Browsing by Author "Floyd, Scott R"
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Item Open Access B7-H3-redirected chimeric antigen receptor T cells target glioblastoma and neurospheres.(EBioMedicine, 2019-09) Nehama, Dean; Di Ianni, Natalia; Musio, Silvia; Du, Hongwei; Patané, Monica; Pollo, Bianca; Finocchiaro, Gaetano; Park, James JH; Dunn, Denise E; Edwards, Drake S; Damrauer, Jeffrey S; Hudson, Hannah; Floyd, Scott R; Ferrone, Soldano; Savoldo, Barbara; Pellegatta, Serena; Dotti, GianpietroBackground
The dismal survival of glioblastoma (GBM) patients urgently calls for the development of new treatments. Chimeric antigen receptor T (CAR-T) cells are an attractive strategy, but preclinical and clinical studies in GBM have shown that heterogeneous expression of the antigens targeted so far causes tumor escape, highlighting the need for the identification of new targets. We explored if B7-H3 is a valuable target for CAR-T cells in GBM.Methods
We compared mRNA expression of antigens in GBM using TCGA data, and validated B7-H3 expression by immunohistochemistry. We then tested the antitumor activity of B7-H3-redirected CAR-T cells against GBM cell lines and patient-derived GBM neurospheres in vitro and in xenograft murine models.Findings
B7-H3 mRNA and protein are overexpressed in GBM relative to normal brain in all GBM subtypes. Of the 46 specimens analyzed by immunohistochemistry, 76% showed high B7-H3 expression, 22% had detectable, but low B7-H3 expression and 2% were negative, as was normal brain. All 20 patient-derived neurospheres showed ubiquitous B7-H3 expression. B7-H3-redirected CAR-T cells effectively targeted GBM cell lines and neurospheres in vitro and in vivo. No significant differences were found between CD28 and 4-1BB co-stimulation, although CD28-co-stimulated CAR-T cells released more inflammatory cytokines.Interpretation
We demonstrated that B7-H3 is highly expressed in GBM specimens and neurospheres that contain putative cancer stem cells, and that B7-H3-redirected CAR-T cells can effectively control tumor growth. Therefore, B7-H3 represents a promising target in GBM. FUND: Alex's Lemonade Stand Foundation; Il Fondo di Gio Onlus; National Cancer Institute; Burroughs Wellcome Fund.Item Open Access BRD4 Prevents R-Loop Formation and Transcription-Replication Conflicts by Ensuring Efficient Transcription Elongation.(Cell reports, 2020-09) Edwards, Drake S; Maganti, Rohin; Tanksley, Jarred P; Luo, Jie; Park, James JH; Balkanska-Sinclair, Elena; Ling, Jinjie; Floyd, Scott REffective spatio-temporal control of transcription and replication during S-phase is paramount to maintaining genomic integrity and cell survival. Dysregulation of these systems can lead to conflicts between the transcription and replication machinery, causing DNA damage and cell death. BRD4 allows efficient transcriptional elongation by stimulating phosphorylation of RNA polymerase II (RNAPII). We report that bromodomain and extra-terminal domain (BET) protein loss of function (LOF) causes RNAPII pausing on the chromatin and DNA damage affecting cells in S-phase. This persistent RNAPII-dependent pausing leads to an accumulation of RNA:DNA hybrids (R-loops) at sites of BRD4 occupancy, leading to transcription-replication conflicts (TRCs), DNA damage, and cell death. Finally, our data show that the BRD4 C-terminal domain, which interacts with P-TEFb, is required to prevent R-loop formation and DNA damage caused by BET protein LOF.Item Open Access CometChip enables parallel analysis of multiple DNA repair activities.(DNA repair, 2021-10) Ge, Jing; Ngo, Le P; Kaushal, Simran; Tay, Ian J; Thadhani, Elina; Kay, Jennifer E; Mazzucato, Patrizia; Chow, Danielle N; Fessler, Jessica L; Weingeist, David M; Sobol, Robert W; Samson, Leona D; Floyd, Scott R; Engelward, Bevin PDNA damage can be cytotoxic and mutagenic, and it is directly linked to aging, cancer, and other diseases. To counteract the deleterious effects of DNA damage, cells have evolved highly conserved DNA repair pathways. Many commonly used DNA repair assays are relatively low throughput and are limited to analysis of one protein or one pathway. Here, we have explored the capacity of the CometChip platform for parallel analysis of multiple DNA repair activities. Taking advantage of the versatility of the traditional comet assay and leveraging micropatterning techniques, the CometChip platform offers increased throughput and sensitivity compared to the traditional comet assay. By exposing cells to DNA damaging agents that create substrates of Base Excision Repair, Nucleotide Excision Repair, and Non-Homologous End Joining, we show that the CometChip is an effective method for assessing repair deficiencies in all three pathways. With these applications of the CometChip platform, we expand the utility of the comet assay for precise, high-throughput, parallel analysis of multiple DNA repair activities.Item Open Access Dosimetric analysis of the alopecia preventing effect of hippocampus sparing whole brain radiation therapy.