Targeted Alpha Radiation and Gamma Rays Induce Distinct and Mutation Status Specific Transcriptional Responses in Human Leukemic Lymphocytes.
Authors: Martin Simona , Kronenwett Ralf , Vandenbulcke Katia , Offner Fritz , Kliszewski Slawomir , Apostolidis Christos , Morgenstern Alfred , Steidl Ulrich , Raschke Sascha , Neumann Frank , Philippe Jan , Slegers Guido , Haas Rainer 
Published: April 2020

Abstract
Radioimmunotherapy specifically targets malignant cells using radionuclides conjugated with monoclonal antibodies. Alpha-particle emittors such as 213Bi have high linear energy transfer and short path length compared with gamma-rays, which allows them to kill cells by only few nuclear hits. As 213Bi-based targeted alpha-immunotherapy evolves as a new treatment modality currently proved in clinical trials for AML, NHL and preclinically for CLL and multiple myeloma, understanding the molecular effects of 213Bi-immunoconjugates is of relevance. We assessed gene expression profiles of PB lymphocytes from 12 CLL pts using cDNA arrays comprising 1,185 genes. Cells treated in vitro with 213Bi-CD20 or an equivalent dose of gamma radiation (60Co) were intra-individually compared with non-treated cells. According to the two variants of B-CLL, with mutated (6 pts), or unmutated (6 pts) rearranged VH, we also aimed to verify whether radiation effects are mutation status specific. Globally, alpha radiation affected two-fold more genes than gamma. This was true for both cell types. Interestingly, there was little overlap between genes affected by both alpha and gamma radiation: 17% of genes in cells with mutated and 14% in cells with unmutated VH. Common genes mainly were involved in DNA repair: PCNA and CDKN1A acting in the p53-mediated pathway, CDC25B, CLK1 and CRY1 known to mediate repair after UV-induced DNA damage. Compared with alpha, effects of gamma radiation were of narrower range, with 24 genes altered in mutated and 16 in unmutated cells. The 46 genes specifically altered by alpha exposure in mutated cells and the 33 genes in unmutated cells belong to a multitude of functional families, showing that a generalized response was induced including deregulation of cytokines (IL-4, TGF β1, TGF ß2), oncogenes (v-myc, v-ski), cell cycle regulating genes (CDK6, cyclin D3, CDC2-like 10, CDKN2D). A network of intracellular signaling by phospholipases ß2, γ1 and 2, PKCε, CXCR4, extracellular signaling by integrin α7, chondroitin sulfate proteoglycan 5 and transcriptional regulation was involved. According to the divergent clinical behaviour of pts with mutated or unmutated VH, we expected differences at molecular level after radiation exposure. Indeed, 53 genes accounted for differences in response to alpha radiation between the two types of lymphocytes. Affiliation to functional families was mutation status specific. Expression of genes coding for interleukins, oncogenes and DNA synthesis was restricted to mutated cells. Hierarchical cluster analysis after alpha and gamma radiation resulted in two clearly distinguishable array clusters, one consisting of samples from pts with mutated VH, the other of samples from pts with unmutated VH. In conclusion, transcriptional changes induced by alpha and gamma radiation encompass molecularly distinct mechanisms and reflect the higher complexity of DNA damage produced by alpha radiation. The two subtypes of B-CLL respond to cytotoxic damage by different pathways which might explain the divergent clinical behaviour of these subgroups of patients.