Using mouse liver cancer models based on somatic genome editing to predict immune checkpoint inhibitor responses.

Tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) are the only two classes of FDA-approved drugs for individuals with advanced hepatocellular carcinoma (HCC). While TKIs confer only modest survival benefits, ICIs have been associated with remarkable outcomes but only in the minority of patients who respond. Understanding the mechanisms that determine the efficacy of ICIs in HCC will help to stratify patients likely to respond to ICIs. This study aims to elucidate how genetic composition and specific oncogenic pathways regulate the immune composition of HCC, which directly affects response to ICIs. A collection of mouse HCCs with genotypes that closely simulate the genetic composition found in human HCCs were established using genome-editing approaches involving the delivery of transposon and CRISPR-Cas9 systems by hydrodynamic tail vein injection. Mouse HCC tumors were analyzed by RNA-sequencing while tumor-infiltrating T cells were analyzed by flow cytometry and single-cell RNA-sequencing. Based on the CD8+ T cell-infiltration level, we characterized tumors with different genotypes into cold and hot tumors. Anti-PD-1 treatment had no effect in cold tumors but was greatly effective in hot tumors. As proof-of-concept, a cold tumor (Trp53KO/MYCOE) and a hot tumor (Keap1KO/MYCOE) were further characterized. Tumor-infiltrating CD8+ T cells from Keap1KO/MYCOE HCCs expressed higher levels of proinflammatory chemokines and exhibited enrichment of a progenitor exhausted CD8+ T-cell phenotype compared to those in Trp53KO/MYCOE HCCs. The TKI sorafenib sensitized Trp53KO/MYCOE HCCs to anti-PD-1 treatment. Single anti-PD-1 treatment appears to be effective in HCCs with genetic mutations driving hot tumors, while combined anti-PD-1 and sorafenib treatment may be more appropriate in HCCs with genetic mutations driving cold tumors. Genetic alterations of different driver genes in mouse liver cancers are associated with tumor-infiltrating CD8+ T cells and anti-PD-1 response. Mouse HCCs with different genetic compositions can be grouped into hot and cold tumors based on the level of tumor-infiltrating CD8+ T cells. This study provides proof-of-concept evidence to show that hot tumors are responsive to anti-PD-1 treatment while cold tumors are more suitable for combined treatment with anti-PD-1 and sorafenib. Our study might help to guide the design of patient stratification systems for single or combined treatments involving anti-PD-1.

Yuen VW ，Chiu DK ，Law CT ，Cheu JW ，Chan CY ，Wong BP ，Goh CC ，Zhang MS ，Xue HD ，Tse AP ，Zhang Y ，Lau HY ，Lee D ，Au-Yeung RKH ，Wong CM ，Wong CC ... -  《-》

Hepatocellular Carcinoma Cells Up-regulate PVRL1, Stabilizing PVR and Inhibiting the Cytotoxic T-Cell Response via TIGIT to Mediate Tumor Resistance to PD1 Inhibitors in Mice.

