Most DNA repair defects do not modify the relationship between relative biological effectiveness and linear energy transfer in CRISPR‐edited cells
Background Cancer is a highly heterogeneous disease, driven by frequent genetic alterations which have significant effects on radiosensitivity. However, radiotherapy for a given cancer type is typically given with a standard dose determined from population‐level trials. As a result, a proportion of...
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| description | Background
Cancer is a highly heterogeneous disease, driven by frequent genetic alterations which have significant effects on radiosensitivity. However, radiotherapy for a given cancer type is typically given with a standard dose determined from population‐level trials. As a result, a proportion of patients are under‐ or over‐dosed, reducing the clinical benefit of radiotherapy. Biological optimization would not only allow individual dose prescription but also a more efficient allocation of limited resources, such as proton and carbon ion therapy.
Proton and ion radiotherapy offer an advantage over photons due to their elevated Relative Biological Effectiveness (RBE) resulting from their elevated Linear Energy Transfer (LET). Despite significant interest in optimizing LET by tailoring radiotherapy plans, RBE's genetic dependence remains unclear.
Purpose
The aim of this study is to better define the RBE/LET relationship in a panel of cell lines with different defects in DSB repair pathways, but otherwise identical biological features and genetic background to isolate these effects.
Methods
Normal human cells (RPE1), genetically modified to introduce defects in DNA double‐strand break (DSB) repair genes, ATM, BRCA1, DCLRE1C, LIG4, PRKDC and TP53, were used to map the RBE‐LET relationship. Cell survival was measured with clonogenic assays after exposure to photons, protons (LET 1 and 12 keV/µm) and alpha particles (129 keV/µm). Gene knockout sensitizer enhancement ratio (SER) values were calculated as the ratio of the mean inactivation dose (MID) of wild‐type cells to repair‐deficient cells, and RBE values were calculated as the ratio of the MID of X‐ray and particle irradiated cells. 53BP1 foci were used to quantify radiation‐induced DSBs and their repair following irradiation.
Results
Deletion of NHEJ genes had the greatest impact on photon sensitivity (ATM−/− SER = 2.0 and Lig4−/− SER = 1.8), with genes associated with HR having smaller effects (BRCA1−/− SER = 1.2). Wild‐type cells showed RBEs of 1.1, 1.3, 5.0 for low‐ and high‐LET protons and alpha particles respectively. SERs for different genes were independent of LET, apart from NHEJ knockouts which proved to be markedly hypersensitive across all tested LETs. Due to this hypersensitivity, the impact of high LET was reduced in cell models lacking the NHEJ repair pathway. HR‐defective cells had moderately increased sensitivity across all tested LETs, but, notably, the contribution of HR pathway to surviv |
| doi_str_mv | 10.1002/mp.16764 |
| format | Article |
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Cancer is a highly heterogeneous disease, driven by frequent genetic alterations which have significant effects on radiosensitivity. However, radiotherapy for a given cancer type is typically given with a standard dose determined from population‐level trials. As a result, a proportion of patients are under‐ or over‐dosed, reducing the clinical benefit of radiotherapy. Biological optimization would not only allow individual dose prescription but also a more efficient allocation of limited resources, such as proton and carbon ion therapy.
Proton and ion radiotherapy offer an advantage over photons due to their elevated Relative Biological Effectiveness (RBE) resulting from their elevated Linear Energy Transfer (LET). Despite significant interest in optimizing LET by tailoring radiotherapy plans, RBE's genetic dependence remains unclear.
Purpose
The aim of this study is to better define the RBE/LET relationship in a panel of cell lines with different defects in DSB repair pathways, but otherwise identical biological features and genetic background to isolate these effects.
Methods
Normal human cells (RPE1), genetically modified to introduce defects in DNA double‐strand break (DSB) repair genes, ATM, BRCA1, DCLRE1C, LIG4, PRKDC and TP53, were used to map the RBE‐LET relationship. Cell survival was measured with clonogenic assays after exposure to photons, protons (LET 1 and 12 keV/µm) and alpha particles (129 keV/µm). Gene knockout sensitizer enhancement ratio (SER) values were calculated as the ratio of the mean inactivation dose (MID) of wild‐type cells to repair‐deficient cells, and RBE values were calculated as the ratio of the MID of X‐ray and particle irradiated cells. 53BP1 foci were used to quantify radiation‐induced DSBs and their repair following irradiation.
