PROJECT SUMMARY ALT (Alternative Lengthening of Telomeres) cancers encompass a significant fraction (15%) of human cancers. These immortalized cells maintain their telomeres using homology-directed recombination. The observation that ALT is activated in a significant fraction of tumors, coupled with its potential as an adaptive mechanism to anti-telomerase tumor therapies, make it an important target for anti-cancer strategies. Yet, targeted therapeutics for ALT cancers have not been greatly explored. Recent studies showed that a hallmark of ALT cells is loss of the chromatin remodeler ATRX, but how its loss contributes to telomere recombination had not been elucidated. We found that loss of ATRX suppresses resolution of sister chromatid cohesion at telomeres to promote sister telomere recombination and prevent deleterious non-allelic recombination. Sister telomere cohesion is normally resolved in G2/prophase of the cell cycle by the poly-ADP ribose polymerase tankyrase 1 that localizes to telomeres through the shelterin subunit TRF1. We found that due to loss of ATRX the “normal” system of telomere resolution is defective in ALT cells and telomeres remain cohered into mitosis. In the absence of ATRX its binding partner (the histone variant macroH2A1.1) is free to sequester tankyrase 1, preventing it from resolving telomere cohesion. Persistent telomere cohesion has an essential role, as forced resolution of telomere cohesion by ATRX introduction or tankyrase 1 overexpression in ALT cells leads to loss of recombination between sister telomeres, increased non-sister copying, and cell death. We hypothesize that persistent telomere cohesion assists in telomere maintenance by promoting sister telomere recombination and in safeguarding genome integrity by preventing aberrant and toxic non-sister recombination. In Aim 1 of this proposal, I will elucidate the mechanisms of persistent telomere cohesion and in Aim 2, I will define the proteome of the cohered state in ALT cells. My studies will provide mechanistic insights into telomere recombination and identify potential targets for ALT cancer therapy.