Previously Funded Research

Tom Cunningham, PhD

The University of Cincinnati

Research Project:

Understanding the Dependency of RAS-driven Lung Adenocarcinomas on PRPS enzyme remodeling

Summary:

Alterations in cellular metabolism are a well-established hallmark of lung cancers driven by the oncogene KRAS. Although much is known about how this occurs, we still lack clinically effective therapies targeting these processes. Due to the interconnected nature of metabolic pathways along with the multiple redundancies built in to the system, no “silver bullet” is likely to be found that hits a single target and can produce durable cancer remission. Our strategy leans into this belief, and instead aims to leverage a novel metabolic vulnerability as a safe starting point in order to systematically dismantle the metabolic resiliency of KRAS-driven lung adenocarcinomas.

We discovered that oncogenic KRAS expression increases levels of an inherently more active isoform of the phosphoribosyl pyrophosphate synthetase enzyme – PRPS2 – which we hypothesize drives increased production of nucleotides and/or NADPH necessary to fuel tumor growth. The PRPS enzyme connects the pentose phosphate pathway to downstream nucleotide production, making it a critical step in producing the building blocks for DNA and RNA as well as regulating the production of NADPH used for lipid synthesis and remediation of oxidative damage. Using the mouse models of lung cancer, we find stripping away the PRPS2 isoform decreases tumor incidence, confirming the metabolic dependency of this oncogene-isozyme pair in vivo. Additionally, fibroblasts CRISPR-engineered to lack PRPS2 and solely express the less active PRPS1 isozyme have dramatically diminished ability to be oncogenically transformed by overexpression of mutant RAS.

Thus, we know that increases in PRPS2 are important (but not vital) for RAS-mediated tumorigenesis, but we do not know how this occurs or why it facilitates lung cancer development. Due to the built-in metabolic redundancy afforded by expression of two PRPS isoforms, we are uniquely positioned to leverage the incomplete restriction in enzymatic activity caused by selectively inactivating the PRPS2 isoform in order to uncover resistance mechanisms that mutant RAS expressing cells utilize to circumvent/overcome diminished nucleotide production.

First, this work identifies a novel metabolic vulnerability in lung cancer. Second, because PRPS2 knockout mice are perfectly viable, fertile, and display no overt phenotypic abnormalities, it nominates a path for the development of effective, but less toxic, targeted therapies. Third, because we are interrogating a linchpin of the cell’s metabolic machinery that controls production of all species of nucleotides, we can address key questions in basic lung cancer biology at a broad biochemical level.

The study has two specific aims.

In Aim 1, we will define the mechanistic basis for deregulated PRPS activity in KRAS-driven lung cancer. We will determine how oncogenic KRAS promotes upregulation of the PRPS2 isoform by interrogating whether induction occurs at the level of transcriptional (DNA->RNA step) or translational (RNA->protein) control. We have already identified several factors that may be responsible, and we will employ the CRISPR genetic engineering toolkit and clinically useful inhibitors of nominated target pathways downstream KRAS signaling to identify the mechanism. Through identification of compounds that interfere with this process, our strategy may provide a rapid path and rationale to bring new compounds to the clinic to help lung cancer patients, or help explain the potential responsiveness of tumors to compounds already being utilized.

In aim 2, we will define the metabolic vulnerability triggered by loss of PRPS2 function in KRAS-mutant lung cancer. We will test two hypotheses that could explain the dependency of KRAS-driven lung cancers to loss of PRPS2 function. The first hypothesis posits that KRAS-driven lung adenocarcinomas are susceptible to loss of PRPS2 function due to overworking upstream pathways that leads to an induction of NADPH-dependent reductive stress that can trigger cancer cell death. An equally plausible second hypothesis is that PRPS2 loss of function leads to nucleotide deficiency that interferes with anabolic programs of DNA replication, transcription, and/or protein synthesis. We will optimize a diagnostically-useful biochemical assay to measure PRPS activity in KRAS-mutant lung cancers that express or lack PRPS2 to uncover how these tumors overcome diminished nucleotide production, which may lead to a productive path towards a rational combinatorial therapy. We will also evaluate a novel therapeutic strategy capable of specifically blocking PRPS2, but not PRPS1, function as a new approach to disrupting the metabolism of KRAS-mutant lung cancer.

In summary, this study will deliver new knowledge regarding the mechanisms oncogenic KRAS employs to deregulate gene expression and metabolism, and it will supply new diagnostic assays and therapeutic tools that can translate to the clinic to help patients with lung cancer.