Disruption of polyunsaturated fatty acid biosynthesis drives STING-dependent acute myeloid leukemia cell maturation and death.

The role of polyunsaturated fatty acid (PUFA) biosynthesis in acute myeloid leukemia (AML) remains largely undefined. A comparative expression analysis of 35 genes encoding fatty acid biosynthesis enzymes showed that fatty acid desaturase 1 (FADS1) was highly expressed across multiple AML subtypes relative to healthy controls and that elevated FADS1 expression correlates with worse overall AML patient survival. Functionally, shRNA-mediated inhibition of FADS1 reduced AML cell growth in vitro and significantly delayed leukemia onset in an AML mouse model. AML cell lines depleted of FADS1 arrested in the G1/S-phase of the cell cycle, acquired characteristics of myeloid maturation and subsequently died. To understand the molecular consequences of FADS1 inhibition, a combination of mass spectrometry-based analysis of complex lipids and gene expression analysis (RNA-seq) was performed. FADS1 inhibition caused AML cells to exhibit significant lipidomic remodeling, including depletion of PUFAs from the phospholipids, phosphatidylserine, and phosphatidylethanolamine. These lipidomic alterations were accompanied by an increase induction of inflammatory and stimulator of interferon genes (STING)-mediated type-1 interferon signaling. Remarkably, genetic deletion of STING largely prevented the AML cell maturation and death phenotypes mediated by FADS1 inhibition. Highlighting the therapeutic implications of these findings, pharmacological blockade of PUFA biosynthesis reduced patient-derived AML cell numbers ex vivo but not that of healthy donor cells. Similarly, STING agonism attenuated patient-derived-AML survival; however, STING activation also reduced healthy granulocyte numbers. Collectively, these data unveil a previously unrecognized importance of PUFA biosynthesis in leukemogenesis and that imbalances in PUFA metabolism can drive STING-mediated AML maturation and death.

4'-Ethynyl-2'-Deoxycytidine (EdC) Preferentially Targets Lymphoma and Leukemia Subtypes by Inducing Replicative Stress.

Anticancer nucleosides are effective against solid tumors and hematological malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induced replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å co-crystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared to FDA approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a pre-clinical nucleoside prodrug candidate for DLBCL and ALL.

Targeting Fatty Acid Reprogramming Suppresses CARM1-expressing Ovarian Cancer.

The arginine methyltransferase CARM1 exhibits high expression levels in several human cancers, with the trend also observed in ovarian cancer. However, therapeutic approaches targeting tumors that overexpress CARM1 have not been explored. Cancer cells exploit metabolic reprogramming such as fatty acids for their survival. Here we report that CARM1 promotes monounsaturated fatty acid synthesis and fatty acid reprogramming represents a metabolic vulnerability for CARM1-expressing ovarian cancer. CARM1 promotes the expression of genes encoding rate-limiting enzymes of de novo fatty acid metabolism such as acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN). In addition, CARM1 upregulates stearoyl-CoA desaturase 1 (SCD1) that produces monounsaturated fatty acid by desaturation. Thus, CARM1 enhances de novo fatty acids synthesis which was subsequently utilized for synthesis of monounsaturated fatty acids. Consequently, inhibition of SCD1 suppresses the growth of ovarian cancer cells in a CARM1 status-dependent manner, which was rescued by the addition of monounsaturated fatty acids. Consistently, CARM1-expressing cells were more tolerant to the addition of saturated fatty acids. Indeed, SCD1 inhibition demonstrated efficacy against ovarian cancer in both orthotopic xenograft and syngeneic mouse models in a CARM1-dependent manner. In summary, our data show that CARM1 reprograms fatty acid metabolism and targeting SCD1 through pharmacological inhibition can serve as a potent therapeutic approach for CARM1-expressing ovarian cancers. Significance: CARM1 reprograms fatty acid metabolism transcriptionally to support ovarian cancer growth by producing monounsaturated fatty acids, supporting SCD1 inhibition as a rational strategy for treating CARM1-expressing ovarian cancer.

Targeting the mevalonate pathway suppresses ARID1A-inactivated cancers by promoting pyroptosis.

