Inhibition of CAF-1 histone chaperone complex triggers cytosolic DNA and dsRNA sensing pathways and induces intrinsic immunity of hepatocellular carcinoma.

BACKGROUND AND AIMS: Chromatin assembly factor 1 (CAF-1) is a replication-dependent epigenetic regulator that controls cell cycle progression and chromatin dynamics. In this study, we aim to investigate the immunomodulatory role and therapeutic potential of the CAF-1 complex in HCC. APPROACH AND RESULTS: CAF-1 complex knockout cell lines were established using the CRISPR/Cas9 system. The effects of CAF-1 in HCC were studied in HCC cell lines, nude mice, and immunocompetent mice. RNA-sequencing, ChIP-Seq, and assay for transposase accessible chromatin with high-throughput sequencing (ATAC-Seq) were used to explore the changes in the epigenome and transcriptome. CAF-1 complex was significantly upregulated in human and mouse HCCs and was associated with poor prognosis in patients with HCC. Knockout of CAF-1 remarkably suppressed HCC growth in both in vitro and in vivo models. Mechanistically, depletion of CAF-1 induced replicative stress and chromatin instability, which eventually led to cytoplasmic DNA leakage as micronuclei. Also, chromatin immunoprecipitation sequencing analyses revealed a massive H3.3 histone variant replacement upon CAF-1 knockout. Enrichment of euchromatic H3.3 increased chromatin accessibility and activated the expression of endogenous retrovirus elements, a phenomenon known as viral mimicry. However, cytosolic micronuclei and endogenous retroviruses are recognized as ectopic elements by the stimulator of interferon genes and dsRNA viral sensing pathways, respectively. As a result, the knockout of CAF-1 activated inflammatory response and antitumor immune surveillance and thereby significantly enhanced the anticancer effect of immune checkpoint inhibitors in HCC. CONCLUSIONS: Our findings suggest that CAF-1 is essential for HCC development; targeting CAF-1 may awaken the anticancer immune response and may work cooperatively with immune checkpoint inhibitor treatment in cancer therapy.

Hypoxia-inducible factor orchestrates adenosine metabolism to promote liver cancer development.

Hypoxia-induced adenosine creates an immunosuppressive tumor microenvironment (TME) and dampens the efficacy of immune checkpoint inhibitors (ICIs). We found that hypoxia-inducible factor 1 (HIF-1) orchestrates adenosine efflux through two steps in hepatocellular carcinoma (HCC). First, HIF-1 activates transcriptional repressor MXI1, which inhibits adenosine kinase (ADK), resulting in the failure of adenosine phosphorylation to adenosine monophosphate. This leads to adenosine accumulation in hypoxic cancer cells. Second, HIF-1 transcriptionally activates equilibrative nucleoside transporter 4, pumping adenosine into the interstitial space of HCC, elevating extracellular adenosine levels. Multiple in vitro assays demonstrated the immunosuppressive role of adenosine on T cells and myeloid cells. Knockout of ADK in vivo skewed intratumoral immune cells to protumorigenic and promoted tumor progression. Therapeutically, combination treatment of adenosine receptor antagonists and anti-PD-1 prolonged survival of HCC-bearing mice. We illustrated the dual role of hypoxia in establishing an adenosine-mediated immunosuppressive TME and offered a potential therapeutic approach that synergizes with ICIs in HCC.

Ferroptosis Suppressor Protein 1 Inhibition Promotes Tumor Ferroptosis and Anti-tumor Immune Responses in Liver Cancer.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with dreadful clinical outcome. Tyrosine kinase inhibitors and immune checkpoint inhibitors are the only United States Food and Drug Administration-approved therapeutic options for patients with advanced HCC with limited therapeutic success. Ferroptosis is a form of immunogenic and regulated cell death caused by chain reaction of iron-dependent lipid peroxidation. Coenzyme Q10 (CoQ10)/ferroptosis suppressor protein 1 (FSP1) axis was recently identified as a novel protective mechanism against ferroptosis. We would like to explore whether FSP1 could be a potential therapeutic target for HCC. METHODS: FSP1 expression in human HCC and paired non-tumorous tissue samples were determined by reverse transcription-quantitative polymerase chain reaction, followed by clinicopathologic correlation and survival studies. Regulatory mechanism for FSP1 was determined using chromatin immunoprecipitation. The hydrodynamic tail vein injection model was used for HCC induction to evaluate the efficacy of FSP1 inhibitor (iFSP1) in vivo. Single-cell RNA sequencing revealed the immunomodulatory effects of iFSP1 treatment. RESULTS: We showed that HCC cells greatly rely on the CoQ10/FSP1 system to overcome ferroptosis. We found that FSP1 was significantly overexpressed in human HCC and is regulated by kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 pathway. FSP1 inhibitor iFSP1 effectively reduced HCC burden and profoundly increased immune infiltrates including dendritic cells, macrophages, and T cells. We also demonstrated that iFSP1 worked synergistically with immunotherapies to suppress HCC progression. CONCLUSIONS: We identified FSP1 as a novel, vulnerable therapeutic target in HCC. The inhibition of FSP1 potently induced ferroptosis, which promoted innate and adaptive anti-tumor immune responses and effectively suppressed HCC tumor growth. FSP1 inhibition therefore represents a new therapeutic strategy for HCC.

