Small Molecule Inhibitor of CBFbeta-RUNX Binding for RUNX Transcription Factor Driven Cancers

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 CBFbeta binding partner. CBFbeta enhances DNA binding of RUNX subunits by relieving auto-inhibition. Both RUNX1 and CBFbeta 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 CBFbeta 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

Core binding factor leukemia hijacks T-cell prone PU.1 antisense promoter [preprint]

The blood system serves as a key model for cell differentiation and cancer. It is orchestrated by precise spatiotemporal expression of the hematopoietic master regulator PU.11–4. PU.1 gene expression is regulated through enhancer-promoter interactions within a topologically associated domain (TAD)5,6. PU.1 levels increase during myeloid differentiation while failure to do so results in myeloid leukemia7. In contrast, T-cell differentiation requires PU.1 to be completely switched off8–10. Little is known about the precise mechanisms of PU.1 repression, physiological as in T-cell differentiation, or pathological as in leukemia. Here we demonstrate that the down-regulation of PU.1 mRNA is a dynamic process involving an alternative promoter11 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) acute myeloid leukemia (AML)12, activate the PU.1 antisense promoter, thus shifting from sense towards antisense transcription and blocking myeloid differentiation. In patients with CBF-AML, we found that an elevated antisense/sense ratio represents a hallmark compared to normal karyotype AML or healthy CD34+ cells. Competitive interaction of the enhancer with the proximal or the antisense promoter are at the heart of differential PU.1 expression during myeloid and T-cell development. Leukemic CBF fusions thus utilize a physiologic mechanism employed by T-cells to decrease sense PU.1 transcription. Our results identify the first example of a sense/antisense promoter competition as a crucial functional switch for gene expression perturbation by oncogenes. This novel basic disease mechanism reveals a previously unknown Achilles heel for future precise therapeutic targeting of oncogene-induced chromatin remodeling

Cbfbeta reduces Cbfbeta-SMMHC-associated acute myeloid leukemia in mice

The gene encoding for core-binding factor beta (CBFbeta) is altered in acute myeloid leukemia samples with an inversion in chromosome 16, expressing the fusion protein CBFbeta-SMMHC. Previous studies have shown that this oncoprotein interferes with hematopoietic differentiation and proliferation and participates in leukemia development. In this study, we provide evidence that Cbfbeta modulates the oncogenic function of this fusion protein. We show that Cbfbeta plays an important role in proliferation of hematopoietic progenitors expressing Cbfbeta-SMMHC in vitro. In addition, Cbfbeta-SMMHC-mediated leukemia development is accelerated in the absence of Cbfbeta. These results indicate that the balance between Cbfbeta and Cbfbeta-SMMHC directly affects leukemia development, and suggest that CBF-specific therapeutic molecules should target CBFbeta-SMMHC function while maintaining CBFbeta activity

ATACseqQC: a Bioconductor package for post-alignment quality assessment of ATAC-seq data

BACKGROUND: ATAC-seq (Assays for Transposase-Accessible Chromatin using sequencing) is a recently developed technique for genome-wide analysis of chromatin accessibility. Compared to earlier methods for assaying chromatin accessibility, ATAC-seq is faster and easier to perform, does not require cross-linking, has higher signal to noise ratio, and can be performed on small cell numbers. However, to ensure a successful ATAC-seq experiment, step-by-step quality assurance processes, including both wet lab quality control and in silico quality assessment, are essential. While several tools have been developed or adopted for assessing read quality, identifying nucleosome occupancy and accessible regions from ATAC-seq data, none of the tools provide a comprehensive set of functionalities for preprocessing and quality assessment of aligned ATAC-seq datasets. RESULTS: We have developed a Bioconductor package, ATACseqQC, for easily generating various diagnostic plots to help researchers quickly assess the quality of their ATAC-seq data. In addition, this package contains functions to preprocess aligned ATAC-seq data for subsequent peak calling. Here we demonstrate the utilities of our package using 25 publicly available ATAC-seq datasets from four studies. We also provide guidelines on what the diagnostic plots should look like for an ideal ATAC-seq dataset. CONCLUSIONS: This software package has been used successfully for preprocessing and assessing several in-house and public ATAC-seq datasets. Diagnostic plots generated by this package will facilitate the quality assessment of ATAC-seq data, and help researchers to evaluate their own ATAC-seq experiments as well as select high-quality ATAC-seq datasets from public repositories such as GEO to avoid generating hypotheses or drawing conclusions from low-quality ATAC-seq experiments. The software, source code, and documentation are freely available as a Bioconductor package at https://bioconductor.org/packages/release/bioc/html/ATACseqQC.html