ARID1A, encoding a subunit of the SWI/SNF complex, is mutated in ∼50% of clear cell ovarian carcinoma (OCCC) cases. Here we show that inhibition of the mevalonate pathway synergizes with immune checkpoint blockade (ICB) by driving inflammasome-regulated immunomodulating pyroptosis in ARID1A-inactivated OCCCs. SWI/SNF inactivation downregulates the rate-limiting enzymes in the mevalonate pathway such as HMGCR and HMGCS1, which creates a dependence on the residual activity of the pathway in ARID1A-inactivated cells. Inhibitors of the mevalonate pathway such as simvastatin suppresses the growth of ARID1A mutant, but not wild-type, OCCCs. In addition, simvastatin synergizes with anti-PD-L1 antibody in a genetic OCCC mouse model driven by conditional Arid1a inactivation and in a humanized immunocompetent ARID1A mutant patient-derived OCCC mouse model. Our data indicate that inhibition of the mevalonate pathway simultaneously suppresses tumor cell growth and boosts antitumor immunity by promoting pyroptosis, which synergizes with ICB in suppressing ARID1A-mutated cancers.

Lysosomal lipid peroxidation regulates tumor immunity.

Lysosomal inhibition elicited by palmitoyl-protein thioesterase 1 (PPT1) inhibitors such as DC661 can produce cell death, but the mechanism for this is not completely understood. Programmed cell death pathways (autophagy, apoptosis, necroptosis, ferroptosis, and pyroptosis) were not required to achieve the cytotoxic effect of DC661. Inhibition of cathepsins, or iron or calcium chelation, did not rescue DC661-induced cytotoxicity. PPT1 inhibition induced lysosomal lipid peroxidation (LLP), which led to lysosomal membrane permeabilization and cell death that could be reversed by the antioxidant N-acetylcysteine (NAC) but not by other lipid peroxidation antioxidants. The lysosomal cysteine transporter MFSD12 was required for intralysosomal transport of NAC and rescue of LLP. PPT1 inhibition produced cell-intrinsic immunogenicity with surface expression of calreticulin that could only be reversed with NAC. DC661-treated cells primed naive T cells and enhanced T cell-mediated toxicity. Mice vaccinated with DC661-treated cells engendered adaptive immunity and tumor rejection in "immune hot" tumors but not in "immune cold" tumors. These findings demonstrate that LLP drives lysosomal cell death, a unique immunogenic form of cell death, pointing the way to rational combinations of immunotherapy and lysosomal inhibition that can be tested in clinical trials.

Targeting UGCG Overcomes Resistance to Lysosomal Autophagy Inhibition.

Lysosomal autophagy inhibition (LAI) with hydroxychloroquine or DC661 can enhance cancer therapy, but tumor regrowth is common. To elucidate LAI resistance, proteomics and immunoblotting demonstrated that LAI induced lipid metabolism enzymes in multiple cancer cell lines. Lipidomics showed that LAI increased cholesterol, sphingolipids, and glycosphingolipids. These changes were associated with striking levels of GM1+ membrane microdomains (GMM) in plasma membranes and lysosomes. Inhibition of cholesterol/sphingolipid metabolism proteins enhanced LAI cytotoxicity. Targeting UDP-glucose ceramide glucosyltransferase (UGCG) synergistically augmented LAI cytotoxicity. Although UGCG inhibition decreased LAI-induced GMM and augmented cell death, UGCG overexpression led to LAI resistance. Melanoma patients with high UGCG expression had significantly shorter disease-specific survival. The FDA-approved UGCG inhibitor eliglustat combined with LAI significantly inhibited tumor growth and improved survival in syngeneic tumors and a therapy-resistant patient-derived xenograft. These findings nominate UGCG as a new cancer target, and clinical trials testing UGCG inhibition in combination with LAI are warranted. SIGNIFICANCE: We discovered UGCG-dependent lipid remodeling drives resistance to LAI. Targeting UGCG with a drug approved for a lysosomal storage disorder enhanced LAI antitumor activity without toxicity. LAI and UGCG inhibition could be tested clinically in multiple cancers. This article is highlighted in the In This Issue feature, p. 247.

The microbiome-derived metabolite TMAO drives immune activation and boosts responses to immune checkpoint blockade in pancreatic cancer.

