Evolution of delayed resistance to immunotherapy in a melanoma responder.

Despite initial responses1-3, most melanoma patients develop resistance4 to immune checkpoint blockade (ICB). To understand the evolution of resistance, we studied 37 tumor samples over 9 years from a patient with metastatic melanoma with complete clinical response to ICB followed by delayed recurrence and death. Phylogenetic analysis revealed co-evolution of seven lineages with multiple convergent, but independent resistance-associated alterations. All recurrent tumors emerged from a lineage characterized by loss of chromosome 15q, with post-treatment clones acquiring additional genomic driver events. Deconvolution of bulk RNA sequencing and highly multiplexed immunofluorescence (t-CyCIF) revealed differences in immune composition among different lineages. Imaging revealed a vasculogenic mimicry phenotype in NGFRhi tumor cells with high PD-L1 expression in close proximity to immune cells. Rapid autopsy demonstrated two distinct NGFR spatial patterns with high polarity and proximity to immune cells in subcutaneous tumors versus a diffuse spatial pattern in lung tumors, suggesting different roles of this neural-crest-like program in different tumor microenvironments. Broadly, this study establishes a high-resolution map of the evolutionary dynamics of resistance to ICB, characterizes a de-differentiated neural-crest tumor population in melanoma immunotherapy resistance and describes site-specific differences in tumor-immune interactions via longitudinal analysis of a patient with melanoma with an unusual clinical course.

Epitope spreading toward wild-type melanocyte-lineage antigens rescues suboptimal immune checkpoint blockade responses.

Although immune checkpoint inhibitors (ICIs), such as anti-programmed cell death protein-1 (PD-1), can deliver durable antitumor effects, most patients with cancer fail to respond. Recent studies suggest that ICI efficacy correlates with a higher load of tumor-specific neoantigens and development of vitiligo in patients with melanoma. Here, we report that patients with low melanoma neoantigen burdens who responded to ICI had tumors with higher expression of pigmentation-related genes. Moreover, expansion of peripheral blood CD8+ T cell populations specific for melanocyte antigens was observed only in patients who responded to anti-PD-1 therapy, suggesting that ICI can promote breakdown of tolerance toward tumor-lineage self-antigens. In a mouse model of poorly immunogenic melanomas, spreading of epitope recognition toward wild-type melanocyte antigens was associated with markedly improved anti-PD-1 efficacy in two independent approaches: introduction of neoantigens by ultraviolet (UV) B radiation mutagenesis or the therapeutic combination of ablative fractional photothermolysis plus imiquimod. Complete responses against UV mutation-bearing tumors after anti-PD-1 resulted in protection from subsequent engraftment of melanomas lacking any shared neoantigens, as well as pancreatic adenocarcinomas forcibly overexpressing melanocyte-lineage antigens. Our data demonstrate that somatic mutations are sufficient to provoke strong antitumor responses after checkpoint blockade, but long-term responses are not restricted to these putative neoantigens. Epitope spreading toward T cell recognition of wild-type tumor-lineage self-antigens represents a common pathway for successful response to ICI, which can be evoked in neoantigen-deficient tumors by combination therapy with ablative fractional photothermolysis and imiquimod.

MCL1 Is Required for Maintenance of Intestinal Homeostasis and Prevention of Carcinogenesis in Mice.

