194 research outputs found
A functional role of DOT1L in luminal muscle invasive bladder cancer
No full textThe full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.Medicine, Faculty ofGraduat
Oncofetal chondroitin sulfate as a target in solid tumors
No full textThe malaria parasite Plasmodium falciparum expresses VAR2CSA proteins on the surface of infected red blood cells that bind a distinct type of chondroitin sulfate (CS) glycosaminoglycan (GAG) exclusively present in the placenta. This interaction is the key underlying cause of pregnancy-associated malaria outbreaks in endemic regions of the world. Interestingly, placental-type CS GAGs become re-expressed in multiple human cancers as a secondary oncofetal CS (ofCS) modification to proteoglycans.
I used this specific feature of malaria tropism to develop novel cancer diagnostic and therapeutic strategies. By incorporating the recombinant VAR2CSA protein (rVAR2) sequence into a chimeric antigen receptor (CAR), I was able to develop a universal anti-ofCS CAR (VAR2-CAR) with affinity for tumor cells expressing ofCS. In order to obtain a strong safety profile, I designed a switch-CAR system in which a non-binding CAR could be secondarily ‘armed’ with a rVAR2-switch molecule producing functional ofCS-reactive sVAR2-CAR T cells. The population of armed CAR T cells was controlled by the concentration of the switch molecule and importantly, halting the addition of the switch molecule through clonal expansion, cleared the armed population of sVAR2-CAR T cells from the organism post-treatment.
Co-culturing the sVAR2-CAR T cells with human tumor cells resulted in activation of the CAR T cells as assessed by expression of activation markers and production of cytokines. The sVAR2-CAR T cells were able to engage diverse ofCS-positive tumor cells and effectively eliminate them. Finally, two treatments of sVAR2-CAR T cells were able to markedly decrease tumor burden in a murine xenograft model of bladder cancer. My data provide proof-of-concept for an rVAR2-armed CAR-T cell approach to treat ofCS-positive cancers.
In addition, ofCS is an efficient and specific target for isolating tumor cells from blood samples. A variety of ofCS positive cancer cells spiked into the blood were successfully isolated by employing rVAR2-coated magnetic beads. Combining rVAR2-technology and nucleic acid detection, I was able to develop a new strategy for isolating and identifying circulating tumor cells (CTC). Taken together, the exclusive expression of ofCS on tumors and the high specificity of rVAR2 to ofCS, can be complementary exploited for tumor diagnosis and treatment.Medicine, Faculty ofMedicine, Department ofGraduat
Pulsed photothermal therapy as a precondition for immune checkpoint blockade
No full textPhotothermal therapy (PTT) refers to the use of photothermal agents (plasmonic nanoparticles) to convert electromagnetic radiation in the near infrared region (NIR) to heat and kill the target (tumor) cells. Due to the high efficacy and minimal side effects, this therapy has been studied extensively in recent years and gained significant clinical traction. In addition to cell death, PTT can be used to induce mild hyperthermia in temperatures between 39-45°C, which can trigger certain physiological responses like immune stimulation and cytokine secretion. This immune response can be reinforced in combination with cancer immunotherapies such as immune checkpoint blockade. Although research has demonstrated that pulsed lasers generate more localized heating than continuous wave (CW) lasers, PTT is traditionally administered by CW lasers, and there is a limited amount of research employing pulsed laser equipment. Using the conventional low power CW laser settings requires long treatment times, induces unwanted heat damage to the surrounding healthy tissue, and leads to higher pain sensation in patients; all limiting parameters in clinical translation of PTT. I hypothesized that a novel pulsed laser device could produce a photothermal effect sufficient to stimulate immune cells within the tumor microenvironment and potentially augment the effectiveness of immune checkpoint blockade therapy. Here, we report the engineering and validation of a novel long pulsed laser device able to induce selective and localized mild hyperthermia in tumors while reducing the heat affected zone and unwanted damage to surrounding tissue. Long-pulsed PTT induces acute necrotic cell death in heat affected areas and the release of tumor associated antigens. This antigen release is immunostimulatory and triggers maturation and stimulation of CD80/CD86 in dendritic cells in vivo. In vivo antitumor studies in a MB49 syngeneic bladder cancer model led to significantly reduced tumor growth and increased survival indicating long-pulse PTT can be an effective and promising strategy to enhanced therapeutic responses when combined with anti-PD-L1 immune checkpoint inhibitors.Medicine, Faculty ofMedicine, Department ofGraduat
Regulation and targeting of chondroitin sulfate in pediatric solid tumors
No full textPediatric solid tumors often present with a non-permissive cold immune cell microenvironment that contributes to immunotherapy resistance. Being a major component and active player in the cancer cell glycocalyx and microenvironment, glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) assist in essentially all hallmarks of cancer, making CS an attractive target in cancer. Although abnormal presentation of CS in cancer is well-established, little is known about its regulation in cancer.
