Understanding the meaning of the human genome codes is one of the keys to unlock the secrets of life. Despite having a fairly good grasp of the sequences of the human genome, we are still far from... Show moreUnderstanding the meaning of the human genome codes is one of the keys to unlock the secrets of life. Despite having a fairly good grasp of the sequences of the human genome, we are still far from understanding the functions of most parts of the genome and their involvement in diseases. The application of the CRISPR-Cas9 genome-editing systems revolutionized the way to study the function of the genome, not only the coding genes but also the non-coding genome. In this thesis, multiple CRISPR screening systems were designed and used to study the transport of chemotherapeutic drugs and the functions of non-coding regulatory elements in distinct biological pathways. Using the CRISPR screening systems, we identified previously known drug exporters such as ABCB1 and ABCG2 and a new doxorubicin importer gene SLC2A3 (GLUT3). We used an innovative dual-CRISPR system to study the functions of noncoding regulatory elements (NCREs) in their endogenous genomic environment. We provide the broad research community with a new tool to study the functions of NCREs in different biological aspects, and it is expected many more important NCREs will be discovered in the future, which would not only be relevant to fundamental biology but also to human diseases. Show less
PurposeTissue-specific drug uptake has not been well studied, compared to the deeper understanding of drug resistance mediated by the cellular efflux system such as MDR1 proteins. It has been... Show morePurposeTissue-specific drug uptake has not been well studied, compared to the deeper understanding of drug resistance mediated by the cellular efflux system such as MDR1 proteins. It has been suggested that many drugs need active or defined transporters to pass the cell membrane. In contrast to efflux components induced after anti-cancer drugs reach the intracellular compartment, drug importers are required for initial drug responses. Furthermore, tissue-specific uptake of anti-cancer drugs may directly impact the side effects of many drugs when they accumulate in healthy tissues. Therefore, linking anti-cancer drugs to their respective drug import transporters would directly help to predict drug responses, whilst minimizing side effects.MethodsTo identify drug transporters of the commonly used anti-cancer drug doxorubicin, we performed focused CRISPR activation and knockout genetic screens targeting all potential membrane-associated transporters and proteins. We monitored the direct uptake of doxorubicin by fluorescence-activated cell sorting (FACS) as the screening readout for identifying transporters/proteins directly involved in doxorubicin uptake.ResultsIntegrating the data from these comprehensive CRISPR screenings, we confirmed previously indicated doxorubicin exporters such as ABCB1 and ABCG2 genes, and identified novel doxorubicin importer gene SLC2A3 (GLUT3). Upregulation of SLC2A3 led to higher doxorubicin uptake and better cell killing, indicating SLC2A3 could be a new marker to predict doxorubicin drug response and minimize side effects for the personalized application of this conventional chemotherapeutic drug.ConclusionsOur study provides a comprehensive way for identifying drug transporters, as exemplified by the commonly used anti-cancer drug doxorubicin. The newly identified importers may have direct clinical implications for the personalized application of doxorubicin in treating distinct tumors. Our results also highlight the necessity of combining both CRISPR knockout and CRISPR activation genetic screens to identify drug transporters. Show less
Neo(adjuvant) systemic treatment regimens containing anthracyclines such as doxorubicin cause a significant risk of heart failure. These regimens are one of the corner stones of osteosarcoma... Show moreNeo(adjuvant) systemic treatment regimens containing anthracyclines such as doxorubicin cause a significant risk of heart failure. These regimens are one of the corner stones of osteosarcoma treatment, and therefore several guidelines are in place to steer cardiotoxicity monitoring through baseline risk stratification and cardiac surveillance during and after completion of cancer therapy. Importantly, baseline risk stratification modules are dependent on age, prior cardiovascular disease and cardiovascular risk factors. Because the majority of osteosarcoma patients are below 30 years of age these criteria rarely apply and most patients are assigned to low or medium risk categories, whereas cardiovascular complications have profound impact on morbidity and mortality in this young population. Therefore, cardiac surveillance is very important in this group for timely detection of cardiotoxicity. Moreover, when severe cardiotoxicity that requires advanced heart failure treatment occurs, a cancer diagnosis has significant implications on treatment options, i.e. mechanical circulatory support and heart transplantation. These challenges are presented in this case of a patient without clinical risk factors admitted with cardiogenic shock requiring advanced heart failure treatment within 1 month after completion of doxorubicin containing chemotherapy for the treatment of high grade osteosarcoma. Show less
The anthracycline drug doxorubicin, is one of the most used chemotherapeutic drugs, with over one million patients treated every year. However, the exact molecular mechanism by which this drug kill... Show moreThe anthracycline drug doxorubicin, is one of the most used chemotherapeutic drugs, with over one million patients treated every year. However, the exact molecular mechanism by which this drug kill tumor cells remain unclear. In addition, treatment with anthracyclines coincides with severe adverse effects such as cardiotoxicity, secondary tumor formation and gonadotoxicity. Understanding how these highly effective anticancer drugs function and why they cause these severe toxicities would have tremendous impact on cancer treatment and the quality of life of cancer survivors. Therefore, even today, studying old anticancer drugs has high therapeutic potential and opens new exciting paths to improve currently available treatment options. Show less
Verschoor, A.J.; Litiere, S.; Marreaud, S.; Judson, I.; Toulmonde, M.; Wardelmann, E.; ... ; Gelderblom, H. 2020
