Aberrant microRNA (miRNA) expression is a defining feature of human malignancy. Specific miRNAs have been identified as promoters or suppressors of metastatic progression. miRNAs control metastasis ...through divergent or convergent regulation of metastatic gene pathways. Some miRNA regulatory networks govern cell-autonomous cancer phenotypes, whereas others modulate the cell-extrinsic composition of the metastatic microenvironment. The use of small RNAs as probes into the molecular and cellular underpinnings of metastasis holds promise for the identification of candidate genes for potential therapeutic intervention.
Through in vivo selection of human cancer cell populations, we uncover a convergent and cooperative miRNA network that drives melanoma metastasis. We identify miR-1908, miR-199a-5p, and miR-199a-3p ...as endogenous promoters of metastatic invasion, angiogenesis, and colonization in melanoma. These miRNAs convergently target apolipoprotein E (ApoE) and the heat shock factor DNAJA4. Cancer-secreted ApoE suppresses invasion and metastatic endothelial recruitment (MER) by engaging melanoma cell LRP1 and endothelial cell LRP8 receptors, respectively, while DNAJA4 promotes ApoE expression. Expression levels of these miRNAs and ApoE correlate with human metastatic progression outcomes. Treatment of cells with locked nucleic acids (LNAs) targeting these miRNAs inhibits metastasis to multiple organs, and therapeutic delivery of these LNAs strongly suppresses melanoma metastasis. We thus identify miRNAs with dual cell-intrinsic/cell-extrinsic roles in cancer, reveal convergent cooperativity in a metastatic miRNA network, identify ApoE as an anti-angiogenic and metastasis-suppressive factor, and uncover multiple prognostic miRNAs with synergistic combinatorial therapeutic potential in melanoma.
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► Multiple miRNAs convergently target ApoE signaling in melanoma, promoting metastasis ► ApoE inhibits invasion and endothelial recruitment through LRP1/LRP8 receptors ► These miRNAs have therapeutic and prognostic potential in melanoma
Three miRNAs drive melanoma metastasis by cooperatively targeting ApoE, which suppresses invasion and angiogenesis by engaging the LRP1 and LRP8 receptors. The expression levels of the microRNAs are prognostic of human melanoma metastatic relapse.
Melanoma metastasis is a devastating outcome lacking an effective preventative therapeutic. We provide pharmacologic, molecular, and genetic evidence establishing the liver-X nuclear hormone receptor ...(LXR) as a therapeutic target in melanoma. Oral administration of multiple LXR agonists suppressed melanoma invasion, angiogenesis, tumor progression, and metastasis. Molecular and genetic experiments revealed these effects to be mediated by LXRβ, which elicits these outcomes through transcriptional induction of tumoral and stromal apolipoprotein-E (ApoE). LXRβ agonism robustly suppressed tumor growth and metastasis across a diverse mutational spectrum of melanoma lines. LXRβ targeting significantly prolonged animal survival, suppressed the progression of established metastases, and inhibited brain metastatic colonization. Importantly, LXRβ activation displayed melanoma-suppressive cooperativity with the frontline regimens dacarbazine, B-Raf inhibition, and the anti-CTLA-4 antibody and robustly inhibited melanomas that had acquired resistance to B-Raf inhibition or dacarbazine. We present a promising therapeutic approach that uniquely acts by transcriptionally activating a metastasis suppressor gene.
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•Nuclear hormone receptor LXRβ is a therapeutic target in melanoma•Oral delivery of LXR agonists inhibits melanoma tumor progression and metastasis•LXRβ mediates melanoma suppression by inducing stromal and tumoral ApoE
Activation of the nuclear hormone receptor LXRβ protects against melanoma tumor growth and metastasis through the induction of the lipoprotein ApoE.
