Depies, Olivia M

Abstract Therapeutic inhibition of BCR signaling using Bruton tyrosine kinase inhibitors (BTKis) has remarkable efficacy in the treatment of chronic lymphocytic leukemia (CLL). However, the efficacy of BTKis is limited by the development of drug resistance, which can be mediated through mutations in genes encoding proteins of BCR signaling pathways. Approaches targeting the BCR mutant proteins have been limited by the large heterogeneity of these mutations. In addition, BCR signaling mutations are only detected in up to two-thirds of the CLL patients who progressed with BTKi treatment, suggesting mechanisms beyond genetic mutations. We previously showed that the BCR signaling pathway impacts the chromatin landscapes of CLL B cells (Wang et al., Blood Cancer J. 2022), suggesting that epigenetic mechanisms are exploited by CLL B cells to support their survival. To gain insights into the epigenetic regulation of BTKi treatment in CLL B cells, we analyzed the genome-wide chromatin accessibility (ATAC-seq) and histone modification (H3K4me1, H3K4me3, H3K27ac, H3K27me3) profiles (CUT&Tag) of leukemic cells from 4 CLL patients on BTKi ibrutinib treatment in a sequential fashion (i.e., baseline, on ibrutinib treatment, and at relapse). We also performed the same analysis in a CLL cohort at two stages of ibrutinib treatment (baseline and while on treatment, n=20). Our studies showed that suppressing BCR signaling by BTKi leads to the downregulation of genes associated with mitochondrial translation, a key process of mitochondrial biogenesis. We also found decreased chromatin accessibility and H3K27ac deposition at the promoters of these MT genes. Interestingly, in patients who experienced disease progression on ibrutinib, there was a restoration of MT gene expression, chromatin accessibility, and H3K27ac levels at the MT gene promoters similar to baseline samples, suggesting an epigenetic-regulated MT both in BTKi treatment naïve and relapsed CLL patients. Importantly, MT inhibition led to a decreased survival of primary CLL B cells. These findings point toward an epigenetic mechanism utilized by BCR signaling to promote MT and leukemic cell survival. We then observed that the transcription factor NRF1 is enriched at the MT promoter in CLL B cells. CRISPR-mediated depletion of NRF1 led to reduced mitochondrial translation and cell survival in BCR signaling active malignant B cell lines and primary CLL B cells. These results suggest that NRF1 controls an epigenetic program linking BCR signaling to control MT, which in turn supports BCR signaling-dependent CLL B cell survival. Our findings demonstrated that BCR signaling utilizes epigenetic mechanisms that regulate mitochondrial metabolism to enhance cell survival in both treatment naïve and BTKi-relapsed CLL. These findings may provide new therapeutic directions for untreated and BTKi-relapsed CLL patients. Citation Format: Olivia M. Depies, Sutapa Sinha, Sameer Parikh, Neil Kay, Zhiquan Wang. An epigenetic program that links oncogenic signaling to mitochondrial biogenesis supports leukemic cell survival in chronic lymphocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5842.

Abstract Extrachromosomal DNA (ecDNA) drives the evolution of cancer cells by conferring survival advantages over normal cells. However, the functional importance of ecDNA replication and maintenance is yet to be evaluated, and the molecular components that contributed to these processes remain largely unknown. Here, using CRISPR-C (circularization of genes and chromosomes by CRISPR) technology and synthetic circular DNA mimicking ecDNA identified in tumors, we generated ecDNA-positive (ecDNA+) cell models. Using these models, we identified the proteins involved in ecDNA replication and maintenance and found DNA damage response (DDR)-related pathways were significantly enriched. We further demonstrated that ecDNA replication led to increased DDR and genome instability and DDR was also essential for ecDNA maintenance, consistent with observations that ecDNA+ tumors exhibited higher DDR scores than ecDNA-negative (ecDNA–) tumors in patients. ATM, MDC1, and CHK2, but not ATR, were activated in ecDNA+ cells compared to ecDNA– cells. Moreover, ecDNA+ cells were more sensitive to ATM and CHK2 inhibition. We further found that type II topoisomerase TOP2B and DNA ligase III (LIG3) were critical regulators for ecDNA replication and maintenance and related DDR. In summary, our findings of the reciprocal interactions between ecDNA and DDR provide new insights into detection and therapy for ecDNA+ tumors.

Introduction: Recent studies have shown that clonal B cells derived from patients with chronic lymphocytic leukemia (CLL) have altered histone modification landscapes compared to B cells from healthy controls. We have also shown that the BCR signaling pathway impacts the chromatin landscapes of CLL B cells, implicating the interaction between oncogenic signaling and epigenetic machinery in CLL (Wang et al., Blood Cancer J. 2022; Wang et al., Cancer Res (2023) 83 (7_Supplement): 4725). However, the majority of the chromatin studies in CLL concentrate on the active enhancers associated with oncogene expression; there is a very limited understanding of lost enhancers in CLL: 1) What is the function of lost enhancers; 2) How are these lost enhancers maintained in CLL; 3) Can these lost enhancers be detected at the precursor state of CLL, monoclonal B-cell lymphocytosis (MBL); 4) if this aberrant chromatin signature affected by CLL treatment. Methods: We systematically mapped the genome-wide histone modification profile (histone marks: H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3, and H3K36me3) in 22 previously untreated CLL samples, 6 high count MBL samples, and peripheral blood B cells from 6 healthy donors by Cleavage Under Targets and Tagmentation (CUT&Tag). We also analyzed B cells from 8 patients before and after one year of continuous Bruton tyrosine kinase inhibitor (BTKi) ibrutinib treatment. The genomic regions with a decrease in both H3K4me1 and H3K27ac enrichment (analyzed by Deseq2, fold change > 1, p < 0.01) were named lost enhancers. We used CRISPR-mediated epigenetic editing to study the role of gained H3K9me3 on EBF1 gene expression. Results: Compared to normal B cells, the lost enhancers (genomic regions defined by decreased enrichment of H3K4me1 and H3K27ac) in CLL B cells showed increased heterochromatin marks (H3K9me3 and H3K27me3) enrichment. Importantly, these enhancers are associated with genes downregulated in CLL and involved in the immune response, indicating the source of epigenetic impact on impaired immunity in CLL. We further found that this aberrant chromatin structure also exits in clonal B cells from the precursor stage of MBL, implicating the importance of epigenetic silencing in CLL evolution. In particular, we found the lost enhancer signature across the gene locus EBF1, an important transcription factor that defines B cell lineage and regulates normal B cell activation. Using CRISPR-mediated epigenetic editing, we demonstrated that the gain of H3K9me3 is sufficient to silence the EBF1 enhancer and its gene expression. Finally, we found that while BTKi ibrutinib treatment partially inhibits the CLL-gained enhancers in some patients, it failed to restore the lost enhancers in CLL B cells. Conclusion: In summary, we have elucidated an epigenetic silencing mechanism that may contribute to the loss of physiological function of normal B cells, which places the clonal B cells at a selection advantage during CLL evolution and increases the risk of serious infections in CLL and MBL ( Fig 1).