KRAS Allelic Imbalance Enhances Fitness and Modulates MAP Kinase Dependence in Cancer.

Investigating therapeutic "outliers" that show exceptional responses to anti-cancer treatment can uncover biomarkers of drug sensitivity. We performed preclinical trials investigating primary murine acute myeloid leukemias (AMLs) generated by retroviral insertional mutagenesis in KrasG12D "knockin" mice with the MEK inhibitor PD0325901 (PD901). One outlier AML responded and exhibited intrinsic drug resistance at relapse. Loss of wild-type (WT) Kras enhanced the fitness of the dominant clone and rendered it sensitive to MEK inhibition. Similarly, human colorectal cancer cell lines with increased KRAS mutant allele frequency were more sensitive to MAP kinase inhibition, and CRISPR-Cas9-mediated replacement of WT KRAS with a mutant allele sensitized heterozygous mutant HCT116 cells to treatment. In a prospectively characterized cohort of patients with advanced cancer, 642 of 1,168 (55%) with KRAS mutations exhibited allelic imbalance. These studies demonstrate that serial genetic changes at the Kras/KRAS locus are frequent in cancer and modulate competitive fitness and MEK dependency.

A saturation mutagenesis screen uncovers resistant and sensitizing secondary KRAS mutations to clinical KRASG12C inhibitors.

Mutant-specific inhibitors of KRASG12C, such as AMG510 (sotorasib) and MRTX849 (adagrasib), offer the unprecedented opportunity to inhibit KRAS, the most frequently mutated and heretofore undruggable oncoprotein. While clinical data are still limited, on-target mutations in KRASG12C at position 12 and other sites are emerging as major drivers of clinical relapse. We identified additional mutations in KRASG12C that impact inhibitor sensitivity through a saturation mutagenesis screen in the KRASG12C NCI-H358 non­small-cell lung cancer (NSCLC) cell line. We also identified individuals in population genetic databases harboring these resistance mutations in their germline and in tumors, including a subset that co-occur with KRASG12C, indicating that these mutations may preexist in patients treated with KRASG12C inhibitors. Notably, through structural modeling, we found that one such mutation (R68L) interferes with the critical protein­drug interface, conferring resistance to both inhibitors. Finally, we uncovered a mutant (S17E) that demonstrated a strong sensitizing phenotype to both inhibitors. Functional studies suggest that S17E sensitizes KRASG12C cells to KRASG12C inhibition by impacting signaling through PI3K/AKT/mTOR but not the MAPK signaling pathway. Our studies highlight the utility of unbiased mutation profiling to understand the functional consequences of all variants of a disease-causing genetic mutant and predict acquired resistant mutations in the targeted therapeutics.

AzidoTMT Enables Direct Enrichment and Highly Multiplexed Quantitation of Proteome-Wide Functional Residues.

Recent advances in targeted covalent inhibitors have aroused significant interest for their potential in drug development for difficult therapeutic targets. Proteome-wide profiling of functional residues is an integral step of covalent drug discovery aimed at defining actionable sites and evaluating compound selectivity in cells. A classical workflow for this purpose is called IsoTOP-ABPP, which employs an activity-based probe and two isotopically labeled azide-TEV-biotin tags to mark, enrich, and quantify proteome from two samples. Here we report a novel isobaric 11plex-AzidoTMT reagent and a new workflow, named AT-MAPP, that significantly expands multiplexing power as compared to the original isoTOP-ABPP. We demonstrate its application in identifying cysteine on- and off-targets using a KRAS G12C covalent inhibitor ARS-1620. However, changes in some of these hits can be explained by modulation at the protein and post-translational levels. Thus, it would be crucial to interrogate site-level bona fide changes in concurrence to proteome-level changes for corroboration. In addition, we perform a multiplexed covalent fragment screening using four acrylamide-based compounds as a proof-of-concept. This study identifies a diverse set of liganded cysteine residues in a compound-dependent manner with an average hit rate of 0.07% in intact cell. Lastly, we screened 20 sulfonyl fluoride-based compounds to demonstrate that the AT-MAPP assay is flexible for noncysteine functional residues such as tyrosine and lysine. Overall, we envision that 11plex-AzidoTMT will be a useful addition to the current toolbox for activity-based protein profiling and covalent drug development.