Doxorubicin (doxo) remains the standard of care for patients with advanced soft tissue sarcoma (STS), even though response rates to doxo are only around 14% to 18%. We evaluated enapotamab vedotin ...(EnaV), an AXL-specific antibody-drug conjugate (ADC), in a panel of STS patient-derived xenografts (PDX). Eight models representing multiple STS subtypes were selected from our STS PDX platform (n = 45) by AXL immunostaining on archived passages. Models were expanded by unilateral transplantation of tumor tissue into the left flank of 20 NMRI nu/nu mice. Once tumors were established, mice were randomized into an EnaV treatment group, or a group treated with isotype control ADC. Treatment efficacy was assessed by tumor volume evaluation, survival analysis, and histological evaluation of tumors, and associated with AXL expression. EnaV demonstrated significant tumor growth delay, regression, and/or prolonged survival compared to isotype control ADC in 5/8 STS PDX models investigated. Experimental passages of responding models were all found positive for AXL at varying levels, but no linear relationship could be identified between the level of expression and level of response to EnaV. One model was found negative for AXL on experimental passage and did not respond to EnaV. This study provides a preclinical rationale for the evaluation of AXL-targeting ADCs in the treatment of AXL-expressing sarcomas.
Diffuse and uncontrollable brain invasion is a hallmark of glioblastoma (GBM), but its mechanism is understood poorly. We developed a 3D ex vivo organotypic model to study GBM invasion. We ...demonstrate that invading GBM cells upregulate a network of extracellular matrix (ECM) components, including multiple collagens, whose expression correlates strongly with grade and clinical outcome. We identify interferon regulatory factor 3 (IRF3) as a transcriptional repressor of ECM factors and show that IRF3 acts as a suppressor of GBM invasion. Therapeutic activation of IRF3 by inhibiting casein kinase 2 (CK2)—a negative regulator of IRF3—downregulated the expression of ECM factors and suppressed GBM invasion in ex vivo and in vivo models across a panel of patient-derived GBM cell lines representative of the main molecular GBM subtypes. Our data provide mechanistic insight into the invasive capacity of GBM tumors and identify a potential therapy to inhibit GBM invasion.
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•Invading GBM cells induce a network of collagens and collagen-interacting proteins•IRF3 acts as a transcriptional repressor of pro-invasive ECM genes in GBM•IRF3 activation leads to robust inhibition of GBM invasion•CK2 is a negative regulator of IRF3; CK2 inhibition suppresses GBM invasiveness
Pencheva et al. describe an ex vivo organotypic brain slice model of glioblastoma invasiveness. Using this model, the authors discover a pro-invasive network of extracellular matrix collagens that are transcriptionally repressed by interferon regulatory factor 3. Small-molecule targeting of casein kinase 2 inhibits invasion by activating interferon regulatory factor 3.
BackgroundTriple-negative breast cancer (TNBC) accounts for 10–20% of newly diagnosed breast cancers worldwide, is usually diagnosed in younger women, and has the worst prognosis among breast cancer ...subtypes.1 2 Due to limited treatment options, chemotherapy remains integral to the TNBC treatment regime, with the recent addition of chemotherapy plus PARP or anti-PD1 inhibitors. While these advancements provided a much-needed benefit to a subset of patients, a deeper understanding is necessary to determine which patients will benefit from current and future immunotherapies. To provide insight into alterations in patient response, we sought to characterize the molecular and immune microenvironment in baseline and post standard