N-Myristoyltransferase Inhibition causes Mitochondrial Iron Overload and Parthanatos in TIM17A-dependent Aggressive Lung Carcinoma.

Myristoylation is a type of protein acylation by which the fatty acid myristate is added to the N-terminus of target proteins, a process mediated by N-myristoyltransferases. Myristoylation is emerging as a promising cancer therapeutic target, however the molecular determinants of sensitivity to N-myristoyltransferase inhibition or the mechanism by which it induces cancer cell death are not completely understood. We report that N-myristoyltransferases are a novel therapeutic target in lung carcinoma cells with LKB1 and/or KEAP1 mutations in a KRAS mutant background. Inhibition of myristoylation decreases cell viability in vitro and tumor growth in vivo. Inhibition of myristoylation causes mitochondrial ferrous iron overload, oxidative stress, elevated protein poly (ADP)-ribosylation and death by parthanatos. Furthermore, NMT inhibitors sensitized lung carcinoma cells to platinum-based chemotherapy. Unexpectedly, the mitochondrial transporter Translocase of Inner Mitochondrial Membrane 17 homologue A (TIM17A) is a critical target of myristoylation inhibitors in these cells. TIM17A silencing recapitulated the effects of NMT inhibition at inducing mitochondrial ferrous iron overload and parthanatos. Furthermore, sensitivity of lung carcinoma cells to myristoylation inhibition correlated with their dependency on TIM17A. This study reveals the unexpected connection between protein myristoylation, the mitochondrial import machinery, and iron homeostasis. It also uncovers myristoylation inhibitors as novel inducers of parthanatos in cancer, and the novel axis N-myristoyltransferase-TIM17A as a potential therapeutic target in highly aggressive lung carcinomas.

MYCT1 controls environmental sensing in human haematopoietic stem cells.

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs1-3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.

Wild-type C-Raf gene dosage and dimerization drive prostate cancer metastasis.

Mutated Ras and Raf kinases are well-known to promote cancer metastasis via flux through the Ras/Raf/MEK/ERK (mitogen-activated protein kinase [MAPK]) pathway. A role for non-mutated Raf in metastasis is also emerging, but the key mechanisms remain unclear. Elevated expression of any of the three wild-type Raf family members (C, A, or B) can drive metastasis. We utilized an in vivo model to show that wild-type C-Raf overexpression can promote metastasis of immortalized prostate cells in a gene dosage-dependent manner. Analysis of the transcriptomic and phosphoproteomic landscape indicated that C-Raf-driven metastasis is accompanied by upregulated MAPK signaling. Use of C-Raf mutants demonstrated that the dimerization domain, but not its kinase activity, is essential for metastasis. Endogenous Raf monomer knockouts revealed that C-Raf's ability to form dimers with endogenous Raf molecules is important for promoting metastasis. These data identify wild-type C-Raf heterodimer signaling as a potential target for treating metastatic disease.

Adenovirus E1A binding to DCAF10 targets proteasomal degradation of RUVBL1/2 AAA+ ATPases required for quaternary assembly of multiprotein machines, innate immunity, and responses to metabolic stress.

IMPORTANCE: Inactivation of EP300/CREBB paralogous cellular lysine acetyltransferases (KATs) during the early phase of infection is a consistent feature of DNA viruses. The cell responds by stabilizing transcription factor IRF3 which activates transcription of scores of interferon-stimulated genes (ISGs), inhibiting viral replication. Human respiratory adenoviruses counter this by assembling a CUL4-based ubiquitin ligase complex that polyubiquitinylates RUVBL1 and 2 inducing their proteasomal degradation. This inhibits accumulation of active IRF3 and the expression of anti-viral ISGs, allowing replication of the respiratory HAdVs in the face of inhibition of EP300/CBEBBP KAT activity by the N-terminal region of E1A.

