Lineage-specific canonical and non-canonical activity of EZH2 in advanced prostate cancer subtypes.

Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and emerging therapeutic target that is overexpressed in most castration-resistant prostate cancers and implicated as a driver of disease progression and resistance to hormonal therapies. Here we define the lineage-specific action and differential activity of EZH2 in both prostate adenocarcinoma (PRAD) and neuroendocrine prostate cancer (NEPC) subtypes of advanced prostate cancer to better understand the role of EZH2 in modulating differentiation, lineage plasticity, and to identify mediators of response and resistance to EZH2 inhibitor therapy. Mechanistically, EZH2 modulates bivalent genes that results in upregulation of NEPC-associated transcriptional drivers (e.g., ASCL1) and neuronal gene programs, and leads to forward differentiation after targeting EZH2 in NEPC. Subtype-specific downstream effects of EZH2 inhibition on cell cycle genes support the potential rationale for co-targeting cyclin/CDK to overcome resistance to EZH2 inhibition.

Genomic and Immunophenotypic Landscape of Acquired Resistance to PD-(L)1 Blockade in Non-Small-Cell Lung Cancer.

PURPOSE: Although immune checkpoint inhibitors (ICI) have extended survival in patients with non-small-cell lung cancer (NSCLC), acquired resistance (AR) to ICI frequently develops after an initial benefit. However, the mechanisms of AR to ICI in NSCLC are largely unknown. METHODS: Comprehensive tumor genomic profiling, machine learning-based assessment of tumor-infiltrating lymphocytes, multiplexed immunofluorescence, and/or HLA-I immunohistochemistry (IHC) were performed on matched pre- and post-ICI tumor biopsies from patients with NSCLC treated with ICI at the Dana-Farber Cancer Institute who developed AR to ICI. Two additional cohorts of patients with intervening chemotherapy or targeted therapies between biopsies were included as controls. RESULTS: We performed comprehensive genomic profiling and immunophenotypic characterization on samples from 82 patients with NSCLC and matched pre- and post-ICI biopsies and compared findings with a control cohort of patients with non-ICI intervening therapies between biopsies (chemotherapy, N = 32; targeted therapies, N = 89; both, N = 17). Putative resistance mutations were identified in 27.8% of immunotherapy-treated cases and included acquired loss-of-function mutations in STK11, B2M, APC, MTOR, KEAP1, and JAK1/2; these acquired alterations were not observed in the control groups. Immunophenotyping of matched pre- and post-ICI samples demonstrated significant decreases in intratumoral lymphocytes, CD3e+ and CD8a+ T cells, and PD-L1-PD1 engagement, as well as increased distance between tumor cells and CD8+PD-1+ T cells. There was a significant decrease in HLA class I expression in the immunotherapy cohort at the time of AR compared with the chemotherapy (P = .005) and the targeted therapy (P = .01) cohorts. CONCLUSION: These findings highlight the genomic and immunophenotypic heterogeneity of ICI resistance in NSCLC, which will need to be considered when developing novel therapeutic strategies aimed at overcoming resistance.

TREX1 Inactivation Unleashes Cancer Cell STING-Interferon Signaling and Promotes Antitumor Immunity.

A substantial fraction of cancers evade immune detection by silencing Stimulator of Interferon Genes (STING)-Interferon (IFN) signaling. Therapeutic reactivation of this program via STING agonists, epigenetic, or DNA-damaging therapies can restore antitumor immunity in multiple preclinical models. Here we show that adaptive induction of three prime exonuclease 1 (TREX1) restrains STING-dependent nucleic acid sensing in cancer cells via its catalytic function in degrading cytosolic DNA. Cancer cell TREX1 expression is coordinately induced with STING by autocrine IFN and downstream STAT1, preventing signal amplification. TREX1 inactivation in cancer cells thus unleashes STING-IFN signaling, recruiting T and natural killer (NK) cells, sensitizing to NK cell-derived IFNγ, and cooperating with programmed cell death protein 1 blockade in multiple mouse tumor models to enhance immunogenicity. Targeting TREX1 may represent a complementary strategy to induce cytosolic DNA and amplify cancer cell STING-IFN signaling as a means to sensitize tumors to immune checkpoint blockade (ICB) and/or cell therapies. SIGNIFICANCE: STING-IFN signaling in cancer cells promotes tumor cell immunogenicity. Inactivation of the DNA exonuclease TREX1, which is adaptively upregulated to limit pathway activation in cancer cells, recruits immune effector cells and primes NK cell-mediated killing. Targeting TREX1 has substantial therapeutic potential to amplify cancer cell immunogenicity and overcome ICB resistance. This article is featured in Selected Articles from This Issue, p. 695.

