Friday, April 26, 2024
4/26/2024
ABSTRACT The long-term goal of this project is to define the molecular mechanisms of an error-prone, stress-induced Okazaki fragment maturation (OFM) pathway by which cancer cells counteract replication stress and survive. Replication stress is a hallmark of cancer cells and has been considered the Achilles' heel for cancer treatment such as radio- and chemotherapy. Under elevated temperature stress, yeast cells mutant for flap endonuclease 1 (FEN1 in humans or RAD27 in yeast) activate DNA damage response pathways to block cell proliferation and induce cell senescence and death; however, a subpopulation of cells can overcome these barriers and escape otherwise lethal conditions. Genome-wide mutations and rearrangements have been suggested as a major molecular mechanism that drives this evolution. However, how such spontaneous mutations are acquired in cells under replication stress is a long-standing question. Recently, we identified an error-prone, 3' flap OFM pathway that is activated in response to stress to support cell survival and fuel cellular evolution; its induction leads to genome-wide mutagenesis and suppression of restrictive growth temperature-induced lethality, a process mimicking that of cancer cells acquiring drug resistance. This led us to a model in which OFM can go in two ways, which may dictate the fate of cells, including human cancer cells: a 5' flap-based, error-free process or an alternative 3' flap-based, stress-induced, and error-prone process. However, key components that drive such flap dynamics remain undefined. The objectives of the proposed project are to define the key enzymes that catalyze 3' flap formation and cleavage in mammalian cells and to provide proof of concept that suppressing alternative 3' flap OFM can prevent drug resistance in human cancer cells. Further preliminary data gathered to support this grant application show that 3' flap OFM is conserved in both yeast and human cells. We observed that anti-cancer EGFR tyrosine kinase inhibitors activated the ATM/CHK2 DNA damage checkpoints in human lung cancer cells. Using yeast genetic screening, we identified Pif1 (PIF1 in humans) and Sgs1 (BLM and WRN in humans) as helicases for 5' to 3' flap transformation and Rad1 (XPF in humans) and Mus81 (MUS81 in humans) as 3' nucleases for 3' flap cleavage, in addition to the 3' nuclease activity of Pol ¿. Therefore, our central hypothesis is that unprocessed 5' flaps in mammalian cells activate ATM/ATR and CHK1/2 signaling to recruit and stimulate PIF1, BLM, and WRN and/or other helicases for transforming 5' flaps into 3' flaps for nucleolytic degradation by 3' nucleases including Pol ¿, XPF, and/or MUS81, and that blocking the 3' flap OFM pathway will suppress DNA mutations and thus prevent drug resistance. To test this, we will: i) determine the roles of helicases PIF1, BLM, and WRN in 3' flap formation and induction of alternative OFM; ii) define the functional distribution of 3' nucleases Pol ¿, XPF, and MUS81 in processing 3' flaps with or without secondary structures during 3' flap OFM; and iii) define the extent to which stress-activated ATM/CHK2 signaling induces 3' flap OFM and mutations to support cancer cell survival and promote drug resistance.
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