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Research paper thumbnail of CD19-CAR T cells undergo exhaustion DNA methylation programming in patients with acute lymphoblastic leukemia

Cell Reports, 2021

CD19-CAR T cell therapy has evolved into the standard of care for relapsed/refractory B cell acut... more CD19-CAR T cell therapy has evolved into the standard of care for relapsed/refractory B cell acute lymphoblastic leukemia (ALL); however, limited persistence of the CAR T cells enables tumor relapse for many patients. To gain a deeper understanding of the molecular characteristics associated with CAR T cell differentiation, we performed longitudinal genome-wide DNA methylation profiling of CD8 + CD19-CAR T cells post-infusion in ALL patients. We report that CAR T cells undergo a rapid and broad erasure of repressive DNA methylation reprograms at effector-associated genes. The CAR T cell post-infusion changes are further characterized by repression of genes (e.g., TCF7 and LEF1) associated with memory potential and a DNA methylation signature (e.g., demethylation at CX3CR1, BATF, and TOX) demarcating a transition toward exhaustion-progenitor T cells. Thus, CD19-CAR T cells undergo exhaustion-associated

Research paper thumbnail of CD19-CAR T Cells Develop Exhaustion Epigenetic Programs during a Clinical Response

Blood, 2021

Background: CD19-CAR T-cell therapy has emerged as a curative approach for patients with relapsed... more Background: CD19-CAR T-cell therapy has emerged as a curative approach for patients with relapsed/refractory B-cell malignancies, including lymphoma and acute lymphoblastic leukemia (ALL). However, many patients develop recurrent disease after initial responses. Therapeutic failure is most likely due to T cell extrinsic and intrinsic mechanisms. While CD19-negative relapse has emerged as a major extrinsic mechanism, T cell intrinsic mechanisms remain elusive. T-cell exhaustion is driven by epigenetic programs, however epigenetic changes in CAR T cells pre and post infusion have not been determined longitudinally. Thus, the goal of this study was to determine the epigenetic landscape of CD19-CAR T cells pre and post infusion as an initial step to elucidate intrinsic mechanisms that limit CAR T-cell effector functions in humans. Methods/Results: A longitudinal analysis of CD8+ CD19-CAR T cell epigenetic changes was performed by whole-genome DNA methylation profiling of CAR T cells dur...

Research paper thumbnail of Deleting DNMT3A in CAR T cells prevents exhaustion and enhances antitumor activity

Science Translational Medicine, 2021

DNMT3A deletion preserves CAR T cell functionality during prolonged stimulation.

Research paper thumbnail of Proinflammatory cytokines promote TET2-mediated DNA demethylation during CD8 T cell effector differentiation

Cell Reports, 2021

Highlights d Bystander inflammation promotes effector DNA methylation programs during T cell prim... more Highlights d Bystander inflammation promotes effector DNA methylation programs during T cell priming d The proinflammatory cytokine, IL-12, induces demethylation of IFNg locus CNS elements d IL-12 drives TET2-mediatiated demethylation of the mouse and human IFNg locus d Human memory T cells retain DNA methylation signatures of IL-12 signaling

Research paper thumbnail of Abstract 1368: Simultaneous cell-free RNA PD-L1 expression and MSI from the same single-tube of blood in solid tumors

