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.

Genome-Wide CRISPR/Cas9 Library Screening Revealed Dietary Restriction of Glutamine in Combination with Inhibition of Pyruvate Metabolism as Effective Liver Cancer Treatment.

Hepatocellular carcinoma (HCC) is the second most lethal cancer worldwide. Glutamine is an essential, extracellular nutrient which supports HCC growth. Dietary glutamine deficiency may be a potential therapeutic approach for HCC. HCC cells overcome metabolic challenges by rewiring their metabolic pathways for rapid adaptations. The efficiency of dietary glutamine deficiency as HCC treatment is examined and the adaptation machinery under glutamine depletion in HCC cells is unraveled. Using genome-wide CRISPR/Cas9 knockout library screening, this study identifies that pyruvate dehydrogenase α (PDHA), pyruvate dehydrogenase ß (PDHB), and pyruvate carboxylase (PC) in pyruvate metabolism are crucial to the adaptation of glutamine depletion in HCC cells. Knockout of either PDHA, PDHB or PC induced metabolic reprogramming of the tricarboxylic acid (TCA) cycle, disrupts mitochondrial function, leading to the suppression of HCC cell proliferation under glutamine depletion. Surprisingly, dietary glutamine restriction improves therapeutic responses of HCC to PDH or PC inhibitor in mouse HCC models. Stable isotope carbon tracing confirms that PDH or PC inhibitors further disrupt the metabolic rewiring of the TCA cycle induced by dietary glutamine depletion in HCC. In summary, the results demonstrate that pyruvate metabolism acts as novel targetable metabolic vulnerabilities for HCC treatment in combination with a glutamine-deficient diet.

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.

Histone chaperone FACT complex coordinates with HIF to mediate an expeditious transcription program to adapt to poorly oxygenated cancers.

Cancer cells adapt to hypoxia through HIFs (hypoxia-inducible factors), which initiate the transcription of numerous genes for cancer cell survival in the hypoxia microenvironment. In this study, we find that the FACT (facilitates chromatin transcription) complex works cooperatively with HIFs to facilitate the expeditious expression of HIF targets for hypoxia adaptation. Knockout (KO) of the FACT complex abolishes HIF-mediated transcription by impeding transcription elongation in hypoxic cancer cells. Interestingly, the FACT complex is post-translationally regulated by PHD/VHL-mediated hydroxylation and proteasomal degradation, in similar fashion to HIF-1/2α. Metabolic tracing confirms that FACT KO suppresses glycolytic flux and impairs lactate extrusion, leading to intracellular acidification and apoptosis in cancer cells. Therapeutically, hepatic artery ligation and anti-angiogenic inhibitors adversely induce intratumoral hypoxia, while co-treatment with FACT inhibitor curaxin remarkably hinders the growth of hypoxic tumors. In summary, our findings suggest that the FACT complex is a critical component of hypoxia adaptation and a therapeutic target for hypoxic tumors.

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.

Adaptive and Constitutive Activations of Malic Enzymes Confer Liver Cancer Multilayered Protection Against Reactive Oxygen Species.

BACKGROUND AND AIMS: HCC undergoes active metabolic reprogramming. Reactive oxygen species (ROS) are excessively generated in cancer cells and are neutralized by NADPH. Malic enzymes (MEs) are the less studied NADPH producers in cancer. APPROACH AND RESULTS: We found that ME1, but not ME3, was regulated by the typical oxidative stress response pathway mediated by kelch-like ECH associated protein 1/nuclear factor erythroid 2-related factor (NRF2). Surprisingly, ME3 was constitutively induced by superenhancers. Disruption of any ME regulatory pathways decelerated HCC progression and sensitized HCC to sorafenib. Therapeutically, simultaneous blockade of NRF2 and a superenhancer complex completely impeded HCC growth. We show that superenhancers allow cancer cells to counteract the intrinsically high level of ROS through constitutively activating ME3 expression. When HCC cells encounter further episodes of ROS insult, NRF2 allows cancer cells to adapt by transcriptionally activating ME1. CONCLUSIONS: Our study reveals the complementary regulatory mechanisms which control MEs and provide cancer cells multiple layers of defense against oxidative stress. Targeting both regulatory mechanisms represents a potential therapeutic approach for HCC treatment.

Genome-wide CRISPR-Cas9 knockout library screening identified PTPMT1 in cardiolipin synthesis is crucial to survival in hypoxia in liver cancer.

