Tumor Treating Fields (TTFields) Delay DNA Damage Repair Following Radiation Treatment of Glioma Cells: Implications for Irradiation Through TTFields Transducer Arrays

Abstract Acute Myeloid Leukemia (AML) is most commonly seen in people over the age of 65 and has a median age of 63. Globally there is an increasingly elderly population so the rate of incidence of AML is set to increase. The therapy landscape for AML has changed little over the past four decades. Cytarabine, first approved in 1969, is still the standard of care induction therapy for AML. There has been only modest improvements in survival rates during this time and there is currently no method of determining which patients will or will not respond to Cytarabine treatment. An assay, developed in 2014, used microarray data to determine which breast cancer patients had a DNA Damage Repair Deficiency (DDRD) and therefore would be more susceptible to DNA damaging agents. A negative DDRD (DDRD-) score predicts that patients do not to have a DNA Repair Deficiency whilst patients with a positive DDRD (DDRD+) score are predicted to have a DNA Repair Deficiency. This assay has been adapted to different solid cancer types such as ovarian and oesophageal cancer. This project has assessed the potential of using the DDRD assay for AML patients. The assay was applied to publically available microarray data of >600 AML patients (TCGA AML data &GSE6891), who were classed as DDRD- or DDRD+. Excluding patients not treated with Cytarabine, this left 639 patients, 405 DDRD+ and 234 DDRD-. Kaplan Meier analysis showed the DDRD+ patients survived significantly (p=0.00047) worse than the DDRD- cohort. Whole exome sequencing was available for 183 patients (131 DDRD+) and the mutations associated with each group were identified. As the DDRD+ patients had the worst outcome, we focused on group. The list of genes more commonly mutated in the DDRD+ patients (>2 instances and >50% occurring in this group) were subjected to pathway analysis. Deregulated pathways included "leukemogenisis" and "cell proliferation and regulation"; however, the most deregulated pathway was "metabolism of nucleobase containing compounds". As Cytarabine is a nucleobase-containing compound, this is potentially a contributing factor as to why these patients responded poorly to this treatment. The assay was applied to microarray data of a panel of myeloid cell lines, and DDRD-(NB4 & SKM1) and a DDRD+(HL-60) cell line were chosen as experimental models. Clonogenic assays, used to analyse the effect of Cytarabine on these cell lines, showed that the DDRD- cell lines were more sensitive with a lower colony growth rate than the DDRD+cell line. DNA damage induction and repair, following cytarabine treatment or 2gy radiation, were measured using RAD51 foci counts. Whilst foci counts were high in all cell lines 2hrs and 4hrs following radiation, the DDRD+ cell line continued to show high levels after 24hrs whereas the levels in the DDRD- cell lines returned to a basal level. RAD51 response to radiation treatment showed that a repair defect is present in DDRD+ cells as they fail to repair the damage induced by radiation. Following treatment with Cytarabine however, few foci were seen in the DDRD+ cell line 2hrs, 4hrs or 24hrs following treatment whereas the DDRD- cell lines responded in a similar fashion to radiation treatment. That RAD51 foci are not present following Cytarabine treatment indicates that Cytarabine fails to induce damage in these cells. The DDRD assay has shown to be an effective method for determining cellular response to Cytarabine in vivo. The non-response of the DDRD+ cell line to Cytarabine suggests that these cells do not elicit a DNA damage or an apoptotic response. This perhaps contributes to their poorer outcome and suggests that Cytarabine is not an effective treatment plan for patients deemed to be DDRD+. Although alternative induction treatment options are currently unavailable for DDRD+ AML patients, this DDRD assay could be used as a biomarker for Cytarabine response in the future. Disclosures No relevant conflicts of interest to declare.

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MiR‐320b/RAD21 axis affects hepatocellular carcinoma radiosensitivity to ionizing radiation treatment through DNA damage repair signaling

Cancer Science ◽

10.1111/cas.14751 ◽

2020 ◽

Author(s):

Jianyang Wang ◽

Hong Zhao ◽

Jing Yu ◽

Xin Xu ◽

Hao Jing ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Dna Damage ◽

Ionizing Radiation ◽

Radiation Treatment ◽

Dna Damage Repair ◽

Damage Repair

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EPCO-04. RADIOTHERAPY IS ASSOCIATED WITH GLOBAL METHYLATION ALTERATIONS IN PATIENT DERIVED GLIOBLASTOMA CELL LINES

Neuro-Oncology ◽

10.1093/neuonc/noab196.003 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi2-vi2

Author(s):