BACKGROUND & AIMS: Intestinal epithelial homeostasis depends on a tightly regulated balance between intestinal epithelial cell (IEC) death and proliferation. While the disruption of several IEC death regulating factors result in intestinal inflammation, the loss of the anti-apoptotic BCL2 family members BCL2 and BCL2L1 has no effect on intestinal homeostasis in mice. We investigated the functions of the antiapoptotic protein MCL1, another member of the BCL2 family, in intestinal homeostasis in mice. METHODS: We generated mice with IEC-specific disruption of Mcl1 (Mcl1ΔIEC mice) or tamoxifen-inducible IEC-specific disruption of Mcl1 (i-Mcl1ΔIEC mice); these mice and mice with full-length Mcl1 (controls) were raised under normal or germ-free conditions. Mice were analyzed by endoscopy and for intestinal epithelial barrier permeability. Intestinal tissues were analyzed by histology, in situ hybridization, proliferation assays, and immunoblots. Levels of calprotectin, a marker of intestinal inflammation, were measured in intestinal tissues and feces. RESULTS: Mcl1ΔIEC mice spontaneously developed apoptotic enterocolopathy, characterized by increased IEC apoptosis, hyperproliferative crypts, epithelial barrier dysfunction, and chronic inflammation. Loss of MCL1 retained intestinal crypts in a hyperproliferated state and prevented the differentiation of intestinal stem cells. Proliferation of intestinal stem cells in MCL1-deficient mice required WNT signaling and was associated with DNA damage accumulation. By 1 year of age, Mcl1ΔIEC mice developed intestinal tumors with morphologic and genetic features of human adenomas and carcinomas. Germ-free housing of Mcl1ΔIEC mice reduced markers of microbiota-induced intestinal inflammation but not tumor development. CONCLUSION: The antiapoptotic protein MCL1, a member of the BCL2 family, is required for maintenance of intestinal homeostasis and prevention of carcinogenesis in mice. Loss of MCL1 results in development of intestinal carcinomas, even under germ-free conditions, and therefore does not involve microbe-induced chronic inflammation. Mcl1ΔIEC mice might be used to study apoptotic enterocolopathy and inflammatory bowel diseases.

Platelet GPIbα is a mediator and potential interventional target for NASH and subsequent liver cancer.

Non-alcoholic fatty liver disease ranges from steatosis to non-alcoholic steatohepatitis (NASH), potentially progressing to cirrhosis and hepatocellular carcinoma (HCC). Here, we show that platelet number, platelet activation and platelet aggregation are increased in NASH but not in steatosis or insulin resistance. Antiplatelet therapy (APT; aspirin/clopidogrel, ticagrelor) but not nonsteroidal anti-inflammatory drug (NSAID) treatment with sulindac prevented NASH and subsequent HCC development. Intravital microscopy showed that liver colonization by platelets depended primarily on Kupffer cells at early and late stages of NASH, involving hyaluronan-CD44 binding. APT reduced intrahepatic platelet accumulation and the frequency of platelet-immune cell interaction, thereby limiting hepatic immune cell trafficking. Consequently, intrahepatic cytokine and chemokine release, macrovesicular steatosis and liver damage were attenuated. Platelet cargo, platelet adhesion and platelet activation but not platelet aggregation were identified as pivotal for NASH and subsequent hepatocarcinogenesis. In particular, platelet-derived GPIbα proved critical for development of NASH and subsequent HCC, independent of its reported cognate ligands vWF, P-selectin or Mac-1, offering a potential target against NASH.

A Dual Role of Caspase-8 in Triggering and Sensing Proliferation-Associated DNA Damage, a Key Determinant of Liver Cancer Development.

Concomitant hepatocyte apoptosis and regeneration is a hallmark of chronic liver diseases (CLDs) predisposing to hepatocellular carcinoma (HCC). Here, we mechanistically link caspase-8-dependent apoptosis to HCC development via proliferation- and replication-associated DNA damage. Proliferation-associated replication stress, DNA damage, and genetic instability are detectable in CLDs before any neoplastic changes occur. Accumulated levels of hepatocyte apoptosis determine and predict subsequent hepatocarcinogenesis. Proliferation-associated DNA damage is sensed by a complex comprising caspase-8, FADD, c-FLIP, and a kinase-dependent function of RIPK1. This platform requires a non-apoptotic function of caspase-8, but no caspase-3 or caspase-8 cleavage. It may represent a DNA damage-sensing mechanism in hepatocytes that can act via JNK and subsequent phosphorylation of the histone variant H2AX.

Visualization of hepatitis E virus RNA and proteins in the human liver.

