Understanding Telomere Elongation Mechanisms in Cancer

Project Summary Malignant cancers must activate telomere maintenance mechanisms to achieve replicative immortality. Mutations in the human Protection Of Telomeres 1 (hPOT1) protein are frequently detected in cancers with abnormally long telomeres. This result suggests that the loss of hPOT1 function disrupts regulation of telomere

length homeostasis to promote telomere elongation. The mouse genome encodes two POT1 proteins, POT1a and POT1b. I have generated Pot1b null tumors that display markedly elongated telomeres mediated by telomerase. In Aim 1, I will measure telomerase expression and its recruitment to telomeres to determine if

changes in telomerase dynamics has led to telomere length increase in these tumors. Then I will investigate the mechanism underlying changes in telomerase dynamics by testing the role of DDR observed in Pot1b null tumors and the role of POT1b-TPP1 interactions in telomerase-mediated length maintenance.

Pot1b null tumors also display very heterogeneous telomere length with extremely long ss G-overhang that is reminiscent of telomeres generated by Alternative Lengthening of Telomeres (ALT), a telomere length maintenance pathway through homology directed repair and independent of telomerase function. When Pot1b

loss is combined with telomerase deletion, telomeres rapidly shorten and potentially select for ALT activation. Aim 2 will test for activation of the ALT maintenance in Pot1b-/-; Tert-/- MEFs. I will also determine if the Pot1b-/-; Tert-/-; Atrx-/- genetic background is sufficient to activate ALT. Furthermore, I will reconstitute POT1b domain

mutants in ALT positive cells to investigate the mechanisms utilized by POT1b to repress ALT. Finally, I will perform RNA-seq to identify novel factors contributing to ALT activation in Pot1b-/-; Tert-/- MEFs. Together, these aims explore the central hypothesis that POT1b represses the activation of telomere

maintenance mechanisms. Aim 1 will investigate the mechanism leading to telomerase-mediated telomere hyper-elongation in the absence of POT1b and Aim 2 will determine if the combined loss of POT1b and TERT activates ALT. Completion of these aims will contribute to our knowledge of the telomere maintenance

mechanisms necessary to promote replicative immortality and cancer initiation.