of care matched samples. We used a multimodal approach including immunohistochemistry (IHC), multiplex immunofluorescence (mIF), and single-slide FFPE tumor gene expression to characterize matched pre- and post-TNBC tumor samples.MethodsA cohort of 34 patients diagnosed with TNBC were commercially sourced. Matched tumor biopsies were collected following neoadjuvant chemotherapy and following progression on 2nd line pembrolizumab or nivolumab monotherapy. These matched samples were profiled using IHC (CD3, CD8, PDL1), mIF (CD20, CD163/CD68, CD11c and PanCK) and single-slide FFPE whole transcriptomic profiling. Response to anti-PD1 therapy was scored using RESIST 1.1 (best overall response BOR, progressive disease PD or partial response PR) and progression free survival (PFS).ResultsDifferential gene expression analysis was performed to identify predictors of partial response to anti-PD1 therapy; higher expression of macrophage associated genes CD163 and FOLR2 were associated with PD patients. Prognostic genes, such as IGSF9,3 CERS6 4 and GRHL2,5 were identified in PR patients have been previously implicated in breast cancer. Cell-type signature analysis identified a significant enrichment of adipocytes and macrophages (M2 subtype) in PD patients. Moreover, regardless of patient response, adipocytes and endothelial cells were significantly enriched prior to anti-PD1 therapy. CD3 and CD8 counts were comparable before and after anti-PD1 treatment.ConclusionsConsistent with our findings, M2 macrophages, identified by CD163 expression, have been correlated with a worse prognosis in TNBC patients.6 Additionally, cancer-associated adipocytes7 and endothelial cells8 may be important in breast cancer progression and metastasis. In our cohort, the TNBC tumor microenvironment contained immunosuppressive factors, adipocytes fatty acid metabolism, and endothelial cells angiogenesis that could be used to stratify patients that are more likely to benefit from immunotherapies. Furthermore, directly targeting these immunosuppressive factors prior to immunotherapy could help establish a more responsive tumor microenvironment.ReferencesBauer K, Brown M, Cress R, Parise C, Caggiano V. Descriptive analysis of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype. Cancer. 2007; 109:1721−1728.Lehmann B, Bauer J, Chen X, Sanders M, Chakravarthy A, Shyr Y, and Pietenpol J. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest. 2011; 121: 2750−2767.Li Y, Deng Y, Zhao Y, Zhang W, Zhang S, Zhang L, Wang B, Xu Y, Chen S. Immunoglobulin superfamily 9 (IGSF9) is trans-activated by p53, inhibits breast cancer metastasis via FAK. Oncogene. 2022; 41: 4658−4672.Chen H, He B, Ke F. Ceramide Synthase 6 Mediates Triple-Negative Breast Cancer Response to Chemotherapy Through RhoA- and EGFR-Mediated Signaling Pathways. J Breast Cancer. 2022; 25: 500−512.Xiang X, Bin Deng Z, Zhuang X, Ju S, Mu J, Jiang H, Zhang L, Yan J, Miller D, Zhang H. Grhl2 Determines the Epithelial Phenotype of Breast Cancers and Promotes Tumor Progression. PLoS One. 2012; 7: e50781.Bao X, Shi R, Zhao T, Wang Y, Anastasov N, Rosemann M, Fang W. Integrated analysis of single-cell RNA-seq and bulk RNA-seq unravels tumour heterogeneity plus M2-like tumour-associated macrophage infiltration and aggressiveness in TNBC. Cancer Immunol Immunother. 2021; 70:189−202.Wu Q, Li B, Li Z, Li J, Sun S, Sun S. Cancer-associated adipocytes: key players in breast cancer progression. J Hematol Oncol. 2019; 12: 95.Ma Y,Li Y, Guo P, Zhao J, Qin Q, Wang J, , Liang Z, Wei D, Wang Z, Shen J, He S, Tang Q, Lu G, Shiand G, Meng Endothelial Cells Potentially Participate in the Metastasis of Triple-Negative Breast Cancer. J Immunol Res. 2022; 2022: 5412007.Ethics ApprovalSubjects provided informed consent to Capital Biosciences (tissue vendor) for genetic and protein analysis.
