Spatiotemporal regulation of energy usage and sensing in cancer cells

Danielle Schmitt 
Appointment Period: 2017-2018, Grant Year: [32]

A hallmark of cancer is an altered metabolic state, where ATP production is shifted from oxidative phosphorylation to glycolysis. Further contributing to altered metabolism within cancer cells is dysregulation of AMP activated protein kinase (AMPK), a key cellular energy regulator. Investigations into single cell ATP levels revealed cancer cells exhibit diminished ATP levels at the mitochondria, the site of oxidative phosphorylation, as compared to the cytoplasm. AMPK signaling has been found to be spatiotemporally regulated throughout the cell as well. Thus, ATP levels and energy sensing signaling networks are spatiotemporally regulated, but the spatiotemporal dysregulation in cancer cells and the underlying mechanisms have not been fully investigated. Aside from altered energy production, cancer cells also upregulate many biosynthetic pathways. In particular, de novo purine biosynthesis is upregulated in many cancers to meet the demands of rapidly dividing cancer cells for purine nucleotides. The upregulation of de novo purine biosynthesis makes the pathway a validated chemotherapeutic target. De novo purine biosynthesis converts phosphoribosyl pyrophosphate into inosine monophosphate (IMP) in 10 steps, catalyzed by six enzymes. Advances in the understanding of de novo purine biosynthesis have revealed under conditions of purine deprivation, in both normal and cancerous cells, the six enzymes catalyzing purine biosynthesis compartmentalize together into a multienzyme compartment, “the purinosome”. Furthermore, purinosome assembly is associated with increased rates of de novo purine biosynthesis and IMP production. However, de novo purine biosynthesis is a very energetically taxing process, requiring the input of five ATP molecules to produce one IMP molecule. AMPK has been implicated in the regulation of purine biosynthesis and sequestration of a purine biosynthetic enzyme. Aside from this, it is unknown how ATP levels are spatiotemporally regulated within the context of purinosomes, especially to provide the necessary energetics for de novo purine biosynthesis.

A key tool set in the ever-expanding chemical biology toolbox is genetically-encoded fluorescent protein- based biosensors. Genetically encoded biosensors have allowed for the study of kinase signaling and small molecule dynamics in single live cells, revealing spatial regulation within subcellular compartments as well as heterogeneity in cell populations. In particular, biosensors have been made for sensing ATP, ADP:ATP ratio, and AMPK. Moreover, targeting biosensors to specific areas within the cell can shed light on localized signaling and small molecule dynamics. With the exception of ADP:ATP ratio biosensors, all developed biosensors for ATP and AMPK in mammalian cells are Förester resonance energy transfer (FRET)-based. However, the dynamic range of FRET-based biosensors has limited their application. Indeed, ATP biosensors developed for bacteria using a circularly permuted fluorescent protein demonstrated significant enhancement in the dynamic range compared to the FRET-based probe. To overcome the limitations of FRET-based biosensors, we have recently developed a new class of genetically encoded kinase biosensors which use a phosphorylationdependent change in the excitation ratio of a circularly permuted fluorescent protein to report kinase activity. This new biosensor overcomes the limited dynamic range of typical FRET-based biosensors, allowing for wider applications.

I will be investigating four specific aims: 1. Develop high-sensitivity excitation-ratiometric AMPK biosensors for use in human cells; 2. Survey cellular energy compartmentalization in both cancerous and normal cell lines; 3. Probe local energy utilization for de novo purine biosynthesis; and 4. Develop a computational model to predict cellular behavior in response to energy alterations.

Cancer cells have an increased demand for ATP and purine nucleotides to meet requirements for rapid growth. A popular avenue for chemotherapeutic development is cancer metabolism, especially as it differs greatly from the metabolism of normal cells. Metabolic-focused treatments have been successful in the past, but there is a need for improved therapeutics, particularly as altered energy metabolism has been linked to drug resistance in cancer. Therefore, mapping the spatiotemporal regulation and compartmentalization of energy utilization and sensing in cancer cells versus normal cells will allow for critical insights into metabolism on a single cell level. These advances will lead to the development of future chemotherapeutics which may overcome the limitations of current treatments. Additionally, by improving upon current and developing new chemical biological tools, we will also allow for further advances in the understanding of cellular processes as they pertain to cancer biology.

PUBLICATIONS (resulting from this training): 

Schmitt DL, Dranchak P, MacArthur R, Kohnhorst CL, Kyoung M, Inglese J, An S. High content screening identifies the association of the cell cycle with the multienzyme metabolic assembly of glucose metabolism. In preparation.

Schmitt DL, Sundaram A, Jeon M, Luu BT, An S. Spatial regulation of de novo purine biosynthesis by Akt-independent PDK1 signaling pathways. PLoS ONE. 2018, 13(4):e0195989. PMID: 29668719