Abstract DNA mismatch repair (dMMR) defects occur in a broad range of cancers and are characterized by genomic instability due to TA repeat expansion, a condition known as microsatellite instability-high (MSI-H). The use of immune checkpoint inhibitors (ICI) in MSI-H cancers has improved patient outcomes, but ∼30-40% of patients do not respond and ∼20-25% become refractory to ICI. Genetic ablation or pharmacological inhibition of Werner RecQ like, ATP-dependent helicase (WRN) activity has been shown to induce growth arrest and apoptosis in MSI-H cancer cells, but not in microsatellite stable (MSS) cells. Early clinical data with RO7589831 (covalent) and HRO761 (non-covalent) WRN inhibitors demonstrated clinical responses, validating WRN as an actionable target, but uncertainty remains on their ability to fully engage WRN. ETX-880 is a novel, potent and selective small molecule inhibitor of WRN for the treatment of MSI-H cancers. ETX-880 acts through an allosteric, ATP-cooperative binding mechanism and covalent ligation of WRN at cysteine 727 (C727). The preclinical activity of ETX-880 was characterized and compared to clinical stage WRN inhibitors, including RO7589831, HRO761, and GSK4418959 (non-covalent). ETX-880 demonstrated >2-fold tighter binding to WRN than RO7589831 (ETX-880 KI = 0.44 µM versus RO7589831 KI = 1.05 µM) with comparable reactivity for WRN C727. ETX-880 selectively inhibits the ATPase and DNA unwinding activities of WRN with 2-fold greater potency than RO7589831, excellent selectivity against other RecQ helicases, and no activity for the WRNC727A mutant protein. ETX-880 shows strong anti-proliferative effects in a broad panel of MSI-H cancer cell lines with no measurable effects in MSS cells. In MSI-H cancer cells, ETX-880 shows 4-fold more potent anti-proliferative activity than RO7589831 and HRO761 and is ∼30-fold more potent than GSK4418959. In vivo, ETX-880 depletes WRN protein and induces DNA damage markers in tumors and leads to deeper and more sustained tumor regressions than RO7589831 in an MSI-H colorectal cancer xenograft model. At the efficacious dose, ETX-880 achieves 100% target occupancy (TO) at 2 hours post dose and maintains ∼85% TO at 24 hours, in contrast to RO7589831 which shows substantial loss (∼40%) of TO by 24 hours. ETX-880 displays favorable drug-like properties, including good in vitro ADMET, excellent metabolic stability with low in vivo clearance and high oral bioavailability and exposures across animal species. Importantly, human PK predictions reveal low clearance, high oral bioavailability and long half-life, supporting a low once daily oral efficacious dose. Overall, ETX-880 is a potent, selective, covalent WRN inhibitor with excellent ADMET properties leading to deep and sustained target coverage, highlighting its best-in-class potential in MSI-H cancers. Citation Format: Robert F. Koncar, Daliya Banerjee, Mingzong Li, Tao Liu, Upul Bandarage, Alexandra Weinheimer, Jingyan Gao, Fei Peng, Ying Lin, Yong Tang, Karan Kapoor, Minghong Hao, Robbie Chen, Eric Simone, Raj Nagaraja, Shengfang Jin, Meghana M. Kulkarni, Jeffery Kutok. ETX-880, a potential best-in-class, oral, highly potent and selective covalent inhibitor of Werner helicase for the treatment of microsatellite instability-high (MSI-H) cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 423.