Results
Deletion of NHEJ genes had the greatest impact on photon sensitivity (ATM−/− SER = 2.0 and Lig4−/− SER = 1.8), with genes associated with HR having smaller effects (BRCA1−/− SER = 1.2). Wild‐type cells showed RBEs of 1.1, 1.3, 5.0 for low‐ and high‐LET protons and alpha particles respectively. SERs for different genes were independent of LET, apart from NHEJ knockouts which proved to be markedly hypersensitive across all tested LETs. Due to this hypersensitivity, the impact of high LET was reduced in cell models lacking the NHEJ repair pathway. HR‐defective cells had moderately increased sensitivity across all tested LETs, but, notably, the contribution of HR pathway to survival appeared independent of LET. Analysis of 53BP1 foci shows that NHEJ‐defective cells had the least DSB repair capacity after low LET exposure, and no visible repair after high LET exposure. HR‐defective cells also had slower repair kinetics, but the impact of HR defects is not as severe as NHEJ defects.
Conclusions
DSB repair defects, particularly in NHEJ, conferred significant radiosensitivity across all LETs. This sensitization appeared independent of LET, suggesting that the contribution of different DNA repair pathways to survival does not depend on radiation quality.</description><identifier>ISSN: 0094-2405</identifier><identifier>EISSN: 2473-4209</identifier><identifier>DOI: 10.1002/mp.16764</identifier><identifier>PMID: 37753877</identifier><language>eng</language><publisher>United States</publisher><subject>Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR—Cas9 ; DNA damage ; DNA Repair ; high LET ; homologous recombination ; Humans ; Linear Energy Transfer ; Neoplasms ; non‐homologous end joining ; proton therapy ; Protons ; radiation ; radiosensitivity ; RBE ; Relative Biological Effectiveness</subject><ispartof>Medical physics (Lancaster), 2024-01, Vol.51 (1), p.591-600</ispartof><rights>2023 The Authors. published by Wiley Periodicals LLC on behalf of American Association of Physicists in Medicine.</rights><rights>2023 The Authors. Medical Physics published by Wiley Periodicals LLC on behalf of American Association of Physicists in Medicine.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3554-49d4ed4b550e30d0bdeadf38f7fc6f61029d8cbe70ca7eb8bf0b72ee660031683</citedby><cites>FETCH-LOGICAL-c3554-49d4ed4b550e30d0bdeadf38f7fc6f61029d8cbe70ca7eb8bf0b72ee660031683</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fmp.16764$$EPDF$$P50$$Gwiley$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fmp.16764$$EHTML$$P50$$Gwiley$$Hfree_for_read</linktohtml><link.rule.ids>314,777,781,1412,27905,27906,45555,45556</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/37753877$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Guerra Liberal, Francisco D. C.</creatorcontrib><creatorcontrib>Parsons, Jason L.</creatorcontrib><creatorcontrib>McMahon, Stephen J.</creatorcontrib><title>Most DNA repair defects do not modify the relationship between relative biological effectiveness and linear energy transfer in CRISPR‐edited cells</title><title>Medical physics (Lancaster)</title><addtitle>Med Phys</addtitle><description>Background
Cancer is a highly heterogeneous disease, driven by frequent genetic alterations which have significant effects on radiosensitivity. However, radiotherapy for a given cancer type is typically given with a standard dose determined from population‐level trials. As a result, a proportion of patients are under‐ or over‐dosed, reducing the clinical benefit of radiotherapy. Biological optimization would not only allow individual dose prescription but also a more efficient allocation of limited resources, such as proton and carbon ion therapy.
Proton and ion radiotherapy offer an advantage over photons due to their elevated Relative Biological Effectiveness (RBE) resulting from their elevated Linear Energy Transfer (LET). Despite significant interest in optimizing LET by tailoring radiotherapy plans, RBE's genetic dependence remains unclear.
Purpose
The aim of this study is to better define the RBE/LET relationship in a panel of cell lines with different defects in DSB repair pathways, but otherwise identical biological features and genetic background to isolate these effects.
Methods
Normal human cells (RPE1), genetically modified to introduce defects in DNA double‐strand break (DSB) repair genes, ATM, BRCA1, DCLRE1C, LIG4, PRKDC and TP53, were used to map the RBE‐LET relationship. Cell survival was measured with clonogenic assays after exposure to photons, protons (LET 1 and 12 keV/µm) and alpha particles (129 keV/µm). Gene knockout sensitizer enhancement ratio (SER) values were calculated as the ratio of the mean inactivation dose (MID) of wild‐type cells to repair‐deficient cells, and RBE values were calculated as the ratio of the MID of X‐ray and particle irradiated cells. 53BP1 foci were used to quantify radiation‐induced DSBs and their repair following irradiation.