BackgroundThe composition of T-cell subsets within the tumor microenvironment (TME) can impact TCR diversity and immune therapy response. Studies have shown that the presence of tumor-infiltrating ...lymphocytes (TILs), particularly CD8+ cytotoxic T cells, is associated with improved outcomes in HNSCC patients.1 TCR diversity in tumor is difficult to obtain by T-cell receptor (TCR) sequencing with formalin-fixed paraffin-embedded (FFPE) tissue due to RNA degradation. Spatial biology platforms, such as the NanoString GeoMx Digital Spatial Profiler (DSP), have enabled high resolution transcriptomic studies of TME. We used a new 139 gene TCR spike-in panel combined the current whole transcriptome DSP panel to assess T-cell activity and microenvironment changes associated with checkpoint inhibition.MethodsWe selected a cohort of 22 HNSCC patients whose tumors were resected then treated with chemotherapy. Upon progression, patients were treated with αPD1 immune checkpoint inhibitor therapy (ICI). Matched biopsies collected before and after ICI treatment were profiled with GeoMx DSP whole transcriptome atlas (WTA) panel spiked with the TCR add-on panel.3 4 We stained tissues with fluorophore-labeled antibodies to CD3, CD20 and cytokeratin for region of interest selection. We then used the exquisite targeting features of GeoMx to profile CD3+ T cells and compared them to adjacent tumor and CD3- stroma. Tissues were also stained by immunohistochemistry (IHC) for CD3, CD8 and the positive cells were quantified; whole-genome RNA transcriptomics was also performed on the bulk tissue using HTG EdgeSeq.ResultsSpatially resolved transcriptomics allows probing of TME by cell type. We applied the new capability to assess T-cell diversity in-situ to calculate the Simpson Diversity Metric using α/β V/J gene frequencies. Higher diversity in the TCRα-V genes in T-cells adjacent to tumor was positively associated with partial response to ICI. In contrast, IHC counts of CD3 and CD8 cells alone in the biopsy were not predictive of response. We compared TCR V/J gene frequency within patient samples (pre/post ICI) and between patients; each patient had a distinct pattern of V/J subunit usage, consistent with literature. Tertiary lymphoid structures have been positively associated with response to ICI.2 We profiled T-cells in regions enriched for B-cells and found increased expression of pro-inflammatory markers across the cohort. In non-T-cell stroma, immune cell signature analysis suggested dendritic cells were elevated in partial responders; TGFβ was associated with non-responders.ConclusionsHigh resolution profiling of T-cell diversity in the TME can uncover local mechanisms important to checkpoint inhibitor therapy response.ReferencesAlmangush A, De Keukeleire S, Rottey S, Ferdinande L, Vermassen T, Leivo I, Mäkitie A. Tumor-Infiltrating Lymphocytes in Head and Neck Cancer: Ready for Prime Time? Cancers. 2022;14:1558Ruffin A, Cillo A, Tabib T, Liu A, Onkar S, Kunning S, Lampenfeld C, Atiya H, Abecassis I, Kürten C, Qi Z, Soose R, Duvvuri U, Kim S, Oesterrich S, Lafyatis R, Coffman L, Ferris R, Vignali D, Bruno T. B cell signatures and tertiary lymphoid structures contribute to outcome in head and neck squamous cell carcinoma Nature Communications. 2021;12:3349Fan L, Bachu M, Ma-Edmonds M, Harris A, Muthuswamy A, Couto S, Fereshteh M, Jabado O. In-situ Estimation of T-cell Receptor Diversity and Transcriptomics with NanoString Digital Spatial Profiling in FFPE Tumor Biopsies. AGBT 2023 Poster # 110Raay K, Reeves J, Piazza E, Kaplan H, Vivian J, Fernandez F, Hoang M, Beechem J Spatially resolved expression of T cell receptors elucidates spatial relationships between T cells, immune infiltration, and cancer-associated pathways. AACR Annual Meeting 2023; Poster # 615Ethics ApprovalSubjects provided informed consent to Capital Biosciences, Inc. (Gaithersburg, MD) for genetic and protein analysis
