TGS1 mediates 2,2,7-trimethyl guanosine capping of the human telomerase RNA in lung tumor organoids

Pathways that direct the selection of the telomerase-dependent or recombination-based, alternative lengthening of telomere pathway (ALT) in cancer cells are poorly understood. Using human lung tumor organoids and classic lung cancer cells we show that formation of the 2,2,7- trimethylguanosine (TMG) cap structure at the human telomerase RNA 5' end by the Trimethylguanosine Synthase 1 (TGS1) is central for recruiting telomerase to telomeres and engaging Cajal bodies in telomere maintenance. Treatment of tumor organoids and lung cancer cell lines with the TGS1 inhibitor sinefungin leads to Exonuclease 1 mediated generation of telomere 3' end protrusions that engage in RAD51-dependent, homology directed recombination and the activation of key features of the ALT pathway. This indicates a critical role for 2,2,7-TMG capping of the RNA component of human telomerase (hTR) in enforcing telomerase-dependent telomere maintenance to restrict the formation of telomeric substrates conductive to ALT. Lung tumor organoids shown in this study were obtained from three patients diagnosed with lung cancer. Criteria for patient enrolment to the observational study: Age: = 18; size of lesion: =1cm; absence of documented infectious disease, absence of metastases; absence of neoadjuvant therapy; informed patient consent was obtained from all enrolled individuals. Tumor organoids recapitulate the tissue architecture of the primary lung tumors and maintain key aspects of genomic alterations of the original tumors after expansion in tissue culture. For each patient DNA was obtained from matched lung tumor tissue, normal lung tissue and lung tumor organoid cultures and whole exome sequencing (WES) was performed.