Overall survival in patients with advanced non-small cell lung cancer with KRAS G12C mutation with or without STK11 and/or KEAP1 mutations in a real-world setting.

BACKGROUND: KRAS mutations occur frequently in advanced non-small cell lung cancer (aNSCLC); the G12C mutation is the most prevalent. Alterations in STK11 or KEAP1 commonly co-occur with KRAS mutations in aNSCLC. Using real-world data, we assessed the effect of KRAS G12C mutation with or without STK11 and/or KEAP1 mutations on overall survival (OS) in patients with aNSCLC receiving cancer immunotherapy (CIT), chemotherapy, or both in first line (1L) and second line (2L). METHODS: Patients diagnosed with aNSCLC between January 2011 and March 2020 in a clinico-genomic database were included. Cox proportional hazards models adjusted for left truncation, baseline demographics and clinical characteristics were used to analyze the effect of STK11 and/or KEAP1 co-mutational status on OS in patients with KRAS wild-type (WT) or G12C mutation. RESULTS: Of 2715 patients with aNSCLC without other actionable driver mutations, 1344 (49.5%) had KRAS WT cancer, and 454 (16.7%) had KRAS G12C-positive cancer. At 1L treatment start, significantly more patients with KRAS G12C-positive cancer were female, smokers, and had non-squamous histology, a higher prevalence of metastasis and programmed death-ligand 1 positivity than those with KRAS WT cancer. Median OS was comparable between patients with KRAS G12C-positive and KRAS WT cancer when receiving chemotherapy or combination CIT and chemotherapy in the 1L or 2L. Median OS was numerically longer in patients with KRAS G12C vs KRAS WT cancer treated with 1L CIT (30.2 vs 10.6 months, respectively) or 2L CIT (11.3 vs 7.6 months, respectively). Co-mutation of STK11 and KEAP1 was associated with significantly shorter OS in patients receiving any type of 1L therapy, regardless of KRAS G12C mutational status. CONCLUSIONS: This real-world study showed that patients with KRAS G12C-positive or KRAS WT cancer have similar OS in the 1L or 2L when treated with chemotherapy or combination CIT and chemotherapy. In contrast to aNSCLC patients with EGFR or ALK driver mutations, patients with KRAS G12C-positive cancer may benefit from CIT monotherapy. Co-mutation of STK11 and KEAP1 was associated with significantly shorter survival, independent of KRAS G12C mutational status, reflecting the poor prognosis and high unmet need in this patient population.

Structure-based optimization of hydroxylactam as potent, cell-active inhibitors of lactate dehydrogenase.

Structure-based design was utilized to optimize 6,6-diaryl substituted dihydropyrone and hydroxylactam to obtain inhibitors of lactate dehydrogenase (LDH) with low nanomolar biochemical and single-digit micromolar cellular potencies. Surprisingly the replacement of a phenyl with a pyridyl moiety in the chemical structure revealed a new binding mode for the inhibitors with subtle conformational change of the LDHA active site. This led to the identification of a potent, cell-active hydroxylactam inhibitor exhibiting an in vivo pharmacokinetic profile suitable for mouse tumor xenograft study.

Double genetic disruption of lactate dehydrogenases A and B is required to ablate the "Warburg effect" restricting tumor growth to oxidative metabolism.

