CD19-CAR T cells undergo exhaustion DNA methylation programming in patients with acute lymphoblastic leukemia (original) (raw)
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Leukemia, 2023
Chimeric antigen receptor (CAR) T cells provide new perspectives for treatment of hematological malignancies. Manufacturing of these cellular products includes culture expansion procedures, which may affect cellular integrity and therapeutic outcome. In this study, we investigated culture-associated epigenetic changes in CAR T cells and found continuous gain of DNAm, particularly within genes that are relevant for T cell function. Hypermethylation in many genes, such as TCF7, RUNX1, and TOX, was reflected by transcriptional downregulation. 332 CG dinucleotides (CpGs) showed an almost linear gain in methylation with cell culture time, albeit neighboring CpGs were not coherently regulated on the same DNA strands. An epigenetic signature based on 14 of these culture-associated CpGs predicted cell culture time across various culture conditions. Notably, even in CAR T cell products of similar culture time higher DNAm levels at these CpGs were associated with significantly reduced long-term survival post transfusion. Our data demonstrate that cell culture expansion of CAR T cells evokes DNA hypermethylation at specific sites in the genome and the signature may also reflect loss of potential in CAR T cell products. Hence, reduced cultivation periods are beneficial to avoid dysfunctional methylation programs that seem to be associated with worse therapeutic outcome.
DNA methylation profiles in chronic lymphocytic leukemia patients treated with chemoimmunotherapy
Clinical Epigenetics, 2019
Background: In order to gain insight into the contribution of DNA methylation to disease progression of chronic lymphocytic leukemia (CLL), using 450K Illumina arrays, we determined the DNA methylation profiles in paired pre-treatment/relapse samples from 34 CLL patients treated with chemoimmunotherapy, mostly (n = 31) with the fludarabine-cyclophosphamide-rituximab (FCR) regimen. Results: The extent of identified changes in CLL cells versus memory B cells from healthy donors was termed "epigenetic burden" (EB) whereas the number of changes between the pre-treatment versus the relapse sample was termed "relapse changes" (RC). Significant (p < 0.05) associations were identified between (i) high EB and short time-to-first-treatment (TTFT); and, (ii) few RCs and short time-to-relapse. Both the EB and the RC clustered in specific genomic regions and chromatin states, including regulatory regions containing binding sites of transcription factors implicated in B cell and CLL biology. Conclusions: Overall, we show that DNA methylation in CLL follows different dynamics in response to chemoimmunotherapy. These epigenetic alterations were linked with specific clinical and biological features.
Neoplasia, 2014
We have previously described gene expression changes during spontaneous immortalization of T-cells, thereby identifying cellular processes important for cell growth crisis escape and unlimited proliferation. Here, we analyze the same model to investigate the role of genome-wide methylation in the immortalization process at different time points pre-crisis and post-crisis using high-resolution arrays. We show that over time in culture there is an overall accumulation of methylation alterations, with preferential increased methylation close to transcription start sites (TSSs), islands, and shore regions. Methylation and gene expression alterations did not correlate for the majority of genes, but for the fraction that correlated, gain of methylation close to TSS was associated with decreased gene expression. Interestingly, the pattern of CpG site methylation observed in immortal T-cell cultures was similar to clinical T-cell acute lymphoblastic leukemia (T-ALL) samples classified as CpG island methylator phenotype positive. These sites were highly overrepresented by polycomb target genes and involved in developmental, cell adhesion, and cell signaling processes. The presence of non-random methylation events in in vitro immortalized T-cell cultures and diagnostic T-ALL samples indicates altered methylation of CpG sites with a possible role in malignant hematopoiesis.
Pediatric blood & cancer, 2016
Despite increased knowledge about genetic aberrations in pediatric T-cell acute lymphoblastic leukemia (T-ALL), no clinically feasible treatment-stratifying marker exists at diagnosis. Instead patients are enrolled in intensive induction therapies with substantial side effects. In modern protocols, therapy response is monitored by minimal residual disease (MRD) analysis and used for postinduction risk group stratification. DNA methylation profiling is a candidate for subtype discrimination at diagnosis and we investigated its role as a prognostic marker in pediatric T-ALL. Sixty-five diagnostic T-ALL samples from Nordic pediatric patients treated according to the Nordic Society of Pediatric Hematology and Oncology ALL 2008 (NOPHO ALL 2008) protocol were analyzed by HumMeth450K genome wide DNA methylation arrays. Methylation status was analyzed in relation to clinical data and early T-cell precursor (ETP) phenotype. Two distinct CpG island methylator phenotype (CIMP) groups were iden...
Epigenomics, 2016
To identify regions of aberrant DNA methylation in acute lymphoblastic leukemia (ALL) cells of different subtypes on a genome-wide scale. Whole-genome bisulfite sequencing (WGBS) was used to determine the DNA methylation levels in cells from four pediatric ALL patients of different subtypes. The findings were confirmed by 450k DNA methylation arrays in a large patient set. Compared with mature B or T cells WGBS detected on average 82,000 differentially methylated regions per patient. Differentially methylated regions are enriched to CpG poor regions, active enhancers and transcriptional start sites. We also identified approximately 8000 CpG islands with variable intermediate DNA methylation that seems to occur as a result of stochastic de novo methylation. WGBS provides an unbiased view and novel insights into the DNA methylome of ALL cells.
