2-Hydroxyglutarate modulates histone methylation at specific loci and alters gene expression via Rph1 inhibition.

2-Hydroxyglutarate (2-HG) is an oncometabolite that accumulates in certain cancers. Gain-of-function mutations in isocitrate dehydrogenase lead to 2-HG accumulation at the expense of alpha-ketoglutarate. Elevated 2-HG levels inhibit histone and DNA demethylases, causing chromatin structure and gene regulation changes with tumorigenic consequences. We investigated the effects of elevated 2-HG levels in Saccharomyces cerevisiae, a yeast devoid of DNA methylation and heterochromatin-associated histone methylation. Our results demonstrate genetic background-dependent gene expression changes and altered H3K4 and H3K36 methylation at specific loci. Analysis of histone demethylase deletion strains indicated that 2-HG inhibits Rph1 sufficiently to induce extensive gene expression changes. Rph1 is the yeast homolog of human KDM4 demethylases and, among the yeast histone demethylases, was the most sensitive to the inhibitory effect of 2-HG in vitro. Interestingly, Rph1 deficiency favors gene repression and leads to further down-regulation of already silenced genes marked by low H3K4 and H3K36 trimethylation, but abundant in H3K36 dimethylation. Our results provide novel insights into the genome-wide effects of 2-HG and highlight Rph1 as its preferential demethylase target.

Tumor heterogeneity: preclinical models, emerging technologies, and future applications.

Heterogeneity describes the differences among cancer cells within and between tumors. It refers to cancer cells describing variations in morphology, transcriptional profiles, metabolism, and metastatic potential. More recently, the field has included the characterization of the tumor immune microenvironment and the depiction of the dynamics underlying the cellular interactions promoting the tumor ecosystem evolution. Heterogeneity has been found in most tumors representing one of the most challenging behaviors in cancer ecosystems. As one of the critical factors impairing the long-term efficacy of solid tumor therapy, heterogeneity leads to tumor resistance, more aggressive metastasizing, and recurrence. We review the role of the main models and the emerging single-cell and spatial genomic technologies in our understanding of tumor heterogeneity, its contribution to lethal cancer outcomes, and the physiological challenges to consider in designing cancer therapies. We highlight how tumor cells dynamically evolve because of the interactions within the tumor immune microenvironment and how to leverage this to unleash immune recognition through immunotherapy. A multidisciplinary approach grounded in novel bioinformatic and computational tools will allow reaching the integrated, multilayered knowledge of tumor heterogeneity required to implement personalized, more efficient therapies urgently required for cancer patients.

Microneedle-mediated Intratumoral Delivery of Anti-CTLA-4 Promotes cDC1-dependent Eradication of Oral Squamous Cell Carcinoma with Limited irAEs.

Head and neck squamous cell carcinoma (HNSCC) ranks sixth in cancer incidence worldwide and has a 5-year survival rate of only 63%. Immunotherapies-principally immune checkpoint inhibitors (ICI), such as anti-PD-1 and anti-CTLA-4 antibodies that restore endogenous antitumor T-cell immunity-offer the greatest promise for HNSCC treatment. Anti-PD-1 has been recently approved for first-line treatment of recurrent and metastatic HNSCC; however, less than 20% of patients show clinical benefit and durable responses. In addition, the clinical application of ICI has been limited by immune-related adverse events (irAE) consequent to compromised peripheral immune tolerance. Although irAEs are often reversible, they can become severe, prompting premature therapy termination or becoming life threatening. To address the irAEs inherent to systemic ICI therapy, we developed a novel, local delivery strategy based upon an array of soluble microneedles (MN). Using our recently reported syngeneic, tobacco-signature murine HNSCC model, we found that both systemic and local-MN anti-CTLA-4 therapy lead to >90% tumor response, which is dependent on CD8 T cells and conventional dendritic cell type 1 (cDC1). However, local-MN delivery limited the distribution of anti-CTLA-4 antibody from areas distal to draining lymphatic basins. Employing Foxp3-GFPDTR transgenic mice to interrogate irAEs in vivo, we found that local-MN delivery of anti-CTLA-4 protects animals from irAEs observed with systemic therapy. Taken together, our findings support the exploration of MN-intratumoral ICI delivery as a viable strategy for HNSCC treatment with reduced irAEs, and the opportunity to target cDC1s as part of multimodal treatment options to boost ICI therapy.

The driving role of the Cdk5/Tln1/FAKS732 axis in cancer cell extravasation dissected by human vascularized microfluidic models.