A cellular and spatial atlas of TP53 -associated tissue remodeling in lung adenocarcinoma.

TP53 is the most frequently mutated gene across many cancers and is associated with shorter survival in lung adenocarcinoma (LUAD). To define how TP53 mutations affect the LUAD tumor microenvironment (TME), we constructed a multi-omic cellular and spatial tumor atlas of 23 treatment-naïve human lung tumors. We found that TP53 -mutant ( TP53 mut ) malignant cells lose alveolar identity and upregulate highly proliferative and entropic gene expression programs consistently across resectable LUAD patient tumors, genetically engineered mouse models, and cell lines harboring a wide spectrum of TP53 mutations. We further identified a multicellular tumor niche composed of SPP1 + macrophages and collagen-expressing fibroblasts that coincides with hypoxic, pro-metastatic expression programs in TP53 mut tumors. Spatially correlated angiostatic and immune checkpoint interactions, including CD274 - PDCD1 and PVR - TIGIT , are also enriched in TP53 mut LUAD tumors, which may influence response to checkpoint blockade therapy. Our methodology can be further applied to investigate mutation-specific TME changes in other cancers.

Concurrent PTEN and PDGFRB Alterations Characterize Storiform Collagenoma.

Storiform collagenoma is a rare mesenchymal skin tumor that is composed of thickened collagen bundles arranged in a characteristic storiform pattern with a relatively hypocellular CD34-positive spindle cell component. Storiform collagenoma is most often sporadic, but multiple lesions can occur in Cowden syndrome, which is characterized by germline alterations in PTEN (phosphatase and tensin homolog) on chromosome 10. Here, we investigated the molecular pathogenesis of storiform collagenoma using a targeted next-generation DNA sequencing platform, including 5 sporadic cases and one case associated with Cowden syndrome. Recurrent PTEN alterations were identified in all cases, with biallelic PTEN inactivation observed in the case associated with Cowden syndrome and one sporadic case. Unexpectedly, we also identified recurrent activating mutations in the platelet-derived growth factor receptor beta ( PDGFRB ) gene. This included a missense substitution in the D5 Ig-like domain of PDGFRB in the Cowden syndrome-associated case. In addition, we report missense alterations in the juxtamembrane domain of PDGFRB in 4 of 5 (80%) sporadic cases, including mutations that have been previously described in sporadic myofibroma and myopericytoma. Therefore, we confirm the neoplastic nature of storiform collagenoma, we expand the spectrum of reported PDGFRB alterations in mesenchymal tumors and we suggest a possible collaborative role for PTEN and PDGFRB in the pathogenesis of storiform collagenoma.

Aurora A kinase inhibition induces accumulation of SCLC tumor cells in mitosis with restored interferon signaling to increase response to PD-L1.

Despite small cell lung cancers (SCLCs) having a high mutational burden, programmed death-ligand 1 (PD-L1) immunotherapy only modestly increases survival. A subset of SCLCs that lose their ASCL1 neuroendocrine phenotype and restore innate immune signaling (termed the "inflammatory" subtype) have durable responses to PD-L1. Some SCLCs are highly sensitive to Aurora kinase inhibitors, but early-phase trials show short-lived responses, suggesting effective therapeutic combinations are needed to increase their durability. Using immunocompetent SCLC genetically engineered mouse models (GEMMs) and syngeneic xenografts, we show durable efficacy with the combination of a highly specific Aurora A kinase inhibitor (LSN3321213) and PD-L1. LSN3321213 causes accumulation of tumor cells in mitosis with lower ASCL1 expression and higher expression of interferon target genes and antigen-presentation genes mimicking the inflammatory subtype in a cell-cycle-dependent manner. These data demonstrate that inflammatory gene expression is restored in mitosis in SCLC, which can be exploited by Aurora A kinase inhibition.