The composition of the gut microbiome can control innate and adaptive immunity and has emerged as a key regulator of tumor growth, especially in the context of immune checkpoint blockade (ICB) therapy. However, the underlying mechanisms for how the microbiome affects tumor growth remain unclear. Pancreatic ductal adenocarcinoma (PDAC) tends to be refractory to therapy, including ICB. Using a nontargeted, liquid chromatography-tandem mass spectrometry-based metabolomic screen, we identified the gut microbe-derived metabolite trimethylamine N-oxide (TMAO), which enhanced antitumor immunity to PDAC. Delivery of TMAO intraperitoneally or via a dietary choline supplement to orthotopic PDAC-bearing mice reduced tumor growth, associated with an immunostimulatory tumor-associated macrophage (TAM) phenotype, and activated effector T cell response in the tumor microenvironment. Mechanistically, TMAO potentiated the type I interferon (IFN) pathway and conferred antitumor effects in a type I IFN-dependent manner. Delivering TMAO-primed macrophages intravenously produced similar antitumor effects. Combining TMAO with ICB (anti-PD1 and/or anti-Tim3) in a mouse model of PDAC significantly reduced tumor burden and improved survival beyond TMAO or ICB alone. Last, the levels of bacteria containing CutC (an enzyme that generates trimethylamine, the TMAO precursor) correlated with long-term survival in patients with PDAC and improved response to anti-PD1 in patients with melanoma. Together, our study identifies the gut microbial metabolite TMAO as a driver of antitumor immunity and lays the groundwork for potential therapeutic strategies targeting TMAO.

Ghost mitochondria drive metastasis through adaptive GCN2/Akt therapeutic vulnerability.

Cancer metabolism, including in mitochondria, is a disease hallmark and therapeutic target, but its regulation is poorly understood. Here, we show that many human tumors have heterogeneous and often reduced levels of Mic60, or Mitofilin, an essential scaffold of mitochondrial structure. Despite a catastrophic collapse of mitochondrial integrity, loss of bioenergetics, and oxidative damage, tumors with Mic60 depletion slow down cell proliferation, evade cell death, and activate a nuclear gene expression program of innate immunity and cytokine/chemokine signaling. In turn, this induces epithelial-mesenchymal transition (EMT), activates tumor cell movements through exaggerated mitochondrial dynamics, and promotes metastatic dissemination in vivo. In a small-molecule drug screen, compensatory activation of stress response (GCN2) and survival (Akt) signaling maintains the viability of Mic60-low tumors and provides a selective therapeutic vulnerability. These data demonstrate that acutely damaged, "ghost" mitochondria drive tumor progression and expose an actionable therapeutic target in metastasis-prone cancers.

A cancer ubiquitome landscape identifies metabolic reprogramming as target of Parkin tumor suppression.

Changes in metabolism that affect mitochondrial and glycolytic networks are hallmarks of cancer, but their impact in disease is still elusive. Using global proteomics and ubiquitome screens, we now show that Parkin, an E3 ubiquitin ligase and key effector of mitophagy altered in Parkinson's disease, shuts off mitochondrial dynamics and inhibits the non-oxidative phase of the pentose phosphate pathway. This blocks tumor cell movements, creates metabolic and oxidative stress, and inhibits primary and metastatic tumor growth. Uniformly down-regulated in cancer patients, Parkin tumor suppression requires its E3 ligase function, is reversed by antioxidants, and is independent of mitophagy. These data demonstrate that cancer metabolic networks are potent oncogenes directly targeted by endogenous tumor suppression.

Non-invasive plasma glycomic and metabolic biomarkers of post-treatment control of HIV.

Non-invasive biomarkers that predict HIV remission after antiretroviral therapy (ART) interruption are urgently needed. Such biomarkers can improve the safety of analytic treatment interruption (ATI) and provide mechanistic insights into the host pathways involved in post-ART HIV control. Here we report plasma glycomic and metabolic signatures of time-to-viral-rebound and probability-of-viral-remission using samples from two independent cohorts. These samples include a large number of post-treatment controllers, a rare population demonstrating sustained virologic suppression after ART-cessation. These signatures remain significant after adjusting for key demographic and clinical confounders. We also report mechanistic links between some of these biomarkers and HIV latency reactivation and/or myeloid inflammation in vitro. Finally, machine learning algorithms, based on selected sets of these biomarkers, predict time-to-viral-rebound with 74% capacity and probability-of-viral-remission with 97.5% capacity. In summary, we report non-invasive plasma biomarkers, with potential functional significance, that predict both the duration and probability of HIV remission after treatment interruption.

