Research
Regulation of Growth and Proliferation in Normal and Neoplastic Cells
The broad goal of research in our laboratory is to understand the nature of gene regulatory networks that act as critical drivers of cell growth and the consequences of subverting these networks in neoplastic progression. We have focused on a transcription factor network - the Max-Mlx network- whose interacting components together comprise a transcriptional switching system that has been highly conserved throughout evolution. One of the components of the network is the MYC protein. MYC family proteins are essential for normal cellular functions but when deregulated are profoundly involved in the genesis of many different types of tumors. MYC interacts in a specific manner with its dimerization partner, Max, permitting the MYC-Max heterodimer to bind DNA and regulate gene expression. MYC- Max dimers recruit histone acetyltransferases and other factors that mediate increased DNA accessibility within chromatin and act to augment transcription at least in part through increased transcriptional elongation.
Importantly, Max not only interacts with MYC family proteins but also dimerizes with other bHLHZ class proteins including the Mxd family (Mxd1-4 and Mnt) as well as with Mga, a large protein with two DNA binding domains. Mxd proteins act as transcriptional repressors by interacting with the mSin3-histone deacetylase corepressor complex. This co-repressor complex, which is recruited to specific sites in chromatin through its binding with Mxd:Max heterodimers, causes deacetylation of the N-terminal tails of nucleosomal histones leading to formation of a repressive chromatin structure. A more extended form of the network has also been uncovered. A Max-like protein known as Mlx dimerizes with a subset of the Mxd proteins as well as two MYC-like proteins: Mondo A and ChREBP. These Mlx-Mondo heterodimers are profoundly involved in transcriptional regulation of cellular metabolism in response to environmental changes.
Importantly, Max not only interacts with MYC family proteins but also dimerizes with other bHLHZ class proteins including the Mxd family (Mxd1-4 and Mnt) as well as with Mga, a large protein with two DNA binding domains. Mxd proteins act as transcriptional repressors by interacting with the mSin3-histone deacetylase corepressor complex. This co-repressor complex, which is recruited to specific sites in chromatin through its binding with Mxd:Max heterodimers, causes deacetylation of the N-terminal tails of nucleosomal histones leading to formation of a repressive chromatin structure. A more extended form of the network has also been uncovered. A Max-like protein known as Mlx dimerizes with a subset of the Mxd proteins as well as two MYC-like proteins: Mondo A and ChREBP. These Mlx-Mondo heterodimers are profoundly involved in transcriptional regulation of cellular metabolism in response to environmental changes.
Several current projects in the lab are focused on aspects of the Max/Mlx network:
Coordinate regulation of cancer cell metabolism by MYC-Max and MondoA-Mlx heterodimeric transcription factors
We have found that Mlx- MondoA heterodimers, which are members of the extended Max network, are essential for the survival of tumors driven by deregulated Myc. We have established that the basis for this tumor dependency involves cooperation between deregulated MYC-Max and Mlx-MondoA in the regulation of genes critical for multiple tumor cell metabolic pathways. In collaborative studies employing metabolomic analyses we have shown that neuroblastoma cells with high levels of MYC require Mlx-MondoA for expression of key enzymes mediating fatty acid synthesis. Loss of MondoA-Mlx in MYC-driven neuroblastomas decreases lipogenesis and survival. In fact, such cells can be partially rescued by treatment with oleic acid.
We are investigating the possibility that cancers characterized by high levels of deregulated MYC may be uniquely sensitive to inhibitors of fatty acid biosynthesis. Our studies to date indicate that one function of the Mlx-MondoA branch of the network is to adjust metabolism, at the transcriptional level, in response to the increased demand associated with changes in cell growth and proliferation. Such changes occur during normal development (see below) and also during tumor progression in response to deregulated Myc. More recently we have extended this work to examine the dependency of pancreatic cancers on MondoA. Pancreatic cancers are metabolically distinct from neuroblastomas, yet their growth is also dependent on MondoA expression. We have found that in these cancers decreased levels MondoA influences production of branched chain amino acids and mitochondrial function and, at the same time, appears to suppress the tumor cell's response to stress. Thus, the precise nature of the dependency of MYC-driven tumors on MondoA is likely to be different in different tumor types.
Oncogenic and anti-oncogenic functions are embedded in the Max/Mlx transcriptional network
We are studying the role of the extended Max-Mlx transcriptional network in development and in cancer by creating mice with targeted conditional deletions of Max and Mlx family genes.
Deletion of MLX in mice results in male sterility due to defective spermatogenesis which, in turn, is partly due to altered metabolism during spermatogenesis in the testis. In addition, human male germ cell tumors, many of which express high levels of MYC, are also sensitive to loss of Mlx function. Preliminary evidence suggest that Mlx plays a role in human male sterility. During the last year we have employed chromatin immunoprecipitation to map the genomic occupancies of network members in normal and tumor cells. Our aim is to understand in detail how the network responds to perturbations and how gain or loss of the activities of network members leads to tumorigenesis.
While Mlx deletion has little effect on embryonic development, Max loss of function leads to early embryonic lethality, as expected. Currently we are using conditional deletion of Max to explore Max independent functions of MYC in different developmental contexts. We find that while Max deletion targeted to murine B cells has modest effects on B cell development, it completely abrogates the ability of deregulated MYC to induce B cell lymphomas. Importantly loss of Max or inhibition of MYC-Max heterodimerization results in destabilization of the MYC protein.
Although Max is required for MYC-driven B cell lymphomas, we find that in other contexts Max can act as a tumor suppressor. In collaboration with the laboratory of David MacPherson in the Human Biology Division we have shown that Max loss accelerates tumorigenesis in a murine small cell lung cancer model and that these tumors are not dependent on MYC. We are currently focusing on how the widespread genomic occupancy by members of the Max-Mlx network are specifically altered by Max deletion and how this influences gene expression in Max null tumors.
We also discovered that, Mga, another member of the Max-Mlx network acts as a tumor suppressor. Mga is a very large dual-specificity transcription factor that heterodimerizes with Max and acts as a transcriptional repressor by associating with a variant Polycomb complex (PRC1.6). Together with the MacPherson lab we have shown that loss of function mutations in Mga profoundly affect cell growth and motility and are highly oncogenic. Moreover, Mga recruits members of the PRC1.6 complex to thousands of genes and its interaction with PRC1.6 is required for its growth suppressive ability. We are in the midst of determining how this binding specifically affects expression of genes involved in metastasis.
MYC protein mutations
We have identified a single activating point mutation in MYC that, when introduced into the endogenous MYC gene in mice, results in abnormal self-renewal of progenitor cells and leads to a cancer-prone phenotype. Our mouse models are being used to probe the molecular mechanisms underlying Max network functions and to identify genes regulated by the network. In addition, we are also studying a form of MYC protein found in AML which possesses a duplication of MYC's DNA binding domain. Our ongoing work is aimed at determining the molecular and biological properties of this this unusual mutant form of the protein.
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