Core binding factor (CBF) acute myeloid leukemias typically harbor the translocations t(8;21) or inv(16). As cohesin mutations are less commonly observed with inv(16) than with t(8;21), we hypothesized that they may negatively impact inv(16)-driven AML. Using a mouse model of inv(16) with haploinsufficiency of the cohesin subunit Smc3, we paradoxically found that inv(16); Smc3Δ/+ mice have a reduced leukemic latency compared to inv(16); Smc3+/+ mice, disproving our initial hypothesis and instead suggesting a role for cohesin loss in enhancing inv(16)-driven disease. Consistent with the known role of cohesin haploinsufficiency in altering chromatin accessibility, we demonstrated an increase in chromatin accessibility in inv(16); Smc3Δ/+ hematopoietic stem and progenitor cells (HSPCs) prior to leukemia development, with an enrichment for Fli1 DNA binding motifs. Through scRNA-seq on pre-leukemic HSPCs, we observe an increase in Fli1 expression and enhanced Fli1 target expression in ST-HSCs. We further show that Fli1 is essential for the maintenance stage of inv(16); Smc3Δ/+ AML. Our data demonstrate a role for cohesin loss in enhancing the aggressiveness of inv(16)-driven AML and identify Fli1 as a previously unrecognized therapeutic vulnerability in cohesin-mutated AML.

Leukemic stem cells (LSCs) play a central role in disease progression, therapeutic resistance, and relapse in acute myeloid leukemia (AML). However, the identification and characterization of LSCs remain challenging because of their low abundance and their close phenotypic resemblance to normal hematopoietic stem and progenitor cells. Although patient-derived xenograft (PDX) models have provided important insights into AML biology and LSC heterogeneity, the relative engraftment potential of distinct CEBPA mutation subtypes and the immunophenotypic identity of LSCs in CEBPA N-terminal mutant AML ( CEBPA - N -AML) remain poorly defined. To address these questions, we compared the engraftment characteristics of primary human CEBPA-mutated AML samples representing the major mutational subtypes using the highly permissive NSGS xenograft model. Primary CEBPA -N-AML samples exhibited markedly greater engraftment efficiency and leukemogenic potential than other CEBPA -mutated AML subtypes. Furthermore, we identified a CD366⁺CD73⁺CD123⁺CD117⁺CD371⁺CD247⁺ cell population that is highly enriched for functional LSCs in CEBPA-N -AML, demonstrating enhanced clonogenic activity, leukemia-initiating capacity, and long-term self-renewal. Collectively, our findings demonstrate that the leukemogenic potential of CEBPA -mutated AML is strongly influenced by mutation subtype, with CEBPA-N -AML exhibiting superior leukemia-propagating capacity in vivo. We further define a novel immunophenotypic LSC signature specific to CEBPA-N -AML, providing new insights into LSC heterogeneity in CEBPA -mutated AML and establishing a foundation for the development of LSC-directed therapeutic strategies.

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.