Results
Deletion of NHEJ genes had the greatest impact on photon sensitivity (ATM−/− SER = 2.0 and Lig4−/− SER = 1.8), with genes associated with HR having smaller effects (BRCA1−/− SER = 1.2). Wild‐type cells showed RBEs of 1.1, 1.3, 5.0 for low‐ and high‐LET protons and alpha particles respectively. SERs for different genes were independent of LET, apart from NHEJ knockouts which proved to be markedly hypersensitive across all tested LETs. Due to this hypersensitivity, the impact of high LET was reduced in cell models lacking the NHEJ repair pathway. HR‐defective cells had moderately increased sensitivity across all tested LETs, but, notably, the contribution of HR pathway to survival appeared independent of LET. Analysis of 53BP1 foci shows that NHEJ‐defective cells had the least DSB repair capacity after low LET exposure, and no visible repair after high LET exposure. HR‐defective cells also had slower repair kinetics, but the impact of HR defects is not as severe as NHEJ defects.
Conclusions
DSB repair defects, particularly in NHEJ, conferred significant radiosensitivity across all LETs. This sensitization appeared independent of LET, suggesting that the contribution of different DNA repair pathways to survival does not depend on radiation quality.</description><subject>Clustered Regularly Interspaced Short Palindromic Repeats</subject><subject>CRISPR—Cas9</subject><subject>DNA damage</subject><subject>DNA Repair</subject><subject>high LET</subject><subject>homologous recombination</subject><subject>Humans</subject><subject>Linear Energy Transfer</subject><subject>Neoplasms</subject><subject>non‐homologous end joining</subject><subject>proton therapy</subject><subject>Protons</subject><subject>radiation</subject><subject>radiosensitivity</subject><subject>RBE</subject><subject>Relative Biological Effectiveness</subject><issn>0094-2405</issn><issn>2473-4209</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2024</creationdate><recordtype>article</recordtype><sourceid>24P</sourceid><sourceid>WIN</sourceid><sourceid>EIF</sourceid><recordid>eNp1kctq3DAUhkVpSCYX6BMULbtxcmzJkr0M0-YCuZHL2kjWUaJiS67kaZhdH6GLPmGfJJ7MtFlldeDn4-Pn_IR8yuEwByiO-uEwF1LwD2RWcMkyXkD9kcwAap4VHModspvSdwAQrIRtssOkLFkl5Yz8uQxppF-vjmnEQblIDVpsx0RNoD6MtA_G2SUdn3ACOjW64NOTG6jG8RnRb8KfSLULXXh0reoo2pViCj2mRJU3tHMeVaRTEB8nWVQ-WYzUeTq_Pb-7uf376zcaN6KhLXZd2idbVnUJDzZ3jzycfLufn2UX16fn8-OLrGVlyTNeG46G67IEZGBAG1TGsspK2worcihqU7UaJbRKoq60BS0LRCEAWC4qtke-rL1DDD8WmMamd2nVQHkMi9QUlahFXuYC3tA2hpQi2maIrldx2eTQrDZo-qF53WBCP2-sC92j-Q_-e_oEZGvg2XW4fFfUXN6shS80RpN_</recordid><startdate>202401</startdate><enddate>202401</enddate><creator>Guerra Liberal, Francisco D. C.</creator><creator>Parsons, Jason L.</creator><creator>McMahon, Stephen J.</creator><scope>24P</scope><scope>WIN</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>202401</creationdate><title>Most DNA repair defects do not modify the relationship between relative biological effectiveness and linear energy transfer in CRISPR‐edited cells</title><author>Guerra Liberal, Francisco D. C. ; Parsons, Jason L. ; McMahon, Stephen J.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3554-49d4ed4b550e30d0bdeadf38f7fc6f61029d8cbe70ca7eb8bf0b72ee660031683</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2024</creationdate><topic>Clustered Regularly Interspaced Short Palindromic Repeats</topic><topic>CRISPR—Cas9</topic><topic>DNA damage</topic><topic>DNA Repair</topic><topic>high LET</topic><topic>homologous recombination</topic><topic>Humans</topic><topic>Linear Energy Transfer</topic><topic>Neoplasms</topic><topic>non‐homologous end joining</topic><topic>proton therapy</topic><topic>Protons</topic><topic>radiation</topic><topic>radiosensitivity</topic><topic>RBE</topic><topic>Relative Biological Effectiveness</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Guerra Liberal, Francisco D. C.</creatorcontrib><creatorcontrib>Parsons, Jason L.</creatorcontrib><creatorcontrib>McMahon, Stephen J.</creatorcontrib><collection>Wiley Online Library Open Access</collection><collection>Wiley Free Content</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Medical physics (Lancaster)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Guerra Liberal, Francisco D. C.</au><au>Parsons, Jason L.</au><au>McMahon, Stephen J.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Most DNA repair defects do not modify the relationship between relative biological effectiveness and linear energy transfer in CRISPR‐edited cells</atitle><jtitle>Medical physics (Lancaster)</jtitle><addtitle>Med Phys</addtitle><date>2024-01</date><risdate>2024</risdate><volume>51</volume><issue>1</issue><spage>591</spage><epage>600</epage><pages>591-600</pages><issn>0094-2405</issn><eissn>2473-4209</eissn><abstract>Background