Increased glucose consumption distinguishes cancer cells from normal cells and is known as the "Warburg effect" because of increased glycolysis. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme, a hallmark of aggressive cancers, and believed to be the major enzyme responsible for pyruvate-to-lactate conversion. To elucidate its role in tumor growth, we disrupted both the LDHA and LDHB genes in two cancer cell lines (human colon adenocarcinoma and murine melanoma cells). Surprisingly, neither LDHA nor LDHB knockout strongly reduced lactate secretion. In contrast, double knockout (LDHA/B-DKO) fully suppressed LDH activity and lactate secretion. Furthermore, under normoxia, LDHA/B-DKO cells survived the genetic block by shifting their metabolism to oxidative phosphorylation (OXPHOS), entailing a 2-fold reduction in proliferation rates in vitro and in vivo compared with their WT counterparts. Under hypoxia (1% oxygen), however, LDHA/B suppression completely abolished in vitro growth, consistent with the reliance on OXPHOS. Interestingly, activation of the respiratory capacity operated by the LDHA/B-DKO genetic block as well as the resilient growth were not consequences of long-term adaptation. They could be reproduced pharmacologically by treating WT cells with an LDHA/B-specific inhibitor (GNE-140). These findings demonstrate that the Warburg effect is not only based on high LDHA expression, as both LDHA and LDHB need to be deleted to suppress fermentative glycolysis. Finally, we demonstrate that the Warburg effect is dispensable even in aggressive tumors and that the metabolic shift to OXPHOS caused by LDHA/B genetic disruptions is responsible for the tumors' escape and growth.

Metabolic plasticity underpins innate and acquired resistance to LDHA inhibition.

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.

Metabolite profiling stratifies pancreatic ductal adenocarcinomas into subtypes with distinct sensitivities to metabolic inhibitors.

Although targeting cancer metabolism is a promising therapeutic strategy, clinical success will depend on an accurate diagnostic identification of tumor subtypes with specific metabolic requirements. Through broad metabolite profiling, we successfully identified three highly distinct metabolic subtypes in pancreatic ductal adenocarcinoma (PDAC). One subtype was defined by reduced proliferative capacity, whereas the other two subtypes (glycolytic and lipogenic) showed distinct metabolite levels associated with glycolysis, lipogenesis, and redox pathways, confirmed at the transcriptional level. The glycolytic and lipogenic subtypes showed striking differences in glucose and glutamine utilization, as well as mitochondrial function, and corresponded to differences in cell sensitivity to inhibitors of glycolysis, glutamine metabolism, lipid synthesis, and redox balance. In PDAC clinical samples, the lipogenic subtype associated with the epithelial (classical) subtype, whereas the glycolytic subtype strongly associated with the mesenchymal (QM-PDA) subtype, suggesting functional relevance in disease progression. Pharmacogenomic screening of an additional ∼ 200 non-PDAC cell lines validated the association between mesenchymal status and metabolic drug response in other tumor indications. Our findings highlight the utility of broad metabolite profiling to predict sensitivity of tumors to a variety of metabolic inhibitors.

Structure-Based Design and Evaluation of Reversible KRAS G13D Inhibitors.

Oncogenic KRAS mutations were identified decades ago, yet the selective inhibition of specific KRAS mutant proteins represents an ongoing challenge. Recent progress has been made in targeting certain P-loop mutant proteins, in particular KRAS G12C, for which the covalent inhibition of the GDP state via the Switch II pocket is now a clinically validated strategy. Inhibition of other KRAS mutant proteins such as KRAS G13D, on the other hand, still requires clinical validation. The remoteness of the D13 residue relative to the Switch II pocket in combination with the solvent exposure and conformational flexibility of the D13 side chain, as well as the difficulties of targeting carboxylate residues covalently, renders this specific protein particularly challenging to target selectively. In this report, we describe the design and evaluation of potent and KRAS G13D-selective reversible inhibitors. Subnanomolar binding to the GDP state Switch II pocket and biochemical selectivity over WT KRAS are achieved by leveraging a salt bridge with D13.

An allosteric pan-TEAD inhibitor blocks oncogenic YAP/TAZ signaling and overcomes KRAS G12C inhibitor resistance.