ATF3 coordinates serine and nucleotide metabolism to drive cell cycle progression in acute myeloid leukemia.

Metabolic reprogramming is a common feature of many human cancers, including acute myeloid leukemia (AML). However, the upstream regulators that promote AML metabolic reprogramming and the benefits conferred to leukemia cells by these metabolic changes remain largely unknown. We report that the transcription factor ATF3 coordinates serine and nucleotide metabolism to maintain cell cycling, survival, and the differentiation blockade in AML. Analysis of mouse and human AML models demonstrate that ATF3 directly activates the transcription of genes encoding key enzymatic regulators of serine synthesis, one-carbon metabolism, and de novo purine and pyrimidine synthesis. Total steady-state polar metabolite and heavy isotope tracing analyses show that ATF3 inhibition reduces de novo serine synthesis, impedes the incorporation of serine-derived carbons into newly synthesized purines, and disrupts pyrimidine metabolism. Importantly, exogenous nucleotide supplementation mitigates the anti-leukemia effects of ATF3 inhibition. Together, these findings reveal the dependence of AML on ATF3-regulated serine and nucleotide metabolism.

Measuring MYC-Mediated Metabolism in Tumorigenesis.

The MYC gene regulates normal cell growth and is deregulated in many human cancers, contributing to tumor growth and progression. The MYC transcription factor activates RNA polymerases I, II, and III target genes that are considered housekeeping genes. These target genes are largely involved in ribosome biogenesis, fatty acid, protein and nucleotide synthesis, nutrient influx or metabolic waste efflux, glycolysis, and glutamine metabolism. MYC's function as a driver of cell growth has been revealed through RNA sequencing, genome-wide chromatin immunoprecipitation, proteomics, and importantly metabolomics, which is highlighted in this chapter.

Phospholipid Metabolism Is Associated with Time to HIV Rebound upon Treatment Interruption.

Lipids are biologically active molecules involved in a variety of cellular processes and immunological functions, including inflammation. It was recently shown that phospholipids and their derivatives, lysophospholipids, can reactivate latent (dormant) tumor cells, causing cancer recurrence. However, the potential link between lipids and HIV latency, persistence, and viral rebound after cessation of antiretroviral therapy (ART) has never been investigated. We explored the links between plasma lipids and the burden of HIV during ART. We profiled the circulating lipidome from plasma samples from 24 chronically HIV-infected individuals on suppressive ART who subsequently underwent an analytic treatment interruption (ATI) without concurrent immunotherapies. The pre-ATI viral burden was estimated as time-to-viral-rebound and viral load set points post-ATI. We found that higher pre-ATI levels of lysophospholipids, including the proinflammatory lysophosphatidylcholine, were associated with faster time-to-viral-rebound and higher viral set points upon ART cessation. Furthermore, higher pre-ATI levels of the proinflammatory by-product of intestinal lysophosphatidylcholine metabolism, trimethylamine-N-oxide (TMAO), were also linked to faster viral rebound post-ART. Finally, pre-ATI levels of several phosphatidylcholine species (lysophosphatidylcholine precursors) correlated strongly with higher pre-ATI levels of HIV DNA in peripheral CD4+ T cells. Our proof-of-concept data point to phospholipids and lysophospholipids as plausible proinflammatory contributors to HIV persistence and rapid post-ART HIV rebound. The potential interplay between phospholipid metabolism and both the establishment and maintenance of HIV latent reservoirs during and after ART warrants further investigation.IMPORTANCE The likelihood of HIV rebound after stopping antiretroviral therapy (ART) is a combination of the size of HIV reservoirs that persist despite ART and the host immunological and inflammatory factors that control these reservoirs. Therefore, there is a need to comprehensively understand these host factors to develop a strategy to cure HIV infection and prevent viral rebound post-ART. Lipids are important biologically active molecules that are known to mediate several cellular functions, including reactivating latent tumor cells; however, their role in HIV latency, persistence, and post-ART rebound has never been investigated. We observed significant links between higher levels of the proinflammatory lysophosphatidylcholine and its intestinal metabolic by-product, trimethylamine-N-oxide, and both faster time-to-viral-rebound and higher viral load set point post-ART. These data highlight the need for further studies to understand the potential contribution of phosphatidylcholine and lysophosphatidylcholine metabolism in shaping host immunological and inflammatory milieu during and after ART.