The apoptotic splicing regulators RBM5 and RBM10 are subunits of the U2 snRNP engaged with intron branch sites on chromatin.

Understanding the mechanisms of pre-mRNA splicing and spliceosome assembly is limited by technical challenges to examining spliceosomes in vivo. Here we report the isolation of RNP complexes derived from precatalytic A or B-like spliceosomes solubilized from the chromatin pellet of lysed nuclei. We found that these complexes contain U2 snRNP proteins and a portion of the U2 snRNA, bound with intronic branch sites prior to the first catalytic step of splicing. Sequencing these pre-mRNA fragments allowed the transcriptome-wide mapping of branch sites with high sensitivity. In addition to known U2 snRNP proteins, these complexes contained the proteins RBM5 and RBM10. RBM5 and RBM10 are alternative splicing regulators that control exons affecting apoptosis and cell proliferation in cancer, but were not previously shown to associate with the U2 snRNP or to play roles in branch site selection. We delineate a common segment of RBM5 and RBM10, separate from their known functional domains, that is required for their interaction with the U2 snRNP. We identify a large set of splicing events regulated by RBM5 and RBM10 and find that they predominantly act as splicing silencers. Disruption of their U2 interaction renders the proteins inactive for repression of many alternative exons. We further find that these proteins assemble on branch sites of nearly all exons across the transcriptome, including those whose splicing is not altered by them. We propose a model where RBM5 and RBM10 act as components of the U2 snRNP complex. From within this complex, they sense structural features of branchpoint recognition to either allow progression to functional spliceosome or rejection of the complex to inhibit splicing.

A synthetic coolant (WS-23) in disposable electronic cigarettes impairs cytoskeletal function in EpiAirway microtissues exposed at the air liquid interface.

The design of popular disposable electronic cigarettes (ECs) was analyzed, and the concentrations of WS-23, a synthetic coolant, in EC fluids were determined for 22 devices from 4 different brands. All products contained WS-23 in concentrations that ranged from 1.0 to 40.1 mg/mL (mean = 21.4 ± 9.2 mg/mL). To determine the effects of WS-23 on human bronchial epithelium in isolation of other chemicals, we exposed EpiAirway 3-D microtissues to WS-23 at the air liquid interface (ALI) using a cloud chamber that generated aerosols without heating. Proteomics analysis of exposed tissues revealed that the cytoskeleton was a major target of WS-23. BEAS-2B cells were exposed to WS-23 in submerged culture to validate the main results from proteomics. F-actin, which was visualized with phalloidin, decreased concentration dependently in WS-23 treated BEAS-2B cells, and cells became immotile in concentrations above 1.5 mg/mL. Gap closure, which depends on both cell proliferation and migration, was inhibited by 0.45 mg/mL of WS-23. These data show that WS-23 is being added to popular EC fluids at concentrations that can impair processes dependent on the actin cytoskeleton and disturb homeostasis of the bronchial epithelium. The unregulated use of WS-23 in EC products may harm human health.

The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.

Toxoplasma gondii's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole (PV) in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both acute and chronic infections. Murine macrophages infected with ∆gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro, which was confirmed with reduced IL-12 and interferon-gamma in vivo. This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the nuclear factor-κB (NF-κB) complex. While GRA15 similarly regulates NF-κB, infection with ∆gra83/∆gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labeling experiments to reveal candidate GRA83 interacting T. gondii-derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit the parasite burden. Importance Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma's ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection is important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection.

FBXL12 degrades FANCD2 to regulate replication recovery and promote cancer cell survival under conditions of replication stress.