Abstract Oncogenic KRAS alterations occur in approximately 23% of all human cancers. The most common oncogenic KRAS alterations are G12D (∼31%), G12V (∼24%), and G12C (∼15%) mutations. In addition, ∼10% of KRAS-altered cancers have wild-type (WT) KRAS amplification or harbor multiple oncogenic KRAS alterations, including co-occurring KRAS mutations and amplifications. While there have been drug approvals for KRASG12C targeting covalent inhibitors, there are currently no approved targeted therapies against other oncogenic KRAS alterations. Pan-KRAS inhibitors that target multiple oncogenic KRAS alterations while sparing other RAS isoforms have the potential to treat broad patient populations and address resistance to mutant selective KRAS inhibition while avoiding the on-target toxicities associated with pan-RAS inhibition. ETX-929 is an orally bioavailable, potent, dual GTP (ON)- and GDP (OFF)-state pan-KRAS inhibitor with excellent selectivity over other RAS isoforms. ETX-929 binds WT and mutant KRAS in active (ON) and inactive (OFF) conformations with high affinity [KD(ON-state) = single digit nanomolar; KD(OFF-state) = picomolar] and >100-fold selectivity over NRAS and HRAS. In the KRAS-RAF1 RAS-binding domain biochemical assay, ETX-929 potently inhibited complex formation when KRAS (WT / mutant) was either in the active GMPPNP-bound state (single digit nM IC50) or inactive GDP-bound state (pM IC50). In cells, ETX-929’s dual ON / OFF-state inhibitory activity potently inhibits KRAS mutants that rapidly cycle between GTP- and GDP-bound states (KRASWTamp, KRASG12C) as well as slow cycling mutants (KRASG12V and KRASQ61H) that predominantly reside in the ON state. In a broad panel of KRAS mutant (G12X, G13D, Q61H) and WT amplified cancer cell lines, ETX-929 potently inhibited the KRAS pathway (pERK median IC50 = 0.9 nM) and cell proliferation (median IC50 = 6.23 nM), while showing no viability effects on low copy number WT KRAS or mutant NRAS / HRAS cells. Oral ETX-929 showed dose-dependent exposures with concordant sustained tumor pERK reductions, inducing tumor stasis or regression in colorectal, stomach, and lung cancer cell line-derived xenograft models harboring KRAS alterations (G12D, G12V, G12C, and WT amplified). ETX-929 also shows good cross-species pharmacokinetics including higher species, excellent pre-clinical tolerability, and no meaningful off-target activity or in vitro safety signals, supporting its advancement toward clinical development. Overall, ETX-929 is a potential best-in-class, pan-KRAS inhibitor with superior potency, selectivity over NRAS / HRAS, excellent efficacy and favorable drug-like properties that could improve outcomes for KRAS-driven cancers that currently lack targeted therapeutic options. Citation Format: Meghana M. Kulkarni, Lei Cui, Hongbo Deng, Tao Liu, Jingyan Gao, Fei Peng, Ying Lin, Yong Tang, Karan Kapoor, Minghong Hao, Robert F. Koncar, Robbie Chen, Eric Simone, Raj Nagaraja, Shengfang Jin, Jeffery Kutok. ETX-929, a potential best-In-class, oral, highly potent and selective dual ON / OFF state Pan-KRAS small molecule inhibitor for the treatment of KRAS mutant and wild-type amplified cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 418.