A multi-omics approach to Epstein-Barr virus immortalization of B-cells reveals EBNA1 chromatin pioneering activities targeting nucleotide metabolism.

Epstein-Barr virus (EBV) immortalizes resting B-lymphocytes through a highly orchestrated reprogramming of host chromatin structure, transcription and metabolism. Here, we use a multi-omics-based approach to investigate these underlying mechanisms. ATAC-seq analysis of cellular chromatin showed that EBV alters over a third of accessible chromatin during the infection time course, with many of these sites overlapping transcription factors such as PU.1, Interferon Regulatory Factors (IRFs), and CTCF. Integration of RNA-seq analysis identified a complex transcriptional response and associations with EBV nuclear antigens (EBNAs). Focusing on EBNA1 revealed enhancer-binding activity at gene targets involved in nucleotide metabolism, supported by metabolomic analysis which indicated that adenosine and purine metabolism are significantly altered by EBV immortalization. We further validated that adenosine deaminase (ADA) is a direct and critical target of the EBV-directed immortalization process. These findings reveal that purine metabolism and ADA may be useful therapeutic targets for EBV-driven lymphoid cancers.

Cholesterol Pathway Inhibition Induces TGF-ß Signaling to Promote Basal Differentiation in Pancreatic Cancer.

Oncogenic transformation alters lipid metabolism to sustain tumor growth. We define a mechanism by which cholesterol metabolism controls the development and differentiation of pancreatic ductal adenocarcinoma (PDAC). Disruption of distal cholesterol biosynthesis by conditional inactivation of the rate-limiting enzyme Nsdhl or treatment with cholesterol-lowering statins switches glandular pancreatic carcinomas to a basal (mesenchymal) phenotype in mouse models driven by KrasG12D expression and homozygous Trp53 loss. Consistently, PDACs in patients receiving statins show enhanced mesenchymal features. Mechanistically, statins and NSDHL loss induce SREBP1 activation, which promotes the expression of Tgfb1, enabling epithelial-mesenchymal transition. Evidence from patient samples in this study suggests that activation of transforming growth factor ß signaling and epithelial-mesenchymal transition by cholesterol-lowering statins may promote the basal type of PDAC, conferring poor outcomes in patients.

129Xe NMR-Protein Sensor Reveals Cellular Ribose Concentration.

Dysregulation of cellular ribose uptake can be indicative of metabolic abnormalities or tumorigenesis. However, analytical methods are currently limited for quantifying ribose concentration in complex biological samples. Here, we utilize the highly specific recognition of ribose by ribose-binding protein (RBP) to develop a single-protein ribose sensor detectable via a sensitive NMR technique known as hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST). We demonstrate that RBP, with a tunable ribose-binding site and further engineered to bind xenon, enables the quantitation of ribose over a wide concentration range (nM to mM). Ribose binding induces the RBP "closed" conformation, which slows Xe exchange to a rate detectable by hyper-CEST. Such detection is remarkably specific for ribose, with the minimal background signal from endogenous sugars of similar size and structure, for example, glucose or ribose-6-phosphate. Ribose concentration was measured for mammalian cell lysate and serum, which led to estimates of low-mM ribose in a HeLa cell line. This highlights the potential for using genetically encoded periplasmic binding proteins such as RBP to measure metabolites in different biological fluids, tissues, and physiologic states.

The mitophagy effector FUNDC1 controls mitochondrial reprogramming and cellular plasticity in cancer cells.

Mitochondria are signaling hubs in eukaryotic cells. Here, we showed that the mitochondrial FUN14 domain-containing protein-1 (FUNDC1), an effector of Parkin-independent mitophagy, also participates in cellular plasticity by sustaining oxidative bioenergetics, buffering ROS production, and supporting cell proliferation. Targeting this pathway in cancer cells suppressed tumor growth but rendered transformed cells more motile and invasive in a manner dependent on ROS-mediated mitochondrial dynamics and mitochondrial repositioning to the cortical cytoskeleton. Global metabolomics and proteomics profiling identified a FUNDC1 interactome at the mitochondrial inner membrane, comprising the AAA+ protease, LonP1, and subunits of oxidative phosphorylation, complex V (ATP synthase). Independently of its previously identified role in mitophagy, FUNDC1 enabled LonP1 proteostasis, which in turn preserved complex V function and decreased ROS generation. Therefore, mitochondrial reprogramming by a FUNDC1-LonP1 axis controls tumor cell plasticity by switching between proliferative and invasive states in cancer.