(Radiat Oncol, 2015-11-26) Mahadevan, Anand; Sampson, Carrie; LaRosa, Salvatore; Floyd, Scott R; Wong, Eric T; Uhlmann, Erik J; Sengupta, Soma; Kasper, Ekkehard MBACKGROUND: Whole brain radiation therapy (WBRT) is widely used for the treatment of brain metastases. Cognitive decline and alopecia are recognized adverse effects of WBRT. Recently hippocampus sparing whole brain radiation therapy (HS-WBRT) has been shown to reduce the incidence of memory loss. In this study, we found that multi-field intensity modulated radiation therapy (IMRT), with strict constraints to the brain parenchyma and to the hippocampus, reduces follicular scalp dose and prevents alopecia. METHODS: Suitable patients befitting the inclusion criteria of the RTOG 0933 trial received Hippocampus sparing whole brain radiation. On follow up, they were noticed to have full scalp hair preservation. 5 mm thickness of follicle bearing scalp in the radiation field was outlined in the planning CT scans. Conventional opposed lateral WBRT radiation fields were applied to these patient-specific image sets and planned with the same nominal dose of 30 Gy in 10 fractions. The mean and maximum dose to follicle bearing skin and Dose Volume Histogram (DVH) data were analyzed for conventional and HS-WBRT. Paired t-test was used to compare the means. RESULTS: All six patients had fully preserved scalp hair and remained clinically cognitively intact 1-3 months after HS-WBRT. Compared to conventional WBRT, in addition to the intended sparing of the Hippocampus, HS-WBRT delivered significantly lower mean dose (22.42 cGy vs. 16.33 cGy, p < 0.0001), V24 (9 cc vs. 44 cc, p < 0.0000) and V30 (9 cc vs. 0.096 cc, p = 0.0106) to follicle hair bearing scalp and prevented alopecia. There were no recurrences in the Hippocampus area. CONCLUSIONS: HS-WBRT, with an 11-field set up as described, while attempting to conserve hippocampus radiation and maintain radiation dose to brain inadvertently spares follicle-bearing scalp and prevents alopecia.Item Open Access Enhanced efficacy of combined temozolomide and bromodomain inhibitor therapy for gliomas using targeted nanoparticles.(Nature communications, 2018-05-18) Lam, Fred C; Morton, Stephen W; Wyckoff, Jeffrey; Vu Han, Tu-Lan; Hwang, Mun Kyung; Maffa, Amanda; Balkanska-Sinclair, Elena; Yaffe, Michael B; Floyd, Scott R; Hammond, Paula TEffective treatment for glioblastoma (GBM) is limited by the presence of the blood-brain barrier (BBB) and rapid resistance to single agent therapies. To address these issues, we developed a transferrin-functionalized nanoparticle (Tf-NP) that can deliver dual combination therapies. Using intravital imaging, we show the ability of Tf-NPs to traverse intact BBB in mice as well as achieve direct tumor binding in two intracranial orthotopic models of GBM. Treatment of tumor-bearing mice with Tf-NPs loaded with temozolomide and the bromodomain inhibitor JQ1 leads to increased DNA damage and apoptosis that correlates with a 1.5- to 2-fold decrease in tumor burden and corresponding increase in survival compared to equivalent free-drug dosing. Immunocompetent mice treated with Tf-NP-loaded drugs also show protection from the effects of systemic drug toxicity, demonstrating the preclinical potential of this nanoscale platform to deliver novel combination therapies to gliomas and other central nervous system tumors.Item Open Access Fostering Radiation Oncology Physician Scientist Trainees Within a Diverse Workforce: The Radiation Oncology Research Scholar Track.(International journal of radiation oncology, biology, physics, 2021-06) Salama, Joseph K; Floyd, Scott R; Willett, Christopher G; Kirsch, David GThere is a need to foster future generations of radiation oncology physician scientists, but the number of radiation oncologists with sufficient education, training, and funding to make transformative discoveries is relatively small. A large number of MD/PhD graduates have entered he field of radiation oncology over the past 2 decades, but this has not led to a significant cohort of externally funded physician scientists. Because radiation oncologists leading independent research labs have the potential to make transformative discoveries that advance our field and positively affect patients with cancer, we created the Duke Radiation Oncology Research Scholar (RORS) Program. In crafting this program, we sought to eliminate barriers preventing radiation oncology trainees from becoming independent physician scientists. The RORS program integrates the existing American Board of Radiology Holman Pathway with a 2-year post-graduate medical education instructor position with 80% research effort at the same institution. We use a separate match for RORS and traditional residency pathways, which we hope will increase the diversity of our residency program. Since the inception of the RORS program, we have matched 2 trainees into our program. We encourage other radiation oncology residency programs at peer institutions to consider this training pathway as a means to foster the development of independent physician scientists and a diverse workforce in radiation oncology.Item Open Access Ipilmumab and cranial radiation in metastatic melanoma patients: a case series and review.