Molecular and Pathologic Characterization of YAP1-Expressing Small Cell Lung Cancer Cell Lines Leads to Reclassification as SMARCA4-Deficient Malignancies.

PURPOSE: The classification of small cell lung cancer (SCLC) into distinct molecular subtypes defined by ASCL1, NEUROD1, POU2F3, or YAP1 (SCLC-A, -N, -P, or -Y) expression, paves the way for a personalized treatment approach. However, the existence of a distinct YAP1-expressing SCLC subtype remains controversial. EXPERIMENTAL DESIGN: To better understand YAP1-expressing SCLC, the mutational landscape of human SCLC cell lines was interrogated to identify pathogenic alterations unique to SCLC-Y. Xenograft tumors, generated from cell lines representing the four SCLC molecular subtypes, were evaluated by a panel of pathologists who routinely diagnose thoracic malignancies. Diagnoses were complemented by transcriptomic analysis of primary tumors and human cell line datasets. Protein expression profiles were validated in patient tumor tissue. RESULTS: Unexpectedly, pathogenic mutations in SMARCA4 were identified in six of eight SCLC-Y cell lines and correlated with reduced SMARCA4 mRNA and protein expression. Pathologist evaluations revealed that SMARCA4-deficient SCLC-Y tumors exhibited features consistent with thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT). Similarly, the transcriptional profile SMARCA4-mutant SCLC-Y lines more closely resembled primary SMARCA4-UT, or SMARCA4-deficient non-small cell carcinoma, than SCLC. Furthermore, SMARCA4-UT patient samples were associated with a YAP1 transcriptional signature and exhibited strong YAP1 protein expression. Together, we found little evidence to support a diagnosis of SCLC for any of the YAP1-expressing cell lines originally used to define the SCLC-Y subtype. CONCLUSIONS: SMARCA4-mutant SCLC-Y cell lines exhibit characteristics consistent with SMARCA4-deficient malignancies rather than SCLC. Our findings suggest that, unlike ASCL1, NEUROD1, and POU2F3, YAP1 is not a subtype defining transcription factor in SCLC. See related commentary by Rekhtman, p. 1708.

CRISPR-Cas9 screening identifies an IRF1-SOCS1-mediated negative feedback loop that limits CXCL9 expression and antitumor immunity.

CXCL9 expression is a strong predictor of response to immune checkpoint blockade therapy. Accordingly, we sought to develop therapeutic strategies to enhance the expression of CXCL9 and augment antitumor immunity. To perform whole-genome CRISPR-Cas9 screening for regulators of CXCL9 expression, a CXCL9-GFP reporter line is generated using a CRISPR knockin strategy. This approach finds that IRF1 limits CXCL9 expression in both tumor cells and primary myeloid cells through induction of SOCS1, which subsequently limits STAT1 signaling. Thus, we identify a subset of STAT1-dependent genes that do not require IRF1 for their transcription, including CXCL9. Targeting of either IRF1 or SOCS1 potently enhances CXCL9 expression by intratumoral macrophages, which is further enhanced in the context of immune checkpoint blockade therapy. We hence show a non-canonical role for IRF1 in limiting the expression of a subset of STAT1-dependent genes through induction of SOCS1.

Targeting Menin disrupts the KMT2A/B and polycomb balance to paradoxically activate bivalent genes.

Precise control of activating H3K4me3 and repressive H3K27me3 histone modifications at bivalent promoters is essential for normal development and frequently corrupted in cancer. By coupling a cell surface readout of bivalent MHC class I gene expression with whole-genome CRISPR-Cas9 screens, we identify specific roles for MTF2-PRC2.1, PCGF1-PRC1.1 and Menin-KMT2A/B complexes in maintaining bivalency. Genetic loss or pharmacological inhibition of Menin unexpectedly phenocopies the effects of polycomb disruption, resulting in derepression of bivalent genes in both cancer cells and pluripotent stem cells. While Menin and KMT2A/B contribute to H3K4me3 at active genes, a separate Menin-independent function of KMT2A/B maintains H3K4me3 and opposes polycomb-mediated repression at bivalent genes. Release of KMT2A from active genes following Menin targeting alters the balance of polycomb and KMT2A at bivalent genes, facilitating gene activation. This functional partitioning of Menin-KMT2A/B complex components reveals therapeutic opportunities that can be leveraged through inhibition of Menin.