Acute myeloid leukemia (AML) is a genetically diverse cancer of the bone marrow that affects approximately 20,000 new patients annually in the US alone. Many mutations drive leukemogenesis, including those in the core binding factor (CFB) complex, which is a heterodimeric transcription factor involved in hematopoietic cell development and differentiation (Pulikkan and Castilla 2018). Mutations in the CBF complex are present in 15% of adult AML cases and can affect either the alpha (RUNX1) or the beta (CBFβ) subunit through the chromosomal aberrations t(8;21) and inv(16), respectively. These mutations are necessary but not sufficient for leukemic initiation. CBF mutations co-occur with other mutations, including those affecting the cohesin complex, which is mutated in 10% of all AML cases (Ley et al 2013). The cohesin complex is comprised of four subunits, RAD21, SMC1A, SMC3, and STAG1/2, and regulates chromatin architecture and genomic looping, thereby affecting transcription. Mutations in the cohesin complex in AML affect only one subunit, resulting in haploinsufficiency of the complex and loss of function (Heimbruch et al. 2021). Despite CBF leukemias being classified as one group clinically, t(8;21) and inv(16) have different co-mutational profiles (Faber et al. 2016, Opatz et al. 2020, Duployez et al. 2016, Jahn et al. 2020, Qin et al. 2022). Interestingly, cohesin complex mutations have been observed more commonly in t(8;21) AML than in inv(16) AML. We therefore hypothesized that haploinsufficiency of cohesin would be detrimental to the development or maintenance of inv(16) AML. To test this, we utilized the inv(16) model published by Kuo et al. 2006 and the Smc3 haploinsufficiency model published by Viny et al. 2015. Both inv(16) and the loss of a single Smc3 allele are driven by MX1-Cre activation by PIPC injection. In contrast to our hypothesis, we found that inv(16);Smc3+/- mice developed AML with a shorter latency (14 weeks) than did inv(16);Smc3+/+ mice (20 weeks), suggesting the two mutations cooperate during leukemic initiation. ATAC sequencing of HSPCs revealed increased global accessibility in inv(16);Smc3+/- vs. inv(16);Smc3+/+ cells, particularly at Ets family transcription factor binding sites. qPCR and western blotting confirmed an upregulation the Ets family member Fli1 in inv(16);Smc3+/- cells. Upregulation of several known Fli1 target genes such as Ccng1, Ccl2, Mdm2, and Rab27b, was also observed. Interestingly, upregulation of Fli1 was not observed in leukemic cells from inv(16);Smc3+/- mice, suggesting cohesin mutation and Fli1 upregulation may contribute to leukemic initiation but not maintenance. In support of this, knockdown of Rad21 induced apoptosis in leukemias from inv(16);Smc3+/+ mice. We next performed single cell RNA sequencing on lin-, Sca1+, c-Kit+ (LSK) sorted cells from both genotypes to identify which cell types exhibited elevated Fli1. We found a significant upregulation of Fli1 in several clusters, primarily in LSKs, ST-HSCs, and MPPs, suggesting that Fli1 upregulation occurs in the most primitive stem cells. We are currently testing if Fli1 upregulation drives a shortened latency using inv(16);Smc3+/+ HSPCs. Cohesin mutations have not been readily detected in inv(16) AML, with current data primarily including patients of Chinese and German/Austrian origin (Faber et al. 2016, Opatz et al. 2020, Duployez et al. 2016, Jahn et al. 2020, Qin et al. 2022). Studies that have detected cohesin mutations in inv(16) patients have used a targeted sequencing approach in addition to whole genome or exome sequencing. Collectively, this suggests that cohesin mutations may co-occur with inv(16) at a higher rate than reported, which needs to be confirmed with targeted sequencing of a more diverse population. Another possibility is that cohesin mutations primarily affect the initiation of inv(16) AML but have a negative impact on maintenance. Therefore, selective pressure may no longer exist at the AML stage, leading to mutational loss. It would be interesting to examine the clonal evolution of healthy persons harboring inv(16) mutations in an effort to detect gain or loss of cohesin mutations. Our data support the hypothesis that cohesin mutations drive inv(16) AML initiation through the upregulation of Fli1 and its downstream targets in HSPCs and underline the importance of studying the effect of mutations on both AML initiation and maintenance.

Adenosine to inosine (A-to-I) RNA editing, which is catalyzed by adenosine deaminases acting on RNA (ADAR) family of enzymes ADAR1 and ADAR2, has been shown to contribute to multiple cancers. However, other than chronic myeloid leukemia (CML) blast crisis, relatively little is known about its role in other types of hematological malignancies. Here, we found that ADAR2, but not ADAR1 and ADAR3, was specifically downregulated in the core binding factor (CBF) AML with t(8;21) or inv(16) translocations. In t(8;21) AML, RUNX1-driven transcription of ADAR2 was repressed by the RUNX1-ETO AE9a fusion protein in a dominant negative manner. Further functional studies confirmed that ADAR2 could suppress leukemogenesis specifically in t(8;21) and inv16 AML cells dependent on its RNA editing capability. Expression of two exemplary ADAR2-regulated RNA editing targets COPA and COG3 inhibited clonogenic growth of human t(8;21) AML cells. Our findings support a hitherto unappreciated mechanism leading to ADAR2 dysregulation in CBF AML and highlight the functional relevance of loss of ADAR2-mediated RNA editing to CBF AML.