Fanconi anemia (FA) signaling, a key genomic maintenance pathway, is activated in response to replication stress. Here, we report that phosphorylation of the pivotal pathway protein FANCD2 by CHK1 triggers its FBXL12-dependent proteasomal degradation, facilitating FANCD2 clearance at stalled replication forks. This promotes efficient DNA replication under conditions of CYCLIN E- and drug-induced replication stress. Reconstituting FANCD2-deficient fibroblasts with phosphodegron mutants failed to re-establish fork progression. In the absence of FBXL12, FANCD2 becomes trapped on chromatin, leading to replication stress and excessive DNA damage. In human cancers, FBXL12, CYCLIN E, and FA signaling are positively correlated, and FBXL12 upregulation is linked to reduced survival in patients with high CYCLIN E-expressing breast tumors. Finally, depletion of FBXL12 exacerbated oncogene-induced replication stress and sensitized cancer cells to drug-induced replication stress by WEE1 inhibition. Collectively, our results indicate that FBXL12 constitutes a vulnerability and a potential therapeutic target in CYCLIN E-overexpressing cancers.

The Toxoplasma gondii effector GRA83 modulates the host's innate immune response to regulate parasite infection.

Toxoplasma gondii 's propensity to infect its host and cause disease is highly dependent on its ability to modulate host cell functions. One of the strategies the parasite uses to accomplish this is via the export of effector proteins from the secretory dense granules. Dense granule (GRA) proteins are known to play roles in nutrient acquisition, host cell cycle manipulation, and immune regulation. Here, we characterize a novel dense granule protein named GRA83, which localizes to the parasitophorous vacuole in tachyzoites and bradyzoites. Disruption of GRA83 results in increased virulence, weight loss, and parasitemia during the acute infection, as well as a marked increase in the cyst burden during the chronic infection. This increased parasitemia was associated with an accumulation of inflammatory infiltrates in tissues in both the acute and chronic infection. Murine macrophages infected with Δ gra83 tachyzoites produced less interleukin-12 (IL-12) in vitro , which was confirmed with reduced IL-12 and interferon gamma (IFN-γ) in vivo . This dysregulation of cytokines correlates with reduced nuclear translocation of the p65 subunit of the NF-κB complex. While GRA15 similarly regulates NF-κB, infection with Δ gra83/ Δ gra15 parasites did not further reduce p65 translocation to the host cell nucleus, suggesting these GRAs function in converging pathways. We also used proximity labelling experiments to reveal candidate GRA83 interacting T. gondii derived partners. Taken together, this work reveals a novel effector that stimulates the innate immune response, enabling the host to limit parasite burden. Importance: Toxoplasma gondii poses a significant public health concern as it is recognized as one of the leading foodborne pathogens in the United States. Infection with the parasite can cause congenital defects in neonates, life-threatening complications in immunosuppressed patients, and ocular disease. Specialized secretory organelles, including the dense granules, play an important role in the parasite's ability to efficiently invade and regulate components of the host's infection response machinery to limit parasite clearance and establish an acute infection. Toxoplasma' s ability to avoid early clearance, while also successfully infecting the host long enough to establish a persistent chronic infection, is crucial in allowing for its transmission to a new host. While multiple GRAs directly modulate host signaling pathways, they do so in various ways highlighting the parasite's diverse arsenal of effectors that govern infection. Understanding how parasite-derived effectors harness host functions to evade defenses yet ensure a robust infection are important for understanding the complexity of the pathogen's tightly regulated infection. In this study, we characterize a novel secreted protein named GRA83 that stimulates the host cell's response to limit infection.

The MOM1 complex recruits the RdDM machinery via MORC6 to establish de novo DNA methylation.

MORPHEUS' MOLECULE1 (MOM1) is an Arabidopsis factor previously shown to mediate transcriptional silencing independent of major DNA methylation changes. Here we find that MOM1 localizes with sites of RNA-directed DNA methylation (RdDM). Tethering MOM1 with an artificial zinc finger to an unmethylated FWA promoter leads to establishment of DNA methylation and FWA silencing. This process is blocked by mutations in components of the Pol V arm of the RdDM machinery, as well as by mutation of MICRORCHIDIA 6 (MORC6). We find that at some endogenous RdDM sites, MOM1 is required to maintain DNA methylation and a closed chromatin state. In addition, efficient silencing of newly introduced FWA transgenes is impaired in the mom1 mutant. In addition to RdDM sites, we identify a group of MOM1 peaks at active chromatin near genes that colocalized with MORC6. These findings demonstrate a multifaceted role of MOM1 in genome regulation.

