Browsing by Subject "DNA damage response"
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Item Open Access Dissecting Tumor Response to Radiation Therapy Using Genetically Engineered Mouse Models(2015) Moding, Everett JamesApproximately 50% of all patients with cancer receive radiation therapy at some point during the course of their illness. Despite advances in radiation delivery and treatment planning, normal tissue toxicity often limits the ability of radiation to eradicate tumors. The tumor microenvironment consists of tumor cells and stromal cells such as endothelial cells that contribute to tumor initiation, progression and response to therapy. Although endothelial cells can contribute to normal tissue injury following radiation, the contribution of stromal cells to tumor response to radiation therapy remains controversial. To investigate the contribution of endothelial cells to the radiation response of primary tumors, we have developed the technology to contemporaneously mutate different genes in the tumor cells and stromal cells of a genetically engineered mouse model of soft tissue sarcoma. Using this dual recombinase technology, we deleted the DNA damage response gene Atm in sarcoma and heart endothelial cells. Although deletion of Atm increased cell death of proliferating tumor endothelial cells, Atm deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. In addition, the ATM inhibitor NVP-BEZ235 selectively radiosensitized primary sarcomas, demonstrating a therapeutic window for inhibiting ATM during radiation therapy. Sensitizing tumor endothelial cells to radiation by deleting Atm prolonged tumor growth delay following a non-curative dose of radiation, but failed to increase local control. In contrast, deletion of Atm in tumor parenchymal cells increased the probability of tumor eradication. These results demonstrate that tumor parenchymal cells rather than endothelial cells are the critical targets that regulate tumor eradicaiton by radiation therapy.
Item Open Access DNA Damage Response Suppresses Epstein-Barr Virus-Driven Proliferation of Primary Human B Cells(2012) Nikitin, Pavel AThe interaction of human tumor viruses with host growth suppressive pathways is a fine balance between controlled latent infection and virus-induced oncogenesis. This dissertation elucidates how Epstein-Barr virus interacts with the host growth suppressive DNA damage response signaling pathways (DDR) in order to transform infected human B lymphocytes.
Here I report that the activation of the ATM/Chk2 branch of the DDR in hyper-proliferating infected B cells results in G1/S cell cycle arrest and limits viral-mediated transformation. Similar growth arrest was found in mitogen-driven proliferating of B cells that sets the DDR as a default growth suppressive mechanism in human B cells. Hence, the viral protein EBNA3C functions to attenuate the host DDR and to promote immortalization of a small portion of infected B cells. Additionally, the pharmacological inhibition of the DDR in vitro increases viral immortalization of memory B cells that facilitates the isolation of broadly neutralizing antibodies to various infectious agents. Overall, this work defines early EBV-infected hyper-proliferating B cells as a new stage in viral infection that determines subsequent viral-mediated tumorigenesis.
Item Open Access Overcoming Therapeutic Resistance by Targeting Oncogene-Driven and Targeted-Therapy Induced Cancer Dependencies(2020) Ali, MoiezTargeted therapies rarely yield complete tumor responses, and the residual cancer cells that survive upfront treatment act as a reservoir from which eventual resistant disease emerges. Here, we explore several clinically relevant models of disease resistance, with special attention placed on KRAS-driven colorectal cancer (CRC) and EGFR-driven non-small-cell lung cancer (NSCLC).
First, we note that KRAS mutations drive resistance to diverse targeted therapies, including EGFR inhibitors in colorectal cancer (CRC). Through genetic screens, we unexpectedly find that mutant HRAS, which is rarely found in CRC, is a stronger driver of resistance than mutant KRAS. This difference is ascribed to common codon bias in HRAS, which leads to much higher protein expression, and implies that the inherent poor expression of KRAS due to rare codons must be surmounted during drug resistance. In agreement, we demonstrate that primary resistance to cetuximab is dependent upon both KRAS mutational status and protein expression level, and acquired resistance is often associated with KRASQ61mutations that function even when protein expression is low. Finally, we show that cancer cells upregulate translation to facilitate KRASG12-driven acquired resistance, resulting in hypersensitivity to translational inhibitors. These findings demonstrate that codon bias plays a critical role in KRAS-driven resistance and provide a rationale for targeting translation to overcome resistance.