Introduction: Natural killer (NK) cells, as the major subset of innate lymphocytes, produce proinflammatory cytokines and mediate anti-tumor cytotoxicity (1-3). Since NK cells are not limited by clonotypic receptors, they can be utilized in cell therapies against a broad spectrum of malignancies. Irrespective of the clinical potentials, the transcriptional regulation of the development and functions of human NK cells is far from fully understood. A dysregulated NK cell development is a hallmark of GATA2 haploinsufficiency (4)(eg. GATA2 T354M mutation). The specific loss of the CD56 bright NK cell population is a significant striking feature in GATA2-deficient patients (4), with or without reduced total CD56 dim NK cells (4, 12, 13). Consequently, the effector functions of these NK cells are significantly reduced, including inflammatory cytokine production and anti-tumor cytotoxicity (4, 12). However, a mechanistic understanding of GATA2 functions is lacking. In humans, GATA2 forms a core heptad complex with six other transcription factors (TF), including TAL1, regulating more than 1000 target genes. However, the unique functions of the GATA2-TAL1 axis in immature and mature NK cell has not been established. Based on these, we hypothesize that GATA2-dependent and independent functions of TAL1 play an essential role in the development and functions of human NK cells. Results: To explore the mechanism of co-transcriptional regulation of GATA2 and TAL1, we transduced NK-92 cells with lentiviral vectors containing GATA2 WT or GATA2 T354M plasmid and quantified TAL1 mRNA levels by qPCR and protein levels by WB. TAL1 is upregulated in GATA2 T354M-expressing NK-92 cells both at mRNA ( Fig. 1A) and protein levels ( Fig. 1B). TAL1 is a TF activating GTPase of immunity-associated protein (GIMAP) family super-enhancer regions in HSC and T cells (11). As TAL1 target genes, we further quantify GIMAP mRNA levels by qPCR. The result indicates that genes from the GIMAP family were notably upregulated in GATA2 T354M-expressing NK-92 cells ( Fig. 1C). Knocking down TAL1 results in the significant downregulation of TAL1 and GIMAPs in GATA2 T354M-expressingcells ( Fig. 1D). To further validate the GATA2-TAL1 axis, sorted NK cells from PBMCs of three GATA2 T354M patients and five age-matched healthy controls (HC) were analyzed with single-cell RNA-seq. We identified the genes from GIMAP family were significantly upregulated in all three GATA2 T354M patients. The representative data from one patient is shown in Fig. 1E. To explore how GATA2 and TAL1 mediate transcriptional regulation, we performed GATA2 CUT&Tag experiments with human NK cells, in which TAL1 motif was predominately enriched in 63.2% of GATA2 targets among NK cells through HOMER motif enrichment analyses ( Fig. 1F). On the other hand, the TAL1 ChIP-seq data from the K562 cells (download from ENCFF101DBG) reveals that the GATA2 motif is enriched in approximately 44% of TAL1 targets ( Fig. 1G). Moreover, both GATA2 and TAL1 expression in NK cells are modified by H3K27Me3 instead of H3K4Me3, suggesting a repressive methylenation signature in their expression ( Fig. 1H). Conclusion: Based on this study, we uncover a novel role for the GATA2-TAL1 axis in human NK cells. we define the transcriptional regulation role of GATA2 and TAL1 complex and provide direct evidence for the mechanism of NK cell deficiency among GATA2 T354M patients. Importantly, through our current study, we will determine the unique role of TAL1 in NK development and function and its transcriptional network.

