Arginine Deprivation Induces a Dormient Phenotype in Neoplastic Plasma Cells: The Seed and Soil in Multiple Myeloma

Our previous research has demonstrated that a reduction in arginine (Arg) concentration within the bone marrow microenvironment BMME induces metabolic reprogramming in multiple myeloma (MM) plasma cells (PCs), enhancing their survival both in vitro (Romano, 2020) and in vivo (Trudu, 2022).

To explore how PCs adapt to both acute and chronic arginine deprivation, we employed a combination of mRNA-sequencing (RNA-seq), ribosome profiling (Ribo-seq), in vitro assays, and FACS analysis to evaluate two human myeloma cell lines, U266 and NCI-H929, cultured under arg-deprived conditions to mimic BMME.

Arg deprivation induced oxidative stress as confirmed by the expression and nuclear translocation of HO-1 protein in U266 cell line but not in NCI-H929 cell line. The antioxidant system upregulation reflects a lower sensitivity to treatment with proteasome inhibitors, Bortezomib and Carlfizomib in U266 cell line. Integrative amino acid profiling revealed that, while the H929 cell line maintained intracellular arginine levels after 10 days of arginine deprivation, the U266 cell line exhibited arginine auxotrophy under the same conditions.

The direct pairwise comparison of RNA-seq data revealed 888 differentially expressed genes in both cell lines, disclosing a dormant phenotype for NCI-H929 cell line, confirmed by the up-regulation of Axl gene important for the retention of MM cells in the dormant state (Khoo et al. 2019), and in the autotrophic cell line U266 the