This thesis describes two distinct, but related projects. The first project investigated the regulation and functional impact of GAGs, particularly CS, during endoplasmic reticulum (ER) stress in cancer cells. Quantitative and qualitative analyses of GAGs revealed an altered GAG architecture in osteosarcoma cells in response to ER stress with 4-O-sulfated CS levels increasing upon pharmacologically induced ER stress compared to controls. Using cells engineered to display distinct GAG repertoires, we showed that 4-O-sulfated CS protects cells from apoptosis during prolonged ER stress, regulates cellular oxidative homeostasis, and supports the adaptive expression of GRP78, a master regulator of the unfolded protein response. Altogether, we report that perturbations in the ER homeostasis trigger CS biosynthesis, suggesting that these pathways are linked.
A distinct type of 4-O-sulfated CS in the placenta functions as a receptor for the Plasmodium falciparum lectin, VAR2CSA, during pregnancy-associated malaria. Due to the similarities to 4-O-sulfated CS in solid tumors, termed oncofetal CS (ofCS), the malaria VAR2CSA-4-O-sulfated CS binding-module can be exploited for cancer targeting. The second project describes the development of a novel type of bispecific immunotherapy for pediatric solid tumors based on the ability of recombinant VAR2CSA (rVAR2) to target ofCS in pediatric solid tumors. We engineered an interleukin-2 variant (IL-2v) genetically fused to rVAR2 (ILVAR) to specifically bind ofCS on the surface of cancer cells via rVAR2, while engaging immune effector cells via IL2v. We demonstrated that the distinct molecular constituents of ILVAR are functional, thereby enabling anti-cancer cytotoxicity in pre-clinical tumor models. Collectively, the data serve as a proof-of-concept for further evaluating the feasibility of using ILVAR to activate targeted immune responses within the ofCS-positive tumor microenvironment of pediatric cancers.Medicine, Faculty ofMedicine, Department ofGraduat
Targeted IL-2 immunocytokine therapy directed at oncofetal chondroitin sulfate for pediatric solid tumors
No full textThe full abstract for this thesis is available in the body of the thesis, and will be available when the embargo expires.Medicine, Faculty ofMedicine, Department ofGraduat
Post-translational modifications in bladder cancer: Expanding the tumor target repertoire.