BACKGROUND & AIMS: Although hepatitis E constitutes a substantial disease burden worldwide, surprisingly little is known about the localization of hepatitis E virus (HEV) in the human liver. We therefore aimed to visualize HEV RNA and proteins in situ. METHODS: A panel of 12 different antibodies against HEV open reading frame (ORF) 1-3 proteins was evaluated for immunohistochemistry (IHC) and two probes for in situ hybridization (ISH) in formalin-fixed, paraffin-embedded (FFPE) HuH7 cells transfected with HEV ORF1-3 expression vectors. IHC (and partly ISH) were then applied to Hep293TT cells replicating infectious HEV and liver specimens from patients with hepatitis E (n=20) and controls (n=134). RESULTS: Whereas ORF1-3 proteins were all detectable in transfected, HEV protein-expressing cells, only ORF2 and 3 proteins were traceable in cells replicating infectious HEV. Only the ORF2-encoded capsid protein was also unequivocally detectable in liver specimens from patients with hepatitis E. IHC for ORF2 protein revealed a patchy expression in individual or grouped hepatocytes, generally stronger in chronic compared to acute hepatitis. Besides cytoplasmic and canalicular, ORF2 protein also displayed a hitherto unknown nuclear localization. Positivity for ORF2 protein in defined areas correlated with HEV RNA detection by ISH. IHC was specific and comparably sensitive as PCR for HEV RNA. CONCLUSIONS: ORF2 protein can be reliably visualized in the liver of patients with hepatitis E, allowing for sensitive and specific detection of HEV in FFPE samples. Its variable subcellular distribution in individual hepatocytes of the same liver suggests a redistribution of ORF2 protein during infection and interaction with nuclear components. LAY SUMMARY: The open reading frame (ORF) 2 protein can be used to visualize the hepatitis E virus (HEV) in the human liver. This enabled us to discover a hitherto unknown localization of the HEV ORF2 protein in the nucleus of hepatocytes and to develop a test for rapid histopathologic diagnosis of hepatitis E, the most common cause of acute hepatitis worldwide.

Chemical Hybridization of Glucagon and Thyroid Hormone Optimizes Therapeutic Impact for Metabolic Disease.

Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.

The Beneficial Effects of Valproic Acid in Thyroid Cancer Are Mediated through Promoting Redifferentiation and Reducing Stemness Level: An In Vitro Study.

Valproic acid (VPA) has been identified as a histone deacetylase inhibitor, inducing differentiation in transformed cells. However, no study has shown the effect of VPA in the redifferentiation induction and stemness of anaplastic thyroid. The main objective of this study was to evaluate the efficacy of VPA as a differentiation therapy agent in human thyroid cancer based on its effect on stemness and differentiation process. Indications for differentiation of 8305C and B-CPAP cell lines following VPA treatment were obtained by analyzing cell proliferation rate, morphological changes, adherent-dependent colony formation, and Hoechst 33342 staining. The expressions of stemness, differentiation, and aggressiveness specific marker genes were measured by quantitative RT-PCR. VPA treatment effectively showed growth inhibition in both cell lines. The high nuclear-cytoplasmic (N : C) ratio of 8305C cells markedly decreased and treated cells became more epithelial-like. Treated cells showed stronger Hoechst 33342 fluorescence compared with control cells. The hTERT and OCT-4 reduction was paralleled with adherent-dependent colony formation decrement in both cell lines. VPA effectively induced NIS and TTF-1 in anaplastic cells, it whereas showed no clear pattern in papillary cell line. VPA treatment also resulted in the reduction of MMP-2 and MMP-9. These finding suggest that VPA could redifferentiate the anaplastic thyroid cancer cells.

Multitargeting and antimetastatic potentials of silibinin in human HepG-2 and PLC/PRF/5 hepatoma cells.

Hepatocellular carcinoma (HCC) is the most common sort of primary liver malignancy with poor prognosis. This study aimed at examining the effects of silibinin (a putative antimetastatic agent) on some transcriptional markers mechanistically related to HCC recurrence and metastasis in HepG-2 [hepatitis B virus (HBV)-negative and P53 intact) and PLC/PRF/5 (HBV-positive and P53 mutated) cells. The expression of 27 genes in response to silibinin was evaluated by real-time RT-PCR. The MMP gelatinolytic assay and microculture tetrazolium test (MTT) were tested. Silibinin was capable of suppressing the transcriptional levels of ANGPT2, ATP6L, CAP2, CCR6, CCR7, CLDN-10, cortactin, CXCR4, GLI2, HK2, ID1, KIAA0101, mortalin, PAK1, RHOA, SPINK1, and STMN1 as well as the enzymatic activity of MMP-2 but promoted the transcripts of CREB3L3, DDX3X, and PROX1 in both cells. Some significant differences between the cells in response to silibinin were detected that might be related to the differences of the cells in terms of HBV infection and/or P53 mutation, suggesting the possible influence of silibinin on HCC through biological functions of these 2 prognostic factors. In conclusion, our findings suggest that silibinin could potentially function as a multitargeting antimetastatic agent and might provide new insights for HCC therapy particularly for HBV-related and/or P53-mutated HCCs.

