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Background: Arginases play essential roles in metabolic pathways, determining the fitness of both immune and tumour cells. Along with the previously validated role of ARG1 in cancer, the particular significance of ARG2 as a therapeutic target has emerged as its levels correlate with malignant phenotype and poor prognosis. These observations unveil arginases, and specifically ARG2, as well-validated and promising therapeutic targets. OATD-02, a new boronic acid derivative, is the only dual inhibitor, which can address the benefits of pharmacological inhibition of arginase 1 and 2 in cancer. Methods: The inhibitory activity of OATD-02 was determined using recombinant ARG1 and ARG2, as well as in a cellular system using primary hepatocytes and macrophages. In vivo antitumor activity was determined in syngeneic models of colorectal and kidney carcinomas (CT26 and Renca, respectively), as well as in an ARG2-dependent xenograft model of leukaemia (K562). Results: OATD-02 was shown to be a potent dual (ARG1/ARG2) arginase inhibitor with a cellular activity necessary for targeting ARG2. Compared to a reference inhibitor with predominant extracellular activity towards ARG1, we have shown improved and statistically significant antitumor efficacy in the CT26 model and an immunomodulatory effect reflected by Treg inhibition in the Renca model. Importantly, OATD-02 had a superior activity when combined with other immunotherapeutics. Finally, OATD-02 effectively inhibited the proliferation of human K562 leukemic cells both in vitro and in vivo. Conclusions: OATD-02 is a potent small-molecule arginase inhibitor with optimal drug-like properties, including PK/PD profile. Excellent activity against intracellular ARG2 significantly distinguishes OATD-02 from other arginase inhibitors. OATD-02 represents a very promising drug candidate for the combined treatment of tumours, and is the only pharmacological tool that can effectively address the benefits of ARG1/ARG2 inhibition. OATD-02 will enter clinical trials in cancer patients in 2022.

The mammalian Grainyhead-like 3 (GRHL3) transcription factor is essential for epithelial development and plays a protective role against squamous cell carcinoma of the skin and of the oral cavity. A single nucleotide polymorphism (SNP) in GRHL3, rs141193530 (p.P455A), is associated with non-melanoma skin cancer in human patients. Moreover, it is known that this SNP, as well as another variant, rs41268753 (p.T454M), are associated with nonsyndromic cleft palate and that rs41268753 negatively affects GRHL3 transcriptional activity. These SNPs are located in adjacent codons of the GRHL3 gene, and the occurrence of either SNP abolishes a putative threonine-proline phosphorylation motif at T454 in the encoded protein. The role of phosphorylation in regulating mammalian GRHL function is currently unknown. In this work we show that GRHL3 is phosphorylated at several residues in a human keratinocyte cell line, among them at T454. This site is essential for the full transcriptional activity of GRHL3. The T454 residue is phosphorylated by p38 MAPK in vitro and activation of p38 signaling in cells causes an increase in GRHL3 activity. The regulation of GRHL3 function by this pathway is dependent on T454, as the substitution of T454 with methionine inhibits the activation of GRHL3. Taken together, our results show that T454 is one of the phosphorylated residues in GRHL3 in keratinocytes and this residue is important for the upregulation of GRHL3 transcriptional activity by the p38 pathway.

The Grainyhead-like (GRHL) family of transcription factors has three mammalian members, which are currently termed Grainyhead-like 1 (GRHL1), Grainyhead-like 2 (GRHL2), and Grainyhead-like 3 (GRHL3). These factors adopt a DNA-binding immunoglobulin fold homologous to the DNA-binding domain of key tumor suppressor p53. Their patterns of expression are tissue and developmentally specific. Earlier studies of the GRHL proteins focused on their functions in mammalian development. In recent years, these factors have been linked to many different types of cancer: squamous cell carcinoma of the skin, breast cancer, gastric cancer, hepatocellular carcinoma, colorectal cancer, clear cell renal cell carcinoma, neuroblastoma, prostate cancer, and cervical cancer. The roles of GRHL proteins in these various types of cancer are complex, and in some cases appear to be contradictory: they can serve to promote cancer development, or they may act as tumor suppressors, depending on the particular GRHL protein involved and on the cancer type. The reasons for obvious discrepancies in results from different studies remain unclear. At the molecular level, the GRHL transcription factors regulate the expression of genes whose products are involved in cellular proliferation, differentiation, adhesion, and polarity. We herein review the roles of GRHL proteins in cancer development, and we critically examine relevant molecular mechanisms, which were proposed by different authors. We also discuss the significance of recent discoveries implicating the involvement of GRHL transcription factors in cancer and highlight potential future applications of this knowledge in cancer treatment.

