Targeting PIKfyve-driven lipid homeostasis as a metabolic vulnerability in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) subsists in a nutrient-deregulated microenvironment, making it particularly susceptible to treatments that interfere with cancer metabolism12. For example, PDAC utilizes and is dependent on high levels of autophagy and other lysosomal processes3-5. Although targeting these pathways has shown potential in preclinical studies, progress has been hampered by the challenge of identifying and characterizing favorable targets for drug development6. Here, we characterize PIKfyve, a lipid kinase integral to lysosomal functioning7, as a novel and targetable vulnerability in PDAC. In human patient and murine PDAC samples, we discovered that PIKFYVE is overexpressed in PDAC cells compared to adjacent normal cells. Employing a genetically engineered mouse model, we established the essential role of PIKfyve in PDAC progression. Further, through comprehensive metabolic analyses, we found that PIKfyve inhibition obligated PDAC to upregulate de novo lipid synthesis, a relationship previously undescribed. PIKfyve inhibition triggered a distinct lipogenic gene expression and metabolic program, creating a dependency on de novo lipid metabolism pathways, by upregulating genes such as FASN and ACACA. In PDAC, the KRAS-MAPK signaling pathway is a primary driver of de novo lipid synthesis, specifically enhancing FASN and ACACA levels. Accordingly, the simultaneous targeting of PIKfyve and KRAS-MAPK resulted in the elimination of tumor burden in a syngeneic orthotopic model and tumor regression in a xenograft model of PDAC. Taken together, these studies suggest that disrupting lipid metabolism through PIKfyve inhibition induces synthetic lethality in conjunction with KRAS-MAPK-directed therapies for PDAC.

Recharacterization of RSL3 reveals that the selenoproteome is a druggable target in colorectal cancer.

Ferroptosis is an iron-dependent, non-apoptotic form of cell death resulting from the accumulation of lipid peroxides. Colorectal cancer (CRC) accumulates high levels of intracellular iron and reactive oxygen species (ROS), thereby sensitizing cells to ferroptosis. The selenoprotein glutathione peroxidase (GPx4) is a key enzyme in the detoxification of lipid peroxides and can be inhibited by the compound (S)-RSL3 ([1S,3R]-RSL3). However, the stereoisomer (R)-RSL3 ([1R,3R]-RSL3), which does not inhibit GPx4, exhibits equipotent activity to (S)-RSL3 across a panel of CRC cell lines. Utilizing CRC cell lines with an inducible knockdown of GPx4, we demonstrate that (S)-RSL3 sensitivity does not align with GPx4 dependency. Subsequently, a biotinylated (S)-RSL3 was then synthesized to perform affinity purification-mass spectrometry (AP-MS), revealing that (S)-RSL3 acts as a pan-inhibitor of the selenoproteome, targeting both the glutathione and thioredoxin peroxidase systems as well as multiple additional selenoproteins. To investigate the therapeutic potential of broadly disrupting the selenoproteome as a therapeutic strategy in CRC, we employed further chemical and genetic approaches to disrupt selenoprotein function. The findings demonstrate that the selenoprotein inhibitor Auranofin can induce ferroptosis and/or oxidative cell death both in-vitro and in-vivo. Consistent with this data we observe that AlkBH8, a tRNA-selenocysteine methyltransferase required for the translational incorporation of selenocysteine, is essential for CRC growth. In summary, our research elucidates the complex mechanisms underlying ferroptosis in CRC and reveals that modulation of the selenoproteome provides multiple new therapeutic targets and opportunities in CRC.

BRD4-mediated epigenetic regulation of endoplasmic reticulum-mitochondria contact sites is governed by the mitochondrial complex III.

Inter-organellar communication is critical for cellular metabolic homeostasis. One of the most abundant inter-organellar interactions are those at the endoplasmic reticulum and mitochondria contact sites (ERMCS). However, a detailed understanding of the mechanisms governing ERMCS regulation and their roles in cellular metabolism are limited by a lack of tools that permit temporal induction and reversal. Through unbiased screening approaches, we identified fedratinib, an FDA-approved drug, that dramatically increases ERMCS abundance by inhibiting the epigenetic modifier BRD4. Fedratinib rapidly and reversibly modulates mitochondrial and ER morphology and alters metabolic homeostasis. Moreover, ERMCS modulation depends on mitochondria electron transport chain complex III function. Comparison of fedratinib activity to other reported inducers of ERMCS revealed common mechanisms of induction and function, providing clarity and union to a growing body of experimental observations. In total, our results uncovered a novel epigenetic signaling pathway and an endogenous metabolic regulator that connects ERMCS and cellular metabolism.

Conditional lethality profiling reveals anticancer mechanisms of action and drug-nutrient interactions.

Chemical screening studies have identified drug sensitivities across hundreds of cancer cell lines but most putative therapeutics fail to translate. Discovery and development of drug candidates in models that more accurately reflect nutrient availability in human biofluids may help in addressing this major challenge. Here we performed high-throughput screens in conventional versus Human Plasma-Like Medium (HPLM). Sets of conditional anticancer compounds span phases of clinical development and include non-oncology drugs. Among these, we characterize a unique dual-mechanism of action for brivudine, an agent otherwise approved for antiviral treatment. Using an integrative approach, we find that brivudine affects two independent targets in folate metabolism. We also traced conditional phenotypes for several drugs to the availability of nucleotide salvage pathway substrates and verified others for compounds that seemingly elicit off-target anticancer effects. Our findings establish generalizable strategies for exploiting conditional lethality in HPLM to reveal therapeutic candidates and mechanisms of action.

CRISPR/Cas9 Screening to Identify Conditionally Essential Genes in Human Cell Lines.

Forward genetic screens across hundreds of cancer cell lines have started to define the genetic dependencies of proliferating human cells. However, most such screens have been performed in vitro with little consideration into how medium composition might affect gene essentiality. This protocol describes a method to use CRISPR/Cas9-based loss-of-function screens to ask how gene essentiality in human cell lines varies with medium composition. First, a single-guide RNA (sgRNA) library is packaged into lentivirus, and an optimal infection titer is determined for the target cells. Following selection, genomic DNA (gDNA) is extracted from an aliquot of the transduced cells. The remaining transduced cells are then screened in at least two distinct cell culture media. At the conclusion of the screening period, gDNA is collected from each cell population. Next, high-throughput sequencing is used to determine sgRNA barcode abundances from the initial and each of the final populations. Finally, an analytical pipeline is used to identify medium-essential candidate genes from these screen results.

CRISPR screens in physiologic medium reveal conditionally essential genes in human cells.

Forward genetic screens across hundreds of cancer cell lines have started to define the genetic dependencies of proliferating human cells and how these vary by genotype and lineage. Most screens, however, have been carried out in culture media that poorly reflect metabolite availability in human blood. Here, we performed CRISPR-based screens in traditional versus human plasma-like medium (HPLM). Sets of conditionally essential genes in human cancer cell lines span several cellular processes and vary with both natural cell-intrinsic diversity and the combination of basal and serum components that comprise typical media. Notably, we traced the causes for each of three conditional CRISPR phenotypes to the availability of metabolites uniquely defined in HPLM versus conventional media. Our findings reveal the profound impact of medium composition on gene essentiality in human cells, and also suggest general strategies for using genetic screens in HPLM to uncover new cancer vulnerabilities and gene-nutrient interactions.