BackgroundDuoBody-PD-L1×4-1BB (GEN1046) is a class-defining, bispecific immunotherapy designed to induce an antitumor immune response by simultaneous and complementary PD-L1 blockade and conditional ...4-1BB stimulation. Encouraging clinical activity and manageable safety were observed during dose escalation in the ongoing phase 1/2a trial in patients with advanced solid tumors (NCT03917381). We report exploratory pharmacodynamic analyses and potential biomarkers of response in an expansion cohort of patients with PD-(L)1–R/R NSCLC.MethodsPatients with metastatic/unresectable NSCLC who had multiple lines of prior systemic therapy, including a checkpoint inhibitor, received flat-dose DuoBody-PD-L1×4-1BB (100 mg) intravenously every 3 weeks. Immunophenotyping of peripheral blood and measurements of soluble immune mediators were evaluated in serial blood samples in cycles 1–2. Tumor PD-L1 and 4-1BB expression and additional immune markers were evaluated by immunohistochemistry in core needle tumor biopsy specimens collected before treatment and at cycle 2.ResultsAs of May 2021, 40 patients with PD-(L)1–R/R NSCLC were enrolled (median age, 63 years). Treatment with DuoBody-PD-L1×4-1BB elicited pharmacodynamic modulation of immune endpoints within the first 2 cycles. Induction of peripheral IFN-y, CXCL9/10, and expansion of peripheral CD8+ effector memory T cells and activated NK cells were observed starting at cycle 1 (>2-fold from baseline) and maintained or increased through cycle 2. Based on 9 paired tumor biopsy samples, increased PD-L1 and 4-1BB expression and cytotoxic CD8+/GZMB+ cell density were detected following treatment. In a subset of patients with clinical response (n=5 confirmed PRs), a trend of greater induction of IFN-y, CXCL9/10, and activated NK cells was observed vs nonresponders. Disease control rates were higher in patients who had progressed on prior anti–PD-1 therapy within 8 months (64% 16/25) from the first dose of DuoBody-PD-L1×4-1BB. As expected, among patients with evaluable baseline tumors (n=26), most with any degree of tumor reduction (best change, <0%) harbored PD-L1+ tumors (≥1% tumor positive score; 7/10) and showed close spatial proximity between PD-L1+ and 4-1BB+ cells. Conversely, most patients without any degree of tumor reduction presented with PD-L1− tumors (12/16).ConclusionsIn patients with NSCLC who progressed on PD-(L)1 therapy, DuoBody-PD-L1×4-1BB elicited pharmacodynamic effects consistent with its proposed mechanism of action. Relationships between disease control and PD-L1 tumoral expression, as well as time from last prior anti–PD-1 therapy, were observed. These findings support that patient selection and/or anti–PD-1 combination therapy may lead to improved clinical efficacy. Further analyses are ongoing and updated results will be presented.AcknowledgementsThe authors thank Hrefna Kristin Johannsdottir, Lei Pang, and Kate Sasser at Genmab A/S and Friederike Gieseke at BioNTech SE for their valuable contributions. This trial was funded by Genmab A/S and BioNTech SE.Trial RegistrationNCT03917381Ethics ApprovalThis trial is undertaken following full approval of the final protocol, amendments, informed consent form, applicable recruiting materials, and subject compensation programs by the Independent Ethics Committee/Institutional Review Board.ConsentWritten informed consent, in accordance with principles that originated in the Declaration of Helsinki 2013, current ICH guidelines including ICH-GCP E6(R2), applicable regulatory requirements, and sponsor policy, was provided by the patients.
BackgroundUnderstanding the dynamics of immune cells in the lung tumor microenvironment following immune checkpoint inhibitor (ICI) therapy is important for developing therapies tailored to patients ...with progressive disease. We sought to characterize protein and mRNA biomarkers in the tumor and stromal microenvironment in such patients with the Nanostring Digital Spatial Profiling (DSP) platform. DSP technology allows highly multiplexed profiling of proteins and RNA in a spatially resolved manner.MethodsFFPE non-small lung adenocarcinoma biopsies from 18 patients were sourced commercially (Capital Biosciences, MD). Patients had surgical resection of tumors then adjuvant chemotherapy. Upon progression, patients received monotherapy ICI (nivolumab or pembrolizumab). Once progressed on ICI, biopsies were performed and patients were then treated with platinum-doublet or single agent chemotherapy and followed until progression and/or death. Best overall response (BOR) and progression free survival (PFS) was available for ICI. FFPE tumors were sectioned and stained with anti-Pan-Cytokeratin, anti-PDL1 and anti-4-1BB (CD137) using