Isoform-Level Long Noncoding RNA Dependencies in Multiple Myeloma

Introduction

Long noncoding RNAs (lncRNAs) are a diverse group of genomic elements that, despite not encoding proteins, play crucial roles in regulating cellular processes. In multiple myeloma (MM), lncRNAs have emerged as significant factors, functioning as clinical biomarkers, tumor drivers, and potential therapeutic targets. The study of lncRNAs is particularly challenging due to their complex genomic organization, frequent overlap with protein coding genes, and the generation of multiple isoforms through extensive splicing. Moreover, lncRNAs can act as DNA regulatory elements, influencing gene expression independently of their RNA products. We, therefore, performed a novel Cas13d screen for an isoform level characterization of lncRNAs in MM.

Methods

To study the isoform level complexity of lncRNAs, we employed CRISPR-Cas13d that specifically degrades RNA transcripts to knock down lncRNA isoforms originating from various genomic contexts and within different sub-cellular compartments. This approach, termed IsoScan, was designed to screen for lncRNA dependencies in MM through guide RNA (gRNA) “drop-out” viability screens.

We constructed a pooled lentiviral library, IsoScan-L1, comprising 55,405 gRNAs targeting the MM lncRNA transcriptome with isoform-selective precision. More than 95% of these gRNAs were predicted to bind exclusively to a single isoform. Cas13d was introduced into five MM cell lines (AMO1, H929, KMS11, OPM2, R8226), and the system’s efficiency was validated by targeting a MALAT1 isoform. Results confirmed significant growth inhibition and downregulation of MALAT1 (R² = 0.94; p < 0.0001).

Results

Viability screens using depletion of gRNAs in cell culture using IsoScan-L1 identified 598 tumor-promoting (tp)-lncRNA isoforms, representing 12% of the MM lncRNA transcriptome, from 479 lncRNA genes. This included known tp-lncRNAs such as isoforms of MIR17HG, MALAT1, and NEAT1. Secondary screens targeting 315 tp-lncRNA isoforms common to at least two cell lines confirmed the depletion of most tp-lncRNA isoforms both in vitro and in an H929-xenograft model.

We further validated the isoform-selectivity of our gRNAs by targeting seven specific isoforms (SNHG6-003, SNHG8-005, GAS5-003, DANCR-001, ASH1L-AS1-001, LINC-PINT-004, and KDM4-AS1-003) and confirming phenotypic changes (inhibition of cell proliferation) and effective downregulation of tp-lncRNAs at the isoform level. Importantly, gRNAs targeting SNHG6-003 or GAS5-003 did not affect the expression of alternative isoforms or antisense transcripts and intronic small RNAs.

To expand the investigation to other malignancies, we used IsoScan-L2 in six additional Cas13d-expressing cancer cell lines from both hematologic and solid cancers (SU-DHL-4, KARPAS-422, RAJI, A549, MCF7, HCT-116). This broader screen identified 42 isoforms as strong hits (LogFC < -1) across all 11 cancer cell lines including MM, and 86 as hits in at least 8 lines, categorized as "cancer-common" tp-lncRNA isoforms. Additionally, eight "MM-selective" tp-lncRNA isoforms were identified, showing strong hits in at least two MM cell lines without affecting other cancers.

Subcellular RNA sequencing revealed that most tp-lncRNA isoforms were predominantly expressed in chromatin-bound or nuclear-soluble fractions, including isoforms of well-characterized nuclear-active lncRNAs. In contrast, a smaller subset of lncRNA isoforms was predominantly cytoplasmic, with 12 common across the MM cell lines tested.

A particularly notable tp-lncRNA in this study was SNHG6 isoform -003. This isoform was uniquely overexpressed in MM compared to healthy donor plasma cells, especially in relapse cases and in patients with del17p. SNHG6-003 was identified as high dependency and predominantly cytoplasmic, crucial for the survival of MM and other cancer cells. Its cytosolic function was validated through cytosolic-directed Cas13d and cytosolic RNA interference approaches. SNHG6-003 specifically localized to the endoplasmic reticulum (ER), interacted with heat shock proteins, and its knockdown induced ER stress. Targeting SNHG6-003 with antisense oligonucleotides killed CD138+ cells in ex vivo MM patient samples.

Conclusion

This study highlights a significant isoform level lncRNA dependency in MM with potential as therapeutic targets.