Inhibition of the CtBP complex and FBXO11 enhances MHC class II expression and anti-cancer immune responses.

There is increasing recognition of the prognostic significance of tumor cell major histocompatibility complex (MHC) class II expression in anti-cancer immunity. Relapse of acute myeloid leukemia (AML) following allogeneic stem cell transplantation (alloSCT) has recently been linked to MHC class II silencing in leukemic blasts; however, the regulation of MHC class II expression remains incompletely understood. Utilizing unbiased CRISPR-Cas9 screens, we identify that the C-terminal binding protein (CtBP) complex transcriptionally represses MHC class II pathway genes, while the E3 ubiquitin ligase complex component FBXO11 mediates degradation of CIITA, the principal transcription factor regulating MHC class II expression. Targeting these repressive mechanisms selectively induces MHC class II upregulation across a range of AML cell lines. Functionally, MHC class II+ leukemic blasts stimulate antigen-dependent CD4+ T cell activation and potent anti-tumor immune responses, providing fundamental insights into the graft-versus-leukemia effect. These findings establish the rationale for therapeutic strategies aimed at restoring tumor-specific MHC class II expression to salvage AML relapse post-alloSCT and also potentially to enhance immunotherapy outcomes in non-myeloid malignancies.

Pharmacologic Reduction of Mitochondrial Iron Triggers a Noncanonical BAX/BAK-Dependent Cell Death.

Cancer cell metabolism is increasingly recognized as providing an exciting therapeutic opportunity. However, a drug that directly couples targeting of a metabolic dependency with the induction of cell death in cancer cells has largely remained elusive. Here we report that the drug-like small-molecule ironomycin reduces the mitochondrial iron load, resulting in the potent disruption of mitochondrial metabolism. Ironomycin promotes the recruitment and activation of BAX/BAK, but the resulting mitochondrial outer membrane permeabilization (MOMP) does not lead to potent activation of the apoptotic caspases, nor is the ensuing cell death prevented by inhibiting the previously established pathways of programmed cell death. Consistent with the fact that ironomycin and BH3 mimetics induce MOMP through independent nonredundant pathways, we find that ironomycin exhibits marked in vitro and in vivo synergy with venetoclax and overcomes venetoclax resistance in primary patient samples. SIGNIFICANCE: Ironomycin couples targeting of cellular metabolism with cell death by reducing mitochondrial iron, resulting in the alteration of mitochondrial metabolism and the activation of BAX/BAK. Ironomycin induces MOMP through a different mechanism to BH3 mimetics, and consequently combination therapy has marked synergy in cancers such as acute myeloid leukemia. This article is highlighted in the In This Issue feature, p. 587.

SP142 PD-L1 Scoring Shows High Interobserver and Intraobserver Agreement in Triple-negative Breast Carcinoma But Overall Low Percentage Agreement With Other PD-L1 Clones SP263 and 22C3.

