Genome-wide synthetic lethal screen unveils novel CAIX-NFS1/xCT axis as a targetable vulnerability in hypoxic solid tumors

The microenvironment of solid tumors is heterogeneous in oxygen availability, and most solid tumor types contain regions of hypoxia. Hypoxia initiates a global adaptive transcriptional response, and engagement of the hypoxia-inducible factor (HIF) transcriptional program triggers a metabolic shift that is critical for cell survival. The decreased availability of oxygen initiates a shift in carbon utilization to increase dependence on glycolysis for energy production. This leads to accumulation of acidic by-products and reliance upon pH regulatory enzymes and transporters to maintain an alkaline intracellular pH (pHi) to sustain proliferation and survival. As a consequence of the increased efflux of lactate and protons, the extracellular environment acidifies. Akin to hypoxia, acidic pH also leads to metabolic rearrangement to meet cellular macromolecular and bioenergetic demands and sustain survival. Identifying strategies to target these features is important for improving therapeutic efficacy in these microenvironments.

Carbonic anhydrases are a family of metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and protons. Carbonic anhydrase IX and XII (CAIX/XII) are hypoxia-induced, cell surface, extracellular facing pH regulatory enzymes. CAIX and CAXII aid in buffering pHi through cooperation with bicarbonate transporters. CAIX, in particular, predicts poor prognosis in a number of solid tumor types and plays a critical role in several features of high-grade tumors including invasion, metastasis, and cancer stem cell survival. While inhibitors of CAIX are under clinical evaluation, identifying survival mechanisms governed by CAIX and combinatorial approaches to improving therapeutic response and limit resistance is of critical importance.

Regulated cell death is a critical determinant in the success of cancer therapy. Hypoxia selects for mutations in TP53 enriching for cells in these niches that are less sensitive to initiation of apoptosis. Fortunately, additional modes of regulated cell death have emerged that can be engaged to overcome resistance to apoptosis. Ferroptosis is a nonapoptotic form of iron-dependent cell death resulting from toxic accumulation of phospholipid peroxidation. Ferroptosis is intimately linked to the metabolic status of the cell being influenced by redox homeostasis, iron, amino acid and lipid metabolism, and mitochondrial activity. Rewired cellular metabolism in response to hypoxia and acidosis influences many of the same pathways. The HIF pathway has even been implicated in both protecting from and inducing ferroptosis. However, the molecular determinants dictating its role and ways to target these features are unclear.

Here, scientists from Department of Integrative Oncology, BC Cancer Research Institute, identified cellular axes compensating for CA9 loss following an unbiased, genome-wide synthetic lethal CRISPR screen in hypoxic triple-negative breast cancer (TNBC) cells. They uncovered a pattern indicative of a vulnerability to perturbations in redox homeostasis, including the gene encoding the cysteine desulfurase, NFS1, an enzyme critical for iron-sulfur cluster biogenesis. During our investigation into the link between CA9 and NFS1, they found that CAIX influenced cell death by ferroptosis. Suppression of CAIX expression or CAIX/XII activities increased cellular reactive oxygen species (ROS) levels, creating a vulnerability to increases in cellular iron levels associated with NFS1 depletion, addition of exogenous iron, or transit in the bloodstream. Furthermore, the CAIX-generated bicarbonate was important in preventing this. In summary, the data described here demonstrate that the role of CAIX in maintaining an alkaline pHi is critical to suppress ferroptosis.

Thus, an alkaline intracellular pH plays a critical role in suppressing ferroptosis, a finding that may lead to the development of innovative therapeutic strategies for solid tumors to overcome hypoxia- and acidosis-mediated tumor progression and therapeutic resistance. This study has been published in Science Advances reently.

 

Sherry