RNA Cytosine Methyltransferases NSUN1 and NSUN2 Mediate the Lineage-Associated Resistance to Venetoclax in Leukemia

Background:

Combinations of venetoclax/ABT-199, a small molecule that selectively inhibits anti-apoptotic protein BCL2, with hypomethylating agents (HMAs), such as 5-azacytidine (5-AZA, azacidtine) and decitabine have demonstrated remarkable synergistic effects and resulted in high response rates and significant overall survivals in patients with refractory MDS/AML (Ram, et al. Annual Hematology 2019; DiNardo et al. Blood 2019). However, resistance to venetoclax-based therapies has emerged as a major therapeutic barrier and been linked to monocytic clones in leukemia (Kuusanmaki et al. Haematologica 2020; Pei et al. Cancer Discovery 2020). Our recent study demonstrated that specific RNA cytosine methyltransferases (RCMTs), namely NSUN1 and NSUN2, mediate the lineage-associated resistance to 5-AZA through formation of a drug-resistant elongating RNA-Polymerase-II (eRNAPII) complex at nascent RNA (Cheng et al. Nature Communications 2018). This study aims to address the role of NSUN1 and NSUN2 in mediating venetoclax resistance in leukemia.

Experimental Design and Methods:

Experiments, including drug-induced cell growth inhibition, western blot, and co-immunoprecipitation, were performed on leukemia cell lines with various lineages to assess lineage-associated venetoclax resistance and identify the key factors/proteins involved in such resistance. Venetoclax-resistant cell lines were established from drug sensitive lines in order to elucidate mechanisms underlying resistance and cell lineage plasticity. Knockdown of NSUN1 and NSUN2 expression was performed to determine their roles in venetoclax-resistant cell lines.

Results:

Our experimental results have demonstrated monocyte differentiation-associated resistance to venetoclax in leukemia cell lines of different lineages (Figure 1A), which is consistent with previous published studies (Kuusanmaki et al. Haematologica 2020; Pei et al. Cancer Discovery 2020). The degree of lineage-associated venetoclax resistance is closely correlated with the expression of eRNAPII, NSUN2 and NSUN1. Importantly, venetoclax strongly induces expression of eRNAPII and NSUN1 (Figure 1B). We established venetoclax-resistant leukemia cell line (K1VR) from original venetoclax-sensitive granulocytic leukemia cell line Kasumi-1 and confirmed the importance of NSUN1 and NSUN2 in mediating venetoclax resistance in those leukemia cells. siRNA knockdown of NSUN1 or NSUN2 expression inhibits growth of leukemia cells and re-sensitizes the venetoclax-resistant K1VR leukemia cells to a low dose of venetoclax (Figure 1C).

Conclusion:

Our study has demonstrated that RNA cytosine methyltransferases NSUN1 and NSUN2 mediate monocyte-associated resistance to venetoclax in leukemia cells. We are currently extending our study to clinical specimens. Our study may lead to development of novel RNA epigenetics-driven strategies to predict and overcome the resistance to venetoclax-based therapies.