Aram Modrek ◽

David Byun ◽

Ravesanker Ezhilarasan ◽

Matija Snuderl ◽

Erik Sulman

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Dna Damage ◽

Radiation Treatment ◽

Cpg Islands ◽

Dna Damage Repair ◽

Differential Methylation ◽

Response To Therapy ◽

Damage Repair ◽

Genomic Regions

Abstract PURPOSE/OBJECTIVE(S) In glioblastoma, DNA methylation states are the most predictive marker of overall survival and response to therapy. Our understanding of how epigenetic states, such as DNA methylation, are “mis-repaired” after DNA damage repair is scant, hampering our ability to understand how treatment associated DNA methylation alterations may drive tumor resistance and growth. MATERIALS AND METHODS Three different patient derived IDH wild-type glioma stem cell (GSC) lines, in duplicates, were treated with radiation (20 Gray in 10 fractions vs. sham control) and allowed to recover prior to DNA methylation analysis with 850K methylation arrays. To analyze the methylation array data via bioinformatic methods we used RnBeads (version 2.4.0) and R (version 3.6.1) packages. We further focused our analysis to specific genomic regions, including CpG islands, promoters, gene bodies and CTCF motifs to understand how methylation alterations may differ between these and other genomic contexts following radiation. RESULTS There were widespread differential methylation (pre-treatment vs. radiation treatment) changes among the genomic regions examined. Interestingly, we found differential methylation changes at CTCF motifs, which play important DNA-methylation dependent roles in gene expression and chromatin architecture regulation. Hierarchical clustering, PCA and MDS analysis of DNA methylation status amongst CpG islands, promoters, gene bodies and CTCF domains revealed strong intra-sample differences, but not inter-sample differences (between GSC lines), suggesting radiation associated methylation alterations maybe loci and context dependent. CONCLUSION Radiation treatment is associated with wide-spread alterations of DNA methylation states in this patient derived glioblastoma model. Such alterations may drive gene expression changes or genomic architecture alterations that lead to treatment resistance, warranting further mechanistic investigation of the interplay between radiation induced DNA damage and local epigenetic state restoration following DNA damage repair.

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Abstract 3217: Newly identified role of tumor treating fields in DNA damage repair and replication stress pathways

10.1158/1538-7445.am2018-3217 ◽

2018 ◽

Cited By ~ 1

Author(s):

Narasimha Kumar Karanam ◽

Lianghao Ding ◽

Brock Sishc ◽

Debabrata Saha ◽

Michael D. Story

Keyword(s):

Dna Damage ◽

Dna Damage Repair ◽

Replication Stress ◽

Tumor Treating Fields ◽

Damage Repair

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P04.03 Different role of integrin a6 in glioblastoma proneural and mesenchymal stem-like cells

Neuro-Oncology ◽

10.1093/neuonc/noab180.060 ◽

2021 ◽

Vol 23 (Supplement_2) ◽

pp. ii18-ii19

Author(s):

E Stanzani ◽

L Pedrosa ◽

G Bourmeau ◽

O Anezo ◽

A Noguera-Castells ◽

...

Keyword(s):

Dna Damage ◽

Radiation Treatment ◽

Molecular Subtype ◽

Dna Damage Repair ◽

Quadratic Model ◽

Molecular Heterogeneity ◽

Linear Quadratic ◽

Enhanced Sensitivity ◽

Damage Repair

Abstract BACKGROUND The plasticity of tumoral cells and the presence of cells displaying stem-like features are two interrelated traits of Glioblastoma (GBM) lesions and both concur in defining its heterogeneity. Particularly, GBM stem-like cells (GSC) can be classified according to trascriptional data in different subgroups, being the Proneural (PN-GSC) and the Mesenchymal (MES-GSC) the most consolidated clusters. GSC are responsible of most of the malignant characteristics of GBM, including therapeutic resistance and tumor recurrence. Therefore, a better understanding of the mechanisms regulating GSC responsiveness to therapy taking into account GSC molecular heterogeneity may help to improve patient’s outcome. Integrin a6 is a commonly used marker for GSC capable to enrich for GSC population and sustain stemness. We investigate the role of integrin a6 in both PN and MES GSC on stemness and radioresistance. MATERIAL AND METHODS The expression of integrin a6 was analyzed in GSC cultures obtained from post-surgical specimens either displaying PN or MES trascriptional traits. Using cell sorting to enrich for integrin a6 expression (integrin a6-high and a6-low) and gene silencing with lentiviral-based shRNA, integrin a6 impact on both GSC cultures was tested. Also, silenced MES-GSC were analysed by means of RNA-seq. The major pathways found altered by integrin a6 silencing were validated at functional level using gliomasphere-based clonogenic assay, extreme limiting dilution assay and gamma-H2AX to monitor DNA damage repair kinetics. RESULTS After sorting GSC cultures by integrin a6 expression, PN-GSC a6-low showed a significant reduction in clonogenic capability and gliomasphere size when compared to a6-high (p<0.0001). On the contrary, sorted MES-GSC did not display any differences. Similar results were obtained following integrin a6 lentiviral silencing. However, RNAseq on silenced MES-GSC revealed a significant impact on cell cycle regulation and DNA damage repair pathways. Indeed, Integrin a6 trascriptional inhibition in MES-GSCs impaired the capacity to clear gamma-H2AX foci after ionizing radiation (p<0.001) and significantly alters MES-GSC capacity to recover from radiation treatment in gliomasphere formation assay (p<0.01). According to the interpretation of the curves with the linear quadratic model, integrin a6 silenced cells displayed higher alpha- and beta- values and lower alpha/beta ratio. The obtained values demonstrate increased radiosensitivity and impaired capacity to repair sublethal DNA damage, in addition to an enhanced sensitivity to fractionated doses. CONCLUSION The data obtained showed that Integrin a6 regulates proliferation and stemness-related features in PN-GSC while supports radioresistance of MES-GSCs. Altogether, we reveal that integrin a6 controls different stem-associated features in GSCs depending on the molecular subtype.

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