The transcription factor C/EBPα is a major regulator of granulopoiesis. Mutations in the gene that encodes C/EBPα are reported in around 10% of acute myeloid leukemia (AML) patients. The mutations reported in CEBPA are point mutations at C-terminus bZIP domain and/ or frame-shift mutations at N- terminus. CEBPA mutations can be mono-allelic or bi-allelic with one mutation located in N-terminus and other mutation in C-terminus. The type of the CEBPA mutation is critical determinants of AML prognosis-overall survival in patients whose AML carries a mono-allelic bZIP or bi-allelic CEBPA mutation is 60%, while the overall survival of those with mono-allelic N-terminal CEBPA mutations is 20%. Patient-derived xenotransplantation (PDX) models represents a great tool for understanding disease biology and pre-clinical drug testing. PDX models utilizing human primary AML samples have provided novel insights on functional heterogeneity across patients, including the identification of phenotypes associated with leukemia-initiating cell populations. NSGS strain which express human stem cell factor, granulocyte-macrophage colony-stimulating factor and interleukin-3 has been reported to enhance engraftment of human primary AML samples. While engraftment characteristics of multiple AML subtypes have been studied in detail, whether CEBPA mutant AML cells engraft in PDX models, and whether there are any differences in engraftment between different subtypes of CEBPA mutations remain unexplored. Understanding the engraftment characteristics of CEBPA mutant AML samples provide us critical knowledge in disease biology, and will deliver a platform for future pre-clinical drug testing. In this study, we investigated engraftment characteristics of human CEBPA mutated primary AML samples in NSGS mice. Male and female NSGS mice 6-8 weeks of age were sublethally irradiated 5 hours prior to cell injections. T-cell depleted human primary AML cells with 3 subclasses of CEBPA mutation - mono-allelic N-terminal mutant, mono-allelic C-terminal mutant and bi-allelic mutant (1 million per mouse, 5 independent AML samples/subclass) were transplanted via tail vein injection into NSGS mice. Human AML engraftment was assessed by flow cytometry for human CD45+CD33+ cells in bone marrow aspirates 5 days after transplantation. The mice were monitored for leukemia incidence, and peripheral blood was collected submandibularly at 4 weeks intervals after transplantation to study progression of AML. Our data demonstrates that primary AML cells with mono-allelic N-terminal CEBPA mutations display superior engraftment than mono-allelic C-terminal and bi-allelic CEBPA mutations. NSGS mice transplanted with N-terminal CEBPA mutations succumbed to leukemia with a median latency of 8 weeks. Bone marrow and peripheral blood from leukemic mice showed presence of higher myeloid blast cells. Histological assessment by hematoxylin and eosin staining showed that AML cells infiltrated into multiple organs such as liver, spleen and lungs. Secondary transplantation of primary mouse leukemic cells in NSGS recipients developed AML with characteristics very similar to leukemia developed in primary recipients. While mono-allelic C-terminal CEBPA mutated samples did not engraft in NSGS mice, bi-allelic CEBPA mutant samples with GATA2 zinc finger-1 mutation engrafted and developed acute erythroid leukemia (AEL). Bone marrow and peripheral blood from these leukemic mice showed presence of both myeloid and erythroid blast cells, consistent with recent studies showing development of AEL in a genetic model for bi-allelic CEBPA mutation and GATA2 zinc finger-1 mutation. We are currently investigating what are the factors contributing to engraftment and AEL development in AML samples with bi-allelic CEBPA mutation and GATA2 zinc finger-1 mutation. Our studies suggests engraftment of CEBPA mutant AML samples depend on the subclass of CEBPA mutation and secondary mutation present in the AML sample. This knowledge of differential engraftment of CEBPA mutant sample in NSGS mice offers a valuable model for testing novel therapies for CEBPA mutant AML.

The blood system serves as a key model for cell differentiation and cancer. It is orchestrated by precise spatiotemporal expression of crucial transcription factors. One of the key master regulators in the hematopoietic systems is PU.1. Reduced levels of PU.1 are characteristic for human acute myeloid leukemia (AML) and are known to induce AML in mouse models. Here, we show that transcriptional downregulation of PU.1 is an active process involving an alternative promoter in intron 3 that is induced by RUNX transcription factors driving noncoding antisense transcription. Core-binding factor (CBF) fusions RUNX1-ETO and CBFβ-MYH11 in t(8;21) and inv(16) AML, respectively, activate the PU.1 antisense promoter that results in a shift from sense toward antisense transcription and myeloid differentiation blockade. In patients with CBF-AML, we found that an elevated antisense/sense transcript and promoter accessibility ratio represents a hallmark compared with normal karyotype AML or healthy CD34+ cells. Competitive interaction of an enhancer with the proximal or the antisense promoter forms a binary on/off switch for either myeloid or T-cell development. Leukemic CBF fusions thus use a physiological mechanism used by T cells to decrease sense transcription. Our study is the first example of a sense/antisense promoter competition as a crucial functional switch for gene expression perturbation by oncogenes. Hence, this disease mechanism reveals a previously unknown Achilles heel for future precise therapeutic targeting of oncogene-induced chromatin remodeling.