Analyzing Iron and Oxygen-Regulated Protein Complex Formation Using Proteomic Mass Spectrometry.

Multicellular organisms have evolved elaborate strategies to sense and adapt to changes in intracellular oxygen. The canonical cellular pathway responsible for oxygen sensing consists of the von Hippel-Lindau (pVHL) tumor suppressor protein, prolyl hydroxylases (PHD), and hypoxia-inducible factors (HIFs), which together regulate expression of downstream genes involved in oxygen homeostasis. In recent years, it has become increasingly clear that oxygen regulatory mechanisms are intertwined with cellular iron-sensing pathways. Key members of these networks such as prolyl-hydroxylases, E3 ubiquitin ligase adaptor protein FBXL5, iron regulatory proteins (IRPs), and Fe-S cluster proteins require both iron and oxygen for their optimal function and/or are tightly regulated by intracellular concentrations of these molecules. Monitoring how protein interactomes are remodeled as a function of intracellular oxygen and iron levels gives insights into the nature and dynamics of these pathways. We have recently described an oxygen-sensitive interaction between FBXL5 and the cytoplasmic Fe-S cluster targeting complex (CIA targeting complex) with implications in the FBXL5-dependent regulation of IRPs. Based on this work, we present a protocol describing the induction and maintenance of hypoxia in mammalian cell cultures and a mass-spectrometry-based proteomics approach aimed at interrogating changes in interactome of key proteins as a function of intracellular oxygen and iron levels. These methods are widely applicable to understanding the dynamics of iron and oxygen signaling.

High-Field Asymmetric Waveform Ion Mobility Spectrometry Interface Enhances Parallel Reaction Monitoring on an Orbitrap Mass Spectrometer.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) enables gas-phase separations on a chromatographic time scale and has become a useful tool for proteomic applications. Despite its emerging utility, however, the molecular determinants underlying peptide separation by FAIMS have not been systematically investigated. Here, we characterize peptide transmission in a FAIMS device across a broad range of compensation voltages (CVs) and used machine learning to identify charge state and three-dimensional (3D) electrostatic peptide potential as major contributors to peptide intensity at a given CV. We also demonstrate that the machine learning model can be used to predict optimized CV values for peptides, which significantly improves parallel reaction monitoring workflows. Together, these data provide insight into peptide separation by FAIMS and highlight its utility in targeted proteomic applications.

The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers.

The human SMC5/6 complex is a conserved guardian of genome stability and an emerging component of antiviral responses. These disparate functions likely require distinct mechanisms of SMC5/6 regulation. In yeast, Smc5/6 is regulated by its Nse5/6 subunits, but such regulatory subunits for human SMC5/6 are poorly defined. Here, we identify a novel SMC5/6 subunit called SIMC1 that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. We isolated SIMC1 from the proteomic environment of SMC5/6 within polyomavirus large T antigen (LT)-induced subnuclear compartments. SIMC1 uses its SIMs and Nse5-like domain to localize SMC5/6 to polyomavirus replication centers (PyVRCs) at SUMO-rich PML nuclear bodies. SIMC1's Nse5-like domain binds to the putative Nse6 orthologue SLF2 to form an anti-parallel helical dimer resembling the yeast Nse5/6 structure. SIMC1-SLF2 structure-based mutagenesis defines a conserved surface region containing the N-terminus of SIMC1's helical domain that regulates SMC5/6 localization to PyVRCs. Furthermore, SLF1, which recruits SMC5/6 to DNA lesions via its BRCT and ARD motifs, binds SLF2 analogously to SIMC1 and forms a separate Nse5/6-like complex. Thus, two Nse5/6-like complexes with distinct recruitment domains control human SMC5/6 localization.