Immune checkpoint inhibitors are effective in the treatment of some hepatocellular carcinomas (HCCs), but these tumors do not always respond to inhibitors of programmed cell death 1 (PDCD1, also called PD1). We investigated mechanisms of resistance of liver tumors in mice to infiltrating T cells. Mice were given hydrodynamic tail vein injections of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) and transposon vectors to disrupt Trp53 and overexpress C-Myc (Trp53KO/C-MycOE mice). Pvrl1 and Pvrl3 were knocked down in Hepa1-6 cells by using short hairpin RNAs. Hepa1-6 cells were injected into livers of C57BL/6 mice; some mice were given intraperitoneal injections of antibodies against PD1, T-cell immunoreceptor with Ig and ITIM domains (TIGIT), or CD8 before the cancer cells were injected. Liver tissues were collected from mice and analyzed by histology, immunohistochemistry, and quantitative real-time polymerase chain reaction; tumors were analyzed by mass cytometry using markers to detect T cells and other lymphocytes. We obtained HCC and nontumorous liver tissues and clinical data from patients who underwent surgery in Hong Kong and analyzed the tissues by immunohistochemistry. Trp53KO/C-MycOE mice developed liver tumors in 3-5 weeks; injections of anti-PD1 did not slow tumor development. Tumors from mice given anti-PD1 had larger numbers of memory CD8+ T cells (CD44+CD62L-KLRG1int) and T cells that expressed PD1, lymphocyte activating 3 (LAG3), and TIGIT compared with mice not given the antibody. HCC tissues from patients had higher levels of PVRL1 messenger RNA and protein than nontumorous tissues. Increased PVRL1 was associated with shorter times of disease-free survival. Knockdown of Pvrl1 in Hepa1-6 cells caused them to form smaller tumors in mice, infiltrated by higher numbers of CD8+ T cells that expressed the inhibitory protein TIGIT; these effects were not observed in mice with depletion of CD8+ T cells. In Hepa1-6 cells, PVRL1 stabilized cell surface PVR, which interacted with TIGIT on CD8+ T cells; knockdown of Pvrl1 reduced cell-surface levels of PVR but not levels of Pvr messenger RNA. In Trp53KO/C-MycOE mice and mice with tumors grown from Hepa1-6 cells, injection of the combination of anti-PD1 and anti-TIGIT significantly reduced tumor growth, increased the ratio of cytotoxic to regulatory T cells in tumors, and prolonged survival. PVRL1, which is up-regulated by HCC cells, stabilizes cell surface PVR, which interacts with TIGIT, an inhibitory molecule on CD8+ effector memory T cells. This suppresses the ant-tumor immune response. Inhibitors of PVRL1/TIGIT, along with anti-PD1 might be developed for treatment of HCC.

Chiu DK ，Yuen VW ，Cheu JW ，Wei LL ，Ting V ，Fehlings M ，Sumatoh H ，Nardin A ，Newell EW ，Ng IO ，Yau TC ，Wong CM ，Wong CC ... -  《-》

CRKL dictates anti-PD-1 resistance by mediating tumor-associated neutrophil infiltration in hepatocellular carcinoma.

The effectiveness of immune checkpoint inhibitor (ICI) therapy for hepatocellular carcinoma (HCC) is limited by treatment resistance. However, the mechanisms underlying immunotherapy resistance remain elusive. We aimed to identify the role of CT10 regulator of kinase-like (CRKL) in resistance to anti-PD-1 therapy in HCC. Gene expression in HCC specimens from 10 patients receiving anti-PD-1 therapy was identified by RNA-sequencing. A total of 404 HCC samples from tissue microarrays were analyzed by immunohistochemistry. Transgenic mice (Alb-Cre/Trp53fl/fl) received hydrodynamic tail vein injections of a CRKL-overexpressing vector. Mass cytometry by time of flight was used to profile the proportion and status of different immune cell lineages in the mouse tumor tissues. CRKL was identified as a candidate anti-PD-1-resistance gene using a pooled genetic screen. CRKL overexpression nullifies anti-PD-1 treatment efficacy by mobilizing tumor-associated neutrophils (TANs), which block the infiltration and function of CD8+ T cells. PD-L1+ TANs were found to be an essential subset of TANs that were regulated by CRKL expression and display an immunosuppressive phenotype. Mechanistically, CRKL inhibits APC (adenomatous polyposis coli)-mediated proteasomal degradation of β-catenin by competitively decreasing Axin1 binding, and thus promotes VEGFα and CXCL1 expression. Using human HCC samples, we verified the positive correlations of CRKL/β-catenin/VEGFα and CXCL1. Targeting CRKL using CRISPR-Cas9 gene editing (CRKL knockout) or its downstream regulators effectively restored the efficacy of anti-PD-1 therapy in an orthotopic mouse model and a patient-derived organotypic tumor spheroid model. Activation of the CRKL/β-catenin/VEGFα and CXCL1 axis is a critical obstacle to successful anti-PD-1 therapy. Therefore, CRKL inhibitors combined with anti-PD-1 could be useful for the treatment of HCC. Here, we found that CRKL was overexpressed in anti-PD-1-resistant hepatocellular carcinoma (HCC) and that CRKL upregulation promotes anti-PD-1 resistance in HCC. We identified that upregulation of the CRKL/β-catenin/VEGFα and CXCL1 axis contributes to anti-PD-1 tolerance by promoting infiltration of tumor-associated neutrophils. These findings support the strategy of bevacizumab-based immune checkpoint inhibitor combination therapy, and CRKL inhibitors combined with anti-PD-1 therapy may be developed for the treatment of HCC.