SP142 programmed cell death ligand 1 (PD-L1) status predicts response to atezolizumab in triple-negative breast carcinoma (TNBC). Prevalence of VENTANA PD-L1 (SP142) Assay positivity, concordance with the VENTANA PD-L1 (SP263) Assay and Dako PD-L1 IHC 22C3 pharmDx assay, and association with clinicopathologic features were assessed in 447 TNBCs. SP142 PD-L1 intraobserver and interobserver agreement was investigated in a subset of 60 TNBCs, with scores enriched around the 1% cutoff. The effect of a 1-hour training video on pretraining and posttraining scores was ascertained. At a 1% cutoff, 34.2% of tumors were SP142 PD-L1 positive. SP142 PD-L1 positivity was significantly associated with tumor-infiltrating lymphocytes (P <0.01), and node negativity (P=0.02), but not with tumor grade (P=0.35), tumor size (P=0.58), or BRCA mutation (P=0.53). Overall percentage agreement (OPA) for intraobserver and interobserver agreement was 95.0% and 93.7%, respectively, among 5 pathologists trained in TNBC SP142 PD-L1 scoring. In 5 TNBC SP142 PD-L1-naive pathologists, significantly higher OPA to the reference score was achieved after video training (posttraining OPA 85.7%, pretraining OPA 81.5%, P<0.05). PD-L1 status at a 1% cutoff was assessed by SP142 and SP263 in 420 cases, and by SP142 and 22C3 in 423 cases, with OPA of 88.1% and 85.8%, respectively. The VENTANA PD-L1 (SP142) Assay is reproducible for classifying TNBC PD-L1 status by trained observers; however, it is not analytically equivalent to the VENTANA PD-L1 (SP263) Assay and Dako PD-L1 IHC 22C3 pharmDx assay.

Selective targeting of BD1 and BD2 of the BET proteins in cancer and immunoinflammation.

The two tandem bromodomains of the BET (bromodomain and extraterminal domain) proteins enable chromatin binding to facilitate transcription. Drugs that inhibit both bromodomains equally have shown efficacy in certain malignant and inflammatory conditions. To explore the individual functional contributions of the first (BD1) and second (BD2) bromodomains in biology and therapy, we developed selective BD1 and BD2 inhibitors. We found that steady-state gene expression primarily requires BD1, whereas the rapid increase of gene expression induced by inflammatory stimuli requires both BD1 and BD2 of all BET proteins. BD1 inhibitors phenocopied the effects of pan-BET inhibitors in cancer models, whereas BD2 inhibitors were predominantly effective in models of inflammatory and autoimmune disease. These insights into the differential requirement of BD1 and BD2 for the maintenance and induction of gene expression may guide future BET-targeted therapies.

HBO1 is required for the maintenance of leukaemia stem cells.

Acute myeloid leukaemia (AML) is a heterogeneous disease characterized by transcriptional dysregulation that results in a block in differentiation and increased malignant self-renewal. Various epigenetic therapies aimed at reversing these hallmarks of AML have progressed into clinical trials, but most show only modest efficacy owing to an inability to effectively eradicate leukaemia stem cells (LSCs)1. Here, to specifically identify novel dependencies in LSCs, we screened a bespoke library of small hairpin RNAs that target chromatin regulators in a unique ex vivo mouse model of LSCs. We identify the MYST acetyltransferase HBO1 (also known as KAT7 or MYST2) and several known members of the HBO1 protein complex as critical regulators of LSC maintenance. Using CRISPR domain screening and quantitative mass spectrometry, we identified the histone acetyltransferase domain of HBO1 as being essential in the acetylation of histone H3 at K14. H3 acetylated at K14 (H3K14ac) facilitates the processivity of RNA polymerase II to maintain the high expression of key genes (including Hoxa9 and Hoxa10) that help to sustain the functional properties of LSCs. To leverage this dependency therapeutically, we developed a highly potent small-molecule inhibitor of HBO1 and demonstrate its mode of activity as a competitive analogue of acetyl-CoA. Inhibition of HBO1 phenocopied our genetic data and showed efficacy in a broad range of human cell lines and primary AML cells from patients. These biological, structural and chemical insights into a therapeutic target in AML will enable the clinical translation of these findings.

An Evolutionarily Conserved Function of Polycomb Silences the MHC Class I Antigen Presentation Pathway and Enables Immune Evasion in Cancer.

Loss of MHC class I (MHC-I) antigen presentation in cancer cells can elicit immunotherapy resistance. A genome-wide CRISPR/Cas9 screen identified an evolutionarily conserved function of polycomb repressive complex 2 (PRC2) that mediates coordinated transcriptional silencing of the MHC-I antigen processing pathway (MHC-I APP), promoting evasion of T cell-mediated immunity. MHC-I APP gene promoters in MHC-I low cancers harbor bivalent activating H3K4me3 and repressive H3K27me3 histone modifications, silencing basal MHC-I expression and restricting cytokine-induced upregulation. Bivalent chromatin at MHC-I APP genes is a normal developmental process active in embryonic stem cells and maintained during neural progenitor differentiation. This physiological MHC-I silencing highlights a conserved mechanism by which cancers arising from these primitive tissues exploit PRC2 activity to enable immune evasion.