Clinical Research (Excluding Clinical Trials), 2019

Background: Liquid biopsy is a minimally invasive technique available in the clinic using next-ge... more Background: Liquid biopsy is a minimally invasive technique available in the clinic using next-generation sequencing (NGS) of plasma circulating cell-free DNA (cfDNA) and cfRNA. Here we report liquid biopsy results from solid tumors using the Circulogene Theranostics Personalized Gene Profile (CGP, 50-gene panel), as well as, microsatellite instability (MSI) testing. CGP uses proprietary in situ enrichment requiring minimal input volume of 20 uL plasma (cfDNA), 400 uL (cfRNA), and 100 uL buffy coat per case from the same single-tube of blood. Methods: cfRNA PD-L1 expression, MSI, and co-occurring cfDNA mutations were retrospectively compiled from CGP ordered at multiple centers. Ct value cutoff for 1 copy PD-L1 (positive) was 42. MSI was performed using paired buffy coat gDNA (normal) and plasma cfDNA (tumor) on 5 microsatellite mononucleotide markers, BAT-25, BAT-26, NR-21, NR-24 and MONO-27 (Promega; current an industry standard that has been used in KEYNOTE pembrolizumab trials) and analyzed by capillary electrophoresis genetic analyzer. Based on batched processing, Circulogene’s turnaround time (TAT) is 5 business days on average. Data was summarized and the correlation between PD-L1 and MSI was measured. Results: 397 patients (median age 67 years, range 27-96; 210 men: 187 women) underwent CGP testing for both PD-L1 and MSI (11/2017-10/2018). The majority of cancer types were lung (n=187), colorectal (n=43), breast (n=40), pancreatic (n=32), and prostate (n=30). 59/397 (14.9%) were PD-L1 positive, in the range of expected PD-L1 positivity across solid tumors. 110/397 (27.7%) were MSI-High (MSI-H). There was no correlation between PD-L1 and MSI results (r^2 Conclusions: Simultaneous cfRNA PD-L1 and MSI testing from the same single-tube blood is feasible in a liquid biopsy and their positivity has frequency in the expected range for solid tumors (Xang et al. Onco Targets Ther 2016, Zhang et al. AMP Conference 2018, Abstract ST009). Despite the low plasma sample input, there were no sample failures, suggesting CGP in situ enrichment is robust and has a rapid TAT. Assay results with linkage to clinical outcomes is warranted. Citation Format: Glen J. Weiss, Paul Walker, Dilek Aktas, Andrew Ford, Charmaine Brown, Chen-Hsiung Yeh, Nader Javadi. Simultaneous cell-free RNA PD-L1 expression and MSI from the same single-tube of blood in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1368.

Research paper thumbnail of CD19-CAR T cells undergo exhaustion DNA methylation programming in patients with acute lymphoblastic leukemia

Cell Reports, 2021

CD19-CAR T cell therapy has evolved into the standard of care for relapsed/refractory B cell acut... more CD19-CAR T cell therapy has evolved into the standard of care for relapsed/refractory B cell acute lymphoblastic leukemia (ALL); however, limited persistence of the CAR T cells enables tumor relapse for many patients. To gain a deeper understanding of the molecular characteristics associated with CAR T cell differentiation, we performed longitudinal genome-wide DNA methylation profiling of CD8 + CD19-CAR T cells post-infusion in ALL patients. We report that CAR T cells undergo a rapid and broad erasure of repressive DNA methylation reprograms at effector-associated genes. The CAR T cell post-infusion changes are further characterized by repression of genes (e.g., TCF7 and LEF1) associated with memory potential and a DNA methylation signature (e.g., demethylation at CX3CR1, BATF, and TOX) demarcating a transition toward exhaustion-progenitor T cells. Thus, CD19-CAR T cells undergo exhaustion-associated

Research paper thumbnail of CD19-CAR T Cells Develop Exhaustion Epigenetic Programs during a Clinical Response

Blood, 2021

Background: CD19-CAR T-cell therapy has emerged as a curative approach for patients with relapsed... more Background: CD19-CAR T-cell therapy has emerged as a curative approach for patients with relapsed/refractory B-cell malignancies, including lymphoma and acute lymphoblastic leukemia (ALL). However, many patients develop recurrent disease after initial responses. Therapeutic failure is most likely due to T cell extrinsic and intrinsic mechanisms. While CD19-negative relapse has emerged as a major extrinsic mechanism, T cell intrinsic mechanisms remain elusive. T-cell exhaustion is driven by epigenetic programs, however epigenetic changes in CAR T cells pre and post infusion have not been determined longitudinally. Thus, the goal of this study was to determine the epigenetic landscape of CD19-CAR T cells pre and post infusion as an initial step to elucidate intrinsic mechanisms that limit CAR T-cell effector functions in humans. Methods/Results: A longitudinal analysis of CD8+ CD19-CAR T cell epigenetic changes was performed by whole-genome DNA methylation profiling of CAR T cells dur...

