The SPPL3-defined glycosphingolipid repertoire orchestrates HLA class I-mediated immune responses (vol 54, 132.e1, 2021)

Chemical Immunolog

New tools for functional mammalian

Knowledge of the function of individual genes that encode components of cellsignalling pathways is crucial to our understanding of normal growth control and its deregulation in cancer, but we have functional information for only ~15% of human genes at present. Several new technologies have recently become available to identify gene function in mammalian cells using high-throughput genetic screens. These new tools will make it possible to identify new and innovative classes of anticancer drugs, including those that show synthetic lethal interactions with cancer-specific mutations

Stable suppression of tumorigenicity by virus-mediated RNA interference

Most human tumors harbor multiple genetic alterations, including dominant mutant oncogenes. It is often not clear which of these oncogenes are continuously required and which, when inactivated, may inhibit tumorigenesis. Recently, we developed a vector that mediates suppression of gene expression through RNA interference. Here, we use a retroviral version of this vector to specifically and stably inhibit expression of only the oncogenic K-RASV12 allele in human tumor cells. Loss of expression of K-RASV12 leads to loss of anchorage-independent growth and tumorigenicity. These results indicate that viral delivery of small interfering RNAs can be used for tumor-specific gene therapy to reverse the oncogenic phenotype of cancer cells

Functional Annotation of Deubiquitinating Enzymes Using RNA Interference

Protein ubiquitination is a dynamic process, depending on a tightly regulated balance between the activity of ubiquitin ligases and their antagonists, the ubiquitin-specific proteases or deubiquitinating enzymes. The family of ubiquitin ligases has been studied intensively and it is well established that their deregulation contributes to diverse disease processes, including cancer. Much less is known about the function and regulation of the large group of deubiquitinating enzymes. This chapter describes how RNA interference against deubiquitinating enzymes can be used to elucidate their function. The application of this technology will greatly improve the functional annotation of this family of proteases

An evaluation of the efficacy of topical application of salicylic acid for the treatment of familial cylindromatosis

Familial cylindromatosis is a rare genetic disorder, giving rise to neoplasms of the skin appendages. We have recently shown that loss of the cylindromatosis tumour suppressor gene leads to activation of NF-κB, a transcription factor having antiapoptotic activity. This provides a possible explanation for the deregulated growth of cylindromas. In cell-based assays, salicylate can prevent NF-κB activation caused by loss of the cylindromatosis gene, suggesting that salicylic acid application might be a potential treatment for cylindromatosis. Objectives  To assess the effectiveness of topical application of salicylic acid on familial cylindromas. Methods  Cylindromas in five patients from four different cylindromatosis families were treated with twice daily and then once daily topical salicylic acid. Clinical response was determined by serial tumour measurements. Results  In total 17 cylindromas in five patients were studied: 12 target lesions and five control lesions. The median size of the cylindromas was 1·0 cm (range, 0·6-2·8 cm). Two of the 12 cylindromas showed a complete remission. Another eight lesions showed some response, but not sufficient to qualify as partial remission. The control lesions remained stable or increased in size. Conclusions  Salicylic acid is a well-tolerated and potential new treatment for cylindromatosis

The Deubiquitinating Enzyme USP1 Regulates the Fanconi Anemia Pathway

Protein ubiquitination and deubiquitination are dynamic processes implicated in the regulation of numerous cellular pathways. Monoubiquitination of the Fanconi anemia (FA) protein FANCD2 appears to be critical in the repair of DNA damage because many of the proteins that are mutated in FA are required for FANCD2 ubiquitination. By screening a gene family RNAi library, we identify the deubiquitinating enzyme USP1 as a novel component of the Fanconi anemia pathway. Inhibition of USP1 leads to hyperaccumulation of monoubiquitinated FANCD2. Furthermore, USP1 physically associates with FANCD2, and the proteins colocalize in chromatin after DNA damage. Finally, analysis of crosslinker-induced chromosomal aberrations in USP1 knockdown cells suggests a role in DNA repair. We propose that USP1 deubiquitinates FANCD2 when cells exit S phase or recommence cycling after a DNA damage insult and may play a critical role in the FA pathway by recycling FANCD2

TBX-3, the Gene Mutated in Ulnar-Mammary Syndrome, Is a Negative Regulator of p19ARF and Inhibits Senescence

Prolonged culturing of rodent cells in vitro activates p19ARF (named p14ARF in man), resulting in a p53-dependent proliferation arrest known as senescence. The p19ARF-Mdm2-p53 pathway also serves to protect primary cells against oncogenic transformation. We have used a genetic screen in mouse neuronal cells, conditionally immortalized by a temperature-sensitive mutant of SV40 large T antigen, to identify genes that allow bypass of senescence. Using retroviral cDNA expression libraries, we have identified TBX-3 as a potent inhibitor of senescence. TBX-3 is a T-box gene, which is found mutated in the human developmental disorder Ulnar- Mammary Syndrome. We have shown that TBX-3 potently represses expression of both mouse p19ARF and human p14ARF. We have also shown here that point mutants of TBX-3, which are found in Ulnar-Mammary Syndrome, have lost the ability to inhibit senescence and fail to repress mouse p19ARF and human p14ARF expression. These data suggest that the hypoproliferative features of this genetic disorder may be caused, at least in part, by deregulated expression of p14ARF

TBX-3, the Gene Mutated in Ulnar-Mammary Syndrome, Is a Negative Regulator of p19ARF and Inhibits Senescence

Prolonged culturing of rodent cells in vitro activates p19ARF (named p14ARF in man), resulting in a p53-dependent proliferation arrest known as senescence. The p19ARF-Mdm2-p53 pathway also serves to protect primary cells against oncogenic transformation. We have used a genetic screen in mouse neuronal cells, conditionally immortalized by a temperature-sensitive mutant of SV40 large T antigen, to identify genes that allow bypass of senescence. Using retroviral cDNA expression libraries, we have identified TBX-3 as a potent inhibitor of senescence. TBX-3 is a T-box gene, which is found mutated in the human developmental disorder Ulnar- Mammary Syndrome. We have shown that TBX-3 potently represses expression of both mouse p19ARF and human p14ARF. We have also shown here that point mutants of TBX-3, which are found in Ulnar-Mammary Syndrome, have lost the ability to inhibit senescence and fail to repress mouse p19ARF and human p14ARF expression. These data suggest that the hypoproliferative features of this genetic disorder may be caused, at least in part, by deregulated expression of p14ARF

SLFN11 can sensitize tumor cells towards IFN-gamma-mediated T cell killing

Experimental cancer immunology and therap