Benzoxazole-derivatives enhance progranulin expression and reverse the aberrant lysosomal proteome caused by GRN haploinsufficiency.

Heterozygous loss-of-function mutations in the GRN gene are a major cause of hereditary frontotemporal dementia. The mechanisms linking frontotemporal dementia pathogenesis to progranulin deficiency are not well understood, and there is currently no treatment. Our strategy to prevent the onset and progression of frontotemporal dementia in patients with GRN mutations is to utilize small molecule positive regulators of GRN expression to boost progranulin levels from the remaining functional GRN allele, thus restoring progranulin levels back to normal within the brain. This work describes a series of blood-brain-barrier-penetrant small molecules which significantly increase progranulin protein levels in human cellular models, correct progranulin protein deficiency in Grn+/- mouse brains, and reverse lysosomal proteome aberrations, a phenotypic hallmark of frontotemporal dementia, more efficiently than the previously described small molecule suberoylanilide hydroxamic acid. These molecules will allow further elucidation of the cellular functions of progranulin and its role in frontotemporal dementia and will also serve as lead structures for further drug development.

A cholesterol-binding bacterial toxin provides a strategy for identifying a specific Scap inhibitor that blocks lipid synthesis in animal cells.

Lipid synthesis is regulated by the actions of Scap, a polytopic membrane protein that binds cholesterol in membranes of the endoplasmic reticulum (ER). When ER cholesterol levels are low, Scap activates SREBPs, transcription factors that upregulate genes for synthesis of cholesterol, fatty acids, and triglycerides. When ER cholesterol levels rise, the sterol binds to Scap, triggering conformational changes that prevent activation of SREBPs and halting synthesis of lipids. To achieve a molecular understanding of how cholesterol regulates the Scap/SREBP machine and to identify therapeutics for dysregulated lipid metabolism, cholesterol-mimetic compounds that specifically bind and inhibit Scap are needed. To accomplish this goal, we focused on Anthrolysin O (ALO), a pore-forming bacterial toxin that binds cholesterol with a specificity and sensitivity that is uncannily similar to Scap. We reasoned that a small molecule that would bind and inhibit ALO might also inhibit Scap. High-throughput screening of a ~300,000-compound library for ALO-binding unearthed one molecule, termed UT-59, which binds to Scap's cholesterol-binding site. Upon binding, UT-59 triggers the same conformation changes in Scap as those induced by cholesterol and blocks activation of SREBPs and lipogenesis in cultured cells. UT-59 also inhibits SREBP activation in the mouse liver. Unlike five previously reported inhibitors of SREBP activation, UT-59 is the only one that acts specifically by binding to Scap's cholesterol-binding site. Our approach to identify specific Scap inhibitors such as UT-59 holds great promise in developing therapeutic leads for human diseases stemming from elevated SREBP activation, such as fatty liver and certain cancers.

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules.

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and new therapeutic leads. In selected cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

Small molecule glucagon release inhibitors with activity in human islets.

Purpose: Type 1 diabetes (T1D) accounts for an estimated 5% of all diabetes in the United States, afflicting over 1.25 million individuals. Maintaining long-term blood glucose control is the major goal for individuals with T1D. In T1D, insulin-secreting pancreatic islet ß-cells are destroyed by the immune system, but glucagon-secreting islet α-cells survive. These remaining α-cells no longer respond properly to fluctuating blood glucose concentrations. Dysregulated α-cell function contributes to hyper- and hypoglycemia which can lead to macrovascular and microvascular complications. To this end, we sought to discover small molecules that suppress α-cell function for their potential as preclinical candidate compounds. Prior high-throughput screening identified a set of glucagon-suppressing compounds using a rodent α-cell line model, but these compounds were not validated in human systems. Results: Here, we dissociated and replated primary human islet cells and exposed them to 24 h treatment with this set of candidate glucagon-suppressing compounds. Glucagon accumulation in the medium was measured and we determined that compounds SW049164 and SW088799 exhibited significant activity. Candidate compounds were also counter-screened in our InsGLuc-MIN6 ß-cell insulin secretion reporter assay. SW049164 and SW088799 had minimal impact on insulin release after a 24 h exposure. To further validate these hits, we treated intact human islets with a selection of the top candidates for 24 h. SW049164 and SW088799 significantly inhibited glucagon release into the medium without significantly altering whole islet glucagon or insulin content. In concentration-response curves SW088799 exhibited significant inhibition of glucagon release with an IC50 of 1.26 µM. Conclusion: Given the set of tested candidates were all top hits from the primary screen in rodent α-cells, this suggests some conservation of mechanism of action between human and rodents, at least for SW088799. Future structure-activity relationship studies of SW088799 may aid in elucidating its protein target(s) or enable its use as a tool compound to suppress α-cell activity in vitro.

