Pb1913: igh 3’rr recombination uncovers a non-gc imprint and c-myc-dependent igh rearrangement activity in unmutated chronic lymphocytic leukemia

Topic: 5. Chronic lymphocytic leukemia and related disorders - Biology & Translational Research Background: Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B-cells that weakly express a B-cell receptor (BCR). The mutational status of the variable region (IGHV) on the immunoglobulin (Ig) heavy-chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLLs (mCLLs) are genetically imprinted by activation induced-cytidine deaminase (AID). In normal condition, AID is required for IGH rearrangements in activated B-cells: the class switch recombination (CSR) and the recombination between switch Mu (Sµ) and the 3’ regulatory region (3’RR) of the IGH locus (Sµ-3’RRrec) (Peron Science 2012). The fact that most CLL cells express an IgM raises the question of CSR blockade in this B-cell cancer. Low levels of CSR can be observed in a small fraction of CLL tumor cells and seems to correlate with AID expression in these intra-clonal switched CLL cells. AID has been repeatedly detected in CLL B-cells independently of the IGHV mutational status. AID likely contributes to CLL evolution and seems to generate intraclonal diversity targeting the IGH locus and non-Ig off-targets. Aims: In this study, we raised the question of IGH switch blockade in CLL. For that purpose, we analyzed both CSR and Sµ-3’RRrec junctions as reflections of putative switch activity. Methods: We analyzed counts of CSR and Sµ-3’RRrec in 47 DNA sample CLLs diagnosis high blood tumor infiltration (>90%). Results were compared to results from mononuclear cells from peripheral blood from 9 healthy volunteers (HVs) and from 24 pediatric tonsils. The project was conducted according to the guidelines of the Declaration of Helsinki. Results: Our results separated CLLs into two groups on the basis of Sµ-3’RRrec counts per sample, Sµ-3’RRrecHigh cases (mostly unmutated IGHV CLLs) and Sµ-3’RRrecLow cases (mostly mCLLs), but not based on CSR junction counts. Sµ-3’RRrec appeared to be ongoing in Sµ-3’RRrecHigh CLL cells and comparison of Sµ-3’RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sµ-3’RRrecHigh CLLs harbor a non- germinal center (GC) experienced B-cell imprint while Sµ-3’RRrecLow CLLs are from AID-experienced B-cells from a secondary lymphoid organ. Moreover, we observed Sµ-3’RRrecHigh patient homogeneously exhibited telomeres that were shorter than HVs, reflecting increased proliferation cycles, and increased MYC expression. Questioning the impact of c-MYC on IGH recombination, we used the murine B-cell lymphoma CH12F3 cell line and its AID knock-out counterpart stably transfected or not with a MYC overexpression vector and stimulated in vitro to undergo CSR and Sµ-3’RRrec. Results show that c-MYC potentiated both CSR and Sµ-3’RRrec when AID was expressed. Even at low frequency, Sµ-3’RRrec was also possible in the absence of AID, in the presence of c-MYC overexpression. Summary/Conclusion In addition to the proposals already made concerning the CLL cell of origin, our study highlights that the IGH recombinatory activity allows the separation of CLL cases from different origins: the CLL Sµ-3’RRrecLow group that would originate from GC-experienced B-cells and the Sµ-3’RRrecHigh CLLs with a probable non-GC experienced B-cell with different prognoses. Finally, on-going Sµ-3’RRrec in Sµ-3’RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sµ-3’RRrec, even in the absence of AID. Keywords: c-myc, IgH rearrangment, B cell, Chronic lymphocytic leukemia