The roles of DNA, RNA and histone methylation in ageing and cancer.

Chromatin is a macromolecular complex predominantly comprising DNA, histone proteins and RNA. The methylation of chromatin components is highly conserved as it helps coordinate the regulation of gene expression, DNA repair and DNA replication. Dynamic changes in chromatin methylation are essential for cell-fate determination and development. Consequently, inherited or acquired mutations in the major factors that regulate the methylation of DNA, RNA and/or histones are commonly observed in developmental disorders, ageing and cancer. This has provided the impetus for the clinical development of epigenetic therapies aimed at resetting the methylation imbalance observed in these disorders. In this Review, we discuss the cellular functions of chromatin methylation and focus on how this fundamental biological process is corrupted in cancer. We discuss methylation-based cancer therapies and provide a perspective on the emerging data from early-phase clinical trial therapies that target regulators of DNA and histone methylation. We also highlight promising therapeutic strategies, including monitoring chromatin methylation for diagnostic purposes and combination epigenetic therapy strategies that may improve immune surveillance in cancer and increase the efficacy of conventional and targeted anticancer drugs.

Targeting enhancer switching overcomes non-genetic drug resistance in acute myeloid leukaemia.

Non-genetic drug resistance is increasingly recognised in various cancers. Molecular insights into this process are lacking and it is unknown whether stable non-genetic resistance can be overcome. Using single cell RNA-sequencing of paired drug naïve and resistant AML patient samples and cellular barcoding in a unique mouse model of non-genetic resistance, here we demonstrate that transcriptional plasticity drives stable epigenetic resistance. With a CRISPR-Cas9 screen we identify regulators of enhancer function as important modulators of the resistant cell state. We show that inhibition of Lsd1 (Kdm1a) is able to overcome stable epigenetic resistance by facilitating the binding of the pioneer factor, Pu.1 and cofactor, Irf8, to nucleate new enhancers that regulate the expression of key survival genes. This enhancer switching results in the re-distribution of transcriptional co-activators, including Brd4, and provides the opportunity to disable their activity and overcome epigenetic resistance. Together these findings highlight key principles to help counteract non-genetic drug resistance.

CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity.

Cancer cells exploit the expression of the programmed death-1 (PD-1) ligand 1 (PD-L1) to subvert T-cell-mediated immunosurveillance. The success of therapies that disrupt PD-L1-mediated tumour tolerance has highlighted the need to understand the molecular regulation of PD-L1 expression. Here we identify the uncharacterized protein CMTM6 as a critical regulator of PD-L1 in a broad range of cancer cells, by using a genome-wide CRISPR-Cas9 screen. CMTM6 is a ubiquitously expressed protein that binds PD-L1 and maintains its cell surface expression. CMTM6 is not required for PD-L1 maturation but co-localizes with PD-L1 at the plasma membrane and in recycling endosomes, where it prevents PD-L1 from being targeted for lysosome-mediated degradation. Using a quantitative approach to profile the entire plasma membrane proteome, we find that CMTM6 displays specificity for PD-L1. Notably, CMTM6 depletion decreases PD-L1 without compromising cell surface expression of MHC class I. CMTM6 depletion, via the reduction of PD-L1, significantly alleviates the suppression of tumour-specific T cell activity in vitro and in vivo. These findings provide insights into the biology of PD-L1 regulation, identify a previously unrecognized master regulator of this critical immune checkpoint and highlight a potential therapeutic target to overcome immune evasion by tumour cells.

MHC class I molecules are preferentially ubiquitinated on endoplasmic reticulum luminal residues during HRD1 ubiquitin E3 ligase-mediated dislocation.