RUNX1 and FOXP3 interplay regulates expression of breast cancer related genes

Runx1 participation in epithelial mammary cells is still under review. Emerging data indicates that Runx1 could be relevant for breast tumor promotion. However, to date no studies have specifically evaluated the functional contribution of Runx1 to control gene expression in mammary epithelial tumor cells. It has been described that Runx1 activity is defined by protein context interaction. Interestingly, Foxp3 is a breast tumor suppressor gene. Here we show that endogenous Runx1 and Foxp3 physically interact in normal mammary cells and this interaction blocks Runx1 transcriptional activity. Furthermore we demonstrate that Runx1 is able to bind to R-spondin 3 (RSPO3) and Gap Junction protein Alpha 1 (GJA1) promoters. This binding upregulates Rspo3 oncogene expression and downregulates GJA1 tumor suppressor gene expression in a Foxp3-dependent manner. Moreover, reduced Runx1 transcriptional activity decreases tumor cell migration properties. Collectively, these data provide evidence of a new mechanism for breast tumor gene expression regulation, in which Runx1 and Foxp3 physically interact to control mammary epithelial cell gene expression fate. Our work suggests for the first time that Runx1 could be involved in breast tumor progression depending on Foxp3 availability

A small-molecule inhibitor of the aberrant transcription factor CBFβ-SMMHC delays leukemia in mice

This is the author’s version of the work. It is posted here by permission of the AAAS for personal use, not for redistribution. The definitive version was published in Science on 2015 February 13; 347(6223): 779–784, DOI: 10.1126/science.aaa0314.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

Plag1 and Plagl2 are oncogenes that induce acute myeloid leukemia in cooperation with Cbfb-MYH11

Recurrent chromosomal rearrangements are associated with the development of acute myeloid leukemia (AML). The frequent inversion of chromosome 16 creates the CBFB-MYH11 fusion gene that encodes the fusion protein CBFbeta-SMMHC. This fusion protein inhibits the core-binding factor (CBF), resulting in a block of hematopoietic differentiation, and induces leukemia upon the acquisition of additional mutations. A recent genetic screen identified Plag1 and Plagl2 as CBF beta-SMMHC candidate cooperating proteins. In this study, we demonstrate that Plag1 and Plagl2 independently cooperate with CBF beta-SMMHC in vivo to efficiently trigger leukemia with short latency in the mouse. In addition, Plag1 and Plagl2 increased proliferation by inducing G1 to S transition that resulted in the expansion of hematopoietic progenitors and increased cell renewal in vitro. Finally, PLAG1 and PLAGL2 expression was increased in 20% of human AML samples. Interestingly, PLAGL2 was preferentially increased in samples with chromosome 16 inversion, suggesting that PLAG1 and PLAGL2 may also contribute to human AML. Overall, this study shows that Plag1 and Plagl2 are novel leukemia oncogenes that act by expanding hematopoietic progenitors expressing CbF beta-SMMHC

C/EBPalpha in leukemogenesis: a matter of being in the right place with the right signals

Leukemia-initiating cells can originate from hematopoietic progenitor cells that have acquired self-renewal capacity upon transformation with leukemic fusion genes. In this issue of Cancer Cell, Kirstetter and colleagues describe a mouse model for the frequent CEBPA mutations in human acute myeloid leukemia that result in the synthesis of only the 30kDa isoform, but not the 42kDa isoform of C/EBPalpha. This mutation uncouples C/EBPalpha\u27s roles in myeloid differentiation and proliferation control. Furthermore, this mutation activates self-renewal in committed myeloid progenitor cells and induces myeloid malignancy with complete penetrance that is sustained by leukemia-initiating cells with a committed myeloid molecular signature

Molecular Basis and Targeted Inhibition of CBFbeta-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 CBFbeta-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 CBFbeta-SMMHC/RUNX1 binding, the first candidate targeted therapy for inv(16) AML

The transcription factor PlagL2 activates Mpl transcription and signaling in hematopoietic progenitor and leukemia cells

Cytokine signaling pathways are frequent targets of oncogenic mutations in acute myeloid leukemia (AML), promoting proliferation and survival. We have previously shown that the transcription factor PLAGL2 promotes proliferation and cooperates with the leukemia fusion protein Cbfbeta-SMMHC in AML development. Here, we show that PLAGL2 upregulates expression of the thrombopoietin receptor Mpl, using two consensus sites in its proximal promoter. We also show that Mpl overexpression efficiently cooperates with Cbfbeta-SMMHC in development of leukemia in mice. Finally, we demonstrate that PlagL2-expressing leukemic cells show hyper-activation of Jak2 and downstream STAT5, Akt and Erk1/2 pathways in response to Thpo ligand. These results show that PlagL2 expression activates expression of Mpl in hematopoietic progenitors, and that upregulation of wild-type Mpl provides an oncogenic signal in cooperation with CBFbeta-SMMHC in mice