Abstract
Children with Down syndrome (DS) have a significantly higher risk of developing acute myeloid leukemia (ML-DS), a distinct leukemia subtype preceded by transient abnormal myelopoiesis. In addition to trisomy 21 and GATA1 mutation, ML-DS is driven by co-operating mutations in signaling pathways such as JAK/STAT and Ras, cohesin complex associated genes (RAD21, STAG2), or epigenetic regulators (CTCF, SUZ12, EZH2). Although most ML-DS patients respond favorably to chemotherapy, outcomes remain poor in relapsed or refractory cases, which highlights the need for novel therapeutic strategies.
We previously demonstrated that dual inhibition of EZH2 and class I histone deacetylases (HDAC) by using GSK126 and romidepsin synergistically suppressed ML-DS cell viability (Cicek et al., 2022). Here, we present mechanistic insight into this dual epigenetic inhibition synergy and identify Bruton's tyrosine kinase (BTK) as an upstream regulator of EZH2 stability.
The combination treatment led to significantly enhanced apoptosis, as determined by Annexin V staining and cleavage of caspase-3, caspase-7, and poly(ADP-ribose) polymerase in numerous ML-DS cell line/patient-derived xenograft (PDX) lines. Western blotting revealed reduced H3K27 trimethylation (H3K27me3) and increased H3K4 trimethylation (H3K4me3) following combination treatment. Transcriptomic profiling by RNA-Seq showed that combination treatment uniquely regulated large number of genes and suppressed proliferation-associated pathways. Gene set enrichment analysis (GSEA) revealed the downregulation of E2F targets, MYC targets, and G2/M checkpoint genes, which also represent the pathways that are aberrantly activated in ML-DS cell line/PDX models (n=5) compared to normal CD34+ hematopoietic stem and progenitor cells (n=4).
Interestingly, the combination treatment also induced EZH2 protein degradation. To further investigate the mechanism underlying EZH2 degradation, we performed immunoprecipitation-mass spectrometry (IP-MS) using anti-EZH2 antibody to identify potential interaction partners that may regulate EZH2 protein stability. As previous studies have identified several kinase interacting partners of EZH2, which may affect its function or stability, we focused on protein kinases that were detected in EZH2 immunoprecipitates. BTK was identified as a prominent interaction partner of EZH2 in ML-DS cells. Co-immunoprecipitation confirmed endogenous interactions between BTK and EZH2 in CMK, a ML-DS cell line. Moreover, RNA-Seq analysis demonstrated that combination treatment downregulated BTK expression and B-cell receptor (BCR) signaling pathways.
Given this, we hypothesized that BTK downregulation by GSK126/romidepsin combination may destabilize EZH2. Notably, BTK inhibition by Pirtobrutinib, a FDA-approved reversible BTK inhibitor, recapitulated the effects of combination treatment by reducing EZH2 protein level and inducing cell death of relapsed ML-DS PDX cells via apoptosis. Proteosome inhibition with MG132 in the presence of Pirtobrutinib rescued EZH2 level, indicating that BTK inhibition reduces EZH2 stability via the ubiquitin-proteasome pathway. Furthermore, BTK inhibition suppressed key E2F-related oncogenic targets, including c-MYC, Bcl-xL and MCL-1, while upregulating the cell cycle inhibitor p21.
Together, these findings highlight BTK as a novel regulator of EZH2 in ML-DS and suggest that BTK inhibition may be an effective therapeutic strategy in ML-DS.