Inducible mismatch repair streamlines forward genetic approaches to target identification of cytotoxic small molecules.

Orphan cytotoxins are small molecules for which the mechanism of action (MoA) is either unknown or ambiguous. Unveiling the mechanism of these compounds may lead to useful tools for biological investigation and in some cases, new therapeutic leads. In select cases, the DNA mismatch repair-deficient colorectal cancer cell line, HCT116, has been used as a tool in forward genetic screens to identify compound-resistant mutations, which have ultimately led to target identification. To expand the utility of this approach, we engineered cancer cell lines with inducible mismatch repair deficits, thus providing temporal control over mutagenesis. By screening for compound resistance phenotypes in cells with low or high rates of mutagenesis, we increased both the specificity and sensitivity of identifying resistance mutations. Using this inducible mutagenesis system, we implicate targets for multiple orphan cytotoxins, including a natural product and compounds emerging from a high-throughput screen, thus providing a robust tool for future MoA studies.

Fast-Killing Tyrosine Amide ((S)-SW228703) with Blood- and Liver-Stage Antimalarial Activity Associated with the Cyclic Amine Resistance Locus (PfCARL).

Current malaria treatments are threatened by drug resistance, and new drugs are urgently needed. In a phenotypic screen for new antimalarials, we identified (S)-SW228703 ((S)-SW703), a tyrosine amide with asexual blood and liver stage activity and a fast-killing profile. Resistance to (S)-SW703 is associated with mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) and P. falciparum acetyl CoA transporter (PfACT), similarly to several other compounds that share features such as fast activity and liver-stage activity. Compounds with these resistance mechanisms are thought to act in the ER, though their targets are unknown. The tyramine of (S)-SW703 is shared with some reported PfCARL-associated compounds; however, we observed that strict S-stereochemistry was required for the activity of (S)-SW703, suggesting differences in the mechanism of action or binding mode. (S)-SW703 provides a new chemical series with broad activity for multiple life-cycle stages and a fast-killing mechanism of action, available for lead optimization to generate new treatments for malaria.

GSK3 inhibition reduces ECM production and prevents age-related macular degeneration-like pathology.

Malattia Leventinese/Doyne Honeycomb Retinal Dystrophy (ML/DHRD) is an age-related macular degeneration (AMD)-like retinal dystrophy caused by an autosomal dominant R345W mutation in the secreted glycoprotein, fibulin-3 (F3). To identify new small molecules that reduce F3 production from retinal pigmented epithelium (RPE) cells, we knocked-in a luminescent peptide tag (HiBiT) into the endogenous F3 locus which enabled simple, sensitive, and high throughput detection of the protein. The GSK3 inhibitor, CHIR99021 (CHIR), significantly reduced F3 burden (expression, secretion, and intracellular levels) in immortalized RPE and non-RPE cells. Low-level, long-term CHIR treatment promoted remodeling of the RPE extracellular matrix (ECM), reducing sub-RPE deposit-associated proteins (e.g., amelotin, complement component 3, collagen IV, and fibronectin), while increasing RPE differentiation factors (e.g., tyrosinase, and pigment epithelium derived factor). In vivo, treatment of 8 mo R345W+/+ knockin mice with CHIR (25 mg/kg i.p., 1 mo) was well tolerated and significantly reduced R345W F3-associated AMD-like basal laminar deposit number and size, thereby preventing the main pathological feature in these mice. This is the first demonstration of small molecule-based prevention of AMD-like pathology in ML/DHRD mice and may herald a rejuvenation of interest in GSK3 inhibition for the treatment of neurodegenerative diseases, including, potentially AMD itself.