Hypoxia, low oxygen (O2), is a key feature of all solid cancers, including hepatocellular carcinoma (HCC). Genome-wide CRISPR-Cas9 knockout library screening is used to identify reliable therapeutic targets responsible for hypoxic survival in HCC. We find that protein-tyrosine phosphatase mitochondrial 1 (PTPMT1), an important enzyme for cardiolipin (CL) synthesis, is the most significant gene and ranks just after hypoxia-inducible factor (HIF)-1α and HIF-1ß as crucial to hypoxic survival. CL constitutes the mitochondrial membrane and ensures the proper assembly of electron transport chain (ETC) complexes for efficient electron transfer in respiration. ETC becomes highly unstable during hypoxia. Knockout of PTPMT1 stops the maturation of CL and impairs the assembly of ETC complexes, leading to further electron leakage and ROS accumulation at ETC in hypoxia. Excitingly, HCC cells, especially under hypoxic conditions, show great sensitivity toward PTPMT1 inhibitor alexidine dihydrochloride (AD). This study unravels the protective roles of PTPMT1 in hypoxic survival and cancer development.

Genome-wide CRISPR/Cas9 library screening identified PHGDH as a critical driver for Sorafenib resistance in HCC.

Sorafenib is the standard treatment for advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common. By using genome-wide CRISPR/Cas9 library screening, we identify phosphoglycerate dehydrogenase (PHGDH), the first committed enzyme in the serine synthesis pathway (SSP), as a critical driver for Sorafenib resistance. Sorafenib treatment activates SSP by inducing PHGDH expression. With RNAi knockdown and CRISPR/Cas9 knockout models, we show that inactivation of PHGDH paralyzes the SSP and reduce the production of αKG, serine, and NADPH. Concomitantly, inactivation of PHGDH elevates ROS level and induces HCC apoptosis upon Sorafenib treatment. More strikingly, treatment of PHGDH inhibitor NCT-503 works synergistically with Sorafenib to abolish HCC growth in vivo. Similar findings are also obtained in other FDA-approved tyrosine kinase inhibitors (TKIs), including Regorafenib or Lenvatinib. In summary, our results demonstrate that targeting PHGDH is an effective approach to overcome TKI drug resistance in HCC.

Assessment of Stabilization and Activity of the HIFs Important for Hypoxia-Induced Signalling in Cancer Cells.

Blood vessels in tumors contain chaotic branching structures and leaky vessel lumens, resulting in uneven supply of oxygen in the tumor microenvironment. High metabolic and proliferation rate of tumor cells further depletes the local oxygen supply. Therefore, hypoxia is a common phenomenon in multiple solid malignancies. Hypoxia-inducible factors (HIFs) regulate the transcription of a spectrum of genes, which are vitally important for tumor cell adaption under hypoxia, and shape the tumor microenvironment to become more favorable for progression. HIFs are involved in almost every step of cancer development through inducing angiogenesis, metabolic reprogramming, metastasis, cancer stemness maintenance, chemoresistance, and immune evasion. Here, we describe methods for the assessment of HIF activity, as well as identification of novel transcriptional targets of HIFs in vitro and in vivo.

Hepatitis transactivator protein X promotes extracellular matrix modification through HIF/LOX pathway in liver cancer.

Hepatocellular carcinoma (HCC), accounting for 90% of primary liver cancer, is a lethal malignancy that is tightly associated with chronic hepatitis B virus (HBV) infection. HBV encodes a viral onco-protein, transactivator protein X (HBx), which interacts with proteins of hepatocytes to promote oncogenesis. Our current study focused on the interaction of HBx with a transcription factor, hypoxia-inducible factor-1α (HIF-1α), which is stabilized by low O2 condition (hypoxia) and is found to be frequently overexpressed in HCC intra-tumorally due to poor blood perfusion. Here, we showed that overexpression of HBx by tetracycline-inducible systems further stabilized HIF-1α under hypoxia in HBV-negative HCC cell lines. Reversely, knockdown of HBx reduced HIF-1α protein stabilization under hypoxia in HBV-positive HCC cell lines. More intriguingly, overexpression of HBx elevated the mRNA and protein expression of a family of HIF-1α target genes, the lysyl oxidase (LOX) family in HCC. The LOX family members function to cross-link collagen in the extracellular matrix (ECM) to promote cancer progression and metastasis. By analyzing the collagens under scanning electron microscope, we found that collagen fibers were significantly smaller in size when incubated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells in vitro. Transwell invasion assay further revealed that less cells were able to invade through the matrigel which was pre-treated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells. Orthotopic and subcutaneous HCC models further showed that knockdown of HBx in HCC cells reduced collagen crosslinking and stiffness in vivo and repressed HCC growth and metastasis. Taken together, our in vitro and in vivo studies showed the HBx remodeled the ECM through HIF-1α/LOX pathway to promote HCC metastasis.