Misfolded MHC class I heavy chains (MHC I HCs) are targeted for endoplasmic reticulum (ER)-associated degradation (ERAD) by the ubiquitin E3 ligase HRD1, and E2 ubiquitin conjugating enzyme UBE2J1, and represent one of the few known endogenous ERAD substrates. The mechanism by which misfolded proteins are dislocated across the ER membrane into the cytosol is unclear. Here, we investigate the requirements for MHC I ubiquitination and degradation and show that endogenous misfolded MHC I HCs are recognized in the ER lumen by EDEM1 in a glycan-dependent manner and targeted to the core SEL1L/HRD1/UBE2J1 complex. A soluble MHC I HC lacking its transmembrane domain and cytosolic tail uses the same ERAD components and is degraded as efficiently as wild-type MHC I. Unexpectedly, HRD1-dependent polyubiquitination is preferentially targeted to the ER luminal domain of full-length MHC I HCs, despite the presence of an exposed cytosolic C-terminal tail. MHC I luminal domain ubiquitination occurs before p97 ATPase-mediated extraction from the ER membrane and can be targeted to nonlysine, as well as lysine, residues. A subset of integral membrane proteins, therefore, requires an early dislocation event to expose part of their luminal domain to the cytosol, before HRD1-mediated polyubiquitination and dislocation.

Sterol metabolism regulates neuroserpin polymer degradation in the absence of the unfolded protein response in the dementia FENIB.

Mutants of neuroserpin are retained as polymers within the endoplasmic reticulum (ER) of neurones to cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. The cellular consequences are unusual in that the ordered polymers activate the ER overload response (EOR) in the absence of the canonical unfolded protein response. We use both cell lines and Drosophila models to show that the G392E mutant of neuroserpin that forms polymers is degraded by UBE2j1 E2 ligase and Hrd1 E3 ligase while truncated neuroserpin, a protein that lacks 132 amino acids, is degraded by UBE2g2 (E2) and gp78 (E3) ligases. The degradation of G392E neuroserpin results from SREBP-dependent activation of the cholesterol biosynthetic pathway in cells that express polymers of neuroserpin (G392E). Inhibition of HMGCoA reductase, the limiting enzyme of the cholesterol biosynthetic pathway, reduced the ubiquitination of G392E neuroserpin in our cell lines and increased the retention of neuroserpin polymers in both HeLa cells and primary neurones. Our data reveal a reciprocal relationship between cholesterol biosynthesis and the clearance of mutant neuroserpin. This represents the first description of a link between sterol metabolism and modulation of the proteotoxicity mediated by the EOR.

Tapasin-related protein TAPBPR is an additional component of the MHC class I presentation pathway.

Tapasin is an integral component of the peptide-loading complex (PLC) important for efficient peptide loading onto MHC class I molecules. We investigated the function of the tapasin-related protein, TAPBPR. Like tapasin, TAPBPR is widely expressed, IFN-γ-inducible, and binds to MHC class I coupled with ß2-microglobulin in the endoplasmic reticulum. In contrast to tapasin, TAPBPR does not bind ERp57 or calreticulin and is not an integral component of the PLC. ß2-microglobulin is essential for the association between TAPBPR and MHC class I. However, the association between TAPBPR and MHC class I occurs in the absence of a functional PLC, suggesting peptide is not required. Expression of TAPBPR decreases the rate of MHC class I maturation through the secretory pathway and prolongs the association of MHC class I on the PLC. The TAPBPR:MHC class I complex trafficks through the Golgi apparatus, demonstrating a function of TAPBPR beyond the endoplasmic reticulum/cis-Golgi. The identification of TAPBPR as an additional component of the MHC class I antigen-presentation pathway demonstrates that mechanisms controlling MHC class I expression remain incompletely understood.

Studying ubiquitination of MHC class I molecules.

