CRISPR/RfxCas13d Screening Identifies the Long Non-Coding RNA (LncRNA) Nfyc-AS1 As a Molecular Vulnerability in Acute Myeloid Leukemia (AML)
Authors: Papaioannou Dimitrios , Gambi Giovanni , Thandapani Palaniraja , Nicolet Deedra , Lin Ziyan , Mrózek Krzysztof , Ivashkiv Olha , Sirenko Maria , Nadorp Bettina , Eisfeld Ann-Kathrin , Tsirigos Aristotelis , Aifantis Iannis 
Published: December 2024

Introduction: LncRNAs are important regulators of multiple cellular functions in health and disease. In AML, aberrant expression levels of lncRNAs are independently associated with the outcome of AML patients. Individual lncRNAs are functionally relevant in distinct molecular subgroups of AML. Large-scale profiling of lncRNAs that could mediate resistance of leukemic blasts to cytotoxic drugs has recently been reported. However, an unbiased functional screening of lncRNAs, which could represent molecular vulnerabilities in AML is currently lacking.

Methods:To study the functional role of clinically relevant lncRNAs in AML, we focused on 176 lncRNAs, previously associated with outcome of AML patients. We designed a pooled guide RNA (gRNA) library, which comprised 1048 lncRNA-targeting gRNAs and performed drop-out screening experiments. We transfected with our libraries four AML cell lines (i.e., KG-1, MOLM-13, OCI-AML3, THP-1), which expressed the CRISPR/RfxCas13d nuclease. Genomic DNA was collected on days 0 and 9 post-transfection and subjected to deep sequencing and gRNA representation analysis. We used antisense oligonucleotides (hereafter named gapmers) to target lncRNAs and validate our findings. Total RNA sequencing (RNA-Seq), H3K27ac ChIP and chromatin conformation assays (i.e., HiChIP) were performed per published protocols. RNA capture experiments were conducted with antisense biotinylated probes, per the RNA antisense purification (RAP) protocol. We used the RNAScope platform and reagents to directly visualize the NFYC-AS1 lncRNA.

Results: We identified 24 lncRNAs, which were essential for all AML cell lines tested and 9, whose essentiality was subtype-specific. We confirmed our findings by conducting competitive growth assays between gRNA transduced (i.e., GFP-positive) and non-transduced (i.e., GFP-negative) blasts. The NFYC-AS1 lncRNA was among the validated targets that generated a potent survival disadvantage in a subset of leukemic cells. In a large cohort of AML patients analyzed with RNA-Seq, high NFYC-AS1 expression associated with myelomonocytic differentiation of the leukemic blasts (M4/M5 versus other FAB subtypes), as well as presence of NPM1 mutations or KMT2A rearrangements (KMT2Ar). Regarding clinical outcome, high NFYC-AS1 expression was associated with shorter disease-free (P=0.01) and event-free survival (P=0.008). We tested 8 AML cell lines and detected aberrant overexpression of NFYC-AS1 in the THP-1 and MONOMAC-6 cells, which were sensitive to NFYC-AS1 depletion. Delivery of NFYC-AS1-targeting gapmers efficiently depleted NFYC-AS1 and recapitulated a growth-inhibitory effect in AML cell lines and patient blasts with high NFYC-AS1 expression. Of note, NFYC-AS1 targeting did not affect expression levels of the protein-coding NFYC transcript and had no functional effect in healthy hematopoietic cells (as evaluated by apoptosis and colony-forming unit assays). In cell fractionation and RNA visualization experiments, we found that NFYC-AS1 was enriched in the nucleus of the leukemic blasts. To gain mechanistic insights, we performed RNA-Seq following NFYC-AS1 depletion and detected a pronounced effect of NFYC-AS1 knockdown on genes located on chromosome 19. Furthermore, in RAP capture experiments followed by deep sequencing, we observed that NFYC-AS1 avidly localized to the p13.3 locus of chromosome 19. In H3K27ac ChIP and HiChIP experiments we detected strong interactions between the NFYC-AS1 binding site and the promoters of genes involved in myeloid differentiation and survival (e.g., AZU1, PRTN3, ELANE, PTBP1, CNN2) via chromatin looping.NFYC-AS1 depletion altered the chromatin conformation of the chr19p13.3 locus, significantly decreased the strength of these long-range DNA-DNA contacts and impacted on the expression levels of essential genes.

Conclusions : CRISPR/RfxCas13d is a valuable tool that can be utilized to detect molecular vulnerabilities in the non-coding transcriptome of malignant cells. CRISPR/RfxCas13d screening has identified NFYC-AS1 as an essential lncRNA, which regulates chromatin conformation and transcriptional activity in the molecular subset of AML with KMT2Ar or NPM1 mutations. Further experiments to assess the therapeutic utility of NFYC-AS1 targeting (alone or in combination with Menin inhibitors) in preclinical AML models are underway.