Lucio H. Castilla

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

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 A small molecule inhibits leukemia in mice by targeting a transcription factor, a class of proteins thought to be undruggable. [Also see Perspective by Koehler and Chen] 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.

Chemical biology. A small-molecule inhibitor of the aberrant transcription factor CBFbeta-SMMHC delays leukemia in mice

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 A small molecule inhibits leukemia in mice by targeting a transcription factor, a class of proteins thought to be undruggable. [Also see Perspective by Koehler and Chen] 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.

NrasG12D oncoprotein inhibits apoptosis of preleukemic cells expressing Cbfβ-SMMHC via activation of MEK/ERK axis

Acute myeloid leukemia (AML) results from the activity of driver mutations that deregulate proliferation and survival of hematopoietic stem cells (HSCs). The fusion protein CBFβ-SMMHC impairs differentiation in hematopoietic stem and progenitor cells and induces AML in cooperation with other mutations. However, the combined function of CBFβ-SMMHC and cooperating mutations in preleukemic expansion is not known. Here, we used Nras(LSL-G12D); Cbfb(56M) knock-in mice to show that allelic expression of oncogenic Nras(G12D) and Cbfβ-SMMHC increases survival of preleukemic short-term HSCs and myeloid progenitor cells and maintains the differentiation block induced by the fusion protein. Nras(G12D) and Cbfβ-SMMHC synergize to induce leukemia in mice in a cell-autonomous manner, with a shorter median latency and higher leukemia-initiating cell activity than that of mice expressing Cbfβ-SMMHC. Furthermore, Nras(LSL-G12D); Cbfb(56M) leukemic cells were sensitive to pharmacologic inhibition of the MEK/ERK signaling pathway, increasing apoptosis and Bim protein levels. These studies demonstrate that Cbfβ-SMMHC and Nras(G12D) promote the survival of preleukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define Nras(LSL-G12D); Cbfb(56M) mice as a valuable genetic model for the study of inversion(16) AML-targeted therapies.

Small Molecule Inhibitor of CBFβ-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 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.

C/EBPα 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/EBPalphas 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.

Cbfβ Reduces Cbfβ-SMMHC–Associated Acute Myeloid Leukemia in Mice

The gene encoding for core-binding factor β (CBFβ) is altered in acute myeloid leukemia samples with an inversion in chromosome 16, expressing the fusion protein CBFβ-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 Cbfβ modulates the oncogenic function of this fusion protein. We show that Cbfβ plays an important role in proliferation of hematopoietic progenitors expressing Cbfβ-SMMHC in vitro. In addition, Cbfβ-SMMHC-mediated leukemia development is accelerated in the absence of Cbfβ. These results indicate that the balance between Cbfβ and Cbfβ-SMMHC directly affects leukemia development, and suggest that CBF-specific therapeutic molecules should target CBFβ-SMMHC function while maintaining CBFβ activity.

The use of alternative polyadenylation sites renders integrin β1 (Itgb1) mRNA isoforms with differential stability during mammary gland development

Integrins are heterodimeric cell-surface adhesion receptors that play a critical role in tissue development. Characterization of the full-length mRNA encoding the β1 subunit (Itgb1) revealed an alternative functional cleavage and polyadenylation site that yields a new Itgb1 mRNA isoform 578 bp shorter than that previously reported. Using a variety of experimental and bioinformatic approaches, we found that the two Itgb1 isoforms are expressed at different levels in a variety of mouse tissues, including the mammary gland, where they are differentially regulated at successive developmental stages. The longer mRNA species is prevelant during lactation, whereas the shorter is induced after weaning. In 3D cultures, where expression of integrin β1 protein is required for normal formation of acini, experimental blockade of the longer isoform induced enhanced expression of the shorter species which allowed normal morphological mammary differentiation. The short isoform lacks AU-rich motifs and miRNA target sequences that are potentially implicated in the regulation of mRNA stability and translation efficiency. We further determined that the AU-binding protein HuR appears to selectively stabilize the longer isoform in the mammary gland. In summary, the results of the present study identify a new regulatory instance involved in the fine-tuning of Itgb1 expression during mammary gland development and function.

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 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.

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

BackgroundATAC-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.ResultsWe 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.ConclusionsThis 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.

Corrigendum to: "Small Molecule Inhibitor of CBFβ-RUNX Binding for RUNX Transcription Factor Driven Cancers" [EBioMedicine 8 (2016) 117-131]

Anuradha Illendula , Jane Gilmour , Jolanta Grembecka , Venkata Sesha Srimath Tirumala , Adam Boulton , Aravinda Kuntimaddi , Charles Schmidt , Lixin Wang , John A. Pulikkan , Hongliang Zong , Mahmut Parlak , Cem Kuscu , Anna Pickin , Yunpeng Zhou , Yan Gao , Lauren Mishra , Mazhar Adli , Lucio H. Castilla , Roger A. Rajewski , Kevin A. Janes , Monica L. Guzman , Constanze Bonifer , John H. Bushweller a,⁎