Kris Wood

The hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx. We show that ABL kinases phosphorylate and interact with CUL4A, a cullin ring ligase adaptor, and compete with CPSF1 for CUL4A binding, leading to increased HIF-1α protein levels. Further, we identified the MYC proto-oncogene protein as a second CPSF1 substrate and show that active ABL kinase protects MYC from CPSF1-mediated degradation. These studies uncover a role for CPSF1 in cancer pathobiology as an E3-ligase antagonizing the expression of the oncogenic transcription factors, HIF-1α and MYC.

During apoptosis, mitochondrial outer membrane permeabilization (MOMP) enables certain mitochondrial matrix macromolecules to escape into the cytosol. However, the fate of mitochondrial RNA (mtRNA) during apoptosis is unknown. Here, we demonstrate that MOMP results in the cytoplasmic release of mtRNA and that executioner caspases-3 and -7 (casp3/7) prevent cytoplasmic mtRNA from triggering inflammatory signaling. In the setting of genetic or pharmacological casp3/7 inhibition, apoptotic insults result in mtRNA activation of the MDA5/MAVS/IRF3 pathway to drive Type I interferon (IFN) signaling. This pathway is sufficient to activate tumor-intrinsic Type I IFN signaling in immunologically cold cancer models that lack an intact cGAS/STING signaling pathway, promote CD8+ T-cell-dependent anti-tumor immunity, and overcome anti-PD1 refractoriness in vivo. Thus, a key function of casp3/7 is to inhibit inflammation caused by the cytoplasmic release of mtRNA, and pharmacological modulation of this pathway increases the immunogenicity of chemotherapy-induced apoptosis.

Targeted therapies have revolutionized cancer chemotherapy. Unfortunately, most patients develop multifocal resistance to these drugs within a matter of months. Here, we used a high-throughput phenotypic small molecule screen to identify MCB-613 as a compound that selectively targets EGFR -mutant, EGFR inhibitor-resistant non-small cell lung cancer (NSCLC) cells harboring diverse resistance mechanisms. Subsequent proteomic and functional genomic screens involving MCB-613 identified its target in this context to be KEAP1, revealing that this gene is selectively essential in the setting of EGFR inhibitor resistance. In-depth molecular characterization demonstrated that (1) MCB-613 binds KEAP1 covalently; (2) a single molecule of MCB-613 is capable of bridging two KEAP1 monomers together; and, (3) this modification interferes with the degradation of canonical KEAP1 substrates such as NRF2. Surprisingly, NRF2 knockout sensitizes cells to MCB-613, suggesting that the drug functions through modulation of an alternative KEAP1 substrate. Together, these findings advance MCB-613 as a new tool for exploiting the selective essentiality of KEAP1 in drug-resistant, EGFR -mutant NSCLC cells.

PTEN is a well-known tumor suppressor that is inactivated or suppressed at a high frequency in cancer. We sought an assay to screen compounds for ones that differentially inhibited proliferation or induced cytotoxicity in PTEN mutated cancer cells. We employed the isogenic pair of cell lines MCF10-A breast cell line (wild type, WT) and the same cell line with PTEN knocked out (KO) by CRISPR. We sought an assay where these PTEN WT and KO isogenic cell lines were co-cultured in the same well for compound testing. The KO cell line, but not the WT, was tagged with the red fluorescent protein mKate2. We employed a real time microscopic imaging instrument to identify cell populations in co-culture based on red fluorescence to obtain a cell count for each cell line. To acquire cytotoxicity data for each population, the dye CellTox Green was added to the media. To assess the assay, we determined the concentration response of paclitaxel. In order to assess the potential for screening, we performed mock screening in 384-well plate format. Thus, we developed a high throughput co-culture cell cytotoxicity and proliferation assay method that could be employed for any pair of cell lines to identify selective compounds.

The intrinsic apoptosis pathway is controlled by the BCL-2 family of proteins. Although the prosurvival members of this family can help cancer cells evade apoptosis, they may also produce apoptotic vulnerabilities that can potentially be exploited therapeutically. Apoptotic vulnerabilities can be driven by endogenous factors, including altered genetics, signaling, metabolism, structure, and lineage or differentiation state, as well as imposed factors, the most prominent being exposure to anticancer agents. The recent development of BH3 mimetics that inhibit prosurvival BCL-2 family proteins has allowed these apoptotic vulnerabilities to be targeted with demonstrable clinical success. Here, we review the key concepts that are vital for understanding, uncovering, and exploiting apoptotic vulnerabilities in cancer for the potential improvement of patient outcomes.