Gatekeeper Derailed

Dysregulated G1/S checkpoint control and the consequent aberrant cell cycle progression are hallmark features of cancer. While this disruption facilitates unchecked tumorcell proliferation, it also exposes cancer cells to the harmful effects of DNA replication stress. The work presented in this thesis sought to identify genes and pathways that cancer cells depend on to sustain proliferative capacity under conditions of DNA replication stress. To this end, we used G1/S-proficient cells derived from both murine and human sources and inactivated the G1/S checkpoint by disrupting the retinoblastoma (Rb) gene family. Previous studies had shown that Rb-protein-deficient mouse embryonic fibroblasts (MEFs) undergo unscheduled S-phase entry in serum-deprived conditions, resulting in heightened sensitivity to apoptosis due to replication stress. These cells were able to proliferate in the absence of mitogens only when apoptosis was suppressed via BCL2 overexpression (in TB MEFs) and p53 was depleted (in TBP MEFs). Although now capable of proliferating mitogen-independently, TBP MEFs still exhibited signs of replication stress, as indicated by reduced replication fork velocity and dependency on ATR/CHK1. These TBP MEFs were utilized throughout our studies to explore the cellular dependencies that enable proliferation in suboptimal growth conditions associated with replication stress. In Chapter 2, we conducted a focused shRNA genetic screen and identified RECQL as a key protein required for the proliferation of TBP MEFs under replication stress. We demonstrated that RECQL plays a critical role in protecting stalled replication forks from MRE11-mediated double-strand break formation. In Chapter 3, we expanded our search for “replication rescue genes” by conducting a genome-wide CRISPR knockout screen. This approach led to the identification of several critical dependencies in TBP cells proliferating under growth-restricting conditions. We demonstrated that suppression of mTORC1 signaling is essential for maintaining proliferative capacity under these conditions. Depletion of negative mTORC1 regulators was detrimental to serum-starved TBP cells, while knockout of positive regulators conferred a proliferative advantage. These results imply that tumor cells proliferating under suboptimal growth conditions benefit from restrained mTORC1 activity. Our observations could thus help explain the limited clinical efficacy of mTOR inhibitors. Another replication rescue gene identified in our study was ADAR1. ADAR1 (adenosine deaminase acting on RNA) has been described as a negative modulator of interferon signaling, by disrupting hairpin structures in endogenous mRNAs. Numerous studies have highlighted its potential as a therapeutic target to boost tumor immunogenicity and improve the effectiveness of immune checkpoint blockade therapies. In Chapter 4, our findings reveal an additional role of ADAR1 in mitigating mitotic catastrophe. We conducted a detailed analysis of ADAR1 depletion in TBP cells cultured under mitogen-stimulated and -deprived conditions. We observed that ADAR1 depletion induced progressive tetraploidization in both murine and human TBP cells, a phenomenon that appeared to be incompatible with proliferation under serum-starved conditions as well as in vivo growth. Our studies led to the identification of a novel regulatory function of ADAR1 in suppressing aberrant mitotic entry. This newly discovered function broadens our understanding of ADAR1’s role in cellular biology, and extends its potential as a therapeutic target in cancer treatment. In Chapter 5, we found that treatment with the presumed ADAR1 inhibitor 8-azaadenosine (8-aza-A) blocked proliferation of serum-deprived TBP cells. We observed that low concentrations of 8-aza-A slowed down S-phase progression and induced whole genome duplications, thereby selectively inhibiting the growth of mitogen-deprived TBP cells. However, we could not convincingly attribute these effects to ADAR inhibition, raising concerns regarding the specificity of 8-aza-A as an ADAR1 inhibitor. Nevertheless, 8-aza-A effectively suppressed the proliferation of mitogen-deprived TBP cells, warranting further investigation into the underlying mechanisms by which 8-aza-A exerts its effects as well as exploring its therapeutic potential.