Cellular abundance of sodium phosphate cotransporter SLC20A1/PiT1 and phosphate uptake are controlled post-transcriptionally by ESCRT.

Inorganic phosphate is essential for human life. The widely expressed mammalian sodium/phosphate cotransporter SLC20A1/PiT1 mediates phosphate uptake into most cell types; however, while SLC20A1 is required for development, and elevated SLC20A1 expression is associated with vascular calcification and aggressive tumor growth, the mechanisms regulating SLC20A1 protein abundance are unknown. Here, we found that SLC20A1 protein expression is low in phosphate-replete cultured cells but is strikingly induced following phosphate starvation, whereas mRNA expression is high in phosphate-replete cells and only mildly increased by phosphate starvation. To identify regulators of SLC20A1 protein levels, we performed a genome-wide CRISPR-based loss-of-function genetic screen in phosphate-replete cells using SLC20A1 protein induction as readout. Our screen revealed that endosomal sorting complexes required for transport (ESCRT) machinery was essential for proper SLC20A1 protein downregulation in phosphate-replete cells. We show that SLC20A1 colocalizes with ESCRT and that ESCRT deficiency increases SLC20A1 protein and phosphate uptake into cells. We also found numerous additional candidate regulators of mammalian phosphate homeostasis, including genes modifying protein ubiquitination and the Krebs cycle and oxidative phosphorylation pathways. Many of these targets have not been previously implicated in this process. We present here a model in which SLC20A1 protein abundance and phosphate uptake are tonically negatively regulated post-transcriptionally in phosphate-replete cells through direct ESCRT-mediated SLC20A1 degradation. Moreover, our screening results provide a comprehensive resource for future studies to elucidate the mechanisms governing cellular phosphate homeostasis. We conclude that genome-wide CRISPR-based genetic screening is a powerful tool to discover proteins and pathways relevant to physiological processes.

Heterogeneous origins and functions of mouse skeletal muscle-resident macrophages.

Tissue-resident macrophages can originate from embryonic or adult hematopoiesis. They play important roles in a wide range of biological processes including tissue remodeling during organogenesis, organ homeostasis, repair following injury, and immune response to pathogens. Although the origins and tissue-specific functions of resident macrophages have been extensively studied in many other tissues, they are not well characterized in skeletal muscle. In the present study, we have characterized the ontogeny of skeletal muscle-resident macrophages by lineage tracing and bone marrow transplant experiments. We demonstrate that skeletal muscle-resident macrophages originate from both embryonic hematopoietic progenitors located within the yolk sac and fetal liver as well as definitive hematopoietic stem cells located within the bone marrow of adult mice. Single-cell-based transcriptome analyses revealed that skeletal muscle-resident macrophages are distinctive from resident macrophages in other tissues as they express a distinct complement of transcription factors and are composed of functionally diverse subsets correlating to their origins. Functionally, skeletal muscle-resident macrophages appear to maintain tissue homeostasis and promote muscle growth and regeneration.

SOX4-mediated repression of specific tRNAs inhibits proliferation of human glioblastoma cells.

Transfer RNAs (tRNAs) are products of RNA polymerase III (Pol III) and essential for mRNA translation and ultimately cell growth and proliferation. Whether and how individual tRNA genes are specifically regulated is not clear. Here, we report that SOX4, a well-known Pol II-dependent transcription factor that is critical for neurogenesis and reprogramming of somatic cells, also directly controls, unexpectedly, the expression of a subset of tRNA genes and therefore protein synthesis and proliferation of human glioblastoma cells. Genome-wide location analysis through chromatin immunoprecipitation-sequencing uncovers specific targeting of SOX4 to a subset of tRNA genes, including those for tRNAiMet Mechanistically, sequence-specific SOX4-binding impedes the recruitment of TATA box binding protein and Pol III to tRNA genes and thereby represses their expression. CRISPR/Cas9-mediated down-regulation of tRNAiMet greatly inhibits growth and proliferation of human glioblastoma cells. Conversely, ectopic tRNAiMet partially rescues SOX4-mediated repression of cell proliferation. Together, these results uncover a regulatory mode of individual tRNA genes to control cell behavior. Such regulation may coordinate codon usage and translation efficiency to meet the demands of diverse tissues and cell types, including cancer cells.

