Rationale and Objective: Our laboratory has discovered a new set of biochemical signals that are deranged in cells altered in TSC patients. The signals form a circuit called the ULK1 signal, which is normally active in cells of our body to provide a quality control mechanism to ensure the health and recycling of cellular parts. In cells defective in the TSC genes such as those found in the lesions, tubers, and tumors of TSC patients, this ULK1 signal is blocked due to elevations in the activity of a protein complex called mTORC1, which is the major thing the TSC genes serve to do in cells: shut off mTORC1. So when TSC genes are defective, mTORC1 activity is high and it shuts off ULK1. This results in a loss of cellular quality control and recycling, which contributes to the aberrant growth and cellular behavior of TSC-deficient cells. These new findings make a number of predictions for better ways to treat and diagnose TSC, starting off with the current use of mTOR inhibitors and trying to improve upon them by combining them with ULK1 drugs and using markers of ULK1 activity to determine when and where mTOR is getting effectively shut off in TSC patients during therapy.

Applicability of Research: Given how we have decoded the wiring of this TSC-mTORC1-ULK1 circuit, it makes a number of predictions for the treatment and diagnosis of TSC patients. The best available treatments for TSC currently are drugs that can suppress the elevated mTOR found in these patients' cells, such as the drug rapamycin and its analogs (called "rapalogs"). Treatment with rapalogs and newer direct mTOR inhibiting drugs will lead to an activation of ULK1 once its blockage by mTOR is relieved. This means that markers of ULK1 activity may be able to be used in biopsies and blood samples from TSC patients to determine how well the mTOR blockade from rapalogs or other mTOR inhibitors are, as the signal from ULK1 will go "up" proportionally to how much mTOR goes down. Moreover, we found that ULK1 normally provides a survival signal to cells to stay alive during times of stress by allowing them to recycle their own parts and metabolites. These findings suggest that in patients treated with rapamycin or other mTOR inhibitors, ULK1 will actually keep the tumor cells and other TSC-deficient cells alive during the treatment, preventing the full eradication of the tumor cells. So if we could combine mTOR inhibitor drugs with ULK1 inhibitor drugs, we may convert the modest but positive effects seen in TSC and LAM with mTOR drug treatment into a much more sustained and robust response. These studies should help all TSC patients as well as any patients with spontaneously arising LAM (lymphangioleiomyomatosis) or AML (angiomyolipomas). Because the ULK1 components are expressed in all cells of the body, combining ULK1 and mTOR drugs should benefit all organs affected by TSC: brain (tubers, SEGAs), kidney (AML), skin fibromas, heart rhabdomyosarcomas, and lung LAM lesions.

Benefits and Risks: For the use of ULK1 as a diagnostic marker, there is a clear benefit as this provides the first known case of a marker that increases when mTOR is being effectively blocked from therapy. All the current markers are all lowered when mTOR is blocked, which is also useful, but sometimes it is easier to quantify a signal that is low in the starting state and then increased with treatment, proportional to how effective treatment is. It should be a very powerful tool for clinicians, and there is no risk to use these as diagnostics. For the therapeutic combination of ULK1 and mTOR inhibitors, the benefit could be far more durable and lasting responses, which hopefully could mean complete eradication of the tumor cells or restored function to TSC-deficient cell types that are not tumorous (e.g., brain tubers), without patients needing to keep rapamycin for the rest of their lives. The risk is that we do not know if the combination of drugs could harm any normal tissues besides harming the TSC-deficient cells. One reason to be optimistic that there will be no major problems is that mice genetically lacking ULK1 are fine with no obvious negative consequences.

Interim Outcomes: The work does have immediate clinical implications, though it will take 1-2 years of testing and validating the ULK1 diagnostics we are developing to see how well they work as markers of mTOR suppression in TSC therapy. It will likely take longer (5 years) to develop our existing ULK1 inhibitors into a form that may be used clinically, though fortunately our collaborator Nick Cosford has 15 years of experience in bringing kinase inhibitors for the clinic while he was at Merck. In the meantime, we will define how well ULK1 drugs combined with mTOR drugs are for TSC outcomes in cells and mouse models.

Contributions: This study is extremely likely to advance the field of TSC research and also have these immediate applications to patient care. The discovery that ULK1 is suppressed by mTOR in all cell types and the fact we can block it with new drugs make this one of the broadest possible new therapeutic options to rationally combine with mTOR drugs since their discovery as a useful treatment in this disease.