A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis.

Clear-cell carcinomas (CCCs) are a histological group of highly aggressive malignancies commonly originating in the kidney and ovary. CCCs are distinguished by aberrant lipid and glycogen accumulation and are refractory to a broad range of anti-cancer therapies. Here we identify an intrinsic vulnerability to ferroptosis associated with the unique metabolic state in CCCs. This vulnerability transcends lineage and genetic landscape, and can be exploited by inhibiting glutathione peroxidase 4 (GPX4) with small-molecules. Using CRISPR screening and lipidomic profiling, we identify the hypoxia-inducible factor (HIF) pathway as a driver of this vulnerability. In renal CCCs, HIF-2α selectively enriches polyunsaturated lipids, the rate-limiting substrates for lipid peroxidation, by activating the expression of hypoxia-inducible, lipid droplet-associated protein (HILPDA). Our study suggests targeting GPX4 as a therapeutic opportunity in CCCs, and highlights that therapeutic approaches can be identified on the basis of cell states manifested by morphological and metabolic features in hard-to-treat cancers.

Small-molecule inhibitors directly target CARD9 and mimic its protective variant in inflammatory bowel disease.

Advances in human genetics have dramatically expanded our understanding of complex heritable diseases. Genome-wide association studies have identified an allelic series of CARD9 variants associated with increased risk of or protection from inflammatory bowel disease (IBD). The predisposing variant of CARD9 is associated with increased NF-κB-mediated cytokine production. Conversely, the protective variant lacks a functional C-terminal domain and is unable to recruit the E3 ubiquitin ligase TRIM62. Here, we used biochemical insights into CARD9 variant proteins to create a blueprint for IBD therapeutics and recapitulated the mechanism of the CARD9 protective variant using small molecules. We developed a multiplexed bead-based technology to screen compounds for disruption of the CARD9-TRIM62 interaction. We identified compounds that directly and selectively bind CARD9, disrupt TRIM62 recruitment, inhibit TRIM62-mediated ubiquitinylation of CARD9, and demonstrate cellular activity and selectivity in CARD9-dependent pathways. Taken together, small molecules targeting CARD9 illustrate a path toward improved IBD therapeutics.

Ubiquitin Ligase TRIM62 Regulates CARD9-Mediated Anti-fungal Immunity and Intestinal Inflammation.

CARD9 is a central component of anti-fungal innate immune signaling via C-type lectin receptors, and several immune-related disorders are associated with CARD9 alterations. Here, we used a rare CARD9 variant that confers protection against inflammatory bowel disease as an entry point to investigating CARD9 regulation. We showed that the protective variant of CARD9, which is C-terminally truncated, acted in a dominant-negative manner for CARD9-mediated cytokine production, indicating an important role for the C terminus in CARD9 signaling. We identified TRIM62 as a CARD9 binding partner and showed that TRIM62 facilitated K27-linked poly-ubiquitination of CARD9. We identified K125 as the ubiquitinated residue on CARD9 and demonstrated that this ubiquitination was essential for CARD9 activity. Furthermore, we showed that similar to Card9-deficient mice, Trim62-deficient mice had increased susceptibility to fungal infection. In this study, we utilized a rare protective allele to uncover a TRIM62-mediated mechanism for regulation of CARD9 activation.

Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices.

Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of ∼ 30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequence-specific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85D(V12) activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer.

The retinoblastoma protein induces apoptosis directly at the mitochondria.

The retinoblastoma protein gene RB-1 is mutated in one-third of human tumors. Its protein product, pRB (retinoblastoma protein), functions as a transcriptional coregulator in many fundamental cellular processes. Here, we report a nonnuclear role for pRB in apoptosis induction via pRB's direct participation in mitochondrial apoptosis. We uncovered this activity by finding that pRB potentiated TNFα-induced apoptosis even when translation was blocked. This proapoptotic function was highly BAX-dependent, suggesting a role in mitochondrial apoptosis, and accordingly, a fraction of endogenous pRB constitutively associated with mitochondria. Remarkably, we found that recombinant pRB was sufficient to trigger the BAX-dependent permeabilization of mitochondria or liposomes in vitro. Moreover, pRB interacted with BAX in vivo and could directly bind and conformationally activate BAX in vitro. Finally, by targeting pRB specifically to mitochondria, we generated a mutant that lacked pRB's classic nuclear roles. This mito-tagged pRB retained the ability to promote apoptosis in response to TNFα and also additional apoptotic stimuli. Most importantly, induced expression of mito-tagged pRB in Rb(-/-);p53(-/-) tumors was sufficient to block further tumor development. Together, these data establish a nontranscriptional role for pRB in direct activation of BAX and mitochondrial apoptosis in response to diverse stimuli, which is profoundly tumor-suppressive.

Direct activation of full-length proapoptotic BAK.