Polo-like kinase 4 inhibitor CFI-400945 suppresses liver cancer through cell cycle perturbation and eliciting antitumor immunity.

BACKGROUND AND AIMS: Prognosis of HCC remains poor due to lack of effective therapies. Immune checkpoint inhibitors (ICIs) have delayed response and are only effective in a subset of patients. Treatments that could effectively shrink the tumors within a short period of time are idealistic to be employed together with ICIs for durable tumor suppressive effects. HCC acquires increased tolerance to aneuploidy. The rapid division of HCC cells relies on centrosome duplication. In this study, we found that polo-like kinase 4 (PLK4), a centrosome duplication regulator, represents a therapeutic vulnerability in HCC. APPROACH AND RESULTS: An orally available PLK4 inhibitor, CFI-400945, potently suppressed proliferating HCC cells by perturbing centrosome duplication. CFI-400945 induced endoreplication without stopping DNA replication, causing severe aneuploidy, DNA damage, micronuclei formation, cytosolic DNA accumulation, and senescence. The cytosolic DNA accumulation elicited the DEAD box helicase 41-stimulator of interferon genes-interferon regulatory factor 3/7-NF-κß cytosolic DNA sensing pathway, thereby driving the transcription of senescence-associated secretory phenotypes, which recruit immune cells. CFI-400945 was evaluated in liver-specific p53/phosphatase and tensin homolog knockout mouse HCC models established by hydrodynamic tail vein injection. Tumor-infiltrated immune cells were analyzed. CFI-400945 significantly impeded HCC growth and increased infiltration of cluster of differentiation 4-positive (CD4 + ), CD8 + T cells, macrophages, and natural killer cells. Combination therapy of CFI-400945 with anti-programmed death-1 showed a tendency to improve HCC survival. CONCLUSIONS: We show that by targeting a centrosome regulator, PLK4, to activate the cytosolic DNA sensing-mediated immune response, CFI-400945 effectively restrained tumor progression through cell cycle inhibition and inducing antitumor immunity to achieve a durable suppressive effect even in late-stage mouse HCC.

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

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. IMPACT AND IMPLICATIONS: 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.

CFI-402257, a TTK inhibitor, effectively suppresses hepatocellular carcinoma.

Deregulation of cell cycle is a typical feature of cancer cells. Normal cells rely on the strictly coordinated spindle assembly checkpoint (SAC) to maintain the genome integrity and survive. However, cancer cells could bypass this checkpoint mechanism. In this study, we showed the clinical relevance of threonine tyrosine kinase (TTK) protein kinase, a central regulator of the SAC, in hepatocellular carcinoma (HCC) and its potential as therapeutic target. Here, we reported that a newly developed, orally active small molecule inhibitor targeting TTK (CFI-402257) effectively suppressed HCC growth and induced highly aneuploid HCC cells, DNA damage, and micronuclei formation. We identified that CFI-402257 also induced cytosolic DNA, senescence-like response, and activated DDX41-STING cytosolic DNA sensing pathway to produce senescence-associated secretory phenotypes (SASPs) in HCC cells. These SASPs subsequently led to recruitment of different subsets of immune cells (natural killer cells, CD4+ T cells, and CD8+ T cells) for tumor clearance. Our mass cytometry data illustrated the dynamic changes in the tumor-infiltrating immune populations after treatment with CFI-402257. Further, CFI-402257 improved survival in HCC-bearing mice treated with anti-PD-1, suggesting the possibility of combination treatment with immune checkpoint inhibitors in HCC patients. In summary, our study characterized CFI-402257 as a potential therapeutic for HCC, both used as a single agent and in combination therapy.

Hypoxia-induced macropinocytosis represents a metabolic route for liver cancer.