Chemical intervention of influenza virus mRNA nuclear export.

Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.

Extrarenal Anaplastic Wilms Tumor: A Case Report With Genomic Analysis and Tumor Models.

Primary extrarenal Wilms tumors are rare neoplasms that are presumed to arise from metanephric or mesonephric remnants outside of the kidney. Their pathogenesis is debated but has not been studied, and there are no reports of genomic descriptions of extrarenal Wilms tumors. We describe a diffusely anaplastic extrarenal Wilms tumor that occurred in the lower abdomen and upper pelvis of a 10-year-old boy. In addition to the clinical, histopathologic, and radiologic features, we describe the cytogenetic changes and exomic profile of the tumor. The tumor showed loss of the tumor suppressor AMER1, loss of chromosome regions 1p, 16q, and 22q, gain of chromosome 8, and loss of function TP53 mutation-findings known to occur in renal Wilms tumors. This is the first description of the exomic profile of a primary extrarenal Wilms tumor. Our data indicate that primary extrarenal Wilms tumors may follow the same pathogenetic pathways that are seen in renal Wilms tumors. Finally, we describe the establishment of first ever tumor models (primary cell line and patient-derived xenograft) from an extrarenal Wilms tumor.

The Lec5 glycosylation mutant links homeobox genes with cholesterol and lipid-linked oligosaccharides.

Discovered 40 years ago, the Lec5 glycosylation mutant cell line has a complex recessive genotype and is characterized by accumulation of lipid-linked oligosaccharide assembly intermediates, reduced conversion of polyprenols to dolichols, and an unusual phenotypic dependence upon cell culture conditions such as temperature, plating density and medium quality. The heritable defect in Lec5 is unknown. Here we demonstrate an unexpected epigenetic basis for Lec5, with a surprising linkage to increased expression of homeobox genes, which in turn is associated with increased transcription of cholesterol biosynthesis genes. These results suggest testable hypotheses for the biochemical abnormalities of the Lec5 mutant.

Transcriptomic meta-analysis reveals ERRα-mediated oxidative phosphorylation is downregulated in Fuchs' endothelial corneal dystrophy.

BACKGROUND: Late-onset Fuchs' endothelial corneal dystrophy (FECD) is a degenerative disease of cornea and the leading indication for corneal transplantation. Genetically, FECD patients can be categorized as with (RE+) or without (RE-) the CTG trinucleotide repeat expansion in the transcription factor 4 gene. The molecular mechanisms underlying FECD remain unclear, though there are plausible pathogenic models proposed for RE+ FECD. METHOD: In this study, we performed a meta-analysis on RNA sequencing datasets of FECD corneal endothelium including 3 RE+ datasets and 2 RE- datasets, aiming to compare the transcriptomic profiles of RE+ and RE- FECD. Gene differential expression analysis, co-expression networks analysis, and pathway analysis were conducted. RESULTS: There was a striking similarity between RE+ and RE- transcriptomes. There were 1,184 genes significantly upregulated and 1,018 genes significantly downregulated in both RE+ and RE- cases. Pathway analysis identified multiple biological processes significantly enriched in both-mitochondrial functions, energy-related processes, ER-nucleus signaling pathway, demethylation, and RNA splicing were negatively enriched, whereas small GTPase mediated signaling, actin-filament processes, extracellular matrix organization, stem cell differentiation, and neutrophil mediated immunity were positively enriched. The translational initiation process was downregulated in the RE+ transcriptomes. Gene co-expression analysis identified modules with relatively distinct biological processes enriched including downregulation of mitochondrial respiratory chain complex assembly. The majority of oxidative phosphorylation (OXPHOS) subunit genes, as well as their upstream regulator gene estrogen-related receptor alpha (ESRRA), encoding ERRα, were downregulated in both RE+ and RE- cases, and the expression level of ESRRA was correlated with that of OXPHOS subunit genes. CONCLUSION: Meta-analysis increased the power of detecting differentially expressed genes. Integrating differential expression analysis with co-expression analysis helped understand the underlying molecular mechanisms. FECD RE+ and RE- transcriptomic profiles are much alike with the hallmark of downregulation of genes in pathways related to ERRα-mediated OXPHOS.