The Hippo pathway is a key growth control pathway that is conserved across species. The downstream effectors of the Hippo pathway, YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif), are frequently activated in cancers to drive proliferation and survival. Based on the premise that sustained interactions between YAP/TAZ and TEADs (transcriptional enhanced associate domain) are central to their transcriptional activities, we discovered a potent small-molecule inhibitor (SMI), GNE-7883, that allosterically blocks the interactions between YAP/TAZ and all human TEAD paralogs through binding to the TEAD lipid pocket. GNE-7883 effectively reduces chromatin accessibility specifically at TEAD motifs, suppresses cell proliferation in a variety of cell line models and achieves strong antitumor efficacy in vivo. Furthermore, we uncovered that GNE-7883 effectively overcomes both intrinsic and acquired resistance to KRAS (Kirsten rat sarcoma viral oncogene homolog) G12C inhibitors in diverse preclinical models through the inhibition of YAP/TAZ activation. Taken together, this work demonstrates the activities of TEAD SMIs in YAP/TAZ-dependent cancers and highlights their potential broad applications in precision oncology and therapy resistance.

Forward chemical genetics in yeast for discovery of chemical probes targeting metabolism.

The many virtues that made the yeast Saccharomyces cerevisiae a dominant model organism for genetics and molecular biology, are now establishing its role in chemical genetics. Its experimental tractability (i.e., rapid doubling time, simple culture conditions) and the availability of powerful tools for drug-target identification, make yeast an ideal organism for high-throughput phenotypic screening. It may be especially applicable for the discovery of chemical probes targeting highly conserved cellular processes, such as metabolism and bioenergetics, because these probes would likely inhibit the same processes in higher eukaryotes (including man). Importantly, changes in normal cellular metabolism are associated with a variety of diseased states (including neurological disorders and cancer), and exploiting these changes for therapeutic purposes has accordingly gained considerable attention. Here, we review progress and challenges associated with forward chemical genetic screening in yeast. We also discuss evidence supporting these screens as a useful strategy for discovery of new chemical probes and new druggable targets related to cellular metabolism.

Improving treatment of severe hypertension in pregnancy and postpartum using a hypertensive pathway.

OBJECTIVE: To assess the implementation of an antihypertensive pathway order set to improve treatment of severe hypertension in pregnancy and the postpartum period in the inpatient setting. STUDY DESIGN: A multi-disciplinary task force created a hypertensive pathway order set and provided staff training. The order set allowed providers to initiate a treatment algorithm, which then gave nurses guidelines to recheck blood pressures and progressively increase short-acting antihypertensive dosage if needed. Pregnant and postpartum patients documented to have ≥2 consecutive severe range blood pressures in the year prior (2017) and the year after (2019) implementation of the pathway were included. Primary outcomes included whether any antihypertensive was given, whether it was given for all instances of severe hypertension, and time to antihypertensive administration. RESULTS: A total of 566 patients with severe hypertension were included-304 in the pre-implementation year and 262 in the post-implementation year. Significantly more patients received an antihypertensive at least once (67 % versus 80 %, p < 0.01) and for all instances of severe hypertension (29 % versus 47 %, p < 0.01) in the post-intervention cohort. There was a significant improvement in time to antihypertensive administration (24 versus 10 min, p < 0.01). CONCLUSION: This study evaluates the efficacy of an antihypertensive intervention in the Southeast United States, which is particularly significant given the region's higher rates of hypertension and hypertension-related mortality. This study provides confirmatory evidence that implementation of a standardized order set along with measuring compliance and staff education is associated with improved treatment rates and time to treatment administration.

Identifying transcriptional programs underlying cancer drug response with TraCe-seq.

