Divergent proteomic landscapes and functional requirements for telomere maintenance in type I and type II ALT mESCs.

Alternative lengthening of telomeres (ALT) is a telomerase-independent, recombination-based pathway utilized by some cancers. Previously, we established two distinct types of ALT survivors derived from mouse embryonic stem cells (mESCs), characterized by either a unique non-telomeric insertion (type I) or canonical telomeric repeats (type II). In this study, we used telomere-specific proteomics to elucidate the divergent telomere maintenance strategies of these ALT types. We identified distinct proteomic profiles enriched in type I versus type II ALT telomeres. ICE1 is a component of the Little Elongation Complex, which is specifically recruited to type I ALT telomeres, where it supports the transcription of telomeric repeat-containing RNA (TERRA) and telomere maintenance. In contrast, the serine/threonine kinase VRK2 is highly enriched in type II ALT telomeres, where it regulates telomere-nuclear envelope interactions. Depletion of ICE1 or VRK2 impairs telomere stability in type I and type II ALT mESCs, respectively. Our results indicate that cells with a common mESC origin can adopt distinct molecular mechanisms to maintain telomere integrity, highlighting the mechanistic heterogeneity of ALT telomere maintenance.