Background: The Grainyhead-like (GRHL) transcription factors have been linked to many different types of cancer. However, no previous study has attempted to investigate potential correlations in expression of different GRHL genes in this context. Furthermore, there is very little information concerning damaging mutations and/or single nucleotide polymorphisms in GRHL genes that may be linked to cancer.Methods: DNA and RNA were extracted from human non-melanoma skin cancers (NMSC) and adjacent normal tissues (n = 33 pairs of samples). The expression of GRHL genes was measured by quantitative real time PCR. Regulation of GRHL expression by miRNA was studied using cell transfection methods and dual-luciferase reporter system. Targeted deep sequencing of GRHL genes in tumor samples and control tissues were employed to search for mutations and single nucleotide polymorphisms. Single marker rs141193530 was genotyped with pyrosequencing in additional NMSC replication cohort (n = 176). Appropriate statistical and bioinformatic methods were used to analyze and interpret results.Results: We discovered that the expression of two genes – GRHL1 and GRHL3 – is reduced in a coordinated manner in tumor samples, in comparison to the control healthy skin samples obtained from the same individuals. It is possible that both GRHL1 and GRHL3 are regulated, at least to some extent, by different strands of the same oncogenic microRNA – miR-21, what would at least partially explain observed correlation. No de novo mutations in the GRHL genes were detected in the examined tumor samples. However, some single nucleotide polymorphisms in the GRHL genes occur at significantly altered frequencies in the examined group of NMSC patients.Conclusions: Non-melanoma skin cancer growth is accompanied by coordinated reduced expression of epidermal differentiation genes: GRHL1 and GRHL3, which may be regulated by miR-21–3p and -5p, respectively. Some potentially damaging single nucleotide polymorphisms in GRHL genes occur with altered frequencies in NMSC patients, and they may in particular impair the expression of GRHL3 gene or functioning of encoded protein. The presence of these polymorphisms may indicate an increased risk of NMSC development in affected people.

The involvement of Grainyhead-like (GRHL) transcription factors in various cancers is well documented. However, little is known about their role in clear cell renal cell carcinoma (ccRCC). We discovered that the expression of two of these factors-GRHL1 and GRHL2-are downregulated in ccRCC samples, and their expression is correlated with the expression of VHL gene. This suggests a functional link between the GRHL transcription factors and one of the best known tumor suppressors. Although the GRHL genes are not mutated in ccRCC, some of the single nucleotide polymorphisms in these genes may indicate an increased risk of ccRCC development and/or may allow to assess patients' prognoses and predict their responses to various forms of therapy. Silencing of GRHL2 expression in non-tumorigenic kidney cell line results in increased cell proliferation, increased resistance to apoptosis, as well as changes in the levels of selected proteins involved in the pathogenesis of ccRCC. These changes support the potential role for GRHL2 as a suppressor of ccRCC.

The LSF/Grainyhead family of transcription factors consists of proteins whose structure and functions have been preserved in the course of eukaryotic evolution--from primitive unicellular life forms to complex multicellular organisms. In the latter, these factors display tissue specificity and are active mainly in the covering epithelium. The roles of GRH factors are associated with regulation of expression of genes essential for correct differentiation and functioning of the epithelia of ectodermal origin. The Grh gene expression profiles are diverse and variable, especially during embryonic development. Research on the role of GRHL transcription factors is carried out on cellular and organismal level. In experimental animals, aberrant Grh gene expression leads to many diseases, including failure of epidermal wound healing and neural tube defects. Changes of these genes' expression levels are also linked to carcinogenesis. GRHL transcription factors participate in signaling pathways involved in cellular proliferation and apoptosis.

Description of individuals with NMSC (fresh tissues, NGS). Table S2. SureSelect HaloPlex Design (analyzable regions). Table S3. Total number of NMSC patients including the replication cohort â general description. Fig. S1. Levels of GRHL1 protein in HaCaT cells transfected with miR-21â 3p mimic or inhibitor. (DOC 240Â kb)

Description of individuals with NMSC (fresh tissues, NGS). Table S2. SureSelect HaloPlex Design (analyzable regions). Table S3. Total number of NMSC patients including the replication cohort â general description. Fig. S1. Levels of GRHL1 protein in HaCaT cells transfected with miR-21â 3p mimic or inhibitor. (DOC 240Â kb)