In a normal cell cycle, there is redundancy in the role of the cell-cycle dependent kinases (CDKs) in regulating G1/S phase transition. In cancer cells, the regulation of G1/S transition can be subverted by (a) amplification and elevated expression of Cyclin E (CCNE), or (b) mutation/loss of the Retinoblastoma 1 (RB1) gene. Cancer cells with these genomic alterations have been shown to exhibit profound sensitivity to CDK2 depletion, validating CDK2 as a potential therapeutic target. Here, we report the discovery and preclinical characterization of ETX-197, a highly potent and selective small molecule inhibitor of CDK2 enzymatic activity. Based on known ligand-CDK2 structures, ETX-197 is designed to induce previously unexplored interactions within the CDK2 ATP binding pocket leading to improved potency and selectivity compared to other known CDK2 inhibitors. ETX-197 is >100-fold selective against other kinases in the CDK family and the selectivity extends more broadly against 385 other kinases. The affinity of ETX-197 for CDK2 results in tight binding (slow off-rate) and high potency in pharmacodynamic modulation and anti-proliferative activity in vitro and in vivo. Treatment of CCNE-amplified cancer cells with ETX-197 results in concentration-dependent inhibition of pRB phosphorylation, G1/S phase cell-cycle arrest and cell proliferation. In addition, ETX-197 treatment phenocopies CDK2 genetic knock-down in cells, as revealed by bulk-RNA Seq analysis of CCNE-amplified or wild-type cells, confirming that the cellular activity of ETX-197 is on-target and highly selective. In mouse xenograft studies using CCNE-amplified ovarian cancer cell line (OVCAR-3) or patient-derived tumors, ETX-197 treatment causes dose-dependent tumor growth inhibition with excellent tolerability. Interestingly, in RB1-deficient small cell lung cancer cell lines, ETX-197 treatment results in G2/M cell cycle arrest, accumulation of DNA damage, and apoptosis. Xenograft studies with small cell lung cancer cell lines and patient-derived tumor cells also show significant tumor growth inhibition with ETX-197. Additionally, ETX-197 has single-agent efficacy in a breast cancer xenograft model that had acquired resistance to a CDK4/6 inhibitor. These data suggest that ETX-197 has the potential to be a best-in class CDK2 inhibitor for the treatment of cancer with CCNE amplification or RB1 deficiency, including breast cancer that has progressed on treatment with a CDK4/6 inhibitor because of these genomic alterations. Currently, ETX-197 is being clinically developed by BeiGene in a first-in-human (FIH), Phase 1a/1b study to assess the safety, tolerability, pharmacokinetics (PK), pharmacodynamics, and preliminary antitumor activity in patients with advanced, nonresectable, or metastatic solid tumors (NCT06257264). Citation Format: Daliya Banerjee, Alexandra Weinheimer, Jingyan Gao, Fei Pang, Ying Lin, Raj Nagaraja, Yong Tang, Zipeng Fan, Zipeng Fan, Minghong Hao, Shengfang Jin, Tao Liu, Tai Wong. ETX-197/BG-68501, a potential best-in-class potent, selective, oral, small molecule CDK2 inhibitor, has anti-tumor activity in cancer models with Cyclin E amplification or deficiency in the Retinoblastoma 1 gene [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-12-29.

PIK3CA, which encodes p110α, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kα), is one of the most frequently mutated oncogenes and dysregulated PI3Kα activity is important for tumor growth. Up to 40% of hormone receptor (HR)-positive breast cancer (BrCA) tumors and 13% of all solid tumors harbor PIK3CA mutations, with the majority of mutations occurring within p110α kinase and helical domains. Orthosteric ATP-competitive inhibitors, alpelisib and inavolisib, which inhibit both Wild-type (WT) and mutant PI3Kα, are approved in combination regimens for treating PIK3CA-mutant, HR+/HER2-, advanced or metastatic BrCA. However, because PI3Kα is critical for insulin signaling, inhibition of WT PI3Kα results in hyperglycemia, and limits the clinical utility of these inhibitors. To overcome this limitation, ETX-636 was designed as an allosteric, pan-mutant-selective PI3Kα inhibitor and degrader by leveraging our Kinetic Ensemble® platform for optimal binding to a unique metastable state of p110α. In addition to greater biochemical selectivity for mutant PI3Kα over WT PI3Kα, ETX-636 has stronger target binding affinity, better on-target potency in biochemical and cellular pharmacodynamic assays, and demonstrates superior anti-tumor activity in vivo when compared to other allosteric, pan-mutant-selective PI3Kα inhibitors (i.e. RLY-2608 and STX-478). In viability assays run on a panel of 65 cancer cell lines, the presence of PIK3CA mutations was a strong predictor of ETX-636 sensitivity and ETX-636 potency was superior to both RLY-2608 and STX-478. In vitro and in vivo mechanistic studies revealed ETX-636 induces significant proteasome-dependent degradation of mutant p110α, while sparing WT protein (a feature not seen with other pan-mutant allosteric inhibitors), resulting in more durable pathway inhibition. ETX-636 dosed orally, once daily, results in deep and durable pathway inhibition and induces tumor regression in kinase and helical domain PIK3CA-mutant xenografts. In an ER-positive, HER2-negative, PI3Kα-mutant BrCA xenograft, ETX-636 is efficacious as a single agent and shows synergistic activity with fulvestrant, inducing tumor regression while being well-tolerated. Blood glucose levels after dosing in multiple species indicate that ETX-636 does not disrupt glucose homeostasis at efficacious exposures, even under non-fasting conditions. In addition, based on pharmacokinetic, pharmacodynamic, efficacy, and toxicology studies, predicted human efficacious doses of ETX-636 are not projected to cause hyperglycemia. The preclinical profile of ETX-636 underscores its best-in-class potential as an allosteric pan-mutant-selective PI3Kα dual inhibitor and degrader, and supports its clinical development both in single agent and combination clinical trials in patients with PIK3CA-mutant tumors, including HR+/PIK3CA-mutant BrCA. Robert F. Koncar, Mingzong Li, Jingyan Gao, Fei Pang, Ying Lin, Raj Nagaraja, Yong Tang, Hannah Szeto, Zipeng Fan, Karan Kapoor, Robbie Chen, Eric Simone, Minghong Hao, Shengfang Jin, Tao Liu, Meghana Kulkarni, Jeffery Kutok. ETX-636, a novel allosteric pan-mutant-selective PI3Kα dual inhibitor and degrader with best-in-class potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1659.