Endothelial ERα promotes glucose tolerance by enhancing endothelial insulin transport to skeletal muscle.

The estrogen receptor (ER) designated ERα has actions in many cell and tissue types that impact glucose homeostasis. It is unknown if these include mechanisms in endothelial cells, which have the potential to influence relative obesity, and processes in adipose tissue and skeletal muscle that impact glucose control. Here we show that independent of impact on events in adipose tissue, endothelial ERα promotes glucose tolerance by enhancing endothelial insulin transport to skeletal muscle. Endothelial ERα-deficient male mice are glucose intolerant and insulin resistant, and in females the antidiabetogenic actions of estradiol (E2) are absent. The glucose dysregulation is due to impaired skeletal muscle glucose disposal that results from attenuated muscle insulin delivery. Endothelial ERα activation stimulates insulin transcytosis by skeletal muscle microvascular endothelial cells. Mechanistically this involves nuclear ERα-dependent upregulation of vesicular trafficking regulator sorting nexin 5 (SNX5) expression, and PI3 kinase activation that drives plasma membrane recruitment of SNX5. Thus, coupled nuclear and non-nuclear actions of ERα promote endothelial insulin transport to skeletal muscle to foster normal glucose homeostasis.

CCR2+ Macrophages Promote Orthodontic Tooth Movement and Alveolar Bone Remodeling.

During mechanical force-induced alveolar bone remodeling, macrophage-mediated local inflammation plays a critical role. Yet, the detailed heterogeneity of macrophages is still unknown. Single-cell RNA sequencing was used to study the transcriptome heterogeneity of macrophages during alveolar bone remodeling. We identified macrophage subclusters with specific gene expression profiles and functions. CellChat and trajectory analysis revealed a central role of the Ccr2 cluster during development, with the CCL signaling pathway playing a crucial role. We further demonstrated that the Ccr2 cluster modulated bone remodeling associated inflammation through an NF-κB dependent pathway. Blocking CCR2 could significantly reduce the Orthodontic tooth movement (OTM) progression. In addition, we confirmed the variation of CCR2+ macrophages in human periodontal tissues. Our findings reveal that mechanical force-induced functional shift of the Ccr2 macrophages cluster mediated by NF-κB pathway, leading to a pro-inflammatory response and bone remodeling. This macrophage cluster may represent a potential target for the manipulation of OTM.

TP53 promotes lineage commitment of human embryonic stem cells through ciliogenesis and sonic hedgehog signaling.

Aneuploidy, defective differentiation, and inactivation of the tumor suppressor TP53 all occur frequently during tumorigenesis. Here, we probe the potential links among these cancer traits by inactivating TP53 in human embryonic stem cells (hESCs). TP53-/- hESCs exhibit increased proliferation rates, mitotic errors, and low-grade structural aneuploidy; produce poorly differentiated immature teratomas in mice; and fail to differentiate into neural progenitor cells (NPCs) in vitro. Genome-wide CRISPR screen reveals requirements of ciliogenesis and sonic hedgehog (Shh) pathways for hESC differentiation into NPCs. TP53 deletion causes abnormal ciliogenesis in neural rosettes. In addition to restraining cell proliferation through CDKN1A, TP53 activates the transcription of BBS9, which encodes a ciliogenesis regulator required for proper Shh signaling and NPC formation. This developmentally regulated transcriptional program of TP53 promotes ciliogenesis, restrains Shh signaling, and commits hESCs to neural lineages.

Sexual Dimorphism in the Extracellular and Pericellular Matrix of Articular Cartilage.

