Novel Mechanisms of Acalabrutinib Resistance in Patients with Chronic Lymphocytic Leukemia By Whole Genome Methylome Sequencing

Background: Bruton's tyrosine kinase (BTK) inhibition is a key component in B-cell receptor signaling and is one of the most prominent therapeutic targets in hematologic malignancies. Acalabrutinib is a highly selective, covalent, potent next-generation inhibitor of BTK (Barf et al, 2017) and is approved for the treatment of mantle cell lymphoma and chronic lymphocytic leukemia (CLL). However, a small subset of patients develop resistance to acalabrutinib over time. While the major mechanism of resistance to covalent BTK inhibitors is explained by acquired mutations in BTK-Cys481 and its downstream target PLCg2, additional mechanisms are under investigation. Previously, we presented that high surface expression of CD49d (VLA-4) and CD79B correlates with acalabrutinib resistance in CLL patients (Bibikova et al, 2019). In the present study, we performed optimized whole genome methylation sequencing on 42 clinical samples from 22 patients in the ACE-CL-001 clinical trial (NCT02029443) to better understand novel mechanisms of acalabrutinib resistance at an epigenetic level in CLL patients.

Methods: DNA was extracted from peripheral blood mononuclear cells of patients with available samples at baseline (n=20), following acalabrutinib treatment at the time of progressive disease (n=8), or at 6 months post treatment start (n=14, used as non-progressor controls). While bisulfite conversion has been the gold standard for DNA methylation analysis for decades, it damages DNA and performs suboptimally with clinical FFPE and cfDNA samples that are often in limited quantity. Enzymatic methods have recently been reported that do not induce DNA damage, making them more suitable to routine clinical samples that are already DNA-damaged (eg, by formalin fixation) and often have limited DNA amounts. Before performing methylome analysis, we conducted a comprehensive comparison of bisulfite and enzymatic DNA methylation assays in reference cell lines and clinically relevant samples . This revealed that enzymatic methods outperformed bisulfite in several library quality metrics and resulted in increased library yield with the same DNA input and PCR amplification cycles. Enzymatic methods also captured a larger fraction of GC-rich regions, including CpG islands, in all comparisons. Prior to interrogating the full CLL 22 patient sample set, the enzymatic methods were validated in a subset of 6 CLL samples. We conducted a comprehensive analysis of methylation data sets with previously established RNA-seq analysis from paired samples.

Results: Analysis of the acalabrutinib posttreatment samples revealed that the non-progressed patients had significantly higher methylation of 440 genes, whereas progressed samples had higher methylation of only 2 genes. CDKN1C, IL15, and AXL were significantly more methylated in non-progressed samples (Figure 1A), and these genes have been shown to be related to proliferation, survival, or response to BTK inhibition in CLL (De Totero et al, 2008; Sinha et al, 2018; Gupta et al, 2019; Lu et al, 2021; Kagiyama et al, 2021). Using RNA-seq data from paired samples, we showed that, as expected, IL15 methylation negatively correlated with IL15 expression (Figure 1B; R=−0.73, p=7.1e-09). IL15 is a proinflammatory cytokine that has downstream signaling targets in the NF-kB pathway, similar to BTK, indicating that alternative methods of NF-kB signaling may affect response. To support this hypothesis, we found that IL15 methylation significantly correlated with BIRC2 (R=0.59, p=1.4e-5) and RELA (R=0.42, p=0.0034) expression. Both genes play a role in NF-kB signaling, where BIRC2 encodes for the protein cIAP1 that regulates NF-kB activation and RELA encodes for p65, a member of the NF-kB transcription factor family. To our knowledge, it is not known if DNA methylation of IL15-induced expression plays a role in CLL resistance to BTK inhibition, and this finding may have important implications for future combination treatments. To note, CDKN1C, IL15, and AXL methylation were also significantly different between the progressed and non-progressed patients before acalabrutinib treatment, indicating they may be playing a role in innate resistance to acalabrutinib treatment.

Conclusions: Using EM-seq to analyze DNA methylation, we found that reduced IL15 methylation and increased IL15 expression correlates with acalabrutinib innate resistance in CLL patients.

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