PLoS ONE, 2013
Background: Treatment of pediatric T-cell acute lymphoblastic leukemia (T-ALL) has improved, but there is a considerable fraction of patients experiencing a poor outcome. There is a need for better prognostic markers and aberrant DNA methylation is a candidate in other malignancies, but its potential prognostic significance in TALL is hitherto undecided. Design and Methods: Genome wide promoter DNA methylation analysis was performed in pediatric TALL samples (n = 43) using arrays covering .27000 CpG sites. Clinical outcome was evaluated in relation to methylation status and compared with a contemporary TALL group not tested for methylation (n = 32). Results: Based on CpG island methylator phenotype (CIMP), TALL samples were subgrouped as CIMP+ (high methylation) and CIMP2 (low methylation). CIMP2 TALL patients had significantly worse overall and event free survival (p = 0.02 and p = 0.001, respectively) compared to CIMP+ cases. CIMP status was an independent factor for survival in multivariate analysis including age, gender and white blood cell count. Analysis of differently methylated genes in the CIMP subgroups showed an overrepresentation of transcription factors, ligands and polycomb target genes. Conclusions: We identified global promoter methylation profiling as being of relevance for subgrouping and prognostication of pediatric TALL .
Cancer research, 2007
This study examined DNA methylation associated with acute lymphoblastic leukemia (ALL) and showed that selected molecular targets can be pharmacologically modulated to reverse gene silencing. A CpG island (CGI) microarray containing more than 3,400 unique clones that span all human chromosomes was used for large-scale discovery experiments and led to 262 unique CGI loci being statistically identified as methylated in ALL lymphoblasts. The methylation status of 10 clones encompassing 11 genes (DCC, DLC-1, DDX51, KCNK2, LRP1B, NKX6-1, NOPE, PCDHGA12, RPIB9, ABCB1, and SLC2A14) identified as differentially methylated between ALL patients and controls was independently verified. Consequently, the methylation status of DDX51 was found to differentiate patients with Band TALL subtypes (P = 0.011, Fisher's exact test). Next, the relationship between methylation and expression of these genes was examined in ALL cell lines (NALM-6 and Jurkat) before and after treatments with 5-aza-2-deoxycytidine and trichostatin A. More than a 10-fold increase in mRNA expression was observed for two previously identified tumor suppressor genes (DLC-1 and DCC) and also for RPIB9 and PCDHGA12. Although the mechanisms that lead to the CGI methylation of these genes are unknown, bisulfite sequencing of the promoter of RPIB9 suggests that expression is inhibited by methylation within SP1 and AP2 transcription factor binding motifs. Finally, specific chromosomal methylation hotspots were found to be associated with ALL. This study sets the stage for acquiring a better biological understanding of ALL and for the identification of epigenetic biomarkers useful for differential diagnosis, therapeutic monitoring, and the detection of leukemic relapse.
DNA methylation and copy number variation profiling of T-cell lymphoblastic leukemia and lymphoma
Blood Cancer Journal, 2020
Despite having common overlapping immunophenotypic and morphological features, T-cell lymphoblastic leukemia (T-ALL) and lymphoma (T-LBL) have distinct clinical manifestations, which may represent separate diseases. We investigated and compared the epigenetic and genetic landscape of adult and pediatric T-ALL ( n = 77) and T-LBL ( n = 15) patient samples by high-resolution genome-wide DNA methylation and Copy Number Variation (CNV) BeadChip arrays. DNA methylation profiling identified the presence of CpG island methylator phenotype (CIMP) subgroups within both pediatric and adult T-LBL and T-ALL. An epigenetic signature of 128 differentially methylated CpG sites was identified, that clustered T-LBL and T-ALL separately. The most significant differentially methylated gene loci included the SGCE/PEG10 shared promoter region, previously implicated in lymphoid malignancies. CNV analysis confirmed overlapping recurrent aberrations between T-ALL and T-LBL, including 9p21.3 ( CDKN2A/CDKN...
Nature genetics, 2016
Charting differences between tumors and normal tissue is a mainstay of cancer research. However, clonal tumor expansion from complex normal tissue architectures potentially obscures cancer-specific events, including divergent epigenetic patterns. Using whole-genome bisulfite sequencing of normal B cell subsets, we observed broad epigenetic programming of selective transcription factor binding sites coincident with the degree of B cell maturation. By comparing normal B cells to malignant B cells from 268 patients with chronic lymphocytic leukemia (CLL), we showed that tumors derive largely from a continuum of maturation states reflected in normal developmental stages. Epigenetic maturation in CLL was associated with an indolent gene expression pattern and increasingly favorable clinical outcomes. We further uncovered that most previously reported tumor-specific methylation events are normally present in non-malignant B cells. Instead, we identified a potential pathogenic role for tra...