Targeting ESR1 mutation-induced transcriptional addiction in breast cancer with BET inhibition.

Acquired mutations in the ligand-binding domain (LBD) of the gene encoding estrogen receptor α (ESR1) are common mechanisms of endocrine therapy resistance in patients with metastatic ER+ breast cancer. The ESR1 Y537S mutation, in particular, is associated with development of resistance to most endocrine therapies used to treat breast cancer. Employing a high-throughput screen of nearly 1,200 Federal Drug Administration-approved (FDA-approved) drugs, we show that OTX015, a bromodomain and extraterminal domain (BET) inhibitor, is one of the top suppressors of ESR1 mutant cell growth. OTX015 was more efficacious than fulvestrant, a selective ER degrader, in inhibiting ESR1 mutant xenograft growth. When combined with abemaciclib, a CDK4/6 inhibitor, OTX015 induced more potent tumor regression than current standard-of-care treatment of abemaciclib + fulvestrant. OTX015 has preferential activity against Y537S mutant breast cancer cells and blocks their clonal selection in competition studies with WT cells. Thus, BET inhibition has the potential to both prevent and overcome ESR1 mutant-induced endocrine therapy resistance in breast cancer.

Small Molecule-mediated Insulin Hypersecretion Induces Transient ER Stress Response and Loss of Beta Cell Function.

Pancreatic islet beta cells require a fine-tuned endoplasmic reticulum (ER) stress response for normal function; abnormal ER stress contributes to diabetes pathogenesis. Here, we identified a small molecule, SW016789, with time-dependent effects on beta cell ER stress and function. Acute treatment with SW016789 potentiated nutrient-induced calcium influx and insulin secretion, while chronic exposure to SW016789 transiently induced ER stress and shut down secretory function in a reversible manner. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or apoptosis, potentially due to a rapid induction of adaptive genes, weak signaling through the eIF2α kinase PERK, and lack of oxidative stress gene Txnip induction. We determined that SW016789 acted upstream of voltage-dependent calcium channels (VDCCs) and potentiated nutrient- but not KCl-stimulated calcium influx. Measurements of metabolomics, oxygen consumption rate, and G protein-coupled receptor signaling did not explain the potentiating effects of SW016789. In chemical cotreatment experiments, we discovered synergy between SW016789 and activators of protein kinase C and VDCCs, suggesting involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as blockade of VDCCs protected human islets and MIN6 beta cells from hypersecretion-induced dysfunction. We conclude that beta cells undergoing this type of pharmacological hypersecretion have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.

Total synthesis of (+)-spiroindimicin A and congeners unveils their antiparasitic activity.

The spiroindimicins are a unique class of chlorinated indole alkaloids characterized by three heteroaromatic rings structured around a congested spirocyclic stereocenter. Here, we report the first total synthesis of (+)-spiroindimicin A, which bears a challenging C-3'/C-5''-linked spiroindolenine. We detail our initial efforts to effect a biomimetic oxidative spirocyclization from its proposed natural precursor, lynamicin D, and describe how these studies shaped our final abiotic 9-step solution to this complex alkaloid built around a key Pd-catalyzed asymmetric spirocyclization. Scalable access to spiroindimicins A, H, and their congeners has enabled discovery of their activity against several parasites relevant to human health, providing potential starting points for new therapeutics for the neglected tropical diseases leishmaniasis and African sleeping sickness.

Concerted cell and in vivo screen for pancreatic ductal adenocarcinoma (PDA) chemotherapeutics.