The TRIPLE PHD FINGERS proteins are required for SWI/SNF complex-mediated +1 nucleosome positioning and transcription start site determination in Arabidopsis.

Eukaryotes have evolved multiple ATP-dependent chromatin remodelers to shape the nucleosome landscape. We recently uncovered an evolutionarily conserved SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler complex in plants reminiscent of the mammalian BAF subclass, which specifically incorporates the MINUSCULE (MINU) catalytic subunits and the TRIPLE PHD FINGERS (TPF) signature subunits. Here we report experimental evidence that establishes the functional relevance of TPF proteins for the complex activity. Our results show that depletion of TPF triggers similar pleiotropic phenotypes and molecular defects to those found in minu mutants. Moreover, we report the genomic location of MINU2 and TPF proteins as representative members of this SWI/SNF complex and their impact on nucleosome positioning and transcription. These analyses unravel the binding of the complex to thousands of genes where it modulates the position of the +1 nucleosome. These targets tend to produce 5'-shifted transcripts in the tpf and minu mutants pointing to the participation of the complex in alternative transcription start site usage. Interestingly, there is a remarkable correlation between +1 nucleosome shift and 5' transcript length change suggesting their functional connection. In summary, this study unravels the function of a plant SWI/SNF complex involved in +1 nucleosome positioning and transcription start site determination.

Elucidation of TRIM25 ubiquitination targets involved in diverse cellular and antiviral processes.

The tripartite motif (TRIM) family of E3 ubiquitin ligases is well known for its roles in antiviral restriction and innate immunity regulation, in addition to many other cellular pathways. In particular, TRIM25-mediated ubiquitination affects both carcinogenesis and antiviral response. While individual substrates have been identified for TRIM25, it remains unclear how it regulates diverse processes. Here we characterized a mutation, R54P, critical for TRIM25 catalytic activity, which we successfully utilized to "trap" substrates. We demonstrated that TRIM25 targets proteins implicated in stress granule formation (G3BP1/2), nonsense-mediated mRNA decay (UPF1), nucleoside synthesis (NME1), and mRNA translation and stability (PABPC4). The R54P mutation abolishes TRIM25 inhibition of alphaviruses independently of the host interferon response, suggesting that this antiviral effect is a direct consequence of ubiquitination. Consistent with that, we observed diminished antiviral activity upon knockdown of several TRIM25-R54P specific interactors including NME1 and PABPC4. Our findings highlight that multiple substrates mediate the cellular and antiviral activities of TRIM25, illustrating the multi-faceted role of this ubiquitination network in modulating diverse biological processes.

Physical and in silico immunopeptidomic profiling of a cancer antigen prostatic acid phosphatase reveals targets enabling TCR isolation.

Tissue-specific antigens can serve as targets for adoptive T cell transfer-based cancer immunotherapy. Recognition of tumor by T cells is mediated by interaction between peptide-major histocompatibility complexes (pMHCs) and T cell receptors (TCRs). Revealing the identity of peptides bound to MHC is critical in discovering cognate TCRs and predicting potential toxicity. We performed multimodal immunopeptidomic analyses for human prostatic acid phosphatase (PAP), a well-recognized tissue antigen. Three physical methods, including mild acid elution, coimmunoprecipitation, and secreted MHC precipitation, were used to capture a thorough signature of PAP on HLA-A*02:01. Eleven PAP peptides that are potentially A*02:01-restricted were identified, including five predicted strong binders by NetMHCpan 4.0. Peripheral blood mononuclear cells (PBMCs) from more than 20 healthy donors were screened with the PAP peptides. Seven cognate TCRs were isolated which can recognize three distinct epitopes when expressed in PBMCs. One TCR shows reactivity toward cell lines expressing both full-length PAP and HLA-A*02:01. Our results show that a combined multimodal immunopeptidomic approach is productive in revealing target peptides and defining the cloned TCR sequences reactive with prostatic acid phosphatase epitopes.

