CBFß-SMMHC Inhibition Triggers Apoptosis by Disrupting MYC Chromatin Dynamics in Acute Myeloid Leukemia.

The fusion oncoprotein CBFß-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 CBFß-SMMHC maintains cell viability by neutralizing RUNX1-mediated repression of MYC expression. Upon pharmacologic inhibition of the CBFß-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 CBFß-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 CBFß-SMMHC drives leukemia maintenance and suggests that inhibitors targeting chromatin activity may prove effective in inv(16) leukemia therapy.

An erythroid-to-myeloid cell fate conversion is elicited by LSD1 inactivation.

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 ß-globinopathies (sickle cell disease and ß-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.

Targeting Runt-Related Transcription Factor 1 Prevents Pulmonary Fibrosis and Reduces Expression of Severe Acute Respiratory Syndrome Coronavirus 2 Host Mediators.

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-ß1 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.

TNF-α signaling regulates RUNX1 function in endothelial cells.

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.

RUNX1-targeted therapy for AML expressing somatic or germline mutation in RUNX1.

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.

Structure of the Complex of an Iminopyridinedione Protein Tyrosine Phosphatase 4A3 Phosphatase Inhibitor with Human Serum Albumin.

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.

RUNX1 and RUNX2 transcription factors function in opposing roles to regulate breast cancer stem cells.

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, TGFß1, 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 CBFß 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.

RUNX1 is required for oncogenic Myb and Myc enhancer activity in T-cell acute lymphoblastic leukemia.

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 CBFß 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.

Small molecule inhibition of the CBFß/RUNX interaction decreases ovarian cancer growth and migration through alterations in genes related to epithelial-to-mesenchymal transition.

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 CBFß/RUNX protein-protein interaction on ovarian cancer cell lines. METHODS: Ovarian cancer cell lines were treated with CBFß/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 CBFß/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 CBFß/RUNX interaction is a viable strategy for the treatment of ovarian cancer.

Structure and Biophysics of CBFß/RUNX and Its Translocation Products.

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 CBFß 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.

Molecular Basis and Targeted Inhibition of CBFß-SMMHC Acute Myeloid Leukemia.

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 CBFß-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 CBFß-SMMHC/RUNX1 binding, the first candidate targeted therapy for inv(16) AML.

NMR solution structure of the integral membrane enzyme DsbB: functional insights into DsbB-catalyzed disulfide bond formation.

We describe the NMR structure of DsbB, a polytopic helical membrane protein. DsbB, a bacterial cytoplasmic membrane protein, plays a key role in disulfide bond formation. It reoxidizes DsbA, the periplasmic protein disulfide oxidant, using the oxidizing power of membrane-embedded quinones. We determined the structure of an interloop disulfide bond form of DsbB, an intermediate in catalysis. Analysis of the structure and interactions with substrates DsbA and quinone reveals functionally relevant changes induced by these substrates. Analysis of the structure, dynamics measurements, and NMR chemical shifts around the interloop disulfide bond suggest how electron movement from DsbA to quinone through DsbB is regulated and facilitated. Our results demonstrate the extraordinary utility of NMR for functional characterization of polytopic integral membrane proteins and provide insights into the mechanism of DsbB catalysis.

Structural and dynamic studies of the transcription factor ERG reveal DNA binding is allosterically autoinhibited.

The Ets-Related Gene (ERG) belongs to the Ets family of transcription factors and is critically important for maintenance of the hematopoietic stem cell population. A chromosomal translocation observed in the majority of human prostate cancers leads to the aberrant overexpression of ERG. We have identified regions flanking the ERG Ets domain responsible for autoinhibition of DNA binding and solved crystal structures of uninhibited, autoinhibited, and DNA-bound ERG. NMR-based measurements of backbone dynamics show that uninhibited ERG undergoes substantial dynamics on the millisecond-to-microsecond timescale but autoinhibited and DNA-bound ERG do not. We propose a mechanism whereby the allosteric basis of ERG autoinhibition is mediated predominantly by the regulation of Ets-domain dynamics with only modest structural changes.

Structural basis for recognition of SMRT/N-CoR by the MYND domain and its contribution to AML1/ETO's activity.

