a block in B cell differentiation by repressing RUNX1 targets. Interestingly, the remaining RUNX1 allele is not mutated, but is instead often amplified suggesting an onco-supportive role of the endogenous RUNX1. Using genetic and pharmacological inhibition, we have discovered that RUNX1/CBF is essential for the growth and survival of B-ALL cells in vitro and in vivo. Global RNA-Seq on CBF-depleted B-ALL cells revealed significant downregulation of E2F and Myc signature genes along with a marked increase of p53 targets, consistent with these cells accumulating in G0 and subsequently becoming apoptotic. Notably, RNA-Seq and mass cytometry analyses of pro-B cells derived from an hiPSC ETV6-RUNX1 knockin model reveal a similar cell cycle trend indicating that ETV6-RUNX1-mediated inhibition of RUNX1 targets may induce a quiescence-like pre-leukaemic state while continued RUNX1 expression is essential for the survival of overt leukaemia. We propose that CBF-dependency is an Achilles heel of ALL cells that could be exploited by targeting either CBF itself or its key downstream nodes. By combining clinically tested compounds targeting multiple CBF effectors we aim to improve efficacy of potential treatments and circumvent resistance.

Acute myeloid leukemia (AML) is characterized by recurrent chromosomal rearrangements that encode for fusion proteins which drive leukemia initiation and maintenance. The inv(16) (p13q22) rearrangement is a founding mutation and the associated CBFβ-SMMHC fusion protein is essential for the survival of inv(16) AML cells. This Chapter will discuss our understanding of the function of this fusion protein in disrupting hematopoietic homeostasis and creating pre-leukemic blasts, in its cooperation with other co-occurring mutations during leukemia initiation, and in leukemia maintenance. In addition, this chapter will discuss the current approaches used for the treatment of inv(16) AML and the recent development of AI-10-49, a selective targeted inhibitor of CBFβ-SMMHC/RUNX1 binding, the first candidate targeted therapy for inv(16) AML.

The core binding factor (CBF) transcription factor is somewhat unique in that it is composed of a DNA binding RUNX subunit (RUNX1, 2, or 3) and a non-DNA binding CBFβ subunit, which modulates RUNX protein activity by modulating the auto-inhibition of the RUNX subunits. Since the discovery of this fascinating transcription factor more than 20 years ago, there has been a robust effort to characterize the structure as well as the biochemical properties of CBF. More recently, these efforts have also extended to the fusion proteins that arise from the subunits of CBF in leukemia. This chapter highlights the work of numerous labs which has provided a detailed understanding of the structure and function of this transcription factor and its fusion proteins.

The mixed lineage leukemia (MLL) protein is a histone methyltransferase that writes the histone H3 lysine 4 trimethyl (H3K4me3) mark at the promoters of target genes such as HOXA9 and MEIS1. MLL is the target of chromosomal translocations that fuse it in frame to one of over 90 partners, leading to acute myeloid and lymphoid leukemias (AML and ALL, respectively) characterized by poor prognoses1. MLL fusions activate transcription by recruiting the AF4 family/ENL family/P-TEFb (AEP) complex and the DOT1L-AF10 family-ENL family complex (DOT1L complex or DotCom). Transcriptional activation via AF4 recruitment and transcriptional maintenance via DOT1L recruitment are required for MLL leukemias. Despite the large number of fusion partners, members of the AEP complex account for nearly 70% of MLL rearrangements1. These fusions constitutively activate MLL targets by bypassing recruitment via ENL (MLLT1) and AF9 (MLLT3) YEATS domain binding to crotonylated or acetylated histone H3. The AF9 ANC1 homology domain (AHD), retained in MLL fusions, is intrinsically disordered, but undergoes coupled folding and binding upon interaction with its binding proteins2. The AHD recruits AF4 and DOT1L, which support transcriptional elongation, as well as the BCL6 corepressor (BCOR) and chromobox homolog 8 (CBX8), which are implicated in transcriptional repression. CBX8 (HPC3) is a mammalian ortholog of Drosophila polycomb that binds trimethylated histone H3 lysine 9 and 27 (H3K9me3 and H3K27me3) with variable affinity. Previous reports indicate CBX8 is required for MLL-AF9 and MLL-ENL. BCOR is a transcriptional corepressor that augments BCL6-mediated repression. The BCL6 POZ domain forms a ternary complex with BCOR and SMRT, repressing targets via recruitment of PRC1.1 and HDAC3. BCOR translocations and mutations have been found in a range of cancers. Although it is broadly expressed throughout the hematopoietic system (Bloodspot), little is known about BCOR function in hematopoiesis. Recently, BCOR was shown to have a role in maintenance of human embryonic stem cell pluripotency. BCOR has also been implicated in regulation of myeloid cell proliferation and differentiation and is necessary for MLL-AF9 leukemogenesis. While the roles of the direct MLL-AF9/AF4 and MLL-AF9/DOT1L interactions have been the subject of previous structural and functional studies2-4, the roles of the direct interactions of MLL-AF9 with CBX8 and BCOR remain relatively uncharacterized. We determined the structures of the AF9 AHD-CBX8 and AF9 AHD-BCOR complexes. Based on the structures, we developed point mutants to increase and decrease affinity of CBX8 for AF9. Increased affinity decreased colony forming ability and induced differentiation of MLL-AF9-transformed cells, while decreased affinity had no effect. An additional point mutant was developed to selectively disrupt BCOR binding to AF9. In the context of MLL-AF9, this mutant increases proliferative ability without an effect on colony formation and is unable to cause leukemia in vivo. RNAseq analysis reveals that this mutant affects a different set of genes than loss of DOT1L or AF4 binding or gain of CBX8 binding, leading to a phenotype distinct from that seen with perturbation of other AF9 interactions, functionally distinguishing proliferative capacity from in vivo leukemogenesis. In particular, substantial effects were observed on EYA1 expression, suggesting a critical role for the EYA1/SIX gene network in MLL-AF9 leukemia. 1 Meyer, C. et al. The MLL recombinome of acute leukemias in 2017. Leukemia32, 273-284, doi:10.1038/leu.2017.213 (2018). 2 Leach, B. I. et al. Leukemia fusion target AF9 is an intrinsically disordered transcriptional regulator that recruits multiple partners via coupled folding and binding. Structure21, 176-183, doi:10.1016/j.str.2012.11.011 (2013). 3 Kuntimaddi, A. et al. Degree of recruitment of DOT1L to MLL-AF9 defines level of H3K79 Di- and tri-methylation on target genes and transformation potential. Cell reports11, 808-820, doi:10.1016/j.celrep.2015.04.004 (2015). 4 Lokken, A. A. et al. Importance of a specific amino acid pairing for murine MLL leukemias driven by MLLT1/3 or AFF1/4. Leukemia research38, 1309-1315, doi:10.1016/j.leukres.2014.08.010 (2014). Disclosures No relevant conflicts of interest to declare.