PDA is a major cause of US cancer-related deaths. Oncogenic Kras presents in 90% of human PDAs. Kras mutations occur early in pre-neoplastic lesions but are insufficient to cause PDA. Other contributing factors early in disease progression include chronic pancreatitis, alterations in epigenetic regulators, and tumor suppressor gene mutation. GPCRs activate heterotrimeric G-proteins that stimulate intracellular calcium and oncogenic Kras signaling, thereby promoting pancreatitis and progression to PDA. By contrast, Rgs proteins inhibit Gi/q-coupled GPCRs to negatively regulate PDA progression. Rgs16::GFP is expressed in response to caerulein-induced acinar cell dedifferentiation, early neoplasia, and throughout PDA progression. In genetically engineered mouse models of PDA, Rgs16::GFP is useful for pre-clinical rapid in vivo validation of novel chemotherapeutics targeting early lesions in patients following successful resection or at high risk for progressing to PDA. Cultured primary PDA cells express Rgs16::GFP in response to cytotoxic drugs. A histone deacetylase inhibitor, TSA, stimulated Rgs16::GFP expression in PDA primary cells, potentiated gemcitabine and JQ1 cytotoxicity in cell culture, and Gem + TSA + JQ1 inhibited tumor initiation and progression in vivo. Here we establish the use of Rgs16::GFP expression for testing drug combinations in cell culture and validation of best candidates in our rapid in vivo screen.

The structure of importin α and the nuclear localization peptide of ChREBP, and small compound inhibitors of ChREBP-importin α interactions.

The carbohydrate response element binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in glucose-mediated induction of genes involved in hepatic glycolysis and lipogenesis. In response to fluctuating blood glucose levels ChREBP activity is regulated mainly by nucleocytoplasmic shuttling of ChREBP. Under high glucose ChREBP binds to importin α and importin ß and translocates into the nucleus to initiate transcription. We have previously shown that the nuclear localization signal site (NLS) for ChREBP is bipartite with the NLS extending from Arg158 to Lys190. Here, we report the 2.5 Šcrystal structure of the ChREBP-NLS peptide bound to importin α. The structure revealed that the NLS binding is monopartite, with the amino acid residues K171RRI174 from the ChREBP-NLS interacting with ARM2-ARM5 on importin α. We discovered that importin α also binds to the primary binding site of the 14-3-3 proteins with high affinity, which suggests that both importin α and 14-3-3 are each competing with the other for this broad-binding region (residues 117-196) on ChREBP. We screened a small compound library and identified two novel compounds that inhibit the ChREBP-NLS/importin α interaction, nuclear localization, and transcription activities of ChREBP. These candidate molecules support developing inhibitors of ChREBP that may be useful in treatment of obesity and the associated diseases.

Simultaneous Control of Endogenous and User-Defined Genetic Pathways Using Unique ecDHFR Pharmacological Chaperones.

Destabilizing domains (DDs), such as a mutated form of Escherichia coli dihydrofolate reductase (ecDHFR), confer instability and promote protein degradation. However, when combined with small-molecule stabilizers (e.g., the antibiotic trimethoprim), DDs allow positive regulation of fusion protein abundance. Using a combinatorial screening approach, we identified and validated 17 unique 2,4-diaminopyrimidine/triazine-based ecDHFR DD stabilizers, at least 15 of which were ineffective antibiotics against E. coli and S. aureus. Identified stabilizers functioned in vivo to control an ecDHFR DD-firefly luciferase in the mouse eye and/or the liver. Next, stabilizers were leveraged to perform synergistic dual functions in vitro (HeLa cell death sensitization) and in vivo (repression of ocular inflammation) by stabilizing a user-defined ecDHFR DD while also controlling endogenous signaling pathways. Thus, these newly identified pharmacological chaperones allow for simultaneous control of compound-specific endogenous and user-defined genetic pathways, the combination of which may provide synergistic effects in complex biological scenarios.

Interaction between the autophagy protein Beclin 1 and Na+,K+-ATPase during starvation, exercise, and ischemia.