The core binding factor composed of CBFβ and RUNX subunits plays a critical role in most hematopoietic lineages and is deregulated in acute myeloid leukemia (AML). The fusion oncogene CBFβ-SMMHC expressed in AML with the chromosome inversion inv(16)(p13q22) acts as a driver oncogene in hematopoietic stem cells and induces AML. This review focuses on novel insights regarding the molecular mechanisms involved in CBFβ-SMMHC-driven leukemogenesis and recent advances in therapeutic approaches to target CBFβ-SMMHC in inv(16) AML.

Introduction: Alterations of core binding factors (CBF), Runx1 and CBFβ are frequent mutational targets in acute myeloid leukemia (AML). Chromosomal translocations t(8;21)(q22;q22) and inv(16)(p13q22), creating the fusion proteins RUNX1-ETO and CBFβ-MYH11 respectively, account for 15% and thus the largest sub-group of AML called CBF-AML. CBF oncogenes induce global changes in chromatin structure and gene regulation, which lead to differentiation blockade. A critical leukemic event could be the inactivation of PU.1 transcription factor. Normal myeloid differentiation needs PU.1 levels to increase, failure to do so leads to a stop of differentiation and AML development. In contrast, T-cell differentiation requires PU.1 to be completely switched off. The exact mechanism of PU.1 suppression, physiological for T-lymphopoiesis or pathological for leukemia, remains elusive. Results: We assessed the activation of the PU.1 locus throughout human hematopoietic differentiation stages using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) and reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Interestingly, we observed high accessibility of a previously identified antisense promoter (AsPr) in intron 3 and antisense transcript (asRNA) expression during early lymphopoiesis which preceded locus shutdown in T cells. The ratio of AsPr/PrPr accessibility and of antisense/sense transcription clearly indicated cellular fate during hematopoiesis (Figure 1A). T-lymphoid differentiation was related to the timely expression of RUNX transcription factors. RUNX1, RUNX3, and the CBF fusions RUNX1-ETO and CBFβ-MYH11 were capable to transactivate PU.1 AsPr. In CBF-AML patient samples we strikingly found elevated asRNA/mRNA ratios compared to normal karyotype AML or healthy CD34+ cells (Figure 1B) and increased AsPr/PrPr ratios unsing DNaseI-seq data in RUNX1-ETO AML patients. Functionally we found that PU.1 asRNA depletion in t(8;21) xenografted immune-deficient (NOD/SCID) mice restored a normal survival (Figure 1C) demonstrating that PU.1 antisense transcripts are required for CBF leukemia outgrow in vivo. To further dissect the mechanism of how CBFs could drive PU.1 antisense transcription we applied active RNA polymerase mapping (PRO-seq) and chromatin accessibility (ATAC-seq) and found a shift from PU.1 antisense to sense transcription after RUNX1-ETO depletion. Using chromosomal conformation capture sequencing (3C, Hi-C and CHiC) in T-lymphoid, myeloid and RUNX1-ETO cells combined with transcript quantification we observed that competitive interaction of an upstream enhancer with the proximal or the antisense promoter are at the heart of differential PU.1 expression during myeloid and T-cell development (Figure 2A). Leukemic CBF fusions thus utilize a physiologic mechanism employed by T-cells to decrease sense PU.1 transcription (Figure 2B). Conclusion: The data suggest that silencing transcription factor PU.1 is an active process that requires a specific chromosome formation that is induced by CBF fusions. Sense/antisense promoter competition represents a crucial functional switch for gene expression perturbation by oncogenes and provide a potential strategy for future precise therapeutic targeting of oncogene-induced chromatin remodeling. Disclosures Valent: Allcyte GmbH: Research Funding; Pfizer: Honoraria; Cellgene: Honoraria, Research Funding. Staber:Roche: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; msd: Consultancy, Honoraria; Celgene/ BMS: Consultancy, Honoraria.