ALK peptide vaccination restores the immunogenicity of ALK-rearranged non-small cell lung cancer.

Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) is treated with ALK tyrosine kinase inhibitors (TKIs), but the lack of activity of immune checkpoint inhibitors (ICIs) is poorly understood. Here, we identified immunogenic ALK peptides to show that ICIs induced rejection of ALK+ tumors in the flank but not in the lung. A single-peptide vaccination restored priming of ALK-specific CD8+ T cells, eradicated lung tumors in combination with ALK TKIs and prevented metastatic dissemination of tumors to the brain. The poor response of ALK+ NSCLC to ICIs was due to ineffective CD8+ T cell priming against ALK antigens and is circumvented through specific vaccination. Finally, we identified human ALK peptides displayed by HLA-A*02:01 and HLA-B*07:02 molecules. These peptides were immunogenic in HLA-transgenic mice and were recognized by CD8+ T cells from individuals with NSCLC, paving the way for the development of a clinical vaccine to treat ALK+ NSCLC.

MET-Induced CD73 Restrains STING-Mediated Immunogenicity of EGFR-Mutant Lung Cancer.

Immunotherapy has shown limited efficacy in patients with EGFR-mutated lung cancer. Efforts to enhance the immunogenicity of EGFR-mutated lung cancer have been unsuccessful to date. Here, we discover that MET amplification, the most common mechanism of resistance to third-generation EGFR tyrosine kinase inhibitors (TKI), activates tumor cell STING, an emerging determinant of cancer immunogenicity (1). However, STING activation was restrained by ectonucleosidase CD73, which is induced in MET-amplified, EGFR-TKI-resistant cells. Systematic genomic analyses and cell line studies confirmed upregulation of CD73 in MET-amplified and MET-activated lung cancer contexts, which depends on coinduction of FOSL1. Pemetrexed (PEM), which is commonly used following EGFR-TKI treatment failure, was identified as an effective potentiator of STING-dependent TBK1-IRF3-STAT1 signaling in MET-amplified, EGFR-TKI-resistant cells. However, PEM treatment also induced adenosine production, which inhibited T-cell responsiveness. In an allogenic humanized mouse model, CD73 deletion enhanced immunogenicity of MET-amplified, EGFR-TKI-resistant cells, and PEM treatment promoted robust responses regardless of CD73 status. Using a physiologic antigen recognition model, inactivation of CD73 significantly increased antigen-specific CD8+ T-cell immunogenicity following PEM treatment. These data reveal that combined PEM and CD73 inhibition can co-opt tumor cell STING induction in TKI-resistant EGFR-mutated lung cancers and promote immunogenicity. SIGNIFICANCE: MET amplification upregulates CD73 to suppress tumor cell STING induction and T-cell responsiveness in TKI-resistant, EGFR-mutated lung cancer, identifying a strategy to enhance immunogenicity and improve treatment.

Mesenchymal and adrenergic cell lineage states in neuroblastoma possess distinct immunogenic phenotypes.

Apart from the anti-GD2 antibody, immunotherapy for neuroblastoma has had limited success due to immune evasion mechanisms, coupled with an incomplete understanding of predictors of response. Here, from bulk and single-cell transcriptomic analyses, we identify a subset of neuroblastomas enriched for transcripts associated with immune activation and inhibition and show that these are predominantly characterized by gene expression signatures of the mesenchymal lineage state. By contrast, tumors expressing adrenergic lineage signatures are less immunogenic. The inherent presence or induction of the mesenchymal state through transcriptional reprogramming or therapy resistance is accompanied by innate and adaptive immune gene activation through epigenetic remodeling. Mesenchymal lineage cells promote T cell infiltration by secreting inflammatory cytokines, are efficiently targeted by cytotoxic T and natural killer cells and respond to immune checkpoint blockade. Together, we demonstrate that distinct immunogenic phenotypes define the divergent lineage states of neuroblastoma and highlight the immunogenic potential of the mesenchymal lineage.

