Adaptation of Chronic Lymphocytic Leukemia to Ibrutinib Is Mediated By Epigenetic Plasticity of Residual Disease and By-Pass Signaling Via MAPK Pathway

Background. Ibrutinib is the first in class inhibitor of BTK, highly active in chronic lymphocytic leukemia (CLL) as monotherapy. The most typical response to ibrutinib is partial remission (PR) with measurable minimal residual disease (MRD) in blood, maintained until genetically driven resistance. We aim to investigate the mechanisms that allows MRD cells to survive despite BTK inhibition.

Methods. Pre-treatment CLL cells and under treatment MRD were systematically collected at six homogeneous timepoints from 33 CLL patients who received ibrutinib in the IOSI-EMA001 or IOSI-EMA003 studies. Samples were characterized by high-dimensional flow cytometry, bulk genomic, transcriptomic, chromatin accessibility, single cell gene expression and chromatin accessibility, and spatial transcriptomics. In vivo validation was performed.

Results. Genetic clonal evolution was observed in 33% (N=11) of the study population, characterized by the expansion of initially subclonal mutations without a consistent pattern in specific genes or pathways. At the level of somatic copy number abnormalities, no clonal shift was detected. BTK or PLCG2 mutations were not present in the MRD phase but emerged during clinical progression.

Ibrutinib induced chromatin dynamics in MRD resulting in a predominantly closed state. Chromatin regions that became more accessible under ibrutinib (N=731) were enriched of binding sites for transcription factor (TF) that lay downstream ERK signaling. Regions that turned into a less accessible state (N=1580) were enriched for the binding sites of TF emanating from the BTK-containing proximal signalosome of the B-cell receptor (BCR) (NF-kB, JUN, NFAT).

Consistently, at the transcriptomic level most of the differentially expressed genes between pre- and post-treatment samples were downregulated in MRD (N=1235), while only a fraction was upregulated (N=114). BCR, IL4, NF-kB, metabolism and proliferation signatures were downregulated in MRD (FDR<0.001), while MAPK and RAS signatures were upregulated (BCR^low/ERK^high) (FDR<0.05). The BCR^low/ERK^high phenotype was also present in residual disease within the tissue, as demonstrated by spatial transcriptomics of residual CLL in lymph nodes excised from patients receiving ibrutinib. This phenotype was further supported by transcriptomic analysis of leukemia splenocytes from TCL1 transgenic mice treated with ibrutinib.

High-dimensional flow cytometry and transcriptomics revealed an upregulation of BCR surface molecules in the MRD. In contrast, receptor tyrosine kinases expression remained unchanged. Using phospho-flow cytometry and immunoblotting of MRD cells, we found that stimulation of the B-cell receptor (BCR) with an anti-IgM— but not stimulation of CD40 and Toll-like receptors— resulted in persistent RAS activation and ERK phosphorylation despite BTK block, while BTK and PLCG2 phosphorylation were expectedly abrogated.

Multiomics matched single-cell RNA-seq and ATAC-seq uncovered the pre-existence of a functional BCR^low/ERK^high clone before ibrutinib treatment with a shared gene regulatory network similar to post treatment samples.

Administration of ulixertinib, an ERK1/2 kinase inhibitor, in combination with ibrutinib significantly delayed tumor growth in vitro and in vivo.

Conclusion. Adaptation to BTK inhibition in CLL is mainly epigenetically driven and selects MRD cells with BCR^low/ERK^high phenotype. RAS-RAF-MEK-ERK proved to be a vulnerability of MRD.