BACKGROUND: Understanding the molecular mechanisms of metastatic dissemination, the leading cause of death in cancer patients, is required to develop novel, effective therapies. Extravasation, an essential rate-limiting process in the metastatic cascade, includes three tightly coordinated steps: cancer cell adhesion to the endothelium, trans-endothelial migration, and early invasion into the secondary site. Focal adhesion proteins, including Tln1 and FAK, regulate the cytoskeleton dynamics: dysregulation of these proteins is often associated with metastatic progression and poor prognosis. METHODS: Here, we studied the previously unexplored role of these targets in each extravasation step using engineered 3D in vitro models, which recapitulate the physiological vascular niche experienced by cancer cells during hematogenous metastasis. RESULTS: Human breast cancer and fibrosarcoma cell lines respond to Cdk5/Tln1/FAK axis perturbation, impairing their metastatic potential. Vascular breaching requires actin polymerization-dependent invadopodia formation. Invadopodia generation requires the structural function of FAK and Tln1 rather than their activation through phosphorylation. Our data support that the inhibition of FAKS732 phosphorylation delocalizes ERK from the nucleus, decreasing ERK phosphorylated form. These findings indicate the critical role of these proteins in driving trans-endothelial migration. In fact, both knock-down experiments and chemical inhibition of FAK dramatically reduces lung colonization in vivo and TEM in microfluidic setting. Altogether, these data indicate that engineered 3D in vitro models coupled to in vivo models, genetic, biochemical, and imaging tools represent a powerful weapon to increase our understanding of metastatic progression. CONCLUSIONS: These findings point to the need for further analyses of previously overlooked phosphorylation sites of FAK, such as the serine 732, and foster the development of new effective antimetastatic treatments targeting late events of the metastatic cascade.

Tipifarnib as a Precision Therapy for HRAS-Mutant Head and Neck Squamous Cell Carcinomas.

Tipifarnib is a potent and highly selective inhibitor of farnesyltransferase (FTase). FTase catalyzes the posttranslational attachment of farnesyl groups to signaling proteins that are required for localization to cell membranes. Although all RAS isoforms are FTase substrates, only HRAS is exclusively dependent upon farnesylation, raising the possibility that HRAS-mutant tumors might be susceptible to tipifarnib-mediated inhibition of FTase. Here, we report the characterization of tipifarnib activity in a wide panel of HRAS-mutant and wild-type head and neck squamous cell carcinoma (HNSCC) xenograft models. Tipifarnib treatment displaced both mutant and wild-type HRAS from membranes but only inhibited proliferation, survival, and spheroid formation of HRAS-mutant cells. In vivo, tipifarnib treatment induced tumor stasis or regression in all six HRAS-mutant xenografts tested but displayed no activity in six HRAS wild-type patient-derived xenograft (PDX) models. Mechanistically, drug treatment resulted in the reduction of MAPK pathway signaling, inhibition of proliferation, induction of apoptosis, and robust abrogation of neovascularization, apparently via effects on both tumor cells and endothelial cells. Bioinformatics and quantitative image analysis further revealed that FTase inhibition induces progressive squamous cell differentiation in tipifarnib-treated HNSCC PDXs. These preclinical findings support that HRAS represents a druggable oncogene in HNSCC through FTase inhibition by tipifarnib, thereby identifying a precision therapeutic option for HNSCCs harboring HRAS mutations.

An high-throughput in vivo screening system to select H3K4-specific histone demethylase inhibitors.

BACKGROUND: Histone demethylases (HDMs) have a prominent role in epigenetic regulation and are emerging as potential therapeutic cancer targets. The search for small molecules able to inhibit HDMs in vivo is very active but at the present few compounds were found to be specific for defined classes of these enzymes. METHODOLOGY/PRINCIPAL FINDINGS: In order to discover inhibitors specific for H3K4 histone demethylation we set up a screening system which tests the effects of candidate small molecule inhibitors on a S.cerevisiae strain which requires Jhd2 demethylase activity to efficiently grow in the presence of rapamycin. In order to validate the system we screened a library of 45 structurally different compounds designed as competitive inhibitors of α -ketoglutarate (α-KG) cofactor of the enzyme, and found that one of them inhibited Jhd2 activity in vitro and in vivo. The same compound effectively inhibits human Jumonji AT-Rich Interactive Domain (JARID) 1B and 1D in vitro and increases H3K4 tri-methylation in HeLa cell nuclear extracts (NEs). When added in vivo to HeLa cells, the compound leads to an increase of tri-methyl-H3K4 (H3K4me3) but does not affect H3K9 tri-methylation. We describe the cytostatic and toxic effects of the compound on HeLa cells at concentrations compatible with its inhibitory activity. CONCLUSIONS/SIGNIFICANCE: Our screening system is proved to be very useful in testing putative H3K4-specific HDM inhibitors for the capacity of acting in vivo without significantly altering the activity of other important 2-oxoglutarate oxygenases.