MPS1 inhibition primes immunogenicity of KRAS-LKB1 mutant lung cancer.

KRAS-LKB1 (KL) mutant lung cancers silence STING owing to intrinsic mitochondrial dysfunction, resulting in T cell exclusion and resistance to programmed cell death (ligand) 1 (PD-[L]1) blockade. Here we discover that KL cells also minimize intracellular accumulation of 2'3'-cyclic GMP-AMP (2'3'-cGAMP) to further avoid downstream STING and STAT1 activation. An unbiased screen to co-opt this vulnerability reveals that transient MPS1 inhibition (MPS1i) potently re-engages this pathway in KL cells via micronuclei generation. This effect is markedly amplified by epigenetic de-repression of STING and only requires pulse MPS1i treatment, creating a therapeutic window compared with non-dividing cells. A single course of decitabine treatment followed by pulse MPS1i therapy restores T cell infiltration in vivo, enhances anti-PD-1 efficacy, and results in a durable response without evidence of significant toxicity.

Genomic and biological study of fusion genes as resistance mechanisms to EGFR inhibitors.

The clinical significance of gene fusions detected by DNA-based next generation sequencing remains unclear as resistance mechanisms to EGFR tyrosine kinase inhibitors in EGFR mutant non-small cell lung cancer. By studying EGFR inhibitor-resistant patients treated with a combination of an EGFR inhibitor and a drug targeting the putative resistance-causing fusion oncogene, we identify patients who benefit and those who do not from this treatment approach. Through evaluation including RNA-seq of potential drug resistance-imparting fusion oncogenes in 504 patients with EGFR mutant lung cancer, we identify only a minority of them as functional, potentially capable of imparting EGFR inhibitor resistance. We further functionally validate fusion oncogenes in vitro using CRISPR-based editing of EGFR mutant cell lines and use these models to identify known and unknown drug resistance mechanisms to combination therapies. Collectively, our results partially reveal the complex nature of fusion oncogenes as potential drug resistance mechanisms and highlight approaches that can be undertaken to determine their functional significance.

Regulation of neuroendocrine plasticity by the RNA-binding protein ZFP36L1.

Some small cell lung cancers (SCLCs) are highly sensitive to inhibitors of the histone demethylase LSD1. LSD1 inhibitors are thought to induce their anti-proliferative effects by blocking neuroendocrine differentiation, but the mechanisms by which LSD1 controls the SCLC neuroendocrine phenotype are not well understood. To identify genes required for LSD1 inhibitor sensitivity in SCLC, we performed a positive selection genome-wide CRISPR/Cas9 loss of function screen and found that ZFP36L1, an mRNA-binding protein that destabilizes mRNAs, is required for LSD1 inhibitor sensitivity. LSD1 binds and represses ZFP36L1 and upon LSD1 inhibition, ZFP36L1 expression is restored, which is sufficient to block the SCLC neuroendocrine differentiation phenotype and induce a non-neuroendocrine "inflammatory" phenotype. Mechanistically, ZFP36L1 binds and destabilizes SOX2 and INSM1 mRNAs, two transcription factors that are required for SCLC neuroendocrine differentiation. This work identifies ZFP36L1 as an LSD1 target gene that controls the SCLC neuroendocrine phenotype and demonstrates that modulating mRNA stability of lineage transcription factors controls neuroendocrine to non-neuroendocrine plasticity.

To Rb or Not to Rb: Uncovering Unique Subsets of Small Cell Lung Carcinoma.