Iron-regulated assembly of the cytosolic iron-sulfur cluster biogenesis machinery.

The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.

Enhancing PD-L1 Degradation by ITCH during MAPK Inhibitor Therapy Suppresses Acquired Resistance.

MAPK inhibitor (MAPKi) therapy in melanoma leads to the accumulation of tumor-surface PD-L1/L2, which may evade antitumor immunity and accelerate acquired resistance. Here, we discover that the E3 ligase ITCH binds, ubiquitinates, and downregulates tumor-surface PD-L1/L2 in MAPKi-treated human melanoma cells, thereby promoting T-cell activation. During MAPKi therapy in vivo, melanoma cell-intrinsic ITCH knockdown induced tumor-surface PD-L1, reduced intratumoral cytolytic CD8+ T cells, and accelerated acquired resistance only in immune-competent mice. Conversely, tumor cell-intrinsic ITCH overexpression reduced MAPKi-elicited PD-L1 accumulation, augmented intratumoral cytolytic CD8+ T cells, and suppressed acquired resistance in BrafV600MUT, NrasMUT, or Nf1MUT melanoma and KrasMUT-driven cancers. CD8+ T-cell depletion and tumor cell-intrinsic PD-L1 overexpression nullified the phenotype of ITCH overexpression, thereby supporting an in vivo ITCH-PD-L1-T-cell regulatory axis. Moreover, we identify a small-molecular ITCH activator that suppresses acquired MAPKi resistance in vivo. Thus, MAPKi-induced PD-L1 accelerates resistance, and a PD-L1-degrading ITCH activator prolongs antitumor response. SIGNIFICANCE: MAPKi induces tumor cell-surface PD-L1 accumulation, which promotes immune evasion and therapy resistance. ITCH degrades PD-L1, optimizing antitumor T-cell immunity. We propose degrading tumor cell-surface PD-L1 and/or activating tumor-intrinsic ITCH as strategies to overcome MAPKi resistance. This article is highlighted in the In This Issue feature, p. 1825.

Porcupine Inhibition Disrupts Mitochondrial Function and Homeostasis in WNT Ligand-Addicted Pancreatic Cancer.

WNT signaling promotes pancreatic ductal adenocarcinoma (PDAC) through diverse effects on proliferation, differentiation, survival, and stemness. A subset of PDAC with inactivating mutations in ring finger protein 43 (RNF43) show growth dependency on autocrine WNT ligand signaling and are susceptible to agents that block WNT ligand acylation by Porcupine O-acyltransferase, which is required for proper WNT ligand processing and secretion. For this study, global transcriptomic, proteomic, and metabolomic analyses were performed to explore the therapeutic response of RNF43-mutant PDAC to the Porcupine inhibitor (PORCNi) LGK974. LGK974 disrupted cellular bioenergetics and mitochondrial function through actions that included rapid mitochondrial depolarization, reduced mitochondrial content, and inhibition of oxidative phosphorylation and tricarboxylic acid cycle. LGK974 also broadly altered transcriptional activity, downregulating genes involved in cell cycle, nucleotide metabolism, and ribosomal biogenesis and upregulating genes involved in epithelial-mesenchymal transition, hypoxia, endocytosis, and lysosomes. Autophagy and lysosomal activity were augmented in response to LGK974, which synergistically inhibited tumor cell viability in combination with chloroquine. Autocrine WNT ligand signaling dictates metabolic dependencies in RNF43-mutant PDAC through a combination of transcription dependent and independent effects linked to mitochondrial health and function. Metabolic adaptations to mitochondrial damage and bioenergetic stress represent potential targetable liabilities in combination with PORCNi for the treatment of WNT ligand-addicted PDAC.