PIK3CA, which encodes p110α, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kα), is one of the most frequently mutated oncogenes, with activating mutations seen in ∼16% of all solid tumors and up to 40% of breast tumors, including hormone receptor-positive/HER2-negative, advanced breast cancer. The most frequent gain-of-function PI3Kα hotspot mutations, H1047R, E542K, and E545K, are well-recognized oncogenic drivers. Cancer cells with PIK3CA activating mutations are dependent on PI3Kα signaling and HR-positive, HER2-negative breast cancer patients with PIK3CA mutations respond to alpelisib, an approved PI3Kα inhibitor. While PI3Kα is important for cancer cell proliferation and tumor growth, it is also a critical component of the insulin signaling pathway. Therefore, the use of orthosteric inhibitors like alpelisib, which inhibit both the wildtype and mutant proteins, often results in significant hyperglycemia limiting their clinical utility. We report the discovery and pre-clinical characterization of an allosteric, pan-mutant-selective PI3Kα inhibitor, ETX-636, which was designed leveraging our Kinetic Ensemble® platform for optimal binding properties. Compared to other allosteric, pan-mutant-selective PI3Kα inhibitors (ie RLY-2608 and STX-478), ETX-636 has stronger target binding affinity, better on-target potency in biochemical and cellular pharmacodynamic and viability assays, and demonstrates superior anti-tumor activity in vivo. More specifically, ETX-636 inhibits both kinase (H1047X) and helical domain (E542K and E545K) mutant PI3Kα biochemical activity with single digit nM potency and 8-10-fold selectivity relative to wildtype PI3Kα protein. ETX-636 shows greater than 1000-fold selectivity over the β, δ, and γ class I PI3K isoforms in biochemical assays and the selectivity extends more broadly across a panel of ∼350 kinases. In cellular assays, ETX-636 potently inhibits proliferation and PI3Kα signaling specifically in PIK3CA-mutant cell lines. Mechanistically, ETX-636 induces proteasome-dependent degradation of mutant p110α protein in vitro and in vivo. Consistent with the compound-mediated decrease of mutant p110α protein and the high binding affinity (slow off-rate) of the compound, ETX-636 achieves sustained PI3Kα pathway inhibition in cellular washout assays and in cell-derived xenograft (CDX) tumor models. Once daily, oral dosing of ETX-636 shows single agent efficacy at well-tolerated doses in both PI3Kα kinase and helical domain-mutant breast cancer CDX models, significantly inhibiting tumor growth or inducing regression, while suppressing PI3K pathway activity in a dose-dependent manner. In an ER-positive, HER2-negative, PI3Kα-mutant breast cancer xenograft model, ETX-636 is efficacious as a single agent and shows enhanced activity in combination with fulvestrant, inducing consistent tumor regression while being well-tolerated. ETX-636 demonstrates superior magnitude and duration of PI3Kα pathway inhibition in vivo, compared to known pan-mutant-selective allosteric PI3Kα inhibitors, as well as orthosteric inhibitors. At efficacious doses, ETX-636 has significantly less of an effect on blood glucose in mice compared to orthosteric inhibitors, demonstrating that ETX-636 can achieve potent anti-tumor activity by targeting mutant PI3Kα protein without significantly affecting the activity of the wildtype protein. In addition, based on pharmacokinetic/pharmacodynamic/efficacy and toxicology studies, ETX-636 is unlikely to pose a significant risk of hyperglycemia at predicted human efficacious doses. These data support clinical exploration of ETX-636 in mutant PI3Kα solid tumors and, potentially, mutant PI3Kα-driven rare diseases. Citation Format: Robert Koncar, Mingzong Li, Jingyan Gao, Fei Pang, Ying Lin, Raj Nagaraja, Yong Tang, Hannah Szeto, Zipeng Fan, Karan Kapoor, Robbie Chen, Eric Simone, Minghong Hao, Shengfang Jin, Tao Liu, Tai Wong, Meghana Kulkarni, Jeffery Kutok. Discovery and characterization of ETX-636, a potential best-in-class, oral, small molecule, pan-mutant-selective PI3Kα inhibitor [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-12-18.

