PS1124 IBRUTINIB TREATMENT ALTERS ENHANCER ACTIVITY PATTERNS IN CHRONIC LYMPHOCYTIC LEUKEMIA

Background: Bruton tyrosine kinase (BTK) has been shown to play a central role in intracellular signalling which influences development, proliferation and survival of leukemic B cells. The first BTK inhibitor (Ibrutinib, ‘Ib’) has shown remarkable efficacy in Chronic Lymphocytic Leukemia (CLL). To date, accumulating evidence support the efficacy of BTK inhibition in diverse disease entities. The emergence of BTK inhibition as a therapeutic strategy for a wide range of B‐cell related diseases has led to an increased interest on in depth understanding of intracellular mechanisms of BTK action. Interestingly, recent studies in a murine genetic knockdown model have shown differential roles of BTK for distinct B cell subsets. Given the fact that human B cells are more dependent on BTK, it remains to be elucidated:(i) which are the intracellular mechanisms for putative differential BTK roles in B cells and (ii) what are the off‐target effects of BTK inhibition which may influence treatment outcome in B‐cell malignancies. Aims: We have hypothesized that BTK could be linked to critical changes to epigenomic dynamics either by a direct involvement in the nucleus or indirectly by a cascade of events starting at the cytoplasm. Our work aimed at providing insights with regard to potential epigenetic alterations upon BTK inhibition in leukemic cells. Methods: We employed pre‐ and post‐Ib treatment samples from 6 CLL patients and we performed a longitudinal analysis of global changes for 5 histone post‐translational modifications (PTMs). This analysis revealed significant heterogeneity of PTMs global changes among patients and between different time points, with H3K27ac showing the most striking differences. Subsequently, we performed genome‐wide mapping of H3K27ac by ChIP‐seq in paired CLL samples under Ib, both at pre‐ and post‐Ib state (4 cases/8 samples). Results: We observed recurrent changes (FDR<0.05) in H3K27ac profiles of post‐Ib state compared to the pre‐Ib. Top‐ranking genomic regions included putative and reported enhancers and super‐enhancers located mostly within gene bodies. Pathway analysis for affected regions indicated also known BTK‐related pathways: BCR, MAPK, mTOR, Calcium influx and NF‐κB. These signalling pathways are linked to BTK signalling and support the hypothesis that the intracellular effects of BTK inhibition leads to altered histone modification profiles. Transcription Factor binding motifs analysis for the list of BTK‐responsive enhancers and target genes provided interesting results suggesting that BTK may act as a key transcription regulator via altering the enhancer architecture of leukemic B‐cells (i.e. SP1, CBFB, MAX motifs). Target genes of the identified BTK‐reponsive enhancer were validated by Real‐Time PCR in an extented cohort of pre‐ and post‐Ib CLL samples. Of note, we observed highly significant changes at expression level for our top candidate active regions/target genes. Summary/Conclusion: In conclusion, our work provides important evidence that BTK inhibition has an impact on intracellular signalling which expands to epigenetic regulation of critical enhancers for B‐cell survival and function. Further investigation could provide valuable insights into the epigenetic and molecular events upon Ib treatment and unravel potential benefits of combinatorial/epigenetic‐relevant treatments.