(J Immunother Cancer, 2015) Schoenfeld, Jonathan D; Mahadevan, Anand; Floyd, Scott R; Dyer, Michael A; Catalano, Paul J; Alexander, Brian M; McDermott, David F; Kaplan, Irving DBACKGROUND: Ipilimumab improves survival in metastatic melanoma patients. This population frequently develops brain metastases, which have been associated with poor survival and are often treated with radiation. Therefore, outcomes following ipilimumab and radiation are of interest, especially given case reports and animal studies suggest combined treatment may generate abscopal responses outside the radiation field. FINDINGS: We reviewed sixteen consecutive melanoma patients who received 1 to 8 courses of radiation, with a sum total of 51, systematically evaluating abscopal responses by following the largest extra-cranial lesion. We also reviewed other series of patients treated with cranial radiation and ipilimumab. Our patients received between 1 and 8 courses of cranial radiation. Four patients received radiation concurrently with ipilimumab. Median survival was 14 months, and 17 months in patients initially treated with SRS. Interestingly, after radiotherapy, there was a 2.8-fold increased likelihood that the rate of extra-cranial index lesion response improved that didn't reach statistical significance (p = 0.07); this was more pronounced when ipilimumab was administered within three months of radiation (p < 0.01). CONCLUSION: Our experience and review of recently published series suggest ipilimumab and cranial radiation is well tolerated and can result in prolonged survival. Timing of ipilimumab administration in relation to radiation may impact outcomes. Additionally, our results demonstrate a trend for favorable systemic response following radiotherapy worthy of further evaluation in studies powered to detect potential synergies between radiation and immunotherapy.Item Open Access Novel Understandings of How Cancer Prevents and Responds to DNA Damage(2020) Edwards, DrakeUnderstanding the differences between normal and malignant tissue is required to find vulnerabilities in cancer that can be exploited. One of the hallmarks of cancer is its ability to sustain proliferative signaling, leading to unbridled cellular replication. This puts an increased pressure on the cell’s ability to maintain genome integrity and creates a potential vulnerability to be targeted by cancer therapies. Targeting how a cancer cell prevents or responds to DNA damage is one way to take advantage of this vulnerability.
My dissertation work aims to better understand this DNA damage response in cancer and tests two hypotheses: The first is whether inhibition of the transcriptional regulator BRD4 leads to an increase in transcription-replication conflicts, DNA damage, and cell death. The second is whether the tumor microenvironment alters the way cancer cells respond to DNA damage induced by radiation therapy in glioblastoma.
Effective spatio-temporal control of transcription and replication during S-phase is paramount to maintain genomic integrity and cell survival. My work shows that BRD4, a BET bromodomain protein and known transcriptional regulator, is important for preventing dysregulation of these systems leading to conflicts between the transcription and replication machinery in S-phase. We demonstrate that inhibition or degradation of BET bromodomain proteins leads to an accumulation of RNA:DNA hybrids, a known cause of transcription-replication conflicts, and causes increased DNA damage and cell death in cancer cells actively undergoing replication. Furthermore,
over-expression of full-length BRD4, which contains a P-TEFb interacting domain known to activate efficient transcription, is necessary and sufficient to rescuing this effect. These results give mechanistic insight into chemotherapeutics that target BRD4 currently in clinical trials.
In complementary work, we explored the effect that the extracellular environment of cancer plays in its response to DNA damage caused by radiation therapy. Standard methods of culturing cancer cells, which do not replicate the extracellular environment of a native tumor, have led to an incomplete understanding of response to therapies such as ionizing radiation in vivo. To understand the role that the tumor environment plays on the radiation response, we used both human and murine glioblastoma cells to show that organotypic brain slice culture was better able to recapitulate the expression profiles of in vivo tumors. Specifically, we saw that pathways involved in multicellular processes, cell morphogenesis, and the extracellular matrix were not only significantly upregulated in glioblastoma cells cultured on brain slices compared to in vitro culture but were also critically important to radiation survival.
Collectively, this dissertation provides novel understandings of how cancer cells prevent and respond to DNA damage as well as a framework for future work in cancer biology.