Changes in Aged Fibroblast Lipid Metabolism Induce Age-Dependent Melanoma Cell Resistance to Targeted Therapy via the Fatty Acid Transporter FATP2.

Older patients with melanoma (>50 years old) have poorer prognoses and response rates to targeted therapy compared with young patients (<50 years old), which can be driven, in part, by the aged microenvironment. Here, we show that aged dermal fibroblasts increase the secretion of neutral lipids, especially ceramides. When melanoma cells are exposed to the aged fibroblast lipid secretome, or cocultured with aged fibroblasts, they increase the uptake of lipids via the fatty acid transporter FATP2, which is upregulated in melanoma cells in the aged microenvironment and known to play roles in lipid synthesis and accumulation. We show that blocking FATP2 in melanoma cells in an aged microenvironment inhibits their accumulation of lipids and disrupts their mitochondrial metabolism. Inhibiting FATP2 overcomes age-related resistance to BRAF/MEK inhibition in animal models, ablates tumor relapse, and significantly extends survival time in older animals. SIGNIFICANCE: These data show that melanoma cells take up lipids from aged fibroblasts, via FATP2, and use them to resist targeted therapy. The response to targeted therapy is altered in aged individuals because of the influences of the aged microenvironment, and these data suggest FATP2 as a target to overcome resistance.See related commentary by Montal and White, p. 1255.This article is highlighted in the In This Issue feature, p. 1241.

Proteome Analysis Using Gel-LC-MS/MS.

This article describes processing of protein samples using 1D SDS gels prior to protease digestion for proteomics workflows that subsequently utilize reversed-phase nanocapillary ultra-high-pressure liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). The resulting LC-MS/MS data are used to identify peptides and thereby infer proteins present in samples ranging from simple mixtures to very complex proteomes. Bottom-up proteome studies usually involve quantitative comparisons across several or many samples. For either situation, 1D SDS gels represent a simple, widely available technique that can be used to either fractionate complex proteomes or rapidly clean up low microgram samples with minimal losses. After gel separation and staining/destaining, appropriate gel slices are excised, and in-gel reduction, alkylation, and protease digestion are performed. Digests are then processed for LC-MS/MS analysis. Protocols are described for either sample fractionation with high-throughput processing of many samples or simple cleanup without fractionation. An optional strategy is to conduct in-solution reduction and alkylation prior to running gels, which is advantageous when a large number of samples will be separated into large numbers of fractions. Optimization of trypsin digestion parameters and comparison to in-solution protease digestion are also described. © 2019 by John Wiley & Sons, Inc.

PPT1 Promotes Tumor Growth and Is the Molecular Target of Chloroquine Derivatives in Cancer.

Clinical trials repurposing lysosomotropic chloroquine (CQ) derivatives as autophagy inhibitors in cancer demonstrate encouraging results, but the underlying mechanism of action remains unknown. Here, we report a novel dimeric CQ (DC661) capable of deacidifying the lysosome and inhibiting autophagy significantly better than hydroxychloroquine (HCQ). Using an in situ photoaffinity pulldown strategy, we identified palmitoyl-protein thioesterase 1 (PPT1) as a molecular target shared across monomeric and dimeric CQ derivatives. HCQ and Lys05 also bound to and inhibited PPT1 activity, but only DC661 maintained activity in acidic media. Knockout of PPT1 in cancer cells using CRISPR/Cas9 editing abrogates autophagy modulation and cytotoxicity of CQ derivatives, and results in significant impairment of tumor growth similar to that observed with DC661. Elevated expression of PPT1 in tumors correlates with poor survival in patients in a variety of cancers. Thus, PPT1 represents a new target in cancer that can be inhibited with CQ derivatives. SIGNIFICANCE: This study identifies PPT1 as the previously unknown lysosomal molecular target of monomeric and dimeric CQ derivatives. Genetic suppression of PPT1 impairs tumor growth, and PPT1 levels are elevated in cancer and associated with poor survival. These findings provide a strong rationale for targeting PPT1 in cancer. This article is highlighted in the In This Issue feature, p. 151.