The use of IHC as a surrogate for specific underlying genomic alterations has allowed for increasingly comprehensive and accurate diagnosis of small cell lung carcinoma (SCLC). This is especially relevant in light of the increasing recognition of the biologic heterogeneity of this aggressive and difficult-to-treat lung tumor. Integrated genomic and IHC profiling of Rb status in SCLC yields new diagnostic insights and has translational implications. See related article by Febres-Aldana et al., p. 4702.

Activation of Tumor-Cell STING Primes NK-Cell Therapy.

Activation of the stimulator of interferon genes (STING) pathway promotes antitumor immunity but STING agonists have yet to achieve clinical success. Increased understanding of the mechanism of action of STING agonists in human tumors is key to developing therapeutic combinations that activate effective innate antitumor immunity. Here, we report that malignant pleural mesothelioma cells robustly express STING and are responsive to STING agonist treatment ex vivo. Using dynamic single-cell RNA sequencing of explants treated with a STING agonist, we observed CXCR3 chemokine activation primarily in tumor cells and cancer-associated fibroblasts, as well as T-cell cytotoxicity. In contrast, primary natural killer (NK) cells resisted STING agonist-induced cytotoxicity. STING agonists enhanced migration and killing of NK cells and mesothelin-targeted chimeric antigen receptor (CAR)-NK cells, improving therapeutic activity in patient-derived organotypic tumor spheroids. These studies reveal the fundamental importance of using human tumor samples to assess innate and cellular immune therapies. By functionally profiling mesothelioma tumor explants with elevated STING expression in tumor cells, we uncovered distinct consequences of STING agonist treatment in humans that support testing combining STING agonists with NK and CAR-NK cell therapies.

Association of High Tumor Mutation Burden in Non-Small Cell Lung Cancers With Increased Immune Infiltration and Improved Clinical Outcomes of PD-L1 Blockade Across PD-L1 Expression Levels.

Importance: Although tumor mutation burden (TMB) has been explored as a potential biomarker of immunotherapy efficacy in solid tumors, there still is a lack of consensus about the optimal TMB threshold that best discriminates improved outcomes of immune checkpoint inhibitor therapy among patients with non-small cell lung cancer (NSCLC). Objectives: To determine the association between increasing TMB levels and immunotherapy efficacy across clinically relevant programmed death ligand-1 (PD-L1) levels in patients with NSCLC. Design, Setting, and Participants: This multicenter cohort study included patients with advanced NSCLC treated with immunotherapy who received programmed cell death-1 (PD-1) or PD-L1 inhibition in the Dana-Farber Cancer Institute (DFCI), Memorial Sloan Kettering Cancer Center (MSKCC), and in the Stand Up To Cancer (SU2C)/Mark Foundation data sets. Clinicopathological and genomic data were collected from patients between September 2013 and September 2020. Data analysis was performed from November 2021 to February 2022. Exposures: Treatment with PD-1/PD-L1 inhibition without chemotherapy. Main Outcomes and Measures: Association of TMB levels with objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). Results: In the entire cohort of 1552 patients with advanced NSCLC who received PD-1/PD-L1 blockade, the median (range) age was 66 (22-92) years, 830 (53.5%) were women, and 1347 (86.8%) had cancer with nonsquamous histologic profile. A regression tree modeling ORR as a function of TMB identified 2 TMB groupings in the discovery cohort (MSKCC), defined as low TMB (≤19.0 mutations per megabase) and high TMB (>19.0 mutations per megabase), which were associated with increasing improvements in ORR, PFS, and OS in the discovery cohort and in 2 independent cohorts (DFCI and SU2C/Mark Foundation). These TMB levels also were associated with significant improvements in outcomes of immunotherapy in each PD-L1 tumor proportion score subgroup of less than 1%, 1% to 49%, and 50% or higher. The ORR to PD-1/PD-L1 inhibition was as high as 57% in patients with high TMB and PD-L1 expression 50% or higher and as low as 8.7% in patients with low TMB and PD-L1 expression less than 1%. Multiplexed immunofluorescence and transcriptomic profiling revealed that high TMB levels were associated with increased CD8-positive, PD-L1-positive T-cell infiltration, increased PD-L1 expression on tumor and immune cells, and upregulation of innate and adaptive immune response signatures. Conclusions and Relevance: These findings suggest that increasing TMB levels are associated with immune cell infiltration and an inflammatory T-cell-mediated response, resulting in increased sensitivity to PD-1/PD-L1 blockade in NSCLC across PD-L1 expression subgroups.