Background Doxorubicin based chemotherapy is standard first line treatment for patients with soft tissue sarcoma. Currently several options to improve survival after doxorubicin based chemotherapy... Show moreBackground Doxorubicin based chemotherapy is standard first line treatment for patients with soft tissue sarcoma. Currently several options to improve survival after doxorubicin based chemotherapy are being studied. This study reports on survival after completing 6 cycles of doxorubicin containing first line treatment, which is important when designing studies trying to improve outcomes of first line treatment. Methods A retrospective database analysis was performed on 2045 patients from 12 EORTC sarcoma trials (inclusion period 1980-2012) receiving first line doxorubicin based chemotherapy for advanced soft tissue sarcoma in order to establish progression free survival and overall survival after completing 6 cycles of first line doxorubicin based chemotherapy. Endpoints were overall survival and progression free survival. Factors studied were histologic subtype and type of doxorubicin chemotherapy. Results 748 of 2045 (36.6%) received at least 6 cycles and did not progress during or at the end of chemotherapy. 475 of 2045 (23.2%) of patients received exactly 6 cycles and did not progress during or at the end of chemotherapy. Median progression free survival after 6 cycles of doxorubicin based chemotherapy was 4.2 months (95% confidence interval 3.7-4.8) and median overall survival 15.7 months (14.0-17.8). Median progression free survival and overall survival from randomisation/registration were 8.7 months (95% confidence interval 8.2-9.1) and 20.1 months (95% confidence interval 18.3-22.3) respectively. Significant differences in progression free survival were found between chemotherapy regimens, but not for overall survival. These data are also reported for patients receiving 7 or more cycles of chemotherapy and for patients with 3 or more cycles of chemotherapy. Conclusion This large retrospective study is the first to report progression free survival and overall survival after completion of 6 cycles of first line doxorubicin containing chemotherapy. These results are important when designing new studies exploring for example maintenance therapy after doxorubicin based chemotherapy. Show less
The aim of my work was to identify novel proteins that are involved in different aspects of disease biology, in the hope that we can target these to treat the disease. Using large-scale screening... Show moreThe aim of my work was to identify novel proteins that are involved in different aspects of disease biology, in the hope that we can target these to treat the disease. Using large-scale screening technologies, I found a novel regulator of chemosensitivity to the anti-cancer drugs etoposide and doxorubicin. Furthermore, novel proteins and drugs that target MHCII antigen presentation were picked up. Interestingly, one of these drugs is currently used in the clinic to treat MHCII-dependent autoimmune diseases, but for which the mechanism of action is unknown. Lastly, these screens yielded enzymes that control the dynamics of endosomes in the cell, which can potentially be targeted in cancers that rely on overactivation of growth signaling receptors. Taken together, my work has provided new potential targets for the treatment or classification of several tumor types, as well as a potential mechanism of action for a widely used drug. Show less
Osteosarcoma is the most frequent malignant bone tumor affecting mainly adolescents and people older than 50years old. Current treatment involves chemotherapy and surgical removal. Despite efforts... Show moreOsteosarcoma is the most frequent malignant bone tumor affecting mainly adolescents and people older than 50years old. Current treatment involves chemotherapy and surgical removal. Despite efforts to find a cure, there is 70% 5year survival rate. For patients that present metastasis at the moment of diagnosis, the prognosis is worse. Additionally, many do not respond well to chemotherapy. The aim of this research was to find new treatment options for patients with osteosarcoma. I searched for inhibitors that could sensitize osteosarcoma cells to chemotherapy. In this thesis, it is shown that targeting cell cycle regulators, proteins involved in apoptosis inhibition, and cellular pathways involved in survival, proliferation and migration are promising candidates for further research. Show less
Gelderblom, H.; Blay, J.Y.; Seddon, B.M.; Leahy, M.; Ray-Coquard, I.; Sleijfer, S.; ... ; Hohenberger, P. 2014
Immunotherapy and chemotherapy are major strategies in current cancer treatments. In the first half of this thesis, a new way of antigen cross-presentation from apoptotic cells to APCs, mediated by... Show moreImmunotherapy and chemotherapy are major strategies in current cancer treatments. In the first half of this thesis, a new way of antigen cross-presentation from apoptotic cells to APCs, mediated by gap junctions, is described. And potential anti-tumor applications of gap junction mediated antigen cross-presentation are discussed in the context of the current knowledge. In the second half of this thesis, an unexpected novel mechanism of action__selective induction of histone eviction__is described, which could explain the long standing clinical observation that anthracycline members, like doxorubicin, daunorubicin and aclarubicin, are efficient but with long term side-effects such as cardiotoxicity Show less
In order to form a distant metastasis, a cancer cell has to migrate out of the primary tumor, intravasate into a blood or a lymphatic vessel, subsequently survive in the absence of cell-cell and... Show moreIn order to form a distant metastasis, a cancer cell has to migrate out of the primary tumor, intravasate into a blood or a lymphatic vessel, subsequently survive in the absence of cell-cell and cell-matrix interactions, extravasate the blood or lymphatic vessel, migrate through the target organ and finally proliferate to grow out into a full metastasis. During all of these processes, specific kinases are involved in the concerted activation of distinct signaling pathways. We hypothesised that the protein tyrosine kinase FAK plays a crucial role in one or multiple of the processes involved in the formation of metastases. Therefore, the overall aim of the studies described in this thesis was to investigate the role of the non-receptor protein tyrosine kinase FAK in the distinct processes involved in tumorigenesis and metastasis and to unravel the involved downstream signaling pathways. Moreover, the potential of a combined therapy of the inhibition of FAK and exposure to the cytostatic doxorubicin was tested, as well as dissection of the intracellular events downstream of FAK. Show less
