BRCA1 and BRCA2 mutation carriers are predisposed to develop breast and ovarian cancers, but the reasons for this tissue specificity are unknown. Breast epithelial cells are known to contain elevated levels of oxidative DNA damage, triggered by hormonally driven growth and its effect on cell metabolism. BRCA1- or BRCA2-deficient cells were found to be more sensitive to oxidative stress, modeled by treatment with patho-physiologic concentrations of hydrogen peroxide. Hydrogen peroxide exposure leads to oxidative DNA damage induced DNA double strand breaks (DSB) in BRCA-deficient cells causing them to accumulate in S-phase. In addition, after hydrogen peroxide treatment, BRCA deficient cells showed impaired Rad51 foci which are dependent on an intact BRCA1-BRCA2 pathway. These DSB resulted in an increase in chromatid-type aberrations, which are characteristic for BRCA1 and BRCA2-deficient cells. The most common result of oxidative DNA damage induced processing of S-phase DSB is an interstitial chromatid deletion, but insertions and exchanges were also seen in BRCA deficient cells. Thus, BRCA1 and BRCA2 are essential for the repair of oxidative DNA damage repair intermediates that persist into S-phase and produce DSB. The implication is that oxidative stress plays a role in the etiology of hereditary breast cancer.
Purpose/Objective(s): CRLX101, an investigational nanoparticle drug conjugate containing the payload camptothecin (CPT), is currently being evaluated clinically in multiple treatment-refractory solid tumors. As a nanoparticle, CRLX101 preferentially accumulates in tumors through enhanced permeability and retention effect. Such differential distribution can result in lower toxicity, especially combined with radiation therapy. Previously, CRLX101 has been shown to be a potent inhibitor of topoisomerase-1 as well as hypoxia-inducible factor-1 alpha (HIF-1a). With these attractive properties, we hypothesized CRLX101 is a potential radiosensitizer with the potential to improve the efficacy of chemoradiation therapy. In this study, we aimed to preclinically evaluate the efficacy and safety of adding CRLX101 to chemoradiation therapy using colorectal cancer as a model. Materials/Methods: To evaluate CRLX101 as a radiosensitizer, it was compared to CPT in vitro using colorectal cancer cell lines (HT-29 and SW480). We first determined CRLX101's direct cytotoxic impact to each cell line and then used the clonogenic assay to compare the efficacy of chemoradiation therapy in vitro. In vivo effects of CRLX101 were examined using mouse xenograft models. We also characterized the mechanism of action of CRLX101 by western blot, immunofluorescence, and immunohistochemistry. Lastly, we examined the hematologic toxicity and skin toxicity profile of CRLX101 and compared it to that of CPT. Results: Radiation survival curves showed that CRLX101 was as potent as CPT as a radiosensitizer in vitro. The addition of CRLX101 to standard chemoradiation therapy significantly increased therapeutic efficacy of 5-Fluorouracil (5-FU) based chemoradiation therapy in a mouse xenograft model of colorectal cancer. More importantly, CRLX101 was superior to oxaliplatin in 5-FU based chemoradiation therapy (P Z .0059), a regimen which has been extensively studied clinically. We demonstrated that CRLX101 improves chemoradiation by delaying repair of DNA double strand breaks, and thus preventing DNA repair. We also found CRLX101 inhibits radiation-induced HIF-1a upregulation as well as its downstream targets VEGF and CAIX. In this xenograft mouse model, and compared to CPT, CRLX101 had minimal hematologic toxicity, and the addition of CRLX101 to radiation did not increase significant hair toxicity or weight loss. Conclusion: Preclinical data suggests CRLX101 is a potent radiosensitizer with the high potential to improve the efficacy of chemoradiation therapy for the treatment of colorectal cancer. Our data supports the ongoing phase 1b/2 clinical investigation utilizing CRLX101 in chemoradiation therapy in the neoadjuvant rectal setting.
Glioblastoma (GBM) is the most common primary malignant neoplasm with poor survival despite treatment. Developing effective therapies remains challenging due to intratumoral heterogeneity, which drives therapeutic resistance and recurrence. To understand how genetic events alter the epigenome to enhance clonal fitness, we developed an isogenic human neural progenitor cell (NPC)-based model of the proneural (PRO) GBM subtype. We introduced TERT promoter (TERTp) C228T and gain-of-function TP53 R248Q mutations in H1 human embryonic stem cells. Wildtype (WT) cells, single TERTp, and double TERTp/TP53 mutants underwent differentiation to NPCs. Lentiviral transduction of double mutants with PDGFRA D842V resulted in triple mutant PRO NPCs. Bulk and single cell transcriptomics of our model system revealed hundreds of gene expression changes with increased mesoderm and human GBM mesenchymal (MES) subtype signatures in single TERTp and double TERTp/TP53 mutants, versus WT cells. TERTp mutation increased telomerase expression and activity, conferring proliferative advantage and immortalization in NPCs and astrocytes. Additionally, TERT expression was further increased in TERTp/TP53 mutants. Surprisingly, triple mutant PRO NPCs, versus WT, displayed < 100 differentially expressed genes, associated with neurodevelopmental and dynamic cytoskeletal processes. Evolution analyses using gene counts signature and splicing dynamics revealed a developmental trajectory model from WT to additive mutants to PRO NPCs. Only triple mutant PRO NPCs formed tumors after intracranial injection in athymic nude mice, with mean survival of 100 days. Tumors presented histopathological features of GBM, and single cell transcriptomic analyses revealed evolution from immune-interacting to both neural progenitor- and neuronal-like subpopulations, with similar cell cycling signatures. Transcription factor genes related to WNT signaling and lineage commitment as well as glial and neuronal cytoskeletal genes exhibited epigenetic selection in vivo, signatures also observed in PRO NPCs in vitro. Our model thus provides opportunity for dissection of epigenetic and functional mechanisms underlying serial mutations during PRO tumor evolution.
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