Hnrnpc and m6a RNA Methylation Control Oncogenic Transcription and Metabolism in T-Cell Leukemia

RNA homeostasis is dysregulated in oncogenesis, impacting cancer progression and therapy resistance. N6-methyladenosine (m⁶A), the predominant epitranscriptomic modification in eukaryotic mRNA, plays a crucial role in RNA biology, affecting transcript stability, translation, and splicing. These diverse effects suggest a complex interaction network among m⁶A machinery components, requiring integrative analyses for a profound understanding of methylated adenosine biology, associated enzymes, and therapeutic implications. Despite extensive research, the prognosis for relapsed patients with T-cell acute lymphoblastic leukemia (T-ALL) remains poor, with a median survival of only 4-8 months (Pui, C. H., Lancet, 2008). T-ALL, with its extensive chromatin and transcriptomic changes (Ntziachristos P. et al., Nature, 2014, Zhou Y. et al., Cancer Discovery, 2020), is an ideal model for studying RNA methylation.

To characterize the m⁶A landscape in T-cell leukemia, we performed ELISA and enhanced UV crosslinking and immunoprecipitation (eCLIP) sequencing on human T-ALL patient samples and healthy thymic controls. Our findings reveal extensive global RNA methylation changes in the T-ALL transcriptome, primarily characterized by hypomethylation. Integration of gene expression and epitranscriptomics data showed a significant positive correlation between RNA methylation and gene expression changes in T-ALL. Hypermethylated transcripts are predominantly upregulated in T-ALL, while hypomethylation is associated with decreased expression. Enrichment analysis indicated that MYC-regulated transcripts and those involved in mevalonate/cholesterol biosynthesis (e.g., 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1)) are highly expressed and enriched with the m⁶A mark in leukemia, showing increased transcript and protein expression levels in T-ALL.

Gene expression analysis, gene essentiality data from the Cancer Dependency Map, and our RNA-binding protein (RBP)-focused CRISPR/Cas9 screen identified that the m⁶A reader heterogeneous nuclear ribonucleoprotein C (HNRNPC) is highly expressed in T-ALL and critical for T-ALL survival compared or cancer types or other m⁶A readers. HNRNPC directly binds and regulates the MYC and cholesterol biosynthesis pathway transcripts. Silencing HNRNPC significantly reduced these pathways via decreased cell proliferation and increased apoptosis. To study m⁶A changes in the T-ALL transcriptome and dynamic HNRNPC positioning, we used METTL3 inhibitors in T-ALL cells. METTL3 inhibition with STM2457 (Yankova E. Nature, 2021) compromised global RNA methylation levels. Contrary to studies in AML, MYC (and the cholesterol biosynthesis pathway) showed hypermethylation coupled to increased HNRNPC binding, intensified translation rates, and elevated transcript and protein levels upon METTL3 inhibition. We further show that HNRNPC interacts with ribosomal proteins, further suggesting a potential role of the protein in translation. Ultimately, metabolomic analyses revealed increased intracellular cholesterol levels following METTL3 inhibition. Treatment with a newer inhibitor STM30006 (Guirguis, A.A. Cancer Discovery, 2023) showed a similar increase in MYC and the cholesterol pathway components.

We then evaluated the levels of RNA methylation machinery components in T-cell leukemia to identify m⁶A-associated therapeutic vulnerabilities. We show that T-ALL cells present elevated FTO levels compared to physiological T cells or AML, due to direct transcriptional regulation by MYC. MYC also controls HNRNPC and the cholesterol biosynthesis pathway transcripts and inhibition of the cholesterol pathway using statins compromises MYC levels, suggesting a feedback loop in T-ALL. The use of the FTO inhibitors CS1 (bisantrene) and CS2 (brequinar, Su R. et al., Cancer Cell, 2020) for the treatment of cell lines, patient samples ex vivo, and a xenograft T-ALL model showed high efficacy of the drugs in inhibiting T-ALL. Further analyses revealed decreased m⁶A levels and repression of MYC and the cholesterol pathway upon FTO inhibition. Combining FTO inhibitors with dexamethasone and other chemotherapeutic agents demonstrated synergy in inhibiting T-ALL.

These findings indicate that T-ALL exhibits dysregulated RNA methylation and demonstrate the potential of targeting HNRNPC and FTO as therapeutic strategies in T-ALL.