Supplementary Figure 1. Synthesis and pharmacokinetic characterization of AG-221. Supplementary Figure 2. Biochemical attributes of AG-221. Supplementary Figure 3. Biochemistry of AG-221 with respect to substrate and cofactor. Supplementary Figure 4. TF-1 IDH2R140Q cells treated with AG-221. Supplementary Figure 5. AG-221 can reverse the block in EPO-induced differentiation caused by the expression of IDH2R140Q in the TF-1 erythroleukemia cell line. Supplementary Figure 6. Pharmacokinetics/pharmacodynamics of AG-221 in IDH2R140Qmutant U87MG xenograft tumor-bearing mice. Supplementary Figure 7. AG-221 does not affect intrinsic hematological parameters or body weight. Supplementary Figure 8. AG-221 strongly reduces the number of human IDH2R140Q blasts in the liver and spleen in AML-1, AML-2, and AML-3. Supplementary Figure 9. Flow cytometry analyses of bone marrow-derived hCD45+ cells in primary human AML xenograft models. Supplementary Figure 10. Affinity of the IDH2R140Q homodimer for NADPH. Supplementary Figure 11. Electron density map diagrams for bound ligands for IDH2R140Q co-complex structures determined by X-ray crystallography.

Supplementary Table 1. Drug metabolism and pharmacokinetic attributes of AG-221. Supplementary Table 2. Selectivity of AG-221 confirmed by testing against a panel of kinases. Supplementary Table 3. Clinical characteristics of patients with IDH2R140Q-mutated AML. Supplementary Table 4. Treated NSG mice (AML-1, AML-2, AML-3) engrafted with human IDH2R140Q mononuclear cells display stable levels of AG-221 in serum. Supplementary Table 5. AG-221 inhibits 2HG production in models AML-1, AML-2, and AML-3. Supplementary Table 6. Summary of pharmacokinetics/pharmacodynamics in primary human acute myeloid leukemia xenograft model (AML-4). Supplementary Table 7. Summary of data collection and refinement statistics. Supplementary Table 8. Percentage of human chimerism in peripheral blood in models AML-1 and AML-2.

Aberrant metabolism of cancer cells is well appreciated, but the identification of cancer subsets with specific metabolic vulnerabilities remains challenging. We conducted a chemical biology screen and identified a subset of neuroendocrine tumors displaying a striking pattern of sensitivity to inhibition of the cholesterol biosynthetic pathway enzyme squalene epoxidase (SQLE). Using a variety of orthogonal approaches, we demonstrate that sensitivity to SQLE inhibition results not from cholesterol biosynthesis pathway inhibition, but rather surprisingly from the specific and toxic accumulation of the SQLE substrate, squalene. These findings highlight SQLE as a potential therapeutic target in a subset of neuroendocrine tumors, particularly small cell lung cancers.