Synergistic effects of arsenic trioxide and silibinin on apoptosis and invasion in human glioblastoma U87MG cell line.

Patients with glioblastoma multiforme (GBM) have poor therapeutic outcomes despite their current therapy. In an attempt to increase the efficacy of therapy for GBM, we studied the efficacy of arsenic trioxide (ATO), a newly introduced treatment for glioma, combined with silibinin, a natural polyphenolic flavonoid, in the GBM cell line, U87MG. The combination therapy synergically inhibited metabolic activity, cell proliferation, and gelatinase A and B activities; it also increased apoptosis. Additionally, it decreased the mRNA level of cathepsin B, uPA, matrix metalloproteinase-2 and 9, membrane type 1-MMP, survivin, BCL2, CA9; it increased mRNA level of caspase-3. Altogether, these results showed that ATO and silibinin in some cases improved and/or complemented the anticancer effects. This study may supply insight into the design of new combination cancer therapies to cells intrinsically less sensitive to routine therapies and suggested a new combination therapy for the highly invasive human glioma treatment.

Multifaceted suppression of aggressive behavior of thyroid carcinoma by all-trans retinoic acid induced re-differentiation.

Since all-trans retinoic acid (ATRA) has shown promising results in differentiation therapy, the present study was designed to investigate the effects of ATRA on thyroid carcinoma and to evaluate the effectiveness of ATRA in redifferentiation induction of thyroid carcinoma. Therefore, we investigated cell growth rate, morphological and nuclear: cytoplasmic ratio, adherent-dependent growth, response to chemotherapy drug following differentiation, T3 and T4 measurement, and critical genes expression pattern. Papillary cell line showed more growth inhibition by ATRA, in addition, mesenchymal and spindle-shape of 8305C cells changed to polygonal. Additionally, high nuclear: cytoplasmic ratio of anaplastic decreased significantly. Redifferentiation significantly suppressed the anchorage-dependent growth in the both cell lines in a dose-dependent manner, potentiated the arsenic trioxide (ATO) effects in anaplastic and papillary cell lines. Furthermore, reduction in the expression of stemness, and invasion related genes was observed in the both cell lines. Altogether, ATRA treatment could hold the aggressive behavior of thyroid carcinoma in restraint and/or potentiate the effect of chemotherapy drug ATO.

Silibinin inhibits invasive properties of human glioblastoma U87MG cells through suppression of cathepsin B and nuclear factor kappa B-mediated induction of matrix metalloproteinase 9.

Glioblastoma multiforme remains one of the most devastating human malignancies because of its high infiltrative capacity. This study aimed to investigate the effects of silibinin on human glioblastoma U87MG cells. The microculture tetrazolium test, bromodeoxyuridine cell proliferation assay, cell-based nuclear factor kappa B (NF-[kappa]B) activation assessment, cathepsin B activity assay, gelatin zymography, and quantitative real-time reverse transcription-PCR were performed to appraise the effects of silibinin on the metabolic activity, DNA synthesis, NF-[kappa]B phosphorylation, cathepsin B activity, and gelatinolytic activity of U87 cells. Silibinin inhibited metabolic activity, cell proliferation, NF-[kappa]B activation, cathepsin B enzymatic levels, and gelatinase B activity in U87 cells. In addition, an expressive decrease in mRNA levels of matrix metalloproteinase-9, cathepsin B, urokinase plasminogen activator receptor, urokinase plasminogen activator, and intercellular adhesion molecule 1 coupled with a significant induction in transcriptional levels of stefin A was observed. Altogether, these issues show for the first time that silibinin treatment could trammel invasive features of a highly invasive human glioma cell line, U87, through suppression of NF-[kappa]B-mediated stimulation of matrix metalloproteinase-9. Furthermore, silibinin might cripple the activation of gelatinase B by cramping transcriptional and enzymatic activities of cathepsin B in U87 cells.