Autosis is a distinct form of cell death that requires both autophagy genes and the Na+,K+-ATPase pump. However, the relationship between the autophagy machinery and Na+,K+-ATPase is unknown. We explored the hypothesis that Na+,K+-ATPase interacts with the autophagy protein Beclin 1 during stress and autosis-inducing conditions. Starvation increased the Beclin 1/Na+,K+-ATPase interaction in cultured cells, and this was blocked by cardiac glycosides, inhibitors of Na+,K+-ATPase. Increases in Beclin 1/Na+,K+-ATPase interaction were also observed in tissues from starved mice, livers of patients with anorexia nervosa, brains of neonatal rats subjected to cerebral hypoxia-ischemia (HI), and kidneys of mice subjected to renal ischemia/reperfusion injury (IRI). Cardiac glycosides blocked the increased Beclin 1/Na+,K+-ATPase interaction during cerebral HI injury and renal IRI. In the mouse renal IRI model, cardiac glycosides reduced numbers of autotic cells in the kidney and improved clinical outcome. Moreover, blockade of endogenous cardiac glycosides increased Beclin 1/Na+,K+-ATPase interaction and autotic cell death in mouse hearts during exercise. Thus, Beclin 1/Na+,K+-ATPase interaction is increased in stress conditions, and cardiac glycosides decrease this interaction and autosis in both pathophysiological and physiological settings. This crosstalk between cellular machinery that generates and consumes energy during stress may represent a fundamental homeostatic mechanism.

1,25-Dihydroxyvitamin D3 enhances glucose-stimulated insulin secretion in mouse and human islets: a role for transcriptional regulation of voltage-gated calcium channels by the vitamin D receptor.

AIM: Vitamin D deficiency in rodents negatively affects glucose-stimulated insulin secretion (GSIS) and human epidemiological studies connect poor vitamin D status with type 2 diabetes. Previous studies performed primarily in rat islets have shown that vitamin D can enhance GSIS. However the molecular pathways linking vitamin D and insulin secretion are currently unknown. Therefore, experiments were undertaken to elucidate the transcriptional role(s) of the vitamin D receptor (VDR) in islet function. METHODS: Human and mouse islets were cultured with vehicle or 1,25-dihydroxyvitamin-D3 (1,25D3) and then subjected to GSIS assays. Insulin expression, insulin content, glucose uptake and glucose-stimulated calcium influx were tested. Microarray analysis was performed. In silico analysis was used to identify VDR response elements (VDRE) within target genes and their activity was tested using reporter assays. RESULTS: Vdr mRNA is abundant in islets and Vdr expression is glucose-responsive. Preincubation of mouse and human islets with 1,25D3 enhances GSIS and increases glucose-stimulated calcium influx. Microarray analysis identified the R-type voltage-gated calcium channel (VGCC) gene, Cacna1e, which is highly upregulated by 1,25D3 in human and mouse islets and contains a conserved VDRE in intron 7. Results from GSIS assays suggest that 1,25D3 might upregulate a variant of R-type VGCC that is resistant to chemical inhibition. CONCLUSION: These results suggest that the role of 1,25D3 in regulating calcium influx acts through the R-Type VGCC during GSIS, thereby modulating the capacity of beta cells to secrete insulin.

HORMAD1 Is a Negative Prognostic Indicator in Lung Adenocarcinoma and Specifies Resistance to Oxidative and Genotoxic Stress.

Cancer testis antigens (CTA) are expressed in testis and placenta and anomalously activated in a variety of tumors. The mechanistic contribution of CTAs to neoplastic phenotypes remains largely unknown. Using a chemigenomics approach, we find that the CTA HORMAD1 correlates with resistance to the mitochondrial complex I inhibitor piericidin A in non-small cell lung cancer (NSCLC). Resistance was due to a reductive intracellular environment that attenuated the accumulation of free radicals. In human lung adenocarcinoma (LUAD) tumors, patients expressing high HORMAD1 exhibited elevated mutational burden and reduced survival. HORMAD1 tumors were enriched for genes essential for homologous recombination (HR), and HORMAD1 promoted RAD51-filament formation, but not DNA resection, during HR. Accordingly, HORMAD1 loss enhanced sensitivity to γ-irradiation and PARP inhibition, and HORMAD1 depletion significantly reduced tumor growth in vivo These results suggest that HORMAD1 expression specifies a novel subtype of LUAD, which has adapted to mitigate DNA damage. In this setting, HORMAD1 could represent a direct target for intervention to enhance sensitivity to DNA-damaging agents or as an immunotherapeutic target in patients.Significance: This study uses a chemigenomics approach to demonstrate that anomalous expression of the CTA HORMAD1 specifies resistance to oxidative stress and promotes HR to support tumor cell survival in NSCLC. Cancer Res; 78(21); 6196-208. ©2018 AACR.

