Long-noncoding vulnerabilities in MM

In this issue of Blood, Grillone et al demonstrate that the long noncoding RNA (lncRNA) RP11-350G8.5 negatively regulates the protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway in multiple myeloma (MM) to prevent the induction of immunogenic cell death (ICD).¹ 

Accounting for approximately 10% of hematological malignancies, MM is a heterogeneous neoplasm caused by the uncontrolled proliferation of plasma cells in the bone marrow.² Despite advancements in treatments, including stem cell transplants and chimeric antigen receptor T-cell therapy, the combination of chemotherapy, immunomodulatory drugs, and proteasome inhibitors only provides a 5-year relative survival rate of 58%. Given its incurable nature and the occurrence of therapy resistance, innovative treatments for MM are urgently needed.

lncRNAs are non-protein-coding RNA transcripts longer than 200 nucleotides, expressed in a cell type-specific manner, and function in cellular homeostasis, differentiation, and development.^3,4 Recently, 989 lncRNAs were identified as selectively expressed in MM, with 89 showing de novo epigenomic activation.⁵ Mapping lncRNAs to genomewide coessentiality modules revealed another 30 high-confidence pan-cancer viability-associated lncRNAs.^6,7 To assess guide RNA and lncRNA essentiality in MM using unbiased genetic perturbations, Grillone and colleagues used a previously reported dual-gRNA CRISPR approach that induces large-fragment deletions and successfully identified lncRNAs required for cancer cell fitness.⁸ Using this technology, the authors perturbed 671 lncRNAs in the MM cell line AMO-1 and its bortezomib-resistant subclone (ABZB), assessed associated cell fitness effects, and integrated gene expression data from patients with MM to prioritize 8 uncharacterized lncRNAs as potentially oncogenic and therapeutic targets for MM. RP11-350G8.5 emerged as the top candidate due to its essentiality in AMO-1 and ABZB and its upregulation and association with poor prognosis in MM.

Noncoding genes can have overlapping and antisense transcripts,⁹ thus ensuring high-confidence genotype-to-phenotype associations for noncoding elements is key. Grillone et al adopted a multifactorial approach to demonstrate that the RP11-350G8.5-antisense gene interleukin 6 receptor (IL-6R) is not a genetic vulnerability in the Cancer Dependency Map, IL-6R expression levels are unaltered in validation experiments, and targeting RP11-350G8.5 by RNA interference phenocopies the effects observed with DNA excisions. Together with extended validations in additional MM cell lines and preliminary in vivo observations, the authors solidified RP11-350G8.5 as a potential clinically relevant vulnerability of MM.

Mechanistically, interfering with RP11-350G8.5 expression downregulated the transcriptional profiles of the endoplasmic reticulum (ER) and unfolded protein response (UPR), and it increased the activity of the PERK phosphorylation cascade. Due to proteostasis stress at the ER, unfolded or misfolded proteins can accumulate and be sensed by the UPR, which adjusts the cell’s protein folding capacity or induces apoptotic cell death. Notably, reducing RP11-350G8.5 expression was accompanied by an increase in cleaved caspase-3, cell surface translocation of calreticulin, and a higher portion of dendritically engulfed MM cells, leading the authors to conclude that RP11-350G8.5 inactivation induces a UPR- and PERK-dependent ICD. To target RP11-350G8.5, the authors opted for antisense oligonucleotides (ASOs) by computationally predicting G-quadruplex secondary structures and performing biophysical characterizations. Targeting regions 840 and 1051 of RP11-350G8.5 increased apoptosis in cellular assays, though further efforts are required to solidify these data.

The findings by Grillone et al are novel on multiple levels. The use of unbiased CRISPR deletion screens to identify noncoding elements as cancer and therapeutic vulnerabilities is intriguing, joining the race to functionally annotate the noncoding genome, which remains largely unexplored. The availability of ever-efficient and precise genetic tools is expected to provide a yet-to-be-seen explosion of functional associations in the dark genome. The identification of RP11-350G8.5 as a regulator of the PERK pathway in MM to prevent the induction of ICD along its exhaustive cellular and biophysical characterization elegantly exemplifies the combination of functional perturbations with mechanistic studies. The study uncovered a new molecular target within the complex regulatory network of MM, thereby enhancing the field and highlighting the role of lncRNAs in cancer cell survival and resistance to therapy. Moreover, these findings have significant clinical implications, as targeting RP11-350G8.5 could provide a new therapeutic strategy for MM, particularly for overcoming resistance to existing treatments.

Several outstanding questions and future directions arise from these findings. First, further validation of RP11-350G8.5 as a therapeutic target in MM is needed. This includes extensive in vivo studies to confirm its role and therapeutic potential across different MM models and patient-derived xenografts. Second, the mechanistic details of how RP11-350G8.5 regulates the PERK pathway and contributes to ICD should be fully elucidated, including interactions with other signaling pathways and molecules. Moreover, developing and optimizing ASOs targeting RP11-350G8.5 require further refinement to enhance their efficacy and specificity while minimizing off-target effects. Furthermore, expanding the unbiased CRISPR deletion screens to a broader range of noncoding RNAs and genomic elements plus using RNA-targeting Cas nucleases could identify additional novel targets for cancer therapy.¹⁰ Finally, translating these findings into clinical applications will involve rigorous preclinical testing, followed by early phase clinical trials to assess the safety and effectiveness of targeting RP11-350G8.5 in patients with MM.

Conflict-of-interest disclosure: M.K. declares no competing financial interests.