Acute myeloid leukemia (AML) is a collection of hematologic malignancies with specific driver mutations that direct the pathology of the disease. The understanding of the origin and function of these mutations at early stages of transformation is critical to understand the etiology of the disease and for the design of effective therapies. The chromosome inversion inv(16) is thought to arise as a founding mutation in a hematopoietic stem cell (HSC) to produce preleukemic HSCs (preL-HSCs) with myeloid bias and differentiation block, and predisposed to AML. Studies in mice and human AML cells have established that inv(16) AML follows a clonal evolution model, in which preL-HSCs expressing the fusion protein CBFβ–SMMHC persist asymptomatic in the bone marrow. The emerging leukemia-initiating cells (LICs) are composed by the inv(16) and a heterogeneous set of mutations. In this review, we will discuss the current understanding of inv(16) preleukemia development, and the function of CBFβ–SMMHC related to preleukemia progression and LIC activity. We also discuss important open mechanistic questions in the etiology of inv(16) AML.

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.

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.

The authors wish to point out that, in some instances of the text in Results section 3.5, the units of ps-ns and μs-ms to define timescales of residue dynamics were inadvertently switched. "Interestingly, these changes occur both in the μs-ms timescale dynamics (principally near the benzimidazole ring) as well as on the ps-ns timescale (in the vicinity of the pyridine ring), with the latter perhaps a result of ring flipping of the pyridine ring." should read "Interestingly, these changes occur both in the ps-ns timescale dynamics (principally near the benzimidazole ring) as well as on the μs-ms timescale (in the vicinity of the pyridine ring), with the latter perhaps a result of ring flipping of the pyridine ring." "For Arg131, we observe increased μs-ms timescale dynamics with compound binding." should read "For Arg131, we observe increased ps-ns timescale dynamics with compound binding." "For Asn104, we observe an increase in ps-ns timescale dynamics with compound binding." should read "For Asn104, we observe an increase in μs-ms timescale dynamics with compound binding." Also, the legend for Fig. 3 contains similar inadvertent unit switching. In the legend for Fig. 3D, "Residues showing increased ps-ns timescale motion are colored red and those showing increased μs-ms timescale motion are colored blue." should read "Residues showing increased μs-ms timescale motion are colored red and those showing increased ps-ns timescale motion are colored blue." In the legend for Fig. 3E, "Red indicates increased ps-ns timescale motion and blue indicates increased μs-ms timescale motion." should read "Red indicates increased μs-ms timescale motion and blue indicates increased ps-ns timescale motion." These errors do not alter the conclusions of the paper. Small Molecule Inhibitor of CBFβ-RUNX Binding for RUNX Transcription Factor Driven CancersTranscription 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. Full-Text PDF Open Access

Myeloid master regulator CCAAT enhancer-binding protein alpha (C/EBPα) is deregulated by multiple mechanisms in leukemia. Inhibition of C/EBPα function plays pivotal roles in leukemogenesis. While much is known about how C/EBPα orchestrates granulopoiesis, our understanding of molecular transformation events, the role(s) of cooperating mutations and clonal evolution during C/EBPα deregulation in leukemia remains elusive. In this review, we will summarize the latest research addressing these topics with special emphasis on CEBPA mutations. We conclude by describing emerging therapeutic strategies to restore C/EBPα function.

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.

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.

Acute myeloid leukemia (AML) is the most common form of adult leukemia. The transcription factor fusion CBFβ-SMMHC (core binding factor β and the smooth-muscle myosin heavy chain), expressed in AML with the chromosome inversion inv(16)(p13q22), outcompetes wild-type CBFβ for binding to the transcription factor RUNX1, deregulates RUNX1 activity in hematopoiesis, and induces AML. Current inv(16) AML treatment with nonselective cytotoxic chemotherapy results in a good initial response but limited long-term survival. Here, we report the development of a protein-protein interaction inhibitor, AI-10-49, that selectively binds to CBFβ-SMMHC and disrupts its binding to RUNX1. AI-10-49 restores RUNX1 transcriptional activity, displays favorable pharmacokinetics, and delays leukemia progression in mice. Treatment of primary inv(16) AML patient blasts with AI-10-49 triggers selective cell death. These data suggest that direct inhibition of the oncogenic CBFβ-SMMHC fusion protein may be an effective therapeutic approach for inv(16) AML, and they provide support for transcription factor targeted therapy in other cancers.