Branched chain amino acid (BCAA) metabolism occurs within the mitochondrial matrix and is comprised of multiple enzymes, some shared, organized into three pathways for the catabolism of leucine, isoleucine, and valine (LEU, ILE, and VAL respectively). Three different acyl-CoA dehydrogenases (ACADs) are active in each catabolic pathway and genetic deficiencies in each have been identified. While characteristic metabolites related to the enzymatic block accumulate in each deficiency, for reasons that are not clear, clinical symptoms are only seen in the context of deficiency of isovaleryl-CoA dehydrogenase (IVDH) in the leucine pathway. Metabolism of fibroblasts derived from patients with mutations in each of the BCAA ACADs were characterized using metabolomics to better understand the flux of BCAA through their respective pathways. Stable isotope labeled LEU, ILE, and VAL in patient and control cell lines revealed that mutations in isobutyryl-CoA dehydrogenase (IBDH in the valine pathway) lead to a significant increase in isobutyrylcarnitine (a surrogate for the enzyme substrate isobutyryl-CoA) leading to metabolism by short-branched chain acyl-CoA dehydrogenase (SBCADH in the isoleucine pathway) and production of the pathway end product propionylcarnitine (a surrogate for propionyl-CoA). Similar cross activity was observed for SBCADH deficient patient cells, leading to a significant increase in propionylcarnitine, presumably by metabolism of 2 methylbutyryl-CoA via IBDH activity. Labeled BCAA studies identified that the majority of the intracellular propionyl-CoA pool in fibroblasts is generated from isoleucine, but heptanoic acid (a surrogate for odd-chain fatty acids) is also efficiently converted to propionate.

Somatic point mutations at a key arginine residue (R132) within the active site of the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) confer a novel gain of function in cancer cells, resulting in the production of d-2-hydroxyglutarate (2-HG), an oncometabolite. Elevated 2-HG levels are implicated in epigenetic alterations and impaired cellular differentiation. IDH1 mutations have been described in an array of hematologic malignancies and solid tumors. Here, we report the discovery of AG-120 (ivosidenib), an inhibitor of the IDH1 mutant enzyme that exhibits profound 2-HG lowering in tumor models and the ability to effect differentiation of primary patient AML samples ex vivo. Preliminary data from phase 1 clinical trials enrolling patients with cancers harboring an IDH1 mutation indicate that AG-120 has an acceptable safety profile and clinical activity.

Abstract Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate–dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation–positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2R140Q-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation–positive advanced hematologic malignancies. Significance: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development. Cancer Discov; 7(5); 478–93. ©2017 AACR. See related commentary by Thomas and Majeti, p. 459. See related article by Shih et al., p. 494. This article is highlighted in the In This Issue feature, p. 443

The most common congenital disorder of glycosylation (CDG), phosphomannomutase 2 (PMM2)-CDG, is caused by mutations in PMM2 that limit availability of mannose precursors required for protein N-glycosylation. The disorder has no therapy and there are no models to test new treatments. We generated compound heterozygous mice with the R137H and F115L mutations in Pmm2 that correspond to the most prevalent alleles found in patients with PMM2-CDG. Many Pmm2R137H/F115L mice died prenatally, while survivors had significantly stunted growth. These animals and cells derived from them showed protein glycosylation deficiencies similar to those found in patients with PMM2-CDG. Growth-related glycoproteins insulin-like growth factor (IGF) 1, IGF binding protein-3 and acid-labile subunit, along with antithrombin III, were all deficient in Pmm2R137H/F115L mice, but their levels in heterozygous mice were comparable to wild-type (WT) littermates. These imbalances, resulting from defective glycosylation, are likely the cause of the stunted growth seen both in our model and in PMM2-CDG patients. Both Pmm2R137H/F115L mouse and PMM2-CDG patient-derived fibroblasts displayed reductions in PMM activity, guanosine diphosphate mannose, lipid-linked oligosaccharide precursor and total cellular protein glycosylation, along with hypoglycosylation of a new endogenous biomarker, glycoprotein 130 (gp130). Over-expression of WT-PMM2 in patient-derived fibroblasts rescued all these defects, showing that restoration of mutant PMM2 activity is a viable therapeutic strategy. This functional mouse model of PMM2-CDG, in vitro assays and identification of the novel gp130 biomarker all shed light on the human disease, and moreover, provide the essential tools to test potential therapeutics for this untreatable disease.