No full textOver the past decade, genomic and transcriptomic analyses have uncovered promising tumor antigens including immunotherapeutic targets in bladder cancer (BCa). Conventional tumor antigens are proteins expressed on the plasma membrane of tumor cells such as EGFR, FGFR3, and ERBB2 in BCa, which can be targeted by antibodies or similar epitope-specific binding reagents. The cellular proteome consists of ∼100,000 proteins but the expression of these proteins is rarely unique to tumor cells. Many tumor-associated proteins are post-translationally modified with phosphorylation, glycosylation, ubiquitination, or SUMOylation moieties. Although these modifications expand the complexity, they potentially offer novel targeting opportunities across tumor sub-populations. Experimental targeting of cancer-specific post-translational modifications (PTMs) has shown encouraging results in pre-clinical models of BCa, which could potentially overcome issues with inherent intra-tumor heterogeneity due to simultaneous expression on different proteins. Here, we review current knowledge on post-translational modifications in BCa and highlight recent efforts in experimental targeting strategies
Abstract B15: Dissecting the molecular mechanism of dianhydrogalactitol (VAL-083) activity in cancer treatment
No full textAbstract
Introduction: Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent causing N7-guanine-methylation and inter-strand DNA crosslinks. In China, VAL-083 is approved to use in the chemotherapeutic treatment of lung cancer and chronic myelogenous leukemia. In the United States, VAL-083 is currently undergoing investigation as a new therapy in the treatment of temozolomide refractory glioblastoma (GBM). VAL-083 is a small water-soluble molecule that readily crosses blood-brain-barrier and accumulates in the tumor tissues in brain, making it a good candidate for targeting brain malignancies, such as GBM and medulloblastoma. Historical data from preclinical studies and clinical trials sponsored by the US National Cancer Institute (NCI) support anti-neoplastic effects of VAL-083 in a variety of cancer types in addition to GBM, including lung cancer, leukemia, cervical cancer, and ovarian cancer. However, the detailed molecular mechanisms mediating VAL-083 sensitivity or resistance in cancer cells is still unclear. Therefore, we investigated the distinct mechanism of action of VAL-083 in different cancer cell lines.
Methods: VAL-083 cytotoxicity was evaluated in a panel of human non-small cell lung cancer (NSCLC) cell lines (A549, H2122, H1792, and H23) and prostate cancer cell lines (PC3 and LNCaP) by crystal violet assays. Cell cycle analysis and DNA damage response were investigated by propidium iodide (PI) and immunofluorescent (IF) staining. Western blot and IF staining analyses were employed to elucidate the DNA repair mechanism involved in VAL-083-treated cancer cells.
Results: In this study, we report new insights into VAL-083's mechanisms of action by showing that VAL-083 induces irreversible cell-cycle arrest and cell death caused by replication-dependent DNA double-strand breaks (DSBs). In all the cancer cells tested, VAL-083 showed broad cytotoxicity with an IC50 range of 3.1 - 25.7 μM. In lung cancer (H2122, H1792, and A549) and prostate cancer (PC3 and LNCaP) cell lines, VAL-083 treatment caused irreversible cell cycle arrest at S/G2 phase as measured by PI and IF staining in synchronized cells, indicating that VAL-083-induced inter-strand crosslinks result in more difficult to repair DNA lesions during replication, including DSBs. Western blot and IF analyses of DNA repair markers were employed to investigate the DNA damage response induced by VAL-083 in cancer cells. The S/G2 phase cell cycle arrest and the increased γH2A.X (an indication of DSB lesions) expression persisted for 48-72 h after treatment with VAL-083, indicating prolonged unrepaired DNA lesions caused by VAL-083. VAL-083 pulse-treatment led to persistent phosphorylation of DSB sensors ataxia telangiectasia mutated (ATM), single-strand DNA-binding replication protein A (RPA32), and H2A.X. Furthermore, Western blot analyses also demonstrated activation of the downstream effectors of ATM and ataxia telangiectasia and Rad3-related protein (ATR) kinases, Chk2 (T68) and Chk1 (S317 and S345). These results suggest that VAL-083-induced persistent and irreversible DNA damage activated the homologous recombination DNA repair signaling pathway in the panel of cancer cells studied.
Conclusions: VAL-083 displayed broad anti-neoplastic activity in different lung and prostate cancer cells through the replication-dependent DSBs. Elucidation of the molecular mechanisms underlying VAL-083 cytotoxicity provides guidance for improved treatment strategies for cancer patients with VAL-083 in either single or combination regimens.
Citation Format: Beibei Zhai, Anne Steino, Jeffrey Bacha, Dennis Brown, Mads Daugaard. Dissecting the molecular mechanism of dianhydrogalactitol (VAL-083) activity in cancer treatment [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B15.</jats:p
Targeting Common but Complex Proteoglycans on Breast Cancer Cells and Stem Cells Using Evolutionary Refined Malaria Proteins
No full textÅh, Instagram [Elektronisk resurs] : Anmeldelse af Mads Eslund imnothererepresentinghardbodies
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