Xie P ，Yu M ，Zhang B ，Yu Q ，Zhao Y ，Wu M ，Jin L ，Yan J ，Zhou B ，Liu S ，Li X ，Zhou C ，Zhu X ，Huang C ，Xu Y ，Xiao Y ，Zhou J ，Fan J ，Hung MC ，Ye Q ，Guo L ，Li H ... -  《-》

Genome-Wide CRISPR Screen Identifies Regulators of Mitogen-Activated Protein Kinase as Suppressors of Liver Tumors in Mice.

It has been a challenge to identify liver tumor suppressors or oncogenes due to the genetic heterogeneity of these tumors. We performed a genome-wide screen to identify suppressors of liver tumor formation in mice, using CRISPR-mediated genome editing. We performed a genome-wide CRISPR/Cas9-based knockout screen of P53-null mouse embryonic liver progenitor cells that overexpressed MYC. We infected p53-/-;Myc;Cas9 hepatocytes with the mGeCKOa lentiviral library of 67,000 single-guide RNAs (sgRNAs), targeting 20,611 mouse genes, and transplanted the transduced cells subcutaneously into nude mice. Within 1 month, all the mice that received the sgRNA library developed subcutaneous tumors. We performed high-throughput sequencing of tumor DNA and identified sgRNAs increased at least 8-fold compared to the initial cell pool. To validate the top 10 candidate tumor suppressors from this screen, we collected data from patients with hepatocellular carcinoma (HCC) using the Cancer Genome Atlas and COSMIC databases. We used CRISPR to inactivate candidate tumor suppressor genes in p53-/-;Myc;Cas9 cells and transplanted them subcutaneously into nude mice; tumor formation was monitored and tumors were analyzed by histology and immunohistochemistry. Mice with liver-specific disruption of p53 were given hydrodynamic tail-vein injections of plasmids encoding Myc and sgRNA/Cas9 designed to disrupt candidate tumor suppressors; growth of tumors and metastases was monitored. We compared gene expression profiles of liver cells with vs without tumor suppressor gene disrupted by sgRNA/Cas9. Genes found to be up-regulated after tumor suppressor loss were examined in liver cancer cell lines; their expression was knocked down using small hairpin RNAs, and tumor growth was examined in nude mice. Effects of the MEK inhibitors AZD6244, U0126, and trametinib, or the multi-kinase inhibitor sorafenib, were examined in human and mouse HCC cell lines. We identified 4 candidate liver tumor suppressor genes not previously associated with liver cancer (Nf1, Plxnb1, Flrt2, and B9d1). CRISPR-mediated knockout of Nf1, a negative regulator of RAS, accelerated liver tumor formation in mice. Loss of Nf1 or activation of RAS up-regulated the liver progenitor cell markers HMGA2 and SOX9. RAS pathway inhibitors suppressed the activation of the Hmga2 and Sox9 genes that resulted from loss of Nf1 or oncogenic activation of RAS. Knockdown of HMGA2 delayed formation of xenograft tumors from cells that expressed oncogenic RAS. In human HCCs, low levels of NF1 messenger RNA or high levels of HMGA2 messenger RNA were associated with shorter patient survival time. Liver cancer cells with inactivation of Plxnb1, Flrt2, and B9d1 formed more tumors in mice and had increased levels of mitogen-activated protein kinase phosphorylation. Using a CRISPR-based strategy, we identified Nf1, Plxnb1, Flrt2, and B9d1 as suppressors of liver tumor formation. We validated the observation that RAS signaling, via mitogen-activated protein kinase, contributes to formation of liver tumors in mice. We associated decreased levels of NF1 and increased levels of its downstream protein HMGA2 with survival times of patients with HCC. Strategies to inhibit or reduce HMGA2 might be developed to treat patients with liver cancer.

Song CQ ，Li Y ，Mou H ，Moore J ，Park A ，Pomyen Y ，Hough S ，Kennedy Z ，Fischer A ，Yin H ，Anderson DG ，Conte D Jr ，Zender L ，Wang XW ，Thorgeirsson S ，Weng Z ，Xue W ... -  《-》

Disruption of SIRT7 Increases the Efficacy of Checkpoint Inhibitor via MEF2D Regulation of Programmed Cell Death 1 Ligand 1 in Hepatocellular Carcinoma Cells.