Intrinsic Immunogenicity of Small Cell Lung Carcinoma Revealed by Its Cellular Plasticity.

Small cell lung carcinoma (SCLC) is highly mutated, yet durable response to immune checkpoint blockade (ICB) is rare. SCLC also exhibits cellular plasticity, which could influence its immunobiology. Here we discover that a distinct subset of SCLC uniquely upregulates MHC I, enriching for durable ICB benefit. In vitro modeling confirms epigenetic recovery of MHC I in SCLC following loss of neuroendocrine differentiation, which tracks with derepression of STING. Transient EZH2 inhibition expands these nonneuroendocrine cells, which display intrinsic innate immune signaling and basally restored antigen presentation. Consistent with these findings, murine nonneuroendocrine SCLC tumors are rejected in a syngeneic model, with clonal expansion of immunodominant effector CD8 T cells. Therapeutically, EZH2 inhibition followed by STING agonism enhances T-cell recognition and rejection of SCLC in mice. Together, these data identify MHC I as a novel biomarker of SCLC immune responsiveness and suggest novel immunotherapeutic approaches to co-opt SCLC's intrinsic immunogenicity. SIGNIFICANCE: SCLC is poorly immunogenic, displaying modest ICB responsiveness with rare durable activity. In profiling its plasticity, we uncover intrinsically immunogenic MHC Ihi subpopulations of nonneuroendocrine SCLC associated with durable ICB benefit. We also find that combined EZH2 inhibition and STING agonism uncovers this cell state, priming cells for immune rejection.This article is highlighted in the In This Issue feature, p. 1861.

Dynamic single-cell RNA sequencing identifies immunotherapy persister cells following PD-1 blockade.

Resistance to oncogene-targeted therapies involves discrete drug-tolerant persister cells, originally discovered through in vitro assays. Whether a similar phenomenon limits efficacy of programmed cell death 1 (PD-1) blockade is poorly understood. Here, we performed dynamic single-cell RNA-Seq of murine organotypic tumor spheroids undergoing PD-1 blockade, identifying a discrete subpopulation of immunotherapy persister cells (IPCs) that resisted CD8+ T cell-mediated killing. These cells expressed Snai1 and stem cell antigen 1 (Sca-1) and exhibited hybrid epithelial-mesenchymal features characteristic of a stem cell-like state. IPCs were expanded by IL-6 but were vulnerable to TNF-α-induced cytotoxicity, relying on baculoviral IAP repeat-containing protein 2 (Birc2) and Birc3 as survival factors. Combining PD-1 blockade with Birc2/3 antagonism in mice reduced IPCs and enhanced tumor cell killing in vivo, resulting in durable responsiveness that matched TNF cytotoxicity thresholds in vitro. Together, these data demonstrate the power of high-resolution functional ex vivo profiling to uncover fundamental mechanisms of immune escape from durable anti-PD-1 responses, while identifying IPCs as a cancer cell subpopulation targetable by specific therapeutic combinations.

Tumor-Derived cGAMP Regulates Activation of the Vasculature.