OBJECTIVE: Women have a higher prevalence and burden of joint injuries and pathologies involving articular cartilage than men. Although knee injuries affecting young women are on the rise, most studies related to sexual dimorphism target postmenopausal women. We hypothesize that sexual dimorphism in cartilage structure and mechanics is present before menopause, which can contribute to sex disparities in cartilage pathologies. DESIGN: Bovine knee was used as a model to study healthy adult cartilage. We compared elastic moduli under compression, abundances of extracellular and pericellular matrix (PCM) proteins using proteomics, and PCM constituency with tissue immunofluorescence. The gene expression of matrix-related genes under basal, anabolic, and catabolic conditions was assessed by quantitative polymerase chain reaction (qPCR). RESULTS: The equilibrium modulus was higher in male cartilage compared with female cartilage. Proteoglycans were not associated with this biomechanical dimorphism. Proteomic and pathway analyses of tissue showed dimorphic enriched pathways in extracellular matrix (ECM)-related proteins in which male cartilage was enriched in matrix interconnectors and crosslinkers that strengthen the ECM network. Moreover, male and female tissue differed in enriched PCM components. Females had more abundance of collagen type VI and decorin, suggesting different PCM mechanics. Furthermore, the activation of regenerative and catabolic function in chondrocytes triggered sex-dependent signatures in gene expression, indicating dimorphic genetic regulation that is dependent on stimulation. CONCLUSIONS: We provide evidence for sexual dimorphism in cartilage before menopause. Some differences are intrinsic to chondrocytes' gene expression defined by their XX versus XY chromosomal constituency.

Global DNA methylation profiling reveals chromosomal instability in IDH-mutant astrocytomas.

Diffusely infiltrating gliomas are among the most common central nervous system tumors in adults. Over the past decade, the subcategorization of these tumors has changed to include both traditional histologic features and more recently identified molecular factors. However, one molecular feature that has yet to be integrated is the presence/absence of chromosomal instability (CIN). Herein, we use global methylation profiling to evaluate a reference cohort of IDH-mutant astrocytomas with and without prior evidence of CIN (n = 42), and apply the resulting methylation-based characteristics to a larger test cohort of publicly-available IDH-mutant astrocytomas (n = 245). We demonstrate that IDH-mutant astrocytomas with evidence of CIN cluster separately from their chromosomally-stable counterparts. CIN cases were associated with higher initial histologic grade, altered expression patterns of genes related to CIN in other cancers, elevated initial total copy number burden, and significantly worse progression-free and overall survival. In addition, in a grade-for-grade analysis, patients with CIN-positive WHO grade 2 and 3 tumors had significantly worse survival. These results suggest that global methylation profiling can be used to discriminate between chromosomally stable and unstable IDH-mutant astrocytomas, and may therefore provide a reliable and cost-effective method for identifying gliomas with chromosomal instability and resultant poor clinical outcome.

Autosomal recessive progeroid syndrome due to homozygosity for a TOMM7 variant.

Multiple genetic loci have been reported for progeroid syndromes. However, the molecular defects in some extremely rare forms of progeria have yet to be elucidated. Here, we report a 21-year-old man of Chinese ancestry who has an autosomal recessive form of progeria, characterized by severe dwarfism, mandibular hypoplasia, hyperopia, and partial lipodystrophy. Analyses of exome sequencing data from the entire family revealed only 1 rare homozygous missense variant (c.86C>T; p.Pro29Leu) in TOMM7 in the proband, while the parents and 2 unaffected siblings were heterozygous for the variant. TOMM7, a nuclear gene, encodes a translocase in the outer mitochondrial membrane. The TOMM complex makes up the outer membrane pore, which is responsible for importing many preproteins into the mitochondria. A proteomic comparison of mitochondria from control and proband-derived cultured fibroblasts revealed an increase in abundance of several proteins involved in oxidative phosphorylation, as well as a reduction in abundance of proteins involved in phospholipid metabolism. We also observed elevated basal and maximal oxygen consumption rates in the fibroblasts from the proband as compared with control fibroblasts. We concluded that altered mitochondrial protein import due to biallelic loss-of-function TOMM7 can cause severe growth retardation and progeroid features.

EGFR ligand shifts the role of EGFR from oncogene to tumour suppressor in EGFR-amplified glioblastoma by suppressing invasion through BIN3 upregulation.

