TGF1 is a potent inhibitor of cell cycle progression and can elicit a growth arrest not only in early-G1, but also when added to cells just before S-phase begins. The goal of our research is to understand this late-G1 TGF1- inhibitory process at the mechanistic level, and determine which enzymes TGF1 targets acutely and how. We then use this information to identify small molecules that mimic inhibition of TGF1 targets and offer clinical utility in suppressing cancer growth. Our group has identified one such target, the CMG replicative helicase, and a potential means to inhibit the helicase. TGF1 acutely blocks activation of the CMG helicase, which is fully formed and ready to function in promoting G1-S transit. Under conditions of TGF1 arrest, the CMG is in a physical complex with the Rb protein, which is required for the helicase to remain inactive. Rb directly interacts with at least one subunit of the CMG, Mcm7, and this interaction occurs via the N-terminus of Rb (RbN) and the C- terminus of Mcm7 (Mcm7CT). Our results indicate that Rb is an inhibitor of the CMG helicase, and we further demonstrate that RbN can inhibit the helicase in the Xenopus cell-free biochemical system. Using this system, we also show that this is derived from Exon7 of RbN, which is lost in human cancers. Inhibiting the CMG helicase presents an innovative opportunity for drug development and cancer intervention. Cancer cells are more sensitive than normal cells to inhibition of CMG function. Inhibiting CMG function also increases the sensitivity of tumor cells to DNA replication-suppressing drugs, of which there are many in the clinical arsenal. Thus, a means to chemically inhibit the CMG has the propensity to increase the therapeutic index of existing anti-neoplastic drugs, and alone can provide for an effective and innovative means to suppress tumor growth.
The Specific Aims of this proposal will extend these important observations by rigorously testing the ability and means by which RbN and Exon7 directly inhibit elongation and/or ATPase activities of the purified CMG helicase. RbN mutants lacking critical domains will be tested for loss of function, and alanine-scanning mutagenesis will be performed to identify important residues within Exon7 that mediate CMG inhibition. These experimental goals will aid in understanding how the CMG is regulated, and specifically how Exon7 achieves inhibition of the CMG. We will also establish effective CMG fluorescence-based assays to be used in targeted library screening for chemical inhibitors of the helicase, and which are compatible with HT approaches. The NCI Diversity Set IV library will be used to identify a small number of inhibitors/probes of the CMG helicase, for future expansion into larger drug discovery projects involving HT analysis.
Our group has used the growth inhibitory properties of TGF1 to determine how this negative growth factor normally blocks cell growth, and which unique targets it suppresses. We have uncovered a novel target of TGF1, the replicative CMG helicase, and we identified a mediator of TGF1 called Rb that binds and inhibits the helicase. Cancer cells are sensitive to inhibition of the helicase, and we will investigate further how Rb inhibits this helicase in a purified system. We will also establish effective helicase assays that are compatible with high-throughput drug screening methods, and perform targeted library screening to validate these assays. The long term goal is to use this information to identify effective chemical inhibitors of the helicase for use in cancer intervention.