D-2-hydroxyglutaric aciduria (D2HGA) type II is a rare neurometabolic disorder caused by germline gain-of-function mutations in isocitrate dehydrogenase 2 (IDH2), resulting in accumulation of D-2-hydroxyglutarate (D2HG). Patients exhibit a wide spectrum of symptoms including cardiomyopathy, epilepsy, developmental delay and limited life span. Currently, there are no effective therapeutic interventions. We generated a D2HGA type II mouse model by introducing the Idh2R140Q mutation at the native chromosomal locus. Idh2R140Q mice displayed significantly elevated 2HG levels and recapitulated multiple defects seen in patients. AGI-026, a potent, selective inhibitor of the human IDH2R140Q-mutant enzyme, suppressed 2HG production, rescued cardiomyopathy, and provided a survival benefit in Idh2R140Q mice; treatment withdrawal resulted in deterioration of cardiac function. We observed differential expression of multiple genes and metabolites that are associated with cardiomyopathy, which were largely reversed by AGI-026. These findings demonstrate the potential therapeutic benefit of an IDH2R140Q inhibitor in patients with D2HGA type II.

Oncogenic isocitrate dehydrogenase (IDH) 1 and IDH2 mutations at three hotspot arginine residues cause an enzymatic gain of function that leads to the production and accumulation of the metabolite 2-hydroxyglutarate (2HG), which contributes to the development of a number of malignancies. In the hematopoietic system, mutations in IDH1 at arginine (R) 132 and in IDH2 at R140 and R172 are commonly observed in acute myeloid leukemia, and elevated 2HG is observed in cells and serum. However, in angioimmunoblastic T-cell lymphoma (AITL), mutations are almost exclusively restricted to IDH2 R172, and levels of 2HG have not been comprehensively measured. In this study, we investigate the expression pattern of mutant IDH2 in the AITL tumor microenvironment and measure levels of 2HG in tissue and serum of AITL patients. We find that mutant IDH2 expression is restricted to the malignant T-cell component of AITL, and that 2HG is elevated in tumor tissue and serum of patients. We also investigate the differences between the three hotspot mutation sites in IDH1 and IDH2 using conditional knock-in mouse models. These studies show that in the lymphoid system, mutations in IDH2 at R172 produce high levels of 2HG compared with mutations at the other two sites and that lymphoid development is impaired in these animals. These data provide evidence that IDH2 R172 mutations may be the only variants present in AITL because of their capacity to produce significant amounts of the oncometabolite 2HG in the cell of origin of this disease.

Although aberrant metabolism in tumors has been well described, the identification of cancer subsets with particular metabolic vulnerabilities has remained challenging. Here, we conducted an siRNA screen focusing on enzymes involved in the tricarboxylic acid (TCA) cycle and uncovered a striking range of cancer cell dependencies on OGDH, the E1 subunit of the alpha-ketoglutarate dehydrogenase complex. Using an integrative metabolomics approach, we identified differential aspartate utilization, via the malate-aspartate shuttle, as a predictor of whether OGDH is required for proliferation in 3D culture assays and for the growth of xenograft tumors. These findings highlight an anaplerotic role of aspartate and, more broadly, suggest that differential nutrient utilization patterns can identify subsets of cancers with distinct metabolic dependencies for potential pharmacological intervention.

Pyruvate kinase deficiency (PKD) is a monogenic metabolic disease caused by mutations in the PKLR gene that leads to hemolytic anemia of variable symptomatology and that can be fatal during the neonatal period. PKD recessive inheritance trait and its curative treatment by allogeneic bone marrow transplantation provide an ideal scenario for developing gene therapy approaches. Here, we provide a preclinical gene therapy for PKD based on a lentiviral vector harboring the hPGK eukaryotic promoter that drives the expression of the PKLR cDNA. This therapeutic vector was used to transduce mouse PKD hematopoietic stem cells (HSCs) that were subsequently transplanted into myeloablated PKD mice. Ectopic RPK expression normalized the erythroid compartment correcting the hematological phenotype and reverting organ pathology. Metabolomic studies demonstrated functional correction of the glycolytic pathway in RBCs derived from genetically corrected PKD HSCs, with no metabolic disturbances in leukocytes. The analysis of the lentiviral insertion sites in the genome of transplanted hematopoietic cells demonstrated no evidence of genotoxicity in any of the transplanted animals. Overall, our results underscore the therapeutic potential of the hPGK-coRPK lentiviral vector and provide high expectations toward the gene therapy of PKD and other erythroid metabolic genetic disorders.

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine Aldh2 locus. These mutants recapitulate essentially all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the ALDH2*2 allele-alcohol interaction may be an even greater human public health hazard than previously appreciated.