Early endosomes represent the first sorting station for vesicular ubiquitylated cargo. Cargo transport is mediated by the endosomal sorting complex required for transport (ESCRT) machinery. Similar to the structural organization of ESCRT-0 proteins, alternative ESCRT-0 (alt-ESCRT-0) proteins, such as Tollip and Tom1, also present multiple ubiquitin-binding domains, including the C2 and CUE (Tollip) and VHS and GAT (Tom1) domains. Tollip localizes the Tollip-Tom1 complex at endosomal compartments by association with phosphatidylinositol 3-phosphate (PtdIns(3)P) through its central C2 domain. Tom1, through its GAT domain, is recruited to endosomes by binding to Tollip's Tom1-binding domain (TBD) through an unknown mechanism. Our NMR data revealed that Tollip TBD is a natively unfolded domain that partially folds at its N-terminus when bound to the first two helices of the Tom1 GAT domain through high affinity hydrophobic contacts. Furthermore, this association abrogates binding of Tollip to PtdIns(3)P by additionally targeting its C2 domain. Binding of the Tollip C2 domain is mediated by the third helix of the Tom1 GAT domain. We also show that the Tom1 GAT domain is able to bind ubiquitin and PtdIns(3)P at overlapping sites, albeit with modest affinity. We propose that association with Tom1 favors Tollip's release from endosomal membranes, allowing Tollip to commit to cargo trafficking. More recently, Tom1 has been shown to associate to signaling endosomal PtdIns(5)P through its VHS domain delaying epidermal growth factor receptor degradation in a bacterial infection model. We found that PtdIns(5)P binding of the Tom1 VHS domain is in the moderately fast-exchange regime at the NMR timescale. We propose that an incremental change in PtdIns(5)P intracellular levels sequesters Tom1 in endosomal cargo-free membrane domains, keeping the protein from its cargo sorting function.

