203 - Mechanisms of Acquired Radioresistance in Diffuse Large B-Cell Lymphoma
Presenter(s)
F. Troschel1, S. Niefind1, T. Habig1, K. A. Brücksken1, L. Reichstein1, G. Poschmann2, E. Korsching3, N. A. Espinoza-Sanchez1, M. Oertel1, S. Hailfinger4, G. Lenz4, B. Greve1, and H. T. Eich5; 1Department of Radiation Oncology, Münster University Hospital, Münster, Germany, 2Institute of Molecular Medicine, Proteome research, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany, 3Cancer and Complex Systems Research Group, Münster University, Münster, Germany, 4Department of Medicine A, Hematology, Oncology, and Pneumology, University Hospital Münster, Münster, Germany, 5Department of Radiation Therapy – Radiation Oncology, University Hospital of Muenster, Muenster, Germany
Purpose/Objective(s): Radiation treatment plays a critical role in the multimodal treatment of Diffuse Large B-Cell Lymphoma (DLBCL). While DLBCL is generally considered sensitive to irradiation and chemo-immunotherapy, post-treatment relapses frequently occur. Relapsed / refractory DLBCL is difficult to treat, and outcomes are much more limited. To improve the understanding of resistance mechanisms, we longitudinally exposed DLBCL cells to irradiation, generating radioresistant DLBCL (rr-DLBCL). We then characterized functional, genomic and proteomic changes in rr-DLBCL compared to wildtype DLBCL (wt-DLBCL).
Materials/Methods: We subjected HT and HBL-1 DLBCL cells to increasing doses of weekly irradiation up to a cumulative dose of 30 Gy. Afterwards, cells received a weekly maintenance dose of 3 Gy. After validating radioresistance with colony-forming assays, we functionally characterized cell cycle (flow cytometry), proliferation activity (BrdU and Real-Time-Glo Assay), apoptosis (Annexin V/PI Assay), DNA damage (?H2AX phosphorylation assay, DNA damage protein phosphorylation), and chemoresistance (MTT assay for doxorubicin and vincristine). We performed both genomic (RT-qPCR, RNA-seq) and proteomic (Western Blot, mass spectrometry-based proteomics) analyses. Finally, we characterized the inflammatory secretome of rr-DLBCL cells and determined their interaction with bystander wt-DLBCL cells.
Results: rr-DLBCL cells showed a more than 50% increase in clonogenic survival after irradiation doses of 2, 4, and 6 Gy compared to wt-DLBCL cells, confirming acquired radioresistance. Cells showed a stronger baseline proliferation rate coupled with a pronounced S phase accumulation while apoptosis was reduced. rr-DLBCL cells also demonstrated a lower DNA damage and an accelerated rate of DNA damage repair as well as increased resistance to doxorubicin and vincristine chemotherapy. DNA repair genes and stemness markers were increased. CD19 marker expression was meaningfully decreased in rr-DLBCL. RNA-seq and proteomic data supported functional enrichment of DNA repair and proliferation-related signaling. Intriguingly, proteomic changes persisted for at least one week after last irradiation. Secretomic analyses showed an increase in pro-tumorigenic inflammation. Importantly, we found that wt-DLBCL cells treated with media from rr-DLBLC cells gained radioresistant properties, suggesting a lateral transmission mechanism.
Conclusion: Repeated exposure to irradiation allowed for the induction of acquired radioresistance in DLBCL. rr-DLBCL cells showed meaningful functional changes including loss of CAR-T target CD19, increase in chemoresistance, and lateral transmission of radioresistance together with corresponding genomic and proteomic alterations. This model of acquired radioresistance may enable the evaluation of approaches to target and reverse resistance mechanisms.