The leukemia fusion protein CBFβ-SMMHC, associated with acute myeloid leukemia (AML) with chromosome inversion inv(16)(p13q22), is a driver mutation in leukemia development. Studies by our laboratory and others have established that CBFβ-SMMHC outcompetes CBFβ for binding to RUNX1, deregulates RUNX1 transcription factor activity in hematopoiesis, and induces AML. Studies in mice and patient AML cells support the concept that CBFβ-SMMHC generates pre-leukemic myeloid progenitors, which acquire cooperating mutations to progress to leukemia. Current inv(16) AML treatment using non-selective cytotoxic chemotherapy results in a good initial response, but long-term survival is approximately 60%. This suggests that additional efforts are necessary for the development of improved therapeutic response for CBF AML patients. We have identified AI-4-57 as the active compound that inhibits CBFβ-SMMHC/RUNX1 binding (IC50= 22 uM), using a screen of the NCI diversity set library. AI-4-57 specifically binds to the CBFβ portion of CBFβ-SMMHC, as determined by NMR. This compound was modified in order to improve its potency and stability, and identified the divalent derivative AI-10-49 for further characterization. AI-10-49 showed increased potency (IC50= 0.26 µM) improved in vivo pharmacokinetics (serum t1/2 = 380 min), and enhanced activity in inv(16) positive ME-1 cells (IC50 = 0.6 uM). Importantly, AI-10-49 showed negligible activity (IC50>25 μM) in normal human bone marrow, defining a robust potential therapeutic window. Co-immunoprecipitation assays of ME-1 cells demonstrated that AI-10-49 (1µM for six hours) effectively and specifically dissociated RUNX1/CBFβ-SMMHC when compared to CBFβ/RUNX1 binding (Meandiss: 90% and 15%, respectively). Expression of RUNX1 target genes RUNX3, CSF1R, and CEBPA is repressed by CBFβ-SMMHC in inv(16) AML. The occupancy of RUNX1 in their promoters was significantly increased by chromatin-immunoprecipitation (8, 2.2, and 8 fold, respectively) in 6 hour treated (1µM AI-10-49) ME-1 cells, suggesting that CBFβ-SMMHC represses RUNX1 targets by blocking RUNX1 binding to target regulatory sites. In addition, RUNX3, CSF1R, and CEBPA expression increased 2 to 8 fold when compared to DMSO treated ME-1 cells. Importantly, RUNX1 occupancy and target expression changes were not observed in inv(16)-negative U937 cells. These data establish AI-10-49 selectivity in inhibiting CBFβ-SMMHC binding to RUNX1 and validate our approach of using bivalent inhibitors to achieve this specificity. To test AI-10-49 activity in vivo, mice were transplanted with leukemic cells expressing CBFβ-SMMHC and NrasG12D (from Cbfb+/MYH11:Ras+/G12Dknock-in mice), and treated, starting at day five post transplantation, with vehicle (DMSO) or 200 mg/kg AI-10-49 for ten days. The median latency of leukemia was delayed one fold in AI-10-49 treated mice (MLAI-10-49= 61 days, MLDMSO= 33.5 days, P=2.7x10-6; Log-rank test). In addition, toxicity assays revealed no detectable cumulative toxicity in mice treated with AI-10-49 for seven days. To test the efficacy of AI-10-49 in human inv(16) AML, the survival of four inv(16) and four normal karyotype AML patient samples were tested in 48 hour dose response assays. The viability of inv(16) patient cells was clearly reduced by AI-10-49 (viability: 50%, 10 μM AI-10-49/DMSO). In contrast, the viability of normal karyotype AML samples was unaffected at concentrations below 20µM. These studies show that AI-10-49 selectively inhibits viability in inv(16) AML blasts, while having negligible effects on AML blasts with normal karyotype or on normal human hematopoietic progenitors. Dysregulated gene expression is a hallmark of cancer and is particularly important for the maintenance of cancer stem cells, such as self-renewal, leading to relapse. The targeting of proteins that drive transcriptional dysregulation, so called “transcription therapy”, represents an avenue for drug development with immense potential. This study reports the development of a small molecule with high efficacy and specific in the inhibition of CBFβ-SMMHC activity while having a minimal effect on CBFβ function. In summary, AI-10-49 is a potent first generation CBFβ-SMMHC inhibitor that induces cell death in inv(16) AML cells and establishes a proof-of-principle that transcription factor fusion oncoproteins can be directly targeted for leukemia treatment. Disclosures No relevant conflicts of interest to declare.