Next, we demonstrate that targeted therapies induce DNA double strand breaks and consequent, ATM-dependent DNA repair in tumor cells that survive upfront treatment. This DNA damage response, observed in both laboratory models and human patients, is driven by a pathway involving the sub-lethal activation of executioner caspases 3 and 7 and the downstream caspase-activated DNase (CAD). As a consequence, tumor cells that survive upfront treatment harbor a synthetic dependence on ATM, and combined treatment with targeted therapies and a selective ATM kinase inhibitor eradicates these cells, leading to more penetrant and durable responses in in vitro and in vivo models of EGFR-mutant NSCLC. Finally, rare patients with EGFR-mutant NSCLC harboring co-occurring, loss-of-function mutations in ATM show evidence of extended progression-free survival relative to patients lacking deleterious ATM mutations. Together, these findings establish a rationale for the mechanism-based integration of ATM inhibitors alongside existing targeted therapy paradigms.
Combined, these studies provide mechanistic-based rationale for pharmacological targeting of tumor-specific processes that may overcome intrinsic and/or acquired resistance states, serving as potential novel therapeutic options for genetically defined subsets of cancer patients.
Item Open Access Post-translational Regulation of RPA32, ATM and Rad17 Controls the DNA Damage Response(2009) Feng, JunjieThe eukaryotic genome integrity is safeguarded by the DNA damage response, which is composed of a network of signal transduction pathways that upon genotoxic stresses, arrest cell cycle progression, motivate repair processes, or induce apoptosis or senescence when cells incur irreparable DNA damage. During this process, DNA damage-induced post-translational modifications, most notably protein phosphorylation, of a variety of DNA damage-responsive proteins has been shown to mediate the initiation, transduction and reception of the DNA damage signals, resulting in alterations of their stability, activities or subcellular localizations, ultimately leading to activation of various downstream effector pathways.
While a lot has been elucidated on the downstream events of the DNA damage response, little is known about how DNA damage is detected. Two still ongoing studies of this dissertation attempt to address this question. Our preliminary work on ATM indicates that serine 2546 is critical for its kinase activity. Substitution of this residue with phosphomimetic aspartate, but not nonphosphorylable alanine, abrogates the kinase activity of ATM and fails to rescue the checkpoint-deficient phenotype exhibited by the ATM-deficient cells, suggesting that removal of an inhibitory phospho group at S2546 might be required for the activation of ATM. In another study, we identified a novel DNA-damage responsive threonine residue (T622) in Rad17, which undergoes ATM/ATR-dependent phosphorylation in vitro and in vivo. Ectopic expression of a phosphodeficient mutant (T622A) of Rad17, but not its wild-type control, shows a pronounced defect in sustaining Chk1 phosphorylation and the corresponding G2/M checkpoint upon DNA damage, suggesting that phosphorylation at T622 might complement that on the two previously reported phosphorylation sites, S635 and S645, to mediate G2/M checkpoint activation while the latter is primarily responsible for intra-S phase checkpoint.
Although a large amount of knowledge has been accumulated about the initiation and activation process of the DNA damage response, how cells recover, the equally important flip side of the response, has remained poorly understood. We have found that in cells recovering from replication stress, RPA32 phosphorylation at ATM/ATR-responsive sites T21 and S33, which reportedly suppresses DNA replication and recruiting other checkpoint and repair proteins to the DNA lesions, is reversed by the serine/threonine protein phosphatase 2A (PP2A). Cells with a RPA32 persistent-phosphorylation mimic (T21D/S33D) exhibit normal checkpoint activation and re-enter the cell cycle normally after recovery, but display a pronounced defect in the repair of DNA breaks. These data indicate that PP2A-mediated RPA32 dephosphorylation may be a required event during the repair process in the DNA damage response.
In summary, these studies in this dissertation highlight the importance of reversible phosphorylation and dephosphorylation in the modulation of the DNA damage response. What's more, they also extend our knowledge and deepen our understanding of this process by revealing that dephosphorylation may positively regulate the activation of cell cycle checkpoints, which is seemingly dominated by protein phosphorylation upon DNA damage, that phosphorylation of certain checkpoint proteins at different sites may result in distinct consequences, and that dephosphorylation of some activated checkpoint/repair proteins may function as an important mechanism for cells to recover from the DNA damage response.