Genetic and non-genetic heterogeneity within cancer cell populations represent major challenges to anticancer therapies. We currently lack robust methods to determine how preexisting and adaptive features affect cellular responses to therapies. Here, by conducting clonal fitness mapping and transcriptional characterization using expressed barcodes and single-cell RNA sequencing (scRNA-seq), we have developed tracking differential clonal response by scRNA-seq (TraCe-seq). TraCe-seq is a method that captures at clonal resolution the origin, fate and differential early adaptive transcriptional programs of cells in a complex population in response to distinct treatments. We used TraCe-seq to benchmark how next-generation dual epidermal growth factor receptor (EGFR) inhibitor-degraders compare to standard EGFR kinase inhibitors in EGFR-mutant lung cancer cells. We identified a loss of antigrowth activity associated with targeted degradation of EGFR protein and an essential role of the endoplasmic reticulum (ER) protein processing pathway in anti-EGFR therapeutic efficacy. Our results suggest that targeted degradation is not always superior to enzymatic inhibition and establish TraCe-seq as an approach to study how preexisting transcriptional programs affect treatment responses.

Conformation-locking antibodies for the discovery and characterization of KRAS inhibitors.

Small molecules that stabilize inactive protein conformations are an underutilized strategy for drugging dynamic or otherwise intractable proteins. To facilitate the discovery and characterization of such inhibitors, we created a screening platform to identify conformation-locking antibodies for molecular probes (CLAMPs) that distinguish and induce rare protein conformational states. Applying the approach to KRAS, we discovered CLAMPs that recognize the open conformation of KRASG12C stabilized by covalent inhibitors. One CLAMP enables the visualization of KRASG12C covalent modification in vivo and can be used to investigate response heterogeneity to KRASG12C inhibitors in patient tumors. A second CLAMP enhances the affinity of weak ligands binding to the KRASG12C switch II region (SWII) by stabilizing a specific conformation of KRASG12C, thereby enabling the discovery of such ligands that could serve as leads for the development of drugs in a high-throughput screen. We show that combining the complementary properties of antibodies and small molecules facilitates the study and drugging of dynamic proteins.

Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast.

Formins have been implicated in the regulation of cytoskeletal structure in animals and fungi. Here we show that the formins Bni1 and Bnr1 of budding yeast stimulate the assembly of actin filaments that function as precursors to tropomyosin-stabilized cables that direct polarized cell growth. With loss of formin function, cables disassemble,whereas increased formin activity causes the hyperaccumulation of cable-like filaments. Unlike the assembly of cortical actin patches, cable assembly requires profilin but not the Arp2/3 complex. Thus formins control a distinct pathway for assembling actin filaments that organize the overall polarity of the cell.

Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast.

Formins have been implicated in the regulation of cytoskeletal structure in animals and fungi. Here we show that the formins Bni1 and Bnr1 of budding yeast stimulate the assembly of actin filaments that function as precursors to tropomyosin-stabilized cables that direct polarized cell growth. With loss of formin function, cables disassemble, whereas increased formin activity causes the hyperaccumulation of cable-like filaments. Unlike the assembly of cortical actin patches, cable assembly requires profilin but not the Arp2/3 complex. Thus formins control a distinct pathway for assembling actin filaments that organize the overall polarity of the cell.

A specificity map for the PDZ domain family.

PDZ domains are protein-protein interaction modules that recognize specific C-terminal sequences to assemble protein complexes in multicellular organisms. By scanning billions of random peptides, we accurately map binding specificity for approximately half of the over 330 PDZ domains in the human and Caenorhabditis elegans proteomes. The domains recognize features of the last seven ligand positions, and we find 16 distinct specificity classes conserved from worm to human, significantly extending the canonical two-class system based on position -2. Thus, most PDZ domains are not promiscuous, but rather are fine-tuned for specific interactions. Specificity profiling of 91 point mutants of a model PDZ domain reveals that the binding site is highly robust, as all mutants were able to recognize C-terminal peptides. However, many mutations altered specificity for ligand positions both close and far from the mutated position, suggesting that binding specificity can evolve rapidly under mutational pressure. Our specificity map enables the prediction and prioritization of natural protein interactions, which can be used to guide PDZ domain cell biology experiments. Using this approach, we predicted and validated several viral ligands for the PDZ domains of the SCRIB polarity protein. These findings indicate that many viruses produce PDZ ligands that disrupt host protein complexes for their own benefit, and that highly pathogenic strains target PDZ domains involved in cell polarity and growth.