Research paper thumbnail of Deleting DNMT3A in CAR T cells prevents exhaustion and enhances antitumor activity

Science Translational Medicine, 2021

DNMT3A deletion preserves CAR T cell functionality during prolonged stimulation.

Research paper thumbnail of Proinflammatory cytokines promote TET2-mediated DNA demethylation during CD8 T cell effector differentiation

Cell Reports, 2021

Highlights d Bystander inflammation promotes effector DNA methylation programs during T cell prim... more Highlights d Bystander inflammation promotes effector DNA methylation programs during T cell priming d The proinflammatory cytokine, IL-12, induces demethylation of IFNg locus CNS elements d IL-12 drives TET2-mediatiated demethylation of the mouse and human IFNg locus d Human memory T cells retain DNA methylation signatures of IL-12 signaling

Research paper thumbnail of Abstract 1368: Simultaneous cell-free RNA PD-L1 expression and MSI from the same single-tube of blood in solid tumors

Clinical Research (Excluding Clinical Trials), 2019

Background: Liquid biopsy is a minimally invasive technique available in the clinic using next-ge... more Background: Liquid biopsy is a minimally invasive technique available in the clinic using next-generation sequencing (NGS) of plasma circulating cell-free DNA (cfDNA) and cfRNA. Here we report liquid biopsy results from solid tumors using the Circulogene Theranostics Personalized Gene Profile (CGP, 50-gene panel), as well as, microsatellite instability (MSI) testing. CGP uses proprietary in situ enrichment requiring minimal input volume of 20 uL plasma (cfDNA), 400 uL (cfRNA), and 100 uL buffy coat per case from the same single-tube of blood. Methods: cfRNA PD-L1 expression, MSI, and co-occurring cfDNA mutations were retrospectively compiled from CGP ordered at multiple centers. Ct value cutoff for 1 copy PD-L1 (positive) was 42. MSI was performed using paired buffy coat gDNA (normal) and plasma cfDNA (tumor) on 5 microsatellite mononucleotide markers, BAT-25, BAT-26, NR-21, NR-24 and MONO-27 (Promega; current an industry standard that has been used in KEYNOTE pembrolizumab trials) and analyzed by capillary electrophoresis genetic analyzer. Based on batched processing, Circulogene’s turnaround time (TAT) is 5 business days on average. Data was summarized and the correlation between PD-L1 and MSI was measured. Results: 397 patients (median age 67 years, range 27-96; 210 men: 187 women) underwent CGP testing for both PD-L1 and MSI (11/2017-10/2018). The majority of cancer types were lung (n=187), colorectal (n=43), breast (n=40), pancreatic (n=32), and prostate (n=30). 59/397 (14.9%) were PD-L1 positive, in the range of expected PD-L1 positivity across solid tumors. 110/397 (27.7%) were MSI-High (MSI-H). There was no correlation between PD-L1 and MSI results (r^2 Conclusions: Simultaneous cfRNA PD-L1 and MSI testing from the same single-tube blood is feasible in a liquid biopsy and their positivity has frequency in the expected range for solid tumors (Xang et al. Onco Targets Ther 2016, Zhang et al. AMP Conference 2018, Abstract ST009). Despite the low plasma sample input, there were no sample failures, suggesting CGP in situ enrichment is robust and has a rapid TAT. Assay results with linkage to clinical outcomes is warranted. Citation Format: Glen J. Weiss, Paul Walker, Dilek Aktas, Andrew Ford, Charmaine Brown, Chen-Hsiung Yeh, Nader Javadi. Simultaneous cell-free RNA PD-L1 expression and MSI from the same single-tube of blood in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1368.