standard immunofluorescence techniques. Twelve circular regions of ~400 um in diameter containing tumor (PanCK+) and stromal (PanCK-) areas were selected per patient (figure 1). The technology uses a photocleavable DNA barcode strategy to multiplex antibodies and RNA in-situ hybridization probes. The GeoMX instrument was used to generate counts for 58 proteins and 84 RNAs on serial sections. Data normalization, linear modeling and survival analysis was conducted in R.ResultsLymphoid and myeloid markers were more abundant in stroma, indicating the microenvironment is diverse and confirming the DSP platform can segment adjacent cells. High levels of PDL1 protein in the tumor were correlated with T cell markers in the stroma (CD3, CD8, ICOS, IDO, OX40L) and inversely correlated with granulocytic (CD66b) and angiogenesis markers (CD34). We focused outcomes analysis on ICI response (9 PD/9 PR). OX40L protein was higher in patients with partial response and associated with delayed progression (figure 2). CD74 protein was associated with progressive disease during ICI therapy. CSF1R, CD4 and PECAM1 mRNA expression levels in stroma trended with progressive disease.Abstract 238 Figure 1Immunofluorescence staining and segmentation of NSCLC tumorAbstract 238 Figure 2Association of OX40L abundance in stroma with PFS using Kaplan-Meier analysisConclusionsIn this study we recapitulated the role of OX40L as a marker for response to ICI1 and CSF1R and PECAM1 in non-response to ICI.2. 3 CD74 is a receptor for the pro-inflammatory cytokine (MIF) however CD74 ectodomain shedding may function as a decoy receptor.4 These findings highlight how DSP can be used to probe the tumor microenvironment to identify pathways specific to NSCLC non-response for therapeutic target and biomarker development.Ethics ApprovalSubjects provided informed consent to Capital Biosciences for genetic and protein analysis.AcknowledgementsLiang Zhang, Adrienne Whitman, Jennifer Hart, JingJing Gong of Nanostring Technologies.ReferencesMassarelli E, Lam VK, Parra ER, et al. High OX-40 expression in the tumor immune infiltrate is a favorable prognostic factor of overall survival in non-small cell lung cancer. J Immunother Cancer 2019;7(1):351.Cannarile MA, Weisser M, Jacob W, Jegg AM, Ries CH, Rüttinger D. Colony-stimulating factor 1 receptor (CSF1R) inhibitors in cancer therapy. J Immunother Cancer 2017;5(1):53.Kuang BH, Wen XZ, Ding Y, et al. The prognostic value of platelet endothelial cell adhesion molecule-1 in non-small-cell lung cancer patients. Med Oncol 2013;30(2):536.Schröder B. The multifaceted roles of the invariant chain CD74-More than just a chaperone. Biochim Biophys Acta 2016;1863(6 Pt A):1269–1281.
BRAF(V600E) mutant melanomas treated with inhibitors of the BRAF and MEK kinases almost invariably develop resistance that is frequently caused by reactivation of the mitogen activated protein kinase ...(MAPK) pathway. To identify novel treatment options for such patients, we searched for acquired vulnerabilities of MAPK inhibitor-resistant melanomas. We find that resistance to BRAF+MEK inhibitors is associated with increased levels of reactive oxygen species (ROS). Subsequent treatment with the histone deacetylase inhibitor vorinostat suppresses SLC7A11, leading to a lethal increase in the already-elevated levels of ROS in drug-resistant cells. This causes selective apoptotic death of only the drug-resistant tumor cells. Consistently, treatment of BRAF inhibitor-resistant melanoma with vorinostat in mice results in dramatic tumor regression. In a study in patients with advanced BRAF+MEK inhibitor-resistant melanoma, we find that vorinostat can selectively ablate drug-resistant tumor cells, providing clinical proof of concept for the novel therapy identified here.
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•BRAF inhibitor-resistant melanomas have elevated ROS levels•Histone deacetylase inhibitors increase ROS levels through suppression of SLC7A11•BRAF inhibitor resistance causes vulnerability to histone deacetylase inhibitors•In patients, histone deacetylase inhibitors selectively kill drug-resistant cells
Collateral sensitivity in BRAF inhibitor-resistant melanoma may confer selective vulnerability to histone deacetylase inhibitors.
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