Browsing by Subject "DNA damage response"

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    Distribution of DNA Damage Response Inhibitors to the Central Nervous System for Brain Tumor Therapy
    (2022-04) Talele, Surabhi
    Brain tumors are associated with a grim prognosis despite the aggressive treatment strategy of surgery, radiation, and chemotherapy. Therefore, there is a need to develop strategies to enhance efficacy of chemo-radiation treatments. DNA damage response (DDR) signaling pathways play a critical role of DNA repair in cancer cells and resistance to radiation and chemotherapy. Inhibition of these pathways can augment tumor kill. Berzosertib, peposertib and AZD1390 are potent small molecule inhibitors of three critical DDR pathways. We evaluated mechanisms limiting central nervous system (CNS) distribution of these molecules and identified their potential for chemo/radiosensitization in brain tumors. CNS distribution of all three molecules is restricted by active efflux mediated by P-gp and/or Bcrp. Moreover, high tissue binding to plasma, brain, and spinal cord, restricts partitioning of unbound drug across the blood-brain barrier (BBB). Additionally, peposertib concentrations in different anatomical regions of the brain were similar, however lower accumulation was observed in the spinal cord. Impact of efflux inhibition using elacridar demonstrated similar increase in peposertib concentrations within each of these regions, indicating that inhibition of efflux activity was functionally similar across these regions. Similarly, no differences were observed in AZD1390 distribution within different anatomical regions of the CNS, and the functional activity of P-gp and Bcrp also remained the same across brain regions. These molecules show heterogeneous tumor distribution in patient-derived xenograft models of brain tumors with drug accumulation maximum within tumor core, slightly lower in adjacent tumor rim, but significantly lower in surrounding normal brain. Berzosertib did not achieve effective chemosensitizing brain and tumor concentrations in vivo. Peposertib, despite its limited CNS delivery, showed potential as a safe yet effective radiosensitizer for brain metastases. Effective radiosensitizing concentrations of AZD1390 were achieved in brain and tumor despite its restricted CNS delivery. Combined, these CNS pharmacokinetic evaluations can guide dosing in pre-clinical efficacy studies and enable future clinical trial design to test these innovative chemo/radiosensitizers with the eventual goal of improving therapy for brain tumors. Integrating knowledge from these studies will aid in determining the potential of DDR inhibitors as effective chemo-radiosensitizing agents in brain tumors.
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    The Multifaceted Role of Calprotectin (S100A8/A9) in Head and Neck Squamous Cell Carcinoma
    (2017-11) Argyris, Prokopios
    Calprotectin (S100A8/A9) is formed as a heterodimeric protein complex of calcium regulating S100A8 and S100A9 encoded by genes mapped to the chromosomal locus 1q21.3 of the epidermal differentiation complex. Whereas extracellular calprotectin presents proinflammatory and antimicrobial properties, intracytoplasmic S100A8/A9 appears to play important roles in cell differentiation, cell cycle progression and proliferation, cell migration and survival. While highly upregulated in a variety of cancers, calprotectin is downregulated in head and neck squamous cell carcinoma (HNSCC); loss of S100A8/A9 is associated with increased DNA methylation and poor overall survival rates in HNSCC patients. Using immunohistochemical analysis for S100A8 and S100A9 we found that S100A8/A9 levels gradually decreased during progression of head and neck tumorigenesis from intra-oral premalignant (precancerous) epithelial dysplasia to invasive HNSCC. Furthermore, S100A8/A9 expression positively correlated with the level of squamous differentiation of the primary tumor. To investigate the localization of the calprotectin complex during cell cycle progression, S100A8/A9-expressing human HNSCC cells and immortalized oral keratinocytes were synchronized at G1/S and G2 phases of the cell cycle. During cell division, S100A8/A9 appeared to translocate from the cytoplasm to the microtubule-organizing centers, decorated the mitotic spindles and co-localized with casein kinase II (CK2). Calprotectin nuclear migration is consistent with a role of S100A8/A9 in the control of the G2/M checkpoint. To probe the role of calprotectin in DNA damage responses (DDR), we exposed S100A8/A9-expressing and S100A8/A9-negative carcinoma cells to genotoxic agents. Interestingly, following low doses of X-radiation and incubation with camptothecin, recruitment of the DNA repair regulatory molecules 53BP1 and γH2AX increased significantly in all calprotectin-positive carcinoma cells but failed to increase in calprotectin-negative