Item Open Access The bromodomain protein Brd4 insulates chromatin from DNA damage signalling.(Nature, 2013-06-13) Floyd, Scott R; Pacold, Michael E; Huang, Qiuying; Clarke, Scott M; Lam, Fred C; Cannell, Ian G; Bryson, Bryan D; Rameseder, Jonathan; Lee, Michael J; Blake, Emily J; Fydrych, Anna; Ho, Richard; Greenberger, Benjamin A; Chen, Grace C; Maffa, Amanda; Del Rosario, Amanda M; Root, David E; Carpenter, Anne E; Hahn, William C; Sabatini, David M; Chen, Clark C; White, Forest M; Bradner, James E; Yaffe, Michael BDNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response. Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage.Item Open Access The effect of MLC leaf width in single-isocenter multi-target radiosurgery with volumetric modulated arc therapy(Journal of Radiosurgery and SBRT, 2019-01-01) Abisheva, Zhanerke; Floyd, Scott R; Salama, Joseph K; Kirkpatrick, John; Yin, Fang-Fang; Moravan, Michael J; Giles, William; Adamson, Justus© 2019 Old City Publishing, Inc. Purpose: Single-isocenter multi-target (SIMT) volumetric modulated arc therapy (VMAT) is primarily limited to linear accelerators utilizing 2.5 mm leaf width MLCs. We explore feasibility of applying this technique to linear accelerators utilizing MLCs with leaf width of 5 mm. Methods: Twenty patients with 3-10 intracranial brain metastases originally treated with 2.5 mm MLCs were re-planned using 5 mm MLCs and relevant dosimetric indices were compared. We also evaluated various strategies of adding VMAT arcs to mitigate degradations of dose quality values. Results: Wider MLCs caused small changes in total MUs (5827 ± 2334 vs 5572 ± 2220, p = 0.006), and conformity index (CI) (2.22% ± 0.05%, p = 0.045), but produced more substantial increases in brain V30%[%] and V50%[%] (27.75% ± 0.16% and 20.04% ± 0.13% respectively, p < 0.001 for both), and V12Gy[cc] (16.91% ± 0.12%, p < 0.001). Conclusion: SIMT radiosurgery delivered via VMAT using 5 mm wide MLCs can achieve similar CI compared to that using 2.5 mm leaf width MLCs but with moderately increased isodose spill, which can be only partially mitigated by increasing the number of VMAT arcs.Item Open Access The role of whole brain radiation therapy in the management of melanoma brain metastases.(Radiat Oncol, 2014-06-22) Dyer, Michael A; Arvold, Nils D; Chen, Yu-Hui; Pinnell, Nancy E; Mitin, Timur; Lee, Eudocia Q; Hodi, F Stephen; Ibrahim, Nageatte; Weiss, Stephanie E; Kelly, Paul J; Floyd, Scott R; Mahadevan, Anand; Alexander, Brian MBACKGROUND: Brain metastases are common in patients with melanoma, and optimal management is not well defined. As melanoma has traditionally been thought of as "radioresistant," the role of whole brain radiation therapy (WBRT) in particular is unclear. We conducted this retrospective study to identify prognostic factors for patients treated with stereotactic radiosurgery (SRS) for melanoma brain metastases and to investigate the role of additional up-front treatment with whole brain radiation therapy (WBRT). METHODS: We reviewed records of 147 patients who received SRS as part of initial management of their melanoma brain metastases from January 2000 through June 2010. Overall survival (OS) and time to distant intracranial progression were calculated using the Kaplan-Meier method. Prognostic factors were evaluated using the Cox proportional hazards model. RESULTS: WBRT was employed with SRS in 27% of patients and as salvage in an additional 22%. Age at SRS > 60 years (hazard ratio [HR] 0.64, p = 0.05), multiple brain metastases (HR 1.90, p = 0.008), and omission of up-front WBRT (HR 2.24, p = 0.005) were associated with distant intracranial progression on multivariate analysis. Extensive extracranial metastases (HR 1.86, p = 0.0006), Karnofsky Performance Status (KPS) ≤ 80% (HR 1.58, p = 0.01), and multiple brain metastases (HR 1.40, p = 0.06) were associated with worse OS on univariate analysis. Extensive extracranial metastases (HR 1.78, p = 0.001) and KPS (HR 1.52, p = 0.02) remained significantly associated with OS on multivariate analysis. In patients with absent or stable extracranial disease, multiple brain metastases were associated with worse OS (multivariate HR 5.89, p = 0.004), and there was a trend toward an association with worse OS when up-front WBRT was omitted (multivariate HR 2.56, p = 0.08). CONCLUSIONS: Multiple brain metastases and omission of up-front WBRT (particularly in combination) are associated with distant intracranial progression. Improvement in intracranial disease control may be especially important in the subset of patients with absent or stable extracranial disease, where the competing risk of death from extracranial disease is low. These results are hypothesis generating and require confirmation from ongoing randomized trials.