AML1/ETO results from the t(8;21) associated with 12%-15% of acute myeloid leukemia. The AML1/ETO MYND domain mediates interactions with the corepressors SMRT and N-CoR and contributes to AML1/ETO's ability to repress proliferation and differentiation of primary bone marrow cells as well as to enhance their self renewal in vitro. We solved the solution structure of the MYND domain and show it to be structurally homologous to the PHD and RING finger families of proteins. We also determined the solution structure of an MYND-SMRT peptide complex. We demonstrated that a single amino acid substitution that disrupts the interaction between the MYND domain and the SMRT peptide attenuated AML1/ETO's effects on proliferation, differentiation, and gene expression.

Functional specificity of CpG DNA-binding CXXC domains in mixed lineage leukemia.

The MLL CXXC domain binds nonmethylated CpG-containing DNA and is essential for the oncogenic properties of MLL fusion proteins. To determine potential functional promiscuity of similar DNA binding domains, we replaced the MLL CXXC domain in the context of the leukemogenic MLL-AF9 fusion with CXXC domains from DNMT1, CGBP (CFP1), and MBD1, or with a methyl-CpG-binding domain (MBD) from MBD1. MLL(DNMT1 CXXC)-AF9 shows robust in vitro colony forming activity and in vivo leukemogenesis, similar to MLL-AF9. However, colony forming ability and leukemogenicity are abrogated in MLL-AF9 containing either the CGBP or MBD1 CXXC domains or the MBD1 MBD domain. Direct comparison of in vitro DNA binding affinity of the isolated CXXC or MBD domains demonstrated that MLL, DNMT1, and CGBP CXXC domains could each bind to unmethylated DNA but with differing affinity. In contrast, the isolated MBD1 CXXC and MBD1 MBD domains were unable to bind to the same DNA. However, all substituted domains still allowed targeting of the MLL fusions to the functionally important Hoxa9 locus in primary bone marrow progenitor cells. In addition to DNA binding activity, it was critical that specific CpG residues in the Hoxa9 locus were protected from methylation for leukemia development. This ultimately prevented histone 3 lysine 9 trimethylation (H3K9me3) of the locus and enabled Hoxa9 expression. These were properties shared by MLL and DNMT1 CXXC domains but not by CGBP CXXC or the other swapped fusions tested. We demonstrate that similar CXXC domains can be mechanistically distinguished by specificity of CpG nucleotides preferentially protected from DNA methylation.

The tetramer structure of the Nervy homology two domain, NHR2, is critical for AML1/ETO's activity.

AML1/ETO is the chimeric protein resulting from the t(8;21) in acute myeloid leukemia. The Nervy homology 2 (NHR2) domain in ETO mediates oligomerization and AML1/ETO's interactions with ETO, MTGR1, and MTG16, and with the corepressor molecules mSin3A and HDAC1 and HDAC3. We solved the NHR2 domain structure and found it to be an alpha-helical tetramer. We show that oligomerization contributes to AML1/ETO's inhibition of granulocyte differentiation, is essential for its ability to enhance the clonogenic potential of primary mouse bone marrow cells, and affects AML1/ETO's activity on several endogenous genes. Oligomerization is also required for AML1/ETO's interactions with ETO, MTGR1, and MTG16, but not with other corepressor molecules.

N-MYC regulates cell survival via eIF4G1 in inv(16) acute myeloid leukemia.

N-MYC (encoded by MYCN) is a critical regulator of hematopoietic stem cell function. While the role of N-MYC deregulation is well established in neuroblastoma, the importance of N-MYC deregulation in leukemogenesis remains elusive. Here, we demonstrate that N-MYC is overexpressed in acute myeloid leukemia (AML) cells with chromosome inversion inv(16) and contributes to the survival and maintenance of inv(16) leukemia. We identified a previously unknown MYCN enhancer, active in multiple AML subtypes, essential for MYCN mRNA levels and survival in inv(16) AML cells. We also identified eukaryotic translation initiation factor 4 gamma 1 (eIF4G1) as a key N-MYC target that sustains leukemic survival in inv(16) AML cells. The oncogenic role of eIF4G1 in AML has not been reported before. Our results reveal a mechanism whereby N-MYC drives a leukemic transcriptional program and provides a rationale for the therapeutic targeting of the N-MYC/eIF4G1 axis in myeloid leukemia.

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.

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.