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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

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.

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

Acute leukemias caused by translocations of the MLL gene at chromosome 11 band q23 (11q23) are characterized by a unique gene expression profile. More recently, data from several laboratories indicate that the most commonly encountered MLL fusion proteins, MLLT1, MLLT3, and AFF1 are found within a molecular complex that facilitates the elongation phase of mRNA transcription. Mutational analyses suggest that interaction between the MLLT1/3 proteins and AFF family proteins are required for experimental transformation of hematopoietic progenitor cells (HPCs). Here, we define a specific pairing of two amino acids that creates a salt bridge between MLLT1/3 and AFF proteins that is critically important for MLL-mediated transformation of HPCs. Our findings, coupled with the newly defined structure of MLLT3 in complex with AFF1, should facilitate the development of small molecules that block this amino acid interaction and interfere with the activity of the most common MLL oncoproteins.

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.

Abstract 4193 The MLL gene encodes a multi-domain protein that is involved in the maintenance of Hox gene expression during development and hematopoiesis, and was first identified through its involvement in chromosome translocations that cause leukemia. The CXXC domain of MLL, which is retained in leukemic MLL fusion proteins, is a cysteine rich DNA binding domain, with specificity for binding nonmethylated CpG-containing DNA, and is essential for MLL fusion proteins' oncogenic properties. We performed domain swap experiments in which CXXC domains from other proteins were swapped in to replace MLL's CXXC domain in the context of an oncogenic MLL fusion. CXXC domains from DNA methyltransferase 1 (DNMT1), CpG binding protein (CGBP), and methyl-CpG binding domain protein 1 (MBD1), as well as a methyl binding domain (MBD) from MBD1 were swapped into the MLL-AF9 fusion. These particular domains were chosen because their described CpG DNA binding capacity is either similar or different from that described for MLL. In vitro colony assays on isolated murine bone marrow progenitor cells infected with domain swapped or wild type MLL-AF9 fusion genes were performed in order to determine whether CpG binding domains from other proteins would affect the ability of MLL-AF9 to give an enhanced proliferative capacity to bone marrow progenitor cells. In vivo murine studies determined whether the different CpG binding domains alter the ability of MLL fusion proteins to cause leukemia. We predicted that the different CpG binding domains would change the strength or specificity of MLL binding to DNA, which would affect the ability of MLL-AF9 to cause leukemia. The results of both in vitro replating assays and in vivo leukemogenesis experiments have shown significant differences between the ability of various CpG DNA binding domains to function in the context of an MLL-AF9 fusion protein. MLL-AF9 containing the DNMT1 CXXC domain shows robust in vitro colony forming activity and in vivo leukemogenesis activity, similar to the oncogenic MLL-AF9 fusion. However, MLL-AF9 containing either the CXXC domain from CGBP or MBD1, or the MBD domain of MBD1 all show reduced colony forming ability and leukemogenicity in vivo. In vitro DNA binding experiments are currently being performed to directly measure and compare the DNA binding affinity of the CXXC domain from MLL to the other domain swap proteins. Preliminary data suggests that MLL CXXC has a stronger DNA binding affinity to non-methylated DNA compared to the other CXXC domains. Furthermore, the DNMT and CGBP CXXC domains both show lower affinity DNA binding compared to MLL CXXC, but they have different effects in MLL-AF9. This suggests that CXXC domain properties in addition to DNA binding affinity, perhaps including protein recruitment, also contribute to an MLL fusion protein's leukemogenic properties. Disclosures: No relevant conflicts of interest to declare.