CLL-384: Proliferation and Unmutated IGHV Status are Associated with Homologous Recombination Activity in CLL Cells

Abstract

In IgHV-unmutated CLL (U-CLL), the proliferation rate is higher compared with IGHV-mutated CLL (M-CLL). Yet, why CLL cells differ in their mutational rates is unknown. Accumulating mutations in the IGHV gene lead to subsequent accumulation of DNA breaks. In rapidly dividing cells, DNA breaks are repaired by the efficient high-fidelity homology-directed DNA (HR) repair apparatus, whereas in slowly dividing cells, they are repaired by the inefficient low-fidelity nonhomologous end-joining (NHEJ) repair mechanism. Since HR is a high-fidelity DNA repair process, we expect that in cells that preferentially utilize HR, the mutations will be substituted by non-mutated (“germline”) nucleotides. We therefore postulate that proliferating U-CLLs utilize HR, which corrects the mutations accumulated during SHM. To test whether U-CLL cells preferentially use HR, we incubated CLL cells from two patients with U-CLL and two patients with M-CLL with antibodies that bind RPA and RAD51, key components in the HR repair pathway. By fluorescence microscopy, we detected these proteins in U-CLL, but not M-CLL, cells. To confirm these findings, we quantified the transcript levels of key players in the DNA repair process by qRT-PCR in four patients with M-CLL and two patients with U-CLL. Consistent with the immunohistochemistry data, the levels of FANCD2, RAD51, BRCA1 and BRCA2, which represent HR, were significantly higher in U-CLL compared with M-CLL. Levels of XRCC5, XRCC6, and PRKDC, which represent the NHEJ machinery, were low across all samples tested. Since HR is only operative in dividing cells, we hypothesized that inducing proliferation will turn on the HR enzymatic machinery. To induce proliferation, we incubated CLL cells from eight patients with CpG oligonucleotide. Using this agonist, proliferation increased by 110% (range: 42 to 250) in M-CLL cells but only by 25% (range 14 to 66) in U-CLL cells. The increase in proliferation rates correlated with upregulation of the HR machinery at the RNA and protein levels as measured by qRT-PCR and immunohistochemistry, respectively. Summary Here, we show that proliferating CLL cells utilize more often HR recombination repair machinery. Whether HR activity contributes to reduced mutational rates in the IGHV gene remains to be determined.