Chromatin complexes regulate gene expression in normal and malignant hematopoiesis. The significance of transcription factor deregulation on the control of epigenetic dynamics in leukemia is poorly understood. The leukemia fusion CBFβ-SMMHC is expressed in inv(16) acute myeloid leukemia (AML), and deregulates the activity of the transcription factor RUNX1. This fusion protein blocks myeloid differentiation, expands pre-leukemic myeloid progenitor cells, and drives AML development. The CBFβ-SMMHC inhibitor, AI-10-49, specifically disrupts its binding to RUNX1 resulting in an acute RUNX1 release, inducing apoptosis in inv(16) AML cells and delaying leukemia latency in mice. However, the mechanism by which AI-10-49 induces apoptosis is unknown. In this study, we utilize pharmacologic, genomic and genetic approaches to demonstrate a RUNX-dependent mechanism by which CBFβ-SMMHC maintains leukemia survival, and that this mechanism can be pharmacologically targeted in the treatment of inv(16) AML. Gene Set Enrichment Analysis of RNA-seq data from inv(16) AML cells treated with AI-10-49 identified the deregulation of a MYC signature, including cell cycle, ribosome biogenesis and metabolism, and the reduction of MYC transcript levels (10-fold). MYC shRNA knockdown induced apoptosis of inv(16) AML cells, and MYC expression rescued AI-10-49 induced apoptosis. Furthermore, mouse leukemia cells transduced with Myc shRNAs showed significant delay in leukemic latency upon transplantation, validating the requirement of MYC in inv(16) AML maintenance in vivo. Similarly, pharmacologic inhibition of MYC activity, using a combined treatment with AI-10-49 and the BET-family bromodomain inhibitor JQ1, revealed a strong synergy in inv(16) AML cells and significant delay in leukemia latency in mice. Analysis of chromatin immunoprecipitation followed by deep-sequencing (ChIP-seq) revealed that AI-10-49 treatment increased RUNX1 occupancy at three MYC distal enhancer elements (including two new enhancers: +0.18 Mb and +0.5 Mb, and the E3 enhancer of the BDME superenhancer at +1.7 Mb) downstream from MYC TSS but not at the MYC TSS or the T-cell leukemia associated N-ME enhancer. The open-chromatin and active nature of these regions was further validated by Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) and H3K27Ac ChIP-seq. Since the MYC locus is occupied by a number of tumor-specific enhancers, we analyzed the enhancer interactions in a 4 Mb region (-1Mb to +3 Mb of MYC TSS) by chromosome conformation capture carbon copy (5C) in inv(16) AML cells. The 5C analysis revealed that the three enhancers are physically connected with each other and with the MYC promoter. Again, the T-cell ALL associated N-ME enhancer was not active in these cells. Furthermore, the six-hour AI-10-49 treatment had not altered these interactions, indicating that MYC repression was probably not due to changes in open chromatin but to alterations in regulatory factors. Therefore, we analyzed the occupancy of chromatin complex components at the three enhancers, N-ME and MYC TSS regions, utilizing ChIP-quantitative PCR. We demonstrate that AI-10-49 treatment results in the repression of the SWI/SNF complex component BRG1 and recruitment of polycomb-repressive complex 1 (PRC1) component RING1B at the three MYC enhancers. This switch was associated with a reduction of active enhancer mark H3K4me1 and an increase of the repressive mark H3K27me3 at these sites. Finally, deletion of a small region in each of these enhancers surrounding the RUNX1 binding site (41 bp, 67 bp and 295 bp) by genome editing (CRISPR/Cas9) reduced MYC transcript levels by 50% and the viability of inv(16) AML cells in vitro, indicating that each one of these enhancers plays a critical role in regulating MYC levels and sustaining the survival of inv(16) AML cells. Our results demonstrate that CBFβ-SMMHC regulates MYC levels by repression of RUNX1 activity at three distal MYC enhancers in inv(16) AML cells. AI-10-49 treatment induces an acute release of RUNX1, increases RUNX1 occupancy at the distal enhancers, and disrupts enhancer chromatin dynamics which in turn induces apoptosis by repressing MYC. Furthermore, this study suggests that combined treatment of inv(16) AML with AI-10-49 and BET-family inhibitors may represent a promising targeted therapy. Disclosures No relevant conflicts of interest to declare.

Transcription factors have traditionally been viewed with skepticism as viable drug targets, but they offer the potential for completely novel mechanisms of action that could more effectively address the stem cell like properties, such as self-renewal and chemo-resistance, that lead to the failure of traditional chemotherapy approaches. Core binding factor is a heterodimeric transcription factor comprised of one of 3 RUNX proteins (RUNX1-3) and a CBFβ binding partner. CBFβ enhances DNA binding of RUNX subunits by relieving auto-inhibition. Both RUNX1 and CBFβ are frequently mutated in human leukemia. More recently, RUNX proteins have been shown to be key players in epithelial cancers, suggesting the targeting of this pathway could have broad utility. In order to test this, we developed small molecules which bind to CBFβ and inhibit its binding to RUNX. Treatment with these inhibitors reduces binding of RUNX1 to target genes, alters the expression of RUNX1 target genes, and impacts cell survival and differentiation. These inhibitors show efficacy against leukemia cells as well as basal-like (triple-negative) breast cancer cells. These inhibitors provide effective tools to probe the utility of targeting RUNX transcription factor function in other cancers., Graphical Abstract Image 10, Highlights • Small molecule inhibitors of CBFβ-RUNX protein-protein interaction developed. • Inhibitors alter occupancy of RUNX1 on target genes and alter their expression. • Inhibitors show efficacy against leukemia cell lines and basal-like (triple negative) breast cancer cell lines. Transcription factors are proteins that bind to DNA and regulate how much of other proteins are made. We describe the development of inhibitors of the interaction between two transcription factors, CBFβ and RUNX. Both these transcription factors are the targets of alterations in human leukemia as well as in a number of solid tumors. The inhibitor changes the behavior of the RUNX transcription factor and alters the levels of proteins it regulates. We show these inhibitors may have potential utility for leukemia as well as one specific type of breast cancer which has a very poor prognosis.