Proapoptotic B-cell lymphoma 2 (BCL-2) antagonist/killer (BAK) and BCL-2-associated X (BAX) form toxic mitochondrial pores in response to cellular stress. Whereas BAX resides predominantly in the cytosol, BAK is constitutively localized to the outer mitochondrial membrane. Select BCL-2 homology domain 3 (BH3) helices activate BAX directly by engaging an α1/α6 trigger site. The inability to express full-length BAK has hampered full dissection of its activation mechanism. Here, we report the production of full-length, monomeric BAK by mutagenesis-based solubilization of its C-terminal α-helical surface. Recombinant BAK autotranslocates to mitochondria but only releases cytochrome c upon BH3 triggering. A direct activation mechanism was explicitly demonstrated using a liposomal system that recapitulates BAK-mediated release upon addition of BH3 ligands. Photoreactive BH3 helices mapped both triggering and autointeractions to the canonical BH3-binding pocket of BAK, whereas the same ligands crosslinked to the α1/α6 site of BAX. Thus, activation of both BAK and BAX is initiated by direct BH3-interaction but at distinct trigger sites. These structural and biochemical insights provide opportunities for developing proapoptotic agents that activate the death pathway through direct but differential engagement of BAK and BAX.

Direct and selective small-molecule activation of proapoptotic BAX.

BCL-2 family proteins are key regulators of the apoptotic pathway. Antiapoptotic members sequester the BCL-2 homology 3 (BH3) death domains of proapoptotic members such as BAX to maintain cell survival. The antiapoptotic BH3-binding groove has been successfully targeted to reactivate apoptosis in cancer. We recently identified a geographically distinct BH3-binding groove that mediates direct BAX activation, suggesting a new strategy for inducing apoptosis by flipping BAX's 'on switch'. Here we applied computational screening to identify a BAX activator molecule that directly and selectively activates BAX. We demonstrate by NMR and biochemical analyses that the molecule engages the BAX trigger site and promotes the functional oligomerization of BAX. The molecule does not interact with the BH3-binding pocket of antiapoptotic proteins or proapoptotic BAK and induces cell death in a BAX-dependent fashion. To our knowledge, we report the first gain-of-function molecular modulator of a BCL-2 family protein and demonstrate a new paradigm for pharmacologic induction of apoptosis.

Development of lipidoid-siRNA formulations for systemic delivery to the liver.

RNA interference therapeutics afford the potential to silence target gene expression specifically, thereby blocking production of disease-causing proteins. The development of safe and effective systemic small interfering RNA (siRNA) delivery systems is of central importance to the therapeutic application of siRNA. Lipid and lipid-like materials are currently the most well-studied siRNA delivery systems for liver delivery, having been utilized in several animal models, including nonhuman primates. Here, we describe the development of a multicomponent, systemic siRNA delivery system, based on the novel lipid-like material 98N(12)-5(1). We show that in vivo delivery efficacy is affected by many parameters, including the formulation composition, nature of particle PEGylation, degree of drug loading, and biophysical parameters such as particle size. In particular, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids can result in significant effects on in vivo efficacy. The lead formulation developed is liver targeted (>90% injected dose distributes to liver) and can induce fully reversible, long-duration gene silencing without loss of activity following repeat administration.

A combinatorial library of lipid-like materials for delivery of RNAi therapeutics.

The safe and effective delivery of RNA interference (RNAi) therapeutics remains an important challenge for clinical development. The diversity of current delivery materials remains limited, in part because of their slow, multi-step syntheses. Here we describe a new class of lipid-like delivery molecules, termed lipidoids, as delivery agents for RNAi therapeutics. Chemical methods were developed to allow the rapid synthesis of a large library of over 1,200 structurally diverse lipidoids. From this library, we identified lipidoids that facilitate high levels of specific silencing of endogenous gene transcripts when formulated with either double-stranded small interfering RNA (siRNA) or single-stranded antisense 2'-O-methyl (2'-OMe) oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids were evaluated in three animal models: mice, rats and nonhuman primates. The studies reported here suggest that these materials may have broad utility for both local and systemic delivery of RNA therapeutics.

Electrostatic ligand coatings of nanoparticles enable ligand-specific gene delivery to human primary cells.

A general method of coating polymer/DNA nanoparticles was developed. Peptide coated nanoparticles were found to have favorable biophysical characteristics including small particle size, near-neutral zeta potential, and stability in serum. At appropriate formulation conditions including near-neutral charge ratio, the coated nanoparticles enabled effective ligand-specific gene delivery to human primary endothelial cells in serum-containing media. As this nanoparticulate drug delivery system has high efficacy, ligand-based specificity, biodegradability, and low cytotoxicity, it may be potentially useful in several clinical applications.

Biodegradable polymeric vectors for gene delivery to human endothelial cells.

Endothelial cells are an important cell type to both cardiovascular disease and cancer, as they play critical roles in vascular function and angiogenesis. However, effective and safe gene delivery to primary endothelial cells in the presence of serum proteins is known to be particularly challenging. A library of biodegradable poly(beta-amino esters) was synthesized for use as potential vectors. Promising vectors were optimized for high efficacy and low cytotoxicity to human umbilical vein endothelial cells (HUVECs) in serum. Vector parameters including polymer type, polymer weight, and DNA loading were varied, and biophysical properties including particle size, zeta potential, and particle stability over time were studied. While many of the poly(beta-amino ester) vectors have similar biophysical properties in the presence of buffer, their biophysical properties changed differentially in the presence of serum proteins, and the properties of these serum-interacting particles correlated to transfection efficacy. Leading poly(beta-amino ester) vectors were found to transfect HUVECs in the presence of serum significantly higher (47 +/- 9% positive, n = 10) than the best commercially available transfection reagents including jetPEI (p < 0.001) and Lipofectamine 2000 (p < 0.01). These results demonstrate the potential of a new class of biomaterials, poly(beta-amino esters), for effective human endothelial cell gene therapy.