Structure/Function Studies of Transcription Factor Drivers in Cancer

Our lab is fundamentally interested in understanding how transcription factors that are drivers in cancer mediate their effects. This basic science understanding is essential to develop new approaches to cancer treatment. Our approach to do this is based on structure/function studies. We determine 3D structures of functional domains from these transcription factors that mediate specific protein-protein or protein-nucleic acid interactions bound to their interaction partner. Based on the structural data, we develop specific point mutations which disrupt this specific interaction but do not affect the structure or stability of the domain. These mutant forms of the transcription factor serve as high quality biological reagents to carry out functional studies with. Furthermore, they recapitulate what a small molecule inhibitor of the interaction would do, so they serve as tools to validate specific interactions for inhibitor development. Using these well-validated biological tools, we probe functional effects including, but not limited to, effects on proliferation, effects on differentiation (flow cytometry), effects on gene expression (RNA-Seq), effects on transcription factor occupancy (ChIP-Seq), effects on epigenetic signaling (ChIP-Seq), and effects in relevant mouse models of the cancer (latter is done with a group of outstanding collaborators at other institutions).

A long-term focus has been structural studies of a novel transcription factor referred to as the core-binding factor (CBF) which is a heterodimeric transcription factor (CBFβ and RUNX1, 2, or 3). CBFβ/RUNX1 is essential for hematopoietic development. Gene translocations associated with the genes coding for CBFβ and RUNX1 produce novel fusion proteins (CBFβ-SMMHC, RUNX1-ETO, TEL-RUNX1) which have been implicated as playing a role in more than 30% of acute leukemias. We have carried out extensive structural and functional studies of the fusion protein forms of CBFβ and RUNX1. We have extended these studies to the MLL protein, a key epigenetic regulator that is the target of chromosomal translocations (MLL-AF9, MLL-ENL, MLL-AF4, etc.) in leukemia which are particularly poor prognosis.

Drug Development Targeting Transcription Drivers in Cancer

Dysregulation of gene expression is a hallmark of all cancers. It is critical for self-renewal and chemo-resistance of cancer cells, which contribute to the inability to completely eradicate cancer cells, thereby leading to relapse. The specific gene expression program that confers these properties derives from the aberrant activity of specific transcription factors that are drivers of disease. Clearly, the most direct and effective approach to alter this gene expression program is to directly target the activity of the transcription factors driving the disease. There are numerous examples of such transcription factor drivers in cancer such as fusion proteins of RUNX1 and CBFβ in leukemia, fusion proteins of ERG in prostate cancer and Ewing’s sarcoma, ETV-1 in melanoma, other members of the Ets family of transcription factors in a variety of different cancers, etc. Transcription factors were traditionally viewed as “undruggable” due to the need to target more challenging protein-protein or protein nucleic acid interactions through which these proteins act. There are still relatively few examples of such agents for cancer treatment, with the MDM2-p53 inhibitors being one example of such an agent that has progressed to the clinic. We are exploring several unique approaches to target this important class of proteins for drug development.

We have developed an inhibitor targeting the CBFβ-SMMHC fusion protein that occurs in inv(16) acute myeloid leukemia (AML). This inhibitor is a protein-protein interaction inhibitor that restores RUNX1 function in these cells. We have shown it is effective in a mouse model of inv(16) AML as well as against human inv(16) AML patient cells. Furthermore, we have shown that much of the effect of the compound is mediated by a dramatic reduction in expression of MYC, a key transcription factor driver in many cancers. This drug has been licensed to Systems Oncology and is progressing toward clinical testing.

Recent Publications

Pulikkan JA, Hegde M, Belaghzal H, Illendula A, Yu J, Ahmed H, O’Hagan K, Ou J, Muller-Tidow C, Wolfe SA, Zhu LJ, Dekker J, Bushweller JH, Castilla LH. CBFβ-SMMHC inhibition leads to alteration of chromatin dynamics at MYC distal enhancers and abrogation of inv(16) leukemia. Cell. June 28, 2018 174(1):172-186. PubMed PMID: 29958106.

Illendula A, Pulikkan JA, Zong H, Grembecka J, Xue L, Sen S, Zhou Y, Boulton A, Kuntimaddi A, Gao Y, Rajewski RA, Guzman ML, Castilla LH, Bushweller JH. Chemical biology. A small-molecule inhibitor of the aberrant transcription factor CBFβ-SMMHC delays leukemia in mice. Science. 2015 Feb 13;347(6223):779-84. PubMed PMID: 25678665

Illendula A, Gilmour J, Grembecka J, Tirumala VSS, Boulton A, Kuntimaddi A, Schmidt C, , Wang L, John A. Pulikkan, Hongliang Zong, Mahmut Parlak, Cem Kuscu, Anna Pickin,  Yunpeng Zhou, Yan Gao Y, Mishra L, Adli M, Castilla LH, Rajewski RA, Janes KA, Guzman ML, Bonifer C, and Bushweller JH. Small Molecule Inhibitor of CBFb-RUNX Binding for RUNX Transcription Factor Driven Cancers. EBioMedicine. 2016 8: 117-131. Pubmed PMID: 27428424

Kuntimaddi A, Achille NJ, Thorpe J, Lokken AA, Singh R, Hemenway CS, Adli M, Zeleznik-Le NJ, Bushweller JH. Degree of recruitment of DOT1L to MLL-AF9 defines level of H3K79 Di- and tri-methylation on target genes and transformation potential. Cell Rep. 2015 May 5;11(5):808-20. PubMed PMID: 25921540

Cierpicki T, Risner LE, Grembecka J, Lukasik SM, Popovic R, Omonkowska M, Shultis DD, Zeleznik-Le NJ, Bushweller JH. Structure of the MLL CXXC domain-DNA complex and its functional role in MLL-AF9 leukemia. Nat Struct Mol Biol. 2010 Jan;17(1):62-8. PubMed PMID: 20010842

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