Immune checkpoint inhibitors have some efficacy in the treatment of hepatocellular carcinoma (HCC). Programmed cell death 1 ligand 1 (PD-L1), expressed on some cancer cells, binds to the receptor programmed cell death 1 (PDCD1, also called PD1) on T cells to prevent their proliferation and reduce the antigen-tumor immune response. Immune cells that infiltrate some types of HCCs secrete interferon gamma (IFNG). Some HCC cells express myocyte enhancer factor 2D (MEF2D), which has been associated with shorter survival times of patients. We studied whether HCC cell expression of MEF2D regulates expression of PD-L1 in response to IFNG. We analyzed immune cells from 20 fresh HCC tissues by flow cytometry. We analyzed 225 fixed HCC tissues (from 2 cohorts) from patients in China by immunohistochemistry and obtained survival data. We created mice with liver-specific knockout of MEF2D (MEF2DLPC-KO mice). We knocked out or knocked down MEF2D, E1A binding protein p300 (p300), or sirtuin 7 (SIRT7) in SMMC-7721, Huh7, H22, and Hepa1-6 HCC cell lines, some incubated with IFNG. We analyzed liver tissues from mice and cell lines by RNA sequencing, immunoblot, dual luciferase reporter, and chromatin precipitation assays. MEF2D protein acetylation and proteins that interact with MEF2D were identified by coimmunoprecipitation and pull-down assays. H22 cells, with MEF2D knockout or without (controls), were transplanted into BALB/c mice, and some mice were given antibodies to deplete T cells. Mice bearing orthotopic tumors grown from HCC cells, with or without knockout of SIRT7, were given injections of an antibody against PD1. Growth of tumors was measured, and tumors were analyzed by immunohistochemistry and flow cytometry. In human HCC specimens, we found an inverse correlation between level of MEF2D and numbers of CD4+ and CD8+ T cells; level of MEF2D correlated with percentages of PD1-positive or TIM3-positive CD8+ T cells. Knockout of MEF2D from H22 cells reduced their growth as allograft tumors in immune-competent mice but not in immune-deficient mice or mice with depletion of CD8+ T cells. When MEF2D-knockout cells were injected into immune-competent mice, they formed smaller tumors that had increased infiltration and activation of T cells compared with control HCC cells. In human and mouse HCC cells, MEF2D knockdown or knockout reduced expression of PD-L1. MEF2D bound the promoter region of the CD274 gene (encodes PD-L1) and activated its transcription. Overexpression of p300 in HCC cells, or knockout of SIRT7, promoted acetylation of MEF2D and increased its binding, along with acetylated histones, to the promoter region of CD274. Exposure of HCC cells to IFNG induced expression of p300 and its binding MEF2D, which reduced the interaction between MEF2D and SIRT7. MEF2D-induced expression of PD-L1 upon IFNG exposure was independent of interferon-regulatory factors 1 or 9. In HCC cells not exposed to IFNG, SIRT7 formed a complex with MEF2D that attenuated expression of PD-L1. Knockout of SIRT7 reduced proliferation of HCC cells and growth of tumors in immune-deficient mice. Compared with allograft tumors grown from control HCC cells, in immune-competent mice, tumors grown from SIRT7-knockout HCC cells expressed higher levels of PD-L1 and had reduced infiltration and activation of T cells. In immune-competent mice given antibodies to PD1, allograft tumors grew more slowly from SIRT7-knockout HCC cells than from control HCC cells. Expression of MEF2D by HCC cells increases their expression of PD-L1, which prevents CD8+ T-cell-mediated antitumor immunity. When HCC cells are exposed to IFNG, p300 acetylates MEF2D, causing it to bind the CD274 gene promoter and up-regulate PD-L1 expression. In addition to promoting HCC cell proliferation, SIRT7 reduced acetylation of MEF2D and expression of PD-L1 in HCC cells not exposed to IFNG. Strategies to manipulate this pathway might increase the efficacy of immune therapies for HCC.

Xiang J ，Zhang N ，Sun H ，Su L ，Zhang C ，Xu H ，Feng J ，Wang M ，Chen J ，Liu L ，Shan J ，Shen J ，Yang Z ，Wang G ，Zhou H ，Prieto J ，Ávila MA ，Liu C ，Qian C ... -  《-》