Loss of the serine/threonine kinase fused results in postnatal growth defects and lethality due to progressive hydrocephalus.

The Drosophila Fused (Fu) kinase is an integral component of the Hedgehog (Hh) pathway that helps promote Hh-dependent gene transcription. Vertebrate homologues of Fu function in the Hh pathway in vitro, suggesting that Fu is evolutionarily conserved. We have generated fused (stk36) knockout mice to address the in vivo function of the mouse Fu (mFu) homologue. fused knockouts develop normally, being born in Mendelian ratios, but fail to thrive within 2 weeks, displaying profound growth retardation with communicating hydrocephalus and early mortality. The fused gene is expressed highly in ependymal cells and the choroid plexus, tissues involved in the production and circulation of cerebral spinal fluid (CSF), suggesting that loss of mFu disrupts CSF homeostasis. Similarly, fused is highly expressed in the nasal epithelium, where fused knockouts display bilateral suppurative rhinitis. No obvious defects were observed in the development of organs where Hh signaling is required (limbs, face, bones, etc.). Specification of neuronal cell fates by Hh in the neural tube was normal in fused knockouts, and induction of Hh target genes in numerous tissues is not affected by the loss of mFu. Furthermore, stimulation of fused knockout cerebellar granule cells to proliferate with Sonic Hh revealed no defect in Hh signal transmission. These results show that the mFu homologue is not required for Hh signaling during embryonic development but is required for proper postnatal development, possibly by regulating the CSF homeostasis or ciliary function.

A CRISPR screen identifies MAPK7 as a target for combination with MEK inhibition in KRAS mutant NSCLC.

Mutant KRAS represents one of the most frequently observed oncogenes in NSCLC, yet no therapies are approved for tumors that express activated KRAS variants. While there is strong rationale for the use of MEK inhibitors to treat tumors with activated RAS/MAPK signaling, these have proven ineffective clinically. We therefore implemented a CRISPR screening approach to identify novel agents to sensitize KRAS mutant NSCLC cells to MEK inhibitor treatment. This approach identified multiple components of the canonical RAS/MAPK pathway consistent with previous studies. In addition, we identified MAPK7 as a novel, strong hit and validated this finding using multiple orthogonal approaches including knockdown and pharmacological inhibition. We show that MAPK7 inhibition attenuates the re-activation of MAPK signaling occurring following long-term MEK inhibition, thereby illustrating that MAPK7 mediates pathway reactivation in the face of MEK inhibition. Finally, genetic knockdown of MAPK7 combined with the MEK inhibitor cobimetinib in a mutant KRAS NSCLC xenograft model to mediate improved tumor growth inhibition. These data highlight that MAPK7 represents a promising target for combination treatment with MEK inhibition in KRAS mutant NSCLC.

Pan-Cancer Metabolic Signature Predicts Co-Dependency on Glutaminase and De Novo Glutathione Synthesis Linked to a High-Mesenchymal Cell State.

The enzyme glutaminase (GLS1) is currently in clinical trials for oncology, yet there are no clear diagnostic criteria to identify responders. The evaluation of 25 basal breast lines expressing GLS1, predominantly through its splice isoform GAC, demonstrated that only GLS1-dependent basal B lines required it for maintaining de novo glutathione synthesis in addition to mitochondrial bioenergetics. Drug sensitivity profiling of 407 tumor lines with GLS1 and gamma-glutamylcysteine synthetase (GCS) inhibitors revealed a high degree of co-dependency on both enzymes across indications, suggesting that redox balance is a key function of GLS1 in tumors. To leverage these findings, we derived a pan-cancer metabolic signature predictive of GLS1/GCS co-dependency and validated it in vivo using four lung patient-derived xenograft models, revealing the additional requirement for expression of GAC above a threshold (log2RPKM + 1 ≥ 4.5, where RPKM is reads per kilobase per million mapped reads). Analysis of the pan-TCGA dataset with our signature identified multiple indications, including mesenchymal tumors, as putative responders to GLS1 inhibitors.