Proposed Topic: Revealing the therapeutic potential of the epitranscriptome and translational regulation at the onset of Leukemia drug resistance

Abstract

B-cell acute lymphoblastic Leukemia (B-ALL) is characterised by prevalent chromosomal translocations leading to the expression of gene fusions. Distinct B-ALL fusions can lead to different proliferative and survival capacities of malignant cells. Consequently, subtype-specific B-ALL fusions exhibit diverse responses to chemotherapy, which can result in therapy resistance through yet unknown mechanisms.

Deep investigation of gene expression control can be an entry point to highlight the subtype-specific drug resistance responses in B-ALL. A preliminary analysis of B-ALL patient samples revealed that B-ALL shows gene expression alterations potentially linked to RNA processing mechanisms, including translation and RNA modifications. Across a panel of 11 common B-ALL subtypes, these alterations occurred as specific to the two most common B-ALL fusion types, ETV6-RUNX1 and KMT2A-MLLT1. Importantly, these subtypes are also associated with highly divergent prognoses, with ETV6-RUNX1 having a more favourable prognoses and KMT2A-MLLT1 known to relapse.

Recent published studies also point to the dynamic involvement of translational control and the epitranscriptome in cancer drug resistance across cancers, but the role of these mechanisms in B-ALL remains unknown. Here, we set out to directly investigate RNA-level signatures of resistance to common chemotherapy in B-ALL. For the first time, we revealed accurate compositional information about the native transcriptome including RNA modification profiles, and the linking functional effects recorded at the level of RNA translatability and stability.

To establish cell models recapitulating the two B-ALL fusion subtypes and subtype-specific resistance development, we used REH (ETV6-RUNX1) and KOPN-8 (KMT2A-MLLT1) cell lines, alongside a non-malignant B-cell line, GM12878. We employed multiple successive rounds of treatment with vincristine (VCR), a prototypical chemotherapy drug used in many haematological malignancy treatment regimens, to progress both B-ALL cell lines to acquired resistance. We then used the advantages of direct RNA long read sequencing to accurately compare the native transcriptomes, RNA modification patterns, stability profiles and per-isoform translatability of the original vs. VCR-resistant cells using innovative in-house developed methods.

At the epitranscriptome level, we showed that METTL3-dependent m6A was upregulated in malignant cells for both B-ALL tumour backgrounds. However, there was no change in METTL3-dependent m6A upon developing VCR resistance for both subtypes. However, for PUS7-dependent pseudouridine, while there was no alteration in malignant B-ALL cells, unexpectedly, we observed a significant increase upon acquiring vincristine resistance in both subtypes. From a translational standpoint, while global translation rates increased in B-ALL malignancy for both subtypes, we observed an overall reduction in translation linked to acquired resistance that may potentially be attributed to epitranscriptomic alterations in PUS7- and DKC1-dependent pseudouridine. In the polysome bound fractions, we observed a shift in PUS7 and DKC1 transcripts from the light polysomal fraction into the heavy polysomal fraction.

Our work is first to provide a deep transcriptomic insight into the RNA-level mechanisms governing drug resistance onset in a prevalent blood cancer. We also highlight a potential mechanism linking the increased gene-level expression and epitranscriptomic signatures of PUS7-dependent pseudouridine on downstream effectors of mammalian targeting of rapamycin (mTOR) signaling pathway as a way of decelerating translation rates in VCR-resistant B-ALL cells. These results not only reveal that RNA modifying proteins potentially serve as novel biomarkers of early VCR resistance, but they also open avenues into using PUS7 and potentially DKC1 as novel therapeutic targets for overcoming VCR resistance in leukemia chemotherapy. Show more