Abstract Tumor cells arise upon escape from two distinct and critical barriers that limit proliferation of human cells, replicative senescence and crisis. Cells in replicative senescence arrest permanently while continuing to metabolize, triggered by short telomeres. Senescence entry however, is avoided by impairment of the main cell cycle checkpoints controlled by the p53 and Rb tumor suppressive pathways. Following senescence bypass and continued proliferation, cells undergo crisis, which is a phase highlighted by substantial telomere deprotection and widespread cell death. Crisis is a stringent tumor-suppressive barrier, as it removes the vast majority of cells that avoid senescence. However, rarely cells overcome this barrier and become neoplastic. The molecular mechanisms and pathways underlying cell death in crisis and spontaneous crisis evasion are not understood. Here, it is proposed to investigate the molecular mechanisms underlying the escape from crisis and crisis bypass, with the expectation that the resulting discoveries will have a strong impact on our understanding of the early steps in cancer development. The preliminary data presented here suggest a novel concept for replicative crisis that implicates autophagy as a major regulator of cell death. Autophagy suppression allowed cells to bypass crisis and continue to proliferate, while accumulating multiple genomic aberrations. This discovery is of profound significance for understanding how genome instability evolves during the early steps of cancer development. Furthermore, the finding suggests that autophagy inhibitors might have counterproductive effects and promote the establishment of neoplastic cells instead of eliminating them. In three specific aims it is proposed to decipher the exact signaling pathways that lead from dysfunctional telomeres to the activation of autophagy-controlled cell death (Aim 1), to determine the consequences of telomere-driven autophagy and of autophagy inhibition during crisis (Aim 2), and to understand the role of autophagy-driven cell death in crisis on tumor development in vivo (Aim 3). In summary, this grant proposal focuses on the mechanisms underlying cell death during replicative crisis, the mechanism of how autophagy is activated and regulated in response to replicative crisis, and how inhibition of autophagy during crisis enables cells with an unstable genome to escape this final barrier against tumor cell establishment and drive malignancy. We will thereby explore our novel hypothesis, in which temporary or permanent resistance to autophagic cell death is the initial event required for the emergence of post-crisis cells and an abrupt rise in genome instability, leading to the establishment of neoplastic cells.