cancer cells, suggesting impaired DDR in the absence of S100A8/A9. Furthermore, post-radiation DNA fragmentation was more prominent in calprotectin-positive cells as assessed by comet assays. S100A8/A9-negative HNSCC cells also appeared more resistant to cisplatin, while S100A8/A9-expressing carcinoma cells were more sensitive even at lower cisplatin doses. TCGA data indicated that more than 363 apoptosis–related genes were significantly upregulated by S100A8/A9–high HNSCCs compared to S100A8/A9–low neoplasms, including CASP1, -3, -4, -5, -7, -8, -9, -10 and -14. Intracellular calprotectin appeared to promote caspase-mediated DNA fragmentation following radio- and chemotherapy, contributing to S100A8/A9-dependent apoptotic death of carcinomatous cells. In addition, in vitro and ex vivo experiments showed that S100A8/A9 levels were inversely correlated to membranous and cytoplasmic EGFR expression, a negative prognosticator for HNSCC. Calprotectin-associated control of DNA damage responses, post-radiation sensitivity and cisplatin cytotoxicity, and EGFR expression could contribute to the increased overall survival rates of patients with S100A8/A9-high HNSCCs. Our current data further supports the tumor-suppressive role of calprotectin in HNSCC and points to new molecular targets for therapy.
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    Systemic And Cns Distributional Pharmacokinetics Of Novel Dna Damage Response Inhibitors: Implications For The Treatment Of Brain Tumors
    (2023-11) Rathi, Sneha
    CNS tumors are among the leading causes of death across all age groups and present a critical unmet need. Despite advancements in CNS tumor diagnostics and use of molecular profiling in tumor classification that have improved our understanding of tumor pathophysiology, there has been limited clinical success in the treatment of brain tumors. This can be attributed in part to the anatomical location of the tumor and challenges associated with brain drug delivery. Numerous failed clinical trials where dose selection was based primarily on observed drug efficacy have highlighted the importance of evaluating exposure-response-effect relationships to inform decisions about dosing regimens. The current standard of care for brain tumors includes surgery, radiation, and chemotherapy. Subsequent activation of DNA damage response pathways induces resistance to DNA damaging radiation and chemotherapy. Therefore, DNA damage response inhibitors are being explored as radio- and chemo-sensitizers to increase the efficacy of DNA damaging radiation and chemotherapy. WSD0628, an ATM inhibitor, and elimusertib, an ATR inhibitor, inhibit the key regulators of the DNA damage response. We evaluated the preclinical systemic pharmacokinetics and CNS distribution of these drugs for evaluating their potential in the treatment of brain tumors. Mechanisms influencing the exposure of pharmacologically active drug in the tissues of interest, such as role of efflux transporters, drug binding to plasma and CNS tissues were evaluated along with in vitro potency and in vivo efficacy in orthotopic tumor bearing mouse models. We used modeling and simulation approaches to predict exposures in tissues of interest and correlate with in vivo efficacy. We observed a greater-than-dose-proportional increase in exposure of WSD0628 in plasma and brain, potentially due to the saturation of the clearance pathways. WSD0628 is homogenously distributed throughout all brain regions and CNS distribution of WSD0628 is not limited by P-gp or Bcrp. Based on our observations, WSD0628 is a potent radiosensitizer with adequate CNS distribution for efficacy in primary and secondary brain tumors and has potential for further exploration. The nonlinear pharmacokinetic behavior of WSD0628 was modeled by simultaneous fitting of the observed concentration time profiles and we propose a model framework for establishing the exposure-response-effect relationship of WSD0628 for the treatment of brain tumors. We also evaluated the potential reasons for lack of preclinical in vivo efficacy of elimusertib in combination with the current standard of care therapies for brain tumors. We observed that the CNS distribution of elimusertib was limited due to rapid clearance from systemic circulation, high extent of binding to CNS tissues, and to a lower extent P-gp mediated active efflux. This resulted in no change in in vivo efficacy in combination with radiation and temozolomide in orthotopic GBM PDX mouse models despite an observed robust in vitro synergy with temozolomide. Acknowledging the potential for species differences, our results indicate that elimusertib may have limited potential for treatment of brain tumors. Taken together, these observations provide critical insights into the potential of these drugs for the treatment of brain tumors and will enable informed decisions in the development of these molecules.