The covalent attachment of ubiquitin to a protein is one of the most common post-translational modifications and regulates diverse eukaryotic cellular processes. Ubiquitination of MHC class I was first described in the context of viral proteins which target MHC class I for degradation in the endoplasmic reticulum and at the cell surface. Study of viral-induced MHC class I degradation has been extremely instructive in elucidating cellular pathways for degradation of membrane and secretory proteins. More recently, ubiquitination of endogenous MHC class I heavy chains which fail to achieve their native conformation and undergo endoplasmic-reticulum associated degradation has been demonstrated.In this chapter we describe methods for identification of endogenous ubiquitinated MHC class I heavy chains by MHC class I-immunoprecipitation and ubiquitin-specific immunoblot or by metabolic labeling and immunoprecipitation.

Long-term stability of anti-cyclic citrullinated peptide antibody status in patients with early inflammatory polyarthritis.

INTRODUCTION: The utility of reassessing anti-cyclic citrullinated peptide (anti-CCP) antibody status later in disease in patients presenting with early undifferentiated inflammatory polyarthritis, particularly in those who test negative for both anti-CCP and rheumatoid factor (RF) at baseline, remains unclear. We aimed therefore to determine the stability of CCP antibody status over time and the prognostic utility of repeated testing in subjects with early inflammatory polyarthritis (IP). METHODS: Anti-CCP and RF were measured at baseline and 5 years in 640 IP patients from the Norfolk Arthritis Register, a primary care-based inception cohort. The relation between change in anti-CCP status/titer and the presence of radiologic erosions, the extent of the Larsen score, and Health Assessment Questionnaire (HAQ) score by 5 years was investigated. RESULTS: With a cut-off of 5 U/ml, 28% subjects tested positive for anti-CCP antibodies, 29% for RF, and 21% for both at baseline. Nine (2%) anti-CCP-negative patients seroconverted to positive, and nine (4.6%) anti-CCP-positive individuals became negative between baseline and 5 years. In contrast, RF status changed in 17% of subjects. However, change in RF status was strongly linked to baseline anti-CCP status and was not independently associated with outcome. Ever positivity for anti-CCP antibodies by 5 years did not improve prediction of radiographic damage compared with baseline status alone (accuracy, 75% versus 74%). A higher baseline anti-CCP titer (but not change in anti-CCP titer) predicted worse radiologic damage at 5 years (P < 0.0001), even at levels below the cut-off for anti-CCP positivity. Thus, a titer of 2 to 5 U/ml was strongly associated with erosions by 5 years (odds ratio, 3.6 (1.5 to 8.3); P = 0.003). CONCLUSIONS: Repeated testing of anti-CCP antibodies or RF in patients with IP does not improve prognostic value and should not be recommended in routine clinical practice.

HRD1 and UBE2J1 target misfolded MHC class I heavy chains for endoplasmic reticulum-associated degradation.

The assembly of MHC class I molecules is governed by stringent endoplasmic reticulum (ER) quality control mechanisms. MHC class I heavy chains that fail to achieve their native conformation in complex with ß2-microglobulin (ß2m) and peptide are targeted for ER-associated degradation. This requires ubiquitination of the MHC class I heavy chain and its dislocation from the ER to the cytosol for proteasome-mediated degradation, although the cellular machinery involved in this process is unknown. Using an siRNA functional screen in ß2m-depleted cells, we identify an essential role for the E3 ligase HRD1 (Synoviolin) together with the E2 ubiquitin-conjugating enzyme UBE2J1 in the ubiquitination and dislocation of misfolded MHC class I heavy chains. HRD1 is also required for the ubiquitination and degradation of the naturally occurring hemochromatosis-associated HFE-C282Y mutant, which is unable to bind ß2m. In the absence of HRD1, misfolded HLA-B27 accumulated in cells with a normal MHC class I assembly pathway, and HRD1 depletion prevented the appearance of low levels of cytosolic unfolded MHC I heavy chains. HRD1 and UBE2J1 associate in a complex together with non-ß2m bound MHC class I heavy chains, Derlin 1, and p97 and discriminate misfolded MHC class I from conformational MHC I-ß2m-peptide heterotrimers. Together these data support a physiological role for HRD1 and UBE2J1 in the homeostatic regulation of MHC class I assembly and expression.