PROJECT SUMMARY/ABSTRACT: Our long term objective is to improve the therapy of tumors that rely on the alternative lengthening of telomere (ALT) mechanism. Approximately 10% of all tumors, including nearly all lower-grade astrocytomas, do not express telomerase but rather use a recombination-based ALT mechanism to extend their telomeric DNA. Because ALT is not used by normal cells, it represents an attractive therapeutic target. Along these lines we recently showed that the telomeres of ALT-dependent cells are uniquely dependent on PARP1 to suppress fusion of their dysfunctional telomeres, and that this ALT-specific protection could be reversed by exposure to PARP inhibitors (PARPi), which in turn led to fusion of telomeric DNA ends, and cell death in vitro and in vivo. This PARPi-induced chromosomal fusion therefore represents a novel telomere-directed action of PARPi as well as a potential new therapeutic avenue for ALT-dependent tumors. In the course of our studies, however, we noted that exposure of ALT-dependent cells to PARPi caused not one but two physically distinct forms of chromosomal fusion: chromosome-type fusions typically noted following TRF2 depletion, and chromatid-type fusions, which are unusual and indicative of dysregulated cNHEJ. Furthermore, PARPi lethality was specifically associated with the appearance of the chromatid-type fusions. We don't currently understand how chromatid-type fusions are triggered by PARPi, or how to enhance their formation. We noted, however, that three proteins reported to suppress aberrant chromatid-type fusion (MRE11, CYREN, and Apollo) all localize to ALT telomeres, and as direct or indirect binding partners of PARP1 could be regulated in a PARP-dependent manner. We also noted that chromatid-type fusions were associated with aneuploidy and non-reciprocal translocations, suggesting that these fusions might be processed in a more lethal manner. Finally, we noted that only a sub-set of PARPi induced chromatid-type fusion, but that this fusion could be enhanced by combined exposure to ATM inhibitors. Based on these observations we hypothesize that PARPi block the function of telomeric proteins that normally limit lethal chromatid-type fusion, and that the use of PARPi/ATMi combinations will maximize chromatid-type fusions and ALT tumor cell killing. This hypothesis will be examined by 1) determining if PARP1 regulates the proteins that inhibit chromatid-type fusion, 2) determining why PARPi-induced chromatid-type fusions are associated with the ALT cell-specific lethality 3) determining how ATMi synergize with PARPi and to identify agents that similarly synergize with PARPi in ALT-dependent cells.