High-Throughput Screens To Identify Autophagy Inducers That Function by Disrupting Beclin 1/Bcl-2 Binding.

Autophagy, a lysosomal degradation pathway, plays a crucial role in cellular homeostasis, development, immunity, tumor suppression, metabolism, prevention of neurodegeneration, and lifespan extension. Thus, pharmacological stimulation of autophagy may be an effective approach for preventing or treating certain human diseases and/or aging. We sought to establish a method for developing new chemical compounds that specifically induce autophagy. To do this, we developed two assays to identify compounds that target a key regulatory node of autophagy induction-specifically, the binding of Bcl-2 (a negative regulator of autophagy) to Beclin 1 (an allosteric modulator of the Beclin 1/VPS34 lipid kinase complex that functions in autophagy initiation). These assays use either a split-luciferase assay to measure Beclin 1/Bcl-2 binding in cells or an AlphaLISA assay to directly measure direct Beclin 1/Bcl-2 binding in vitro. We screened two different chemical compound libraries, comprising ∼300 K compounds, to identify small molecules that disrupt Beclin 1/Bcl-2 binding and induce autophagy. Three novel compounds were identified that directly inhibit Beclin 1/Bcl-2 interaction with an IC50 in the micromolar range and increase autophagic flux. These compounds do not demonstrate significant cytotoxicity, and they exert selectivity for disruption of Bcl-2 binding to the BH3 domain of Beclin 1 compared with the BH3 domain of the pro-apoptotic Bcl-2 family members, Bax and Bim. Thus, we have identified candidate molecules that serve as lead templates for developing potent and selective Beclin 1/Bcl-2 inhibitors that may be clinically useful as autophagy-inducing agents.

Discovery of Cytochrome P450 4F11 Activated Inhibitors of Stearoyl Coenzyme A Desaturase.

Stearoyl-CoA desaturase (SCD) catalyzes the first step in the conversion of saturated fatty acids to unsaturated fatty acids. Unsaturated fatty acids are required for membrane integrity and for cell proliferation. For these reasons, inhibitors of SCD represent potential treatments for cancer. However, systemically active SCD inhibitors result in skin toxicity, which presents an obstacle to their development. We recently described a series of oxalic acid diamides that are converted into active SCD inhibitors within a subset of cancers by CYP4F11-mediated metabolism. Herein, we describe the optimization of the oxalic acid diamides and related N-acyl ureas and an analysis of the structure-activity relationships related to metabolic activation and SCD inhibition.

Author Correction: Small-molecule TFEB pathway agonists that ameliorate metabolic syndrome in mice and extend C. elegans lifespan.

The originally published version of this Article contained an error in the spelling of the author Nathaniel W. Oswald, which was incorrectly given as Nathaniel W. Olswald. This has now been corrected in both the PDF and HTML versions of the Article.

Chemistry-First Approach for Nomination of Personalized Treatment in Lung Cancer.

Diversity in the genetic lesions that cause cancer is extreme. In consequence, a pressing challenge is the development of drugs that target patient-specific disease mechanisms. To address this challenge, we employed a chemistry-first discovery paradigm for de novo identification of druggable targets linked to robust patient selection hypotheses. In particular, a 200,000 compound diversity-oriented chemical library was profiled across a heavily annotated test-bed of >100 cellular models representative of the diverse and characteristic somatic lesions for lung cancer. This approach led to the delineation of 171 chemical-genetic associations, shedding light on the targetability of mechanistic vulnerabilities corresponding to a range of oncogenotypes present in patient populations lacking effective therapy. Chemically addressable addictions to ciliogenesis in TTC21B mutants and GLUT8-dependent serine biosynthesis in KRAS/KEAP1 double mutants are prominent examples. These observations indicate a wealth of actionable opportunities within the complex molecular etiology of cancer.