Hepatocellular carcinoma (HCC) invariably exhibits inadequate O2 (hypoxia) and nutrient supply. Hypoxia-inducible factor (HIF) mediates cascades of molecular events that enable cancer cells to adapt and propagate. Macropinocytosis is an endocytic process initiated by membrane ruffling, causing the engulfment of extracellular fluids (proteins), protein digestion and subsequent incorporation into the biomass. We show that macropinocytosis occurs universally in HCC under hypoxia. HIF-1 activates the transcription of a membrane ruffling protein, EH domain-containing protein 2 (EHD2), to initiate macropinocytosis. Knockout of HIF-1 or EHD2 represses hypoxia-induced macropinocytosis and prevents hypoxic HCC cells from scavenging protein that support cell growth. Germline or somatic deletion of Ehd2 suppresses macropinocytosis and HCC development in mice. Intriguingly, EHD2 is overexpressed in HCC. Consistently, HIF-1 or macropinocytosis inhibitor suppresses macropinocytosis and HCC development. Thus, we show that hypoxia induces macropinocytosis through the HIF/EHD2 pathway in HCC cells, harnessing extracellular protein as a nutrient to survive.

Inhibition of CMTM4 Sensitizes Cholangiocarcinoma and Hepatocellular Carcinoma to T Cell-Mediated Antitumor Immunity Through PD-L1.

Liver cancers consist primarily of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). Immune checkpoint inhibitors have emerged as promising therapeutic agents against liver cancers. Programmed cell death protein 1 (PD-1) is an immunoinhibitory receptor present on T cells that interacts with its ligand programmed death-ligand 1 (PD-L1) found on cancer cells. Blocking PD-1/PD-L1 binding improves T-cell survival, proliferation and cytotoxicity, which enhances their antitumor activity. Better understanding of the molecular mechanisms governing PD-1/PD-L1 response is essential to the development of predictive markers and therapeutic combinations that could improve the efficiency of anti-PD-1/PD-L1 treatment. Chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing 6 (CMTM6) has been recently identified as a major regulator of PD-L1. Another member in the CMTM family, CKLF-like MARVEL transmembrane domain-containing 4 (CMTM4), has been shown to compensate for the effects of CMTM6 when CMTM6 is lost. Interestingly, we found that CMTM4 is the major regulator of PD-L1 in the context of liver cancer. Up-regulated CMTM4 in patients with HCC and ICC is associated with poor patient survival, potentially due to its function in stabilizing PD-L1 expression, hence facilitating escape from T cell-mediated cytotoxicity. We confirmed the role of CMTM4 as a positive regulator of PD-L1 in multiple HCC and ICC cell lines and demonstrated that CMTM4 stabilizes PD-L1 through posttranslational mechanisms. In vivo, suppression of Cmtm4 inhibited HCC growth and increased CD8+ T-cell infiltration in immunocompetent mice. Furthermore, we found that depletion of CMTM4 sensitized HCC tumor to anti-PD-L1 treatment compared with control. This suggests that CMTM4 expression level could be a predictive marker for patient response to anti-PD-L1 treatment, and CMTM4 depletion can potentially be used to enhance the clinical benefits of anti-PD-L1 immunotherapy in patients with liver cancer.

Single-cell RNA sequencing shows the immunosuppressive landscape and tumor heterogeneity of HBV-associated hepatocellular carcinoma.

Interaction between tumor cells and immune cells in the tumor microenvironment is important in cancer development. Immune cells interact with the tumor cells to shape this process. Here, we use single-cell RNA sequencing analysis to delineate the immune landscape and tumor heterogeneity in a cohort of patients with HBV-associated human hepatocellular carcinoma (HCC). We found that tumor-associated macrophages suppress tumor T cell infiltration and TIGIT-NECTIN2 interaction regulates the immunosuppressive environment. The cell state transition of immune cells towards a more immunosuppressive and exhaustive status exemplifies the overall cancer-promoting immunocellular landscape. Furthermore, the heterogeneity of global molecular profiles reveals co-existence of intra-tumoral and inter-tumoral heterogeneity, but is more apparent in the latter. This analysis of the immunosuppressive landscape and intercellular interactions provides mechanistic information for the design of efficacious immune-oncology treatments in hepatocellular carcinoma.

Mechanistic Rationales Guiding Combination Hepatocellular Carcinoma Therapies Involving Immune Checkpoint Inhibitors.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers because of late symptom manifestation leading to delayed diagnosis, which limits patients with HCC in terms of receiving curative surgical treatment. There are only a few therapeutic options for patients with advanced HCC. The emergence of immune checkpoint inhibitors (ICIs) brings HCC treatment to a stage at which nivolumab, an anti-programmed cell death protein 1 monoclonal antibody, achieves a 20% response rate. However, the large proportion of unresponsive patients drives the exploration of therapeutic strategies to improve ICIs' efficacy. Recent preclinical and clinical studies have suggested that ICIs, when used in combinations or when used with other cancer therapies, might elicit synergistic antitumor effects. However, the mechanistic rationales guiding different drug combinations to maximize this synergy remain largely ambiguous. In this review, we discuss different drug combinations used in HCC and the underlying mechanistic rationales, aiming to enhance the understanding of how these treatments can achieve synergy. This knowledge sets the foundation for the development of more effective and promising combination therapies for HCC.

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.

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.