Intratumoral recruitment of immune cells following innate immune activation is critical for anti-tumor immunity and involves cytosolic dsDNA sensing by the cGAS/STING pathway. We have previously shown that KRAS-LKB1 (KL) mutant lung cancer, which is resistant to PD-1 blockade, exhibits silencing of STING, impaired tumor cell production of immune chemoattractants, and T cell exclusion. Since the vasculature is also a critical gatekeeper of immune cell infiltration into tumors, we developed a novel microfluidic model to study KL tumor-vascular interactions. Notably, dsDNA priming of LKB1-reconstituted tumor cells activates the microvasculature, even when tumor cell STING is deleted. cGAS-driven extracellular export of 2'3' cGAMP by cancer cells activates STING signaling in endothelial cells and cooperates with type 1 interferon to increase vascular permeability and expression of E selectin, VCAM-1, and ICAM-1 and T cell adhesion to the endothelium. Thus, tumor cell cGAS-STING signaling not only produces T cell chemoattractants, but also primes tumor vasculature for immune cell escape.

Phosphorylation of RAB7 by TBK1/IKKε Regulates Innate Immune Signaling in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease enriched for mutations in PTEN and dysregulation of innate immune signaling. Here, we demonstrate that Rab7, a recently identified substrate of PTEN phosphatase activity, is also a substrate of the innate immune signaling kinases TANK-binding kinase 1 (TBK1)/IκB kinase ε (IKKε) on the same serine-72 (S72) site. An unbiased search for novel TBK1/IKKε substrates using stable isotope labeling with amino acids in cell culture phosphoproteomic analysis identified Rab7-S72 as a top hit. PTEN-null TNBC cells expressing a phosphomimetic version of Rab7-S72 exhibited diffuse cytosolic Rab7 localization and enhanced innate immune signaling, in contrast to a kinase-resistant version, which localized to active puncta that promote lysosomal-mediated stimulator of interferon genes (STING) degradation. Thus, convergence of PTEN loss and TBK1/IKKε activation on Rab7-S72 phosphorylation limited STING turnover and increased downstream production of IRF3 targets including CXCL10, CCL5, and IFNß. Consistent with this data, PTEN-null TNBC tumors expressed higher levels of STING, and PTEN-null TNBC cell lines were hyperresponsive to STING agonists. Together, these findings begin to uncover how innate immune signaling is dysregulated downstream of TBK1/IKKε in a subset of TNBCs and reveals previously unrecognized cross-talk with STING recycling that may have implications for STING agonism in the clinic. SIGNIFICANCE: These findings identify Rab7 as a substrate for TBK1 for regulation of innate immune signaling, thereby providing important insight for strategies aimed at manipulating the immune response to enhance therapeutic efficacy in TNBC.

A novel GIT2-BRAF fusion in pilocytic astrocytoma.

BACKGROUND: KIAA1549-BRAF fusion is the most common genetic event in pilocytic astrocytoma (PA), and leads to activation of the mitogen activated protein kinase (MAPK) signaling pathway. Fusions of BRAF with other partner genes, as well as other genetic alterations not involving BRAF but also leading to MAPK pathway activation have been described rarely. CASE PRESENTATION: We present a new fusion partner in the low-grade glioma of a 10-year-old male, who presented with headaches and recent episodes of seizures. Magnetic resonance imaging (MRI) demonstrated a right temporal lobe tumor. Histological and immunohistochemical evaluation, and a next generation sequencing assay (Oncopanel, Illumina, 500 genes) including breaKmer analysis for chromosomal rearrangements were performed. Histology was remarkable for a low-grade glioma composed of mildly atypical astrocytes with piloid processes, in a focally microcystic background. Mitoses were not seen; unequivocal Rosenthal fibers or eosinophilic granular bodies were absent. The tumor was positive for OLIG2 and GFAP and negative for BRAF V600E and IDH1 R132H mutant protein immunostains. Oncopanel showed low SOX2 (3q26.33) copy number gain, and no gains at 7q34. There were no significant single nucleotide variants. BreaKmer detected a GIT2-BRAF fusion with loss of BRAF exons 1-8. The integrated diagnosis was low-grade glioma with piloid features, most consistent with pilocytic astrocytoma, WHO grade I. CONCLUSION: GIT2-BRAF fusion has not been reported in the literature in any tumor. Given that the BRAF sequence deleted is identical to that seen in other fusion events in PA, it most likely acts as tumor driver by activation of the MAPK pathway.