Cancer is a highly heterogeneous disease, driven by frequent genetic alterations which have significant effects on radiosensitivity. However, radiotherapy for a given cancer type is typically given with a standard dose determined from population‐level trials. As a result, a proportion of patients are under‐ or over‐dosed, reducing the clinical benefit of radiotherapy. Biological optimization would not only allow individual dose prescription but also a more efficient allocation of limited resources, such as proton and carbon ion therapy.
Proton and ion radiotherapy offer an advantage over photons due to their elevated Relative Biological Effectiveness (RBE) resulting from their elevated Linear Energy Transfer (LET). Despite significant interest in optimizing LET by tailoring radiotherapy plans, RBE's genetic dependence remains unclear.
Purpose
The aim of this study is to better define the RBE/LET relationship in a panel of cell lines with different defects in DSB repair pathways, but otherwise identical biological features and genetic background to isolate these effects.
Methods
Normal human cells (RPE1), genetically modified to introduce defects in DNA double‐strand break (DSB) repair genes, ATM, BRCA1, DCLRE1C, LIG4, PRKDC and TP53, were used to map the RBE‐LET relationship. Cell survival was measured with clonogenic assays after exposure to photons, protons (LET 1 and 12 keV/µm) and alpha particles (129 keV/µm). Gene knockout sensitizer enhancement ratio (SER) values were calculated as the ratio of the mean inactivation dose (MID) of wild‐type cells to repair‐deficient cells, and RBE values were calculated as the ratio of the MID of X‐ray and particle irradiated cells. 53BP1 foci were used to quantify radiation‐induced DSBs and their repair following irradiation.
Results
Deletion of NHEJ genes had the greatest impact on photon sensitivity (ATM−/− SER = 2.0 and Lig4−/− SER = 1.8), with genes associated with HR having smaller effects (BRCA1−/− SER = 1.2). Wild‐type cells showed RBEs of 1.1, 1.3, 5.0 for low‐ and high‐LET protons and alpha particles respectively. SERs for different genes were independent of LET, apart from NHEJ knockouts which proved to be markedly hypersensitive across all tested LETs. Due to this hypersensitivity, the impact of high LET was reduced in cell models lacking the NHEJ repair pathway. HR‐defective cells had moderately increased sensitivity across all tested LETs, but, notably, the contribution of HR pathway to survival appeared independent of LET. Analysis of 53BP1 foci shows that NHEJ‐defective cells had the least DSB repair capacity after low LET exposure, and no visible repair after high LET exposure. HR‐defective cells also had slower repair kinetics, but the impact of HR defects is not as severe as NHEJ defects.
Conclusions
DSB repair defects, particularly in NHEJ, conferred significant radiosensitivity across all LETs. This sensitization appeared independent of LET, suggesting that the contribution of different DNA repair pathways to survival does not depend on radiation quality.</abstract><cop>United States</cop><pmid>37753877</pmid><doi>10.1002/mp.16764</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Medical physics (Lancaster), 2024-01, Vol.51 (1), p.591-600 |
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| source | MEDLINE; Wiley Online Library Journals Frontfile Complete; Alma/SFX Local Collection |
| subjects | Clustered Regularly Interspaced Short Palindromic Repeats CRISPR—Cas9 DNA damage DNA Repair high LET homologous recombination Humans Linear Energy Transfer Neoplasms non‐homologous end joining proton therapy Protons radiation radiosensitivity RBE Relative Biological Effectiveness |
| title | Most DNA repair defects do not modify the relationship between relative biological effectiveness and linear energy transfer in CRISPR‐edited cells |
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