The epidermal growth factor receptor (EGFR) is a prime oncogene that is frequently amplified in glioblastomas. Here we demonstrate a new tumour-suppressive function of EGFR in EGFR-amplified glioblastomas regulated by EGFR ligands. Constitutive EGFR signalling promotes invasion via activation of a TAB1-TAK1-NF-κB-EMP1 pathway, resulting in large tumours and decreased survival in orthotopic models. Ligand-activated EGFR promotes proliferation and surprisingly suppresses invasion by upregulating BIN3, which inhibits a DOCK7-regulated Rho GTPase pathway, resulting in small hyperproliferating non-invasive tumours and improved survival. Data from The Cancer Genome Atlas reveal that in EGFR-amplified glioblastomas, a low level of EGFR ligands confers a worse prognosis, whereas a high level of EGFR ligands confers an improved prognosis. Thus, increased EGFR ligand levels shift the role of EGFR from oncogene to tumour suppressor in EGFR-amplified glioblastomas by suppressing invasion. The tumour-suppressive function of EGFR can be activated therapeutically using tofacitinib, which suppresses invasion by increasing EGFR ligand levels and upregulating BIN3.

Identification of a Putative Enhancer RNA for EGFR in Hyper-Accessible Regions in Esophageal Squamous Cell Carcinoma Cells by Analysis of Chromatin Accessibility Landscapes.

Abnormal genetic and epigenetic modifications play a key role in esophageal cancer. By Assay for Transposase-Accessible Chromatin by sequencing (ATAC-seq), this study compared chromatin accessibility landscapes among two esophageal squamous cell carcinoma (ESCC) cell lines, KYSE-30 and KYSE-150, and a non-cancerous esophageal epithelial cell line, HET-1A. Data showed that hyper-accessible regions in ESCC cells contained genes related with cancer hallmarks, such as epidermal growth factor receptor (EGFR). Multi-omics analysis and digital-droplet PCR results demonstrated that several non-coding RNAs in EGFR upstream were upregulated in ESCC cells. Among them, one appeared to act as an enhancer RNA responsible for EGFR overexpression. Further motif analysis and pharmacological data suggested that AP-1 family transcription factors were able to bind the hyper-accessible regions and thus to regulate cancer cell proliferation and migration. This study discovered a putative enhancer RNA for EGFR gene and the reliance of ESCC on AP-1 transcription factor.

Human cutaneous neurofibroma matrisome revealed by single-cell RNA sequencing.

Neurofibromatosis Type I (NF1) is a neurocutaneous genetic syndrome characterized by a wide spectrum of clinical presentations, including benign peripheral nerve sheath tumor called neurofibroma. These tumors originate from the Schwann cell lineage but other cell types as well as extracellular matrix (ECM) in the neurofibroma microenvironment constitute the majority of the tumor mass. In fact, collagen accounts for up to 50% of the neurofibroma's dry weight. Although the presence of collagens in neurofibroma is indisputable, the exact repertoire of ECM genes and ECM-associated genes (i.e. the matrisome) and their functions are unknown. Here, transcriptome profiling by single-cell RNA sequencing reveals the matrisome of human cutaneous neurofibroma (cNF). We discovered that classic pro-fibrogenic collagen I myofibroblasts are rare in neurofibroma. In contrast, collagen VI, a pro-tumorigenic ECM, is abundant and mainly secreted by neurofibroma fibroblasts. This study also identified potential cell type-specific markers to further elucidate the biology of the cNF microenvironment.

Molecular Signatures of Chromosomal Instability Correlate With Copy Number Variation Patterns and Patient Outcome in IDH-Mutant and IDH-Wildtype Astrocytomas.

Chromosomal instability due to mutations in genes guarding the stability of the genome is a well-known mechanism underlying tumorigenesis and malignant progression in numerous cancers. The effect of this process in gliomas is mostly unknown with relatively little research examining the effects of chromosomal instability on patient outcome and therapeutic efficacy, although studies have shown that overall/total copy number variation (CNV) is elevated in higher histologic grades and in cases with more rapid progression and shorter patient survival. Herein, we examine a 70-gene mRNA expression signature (CIN70), which has been previously shown to correlate tightly with chromosomal instability, in 2 independent cohorts of IDH-mutant astrocytomas (total n = 241), IDH-wildtype astrocytomas (n = 228), and oligodendrogliomas (n = 128). Our results show that CIN70 expression levels correlate with total CNV, as well as higher grade, progression-free survival, and overall survival in both IDH-mutant and IDH-wildtype astrocytomas. In oligodendrogliomas, these mRNA signatures correlate with total CNV but not consistently with clinical outcome. These data suggest that chromosomal instability is an underlying factor in aggressive behavior and progression of a subset of diffuse astrocytomas. In addition, chromosomal instability may in part explain the poor response of diffuse gliomas to treatment and may serve as a future therapeutic target.

Molecular Correlates of Long Survival in IDH-Wildtype Glioblastoma Cohorts.

IDH-wildtype glioblastoma is a relatively common malignant brain tumor in adults. These patients generally have dismal prognoses, although outliers with long survival have been noted in the literature. Recently, it has been reported that many histologically lower-grade IDH-wildtype astrocytomas have a similar clinical outcome to grade IV tumors, suggesting they may represent early or undersampled glioblastomas. cIMPACT-NOW 3 guidelines now recommend upgrading IDH-wildtype astrocytomas with certain molecular criteria (EGFR amplifications, chromosome 7 gain/10 loss, and/or TERT promoter mutations), establishing the concept of a "molecular grade IV" astrocytoma. In this report, we apply these cIMPACT-NOW 3 criteria to 2 independent glioblastoma cohorts, totaling 393 public database and institutional glioblastoma cases: 89 cases without any of the cIMPACT-NOW 3 criteria (GBM-C0) and 304 cases with one or more criteria (GBM-C1-3). In the GBM-C0 groups, there was a trend toward longer recurrence-free survival (median 12-17 vs 6-10 months), significantly longer overall survival (median 32-41 vs 15-18 months), younger age at initial diagnosis, and lower overall mutation burden compared to the GBM-C1-3 cohorts. These data suggest that while histologic features may not be ideal indicators of patient survival in IDH-wildtype astrocytomas, these 3 molecular features may also be important prognostic factors in IDH-wildtype glioblastoma.

EGFR inhibition triggers an adaptive response by co-opting antiviral signaling pathways in lung cancer.

EGFR inhibition is an effective treatment in the minority of non-small cell lung cancer (NSCLC) cases harboring EGFR-activating mutations, but not in EGFR wild type (EGFRwt) tumors. Here, we demonstrate that EGFR inhibition triggers an antiviral defense pathway in NSCLC. Inhibiting mutant EGFR triggers Type I IFN-I upregulation via a RIG-I-TBK1-IRF3 pathway. The ubiquitin ligase TRIM32 associates with TBK1 upon EGFR inhibition, and is required for K63-linked ubiquitination and TBK1 activation. Inhibiting EGFRwt upregulates interferons via an NF-κB-dependent pathway. Inhibition of IFN signaling enhances EGFR-TKI sensitivity in EGFR mutant NSCLC and renders EGFRwt/KRAS mutant NSCLC sensitive to EGFR inhibition in xenograft and immunocompetent mouse models. Furthermore, NSCLC tumors with decreased IFN-I expression are more responsive to EGFR TKI treatment. We propose that IFN-I signaling is a major determinant of EGFR-TKI sensitivity in NSCLC and that a combination of EGFR TKI plus IFN-neutralizing antibody could be useful in most NSCLC patients.

eIF5B drives integrated stress response-dependent translation of PD-L1 in lung cancer.

Cancer cells express high levels of PD-L1, a ligand of the PD-1 receptor on T cells, allowing tumors to suppress T cell activity. Clinical trials utilizing antibodies that disrupt the PD-1/PD-L1 checkpoint have yielded remarkable results, with anti-PD-1 immunotherapy approved as first-line therapy for lung cancer patients. We used CRISPR-based screening to identify regulators of PD-L1 in human lung cancer cells, revealing potent induction of PD-L1 upon disruption of heme biosynthesis. Impairment of heme production activates the integrated stress response (ISR), allowing bypass of inhibitory upstream open reading frames in the PD-L1 5' UTR, resulting in enhanced PD-L1 translation and suppression of anti-tumor immunity. We demonstrated that ISR-dependent PD-L1 translation requires the translation initiation factor eIF5B. eIF5B overexpression, which is frequent in lung adenocarcinomas and associated with poor prognosis, is sufficient to induce PD-L1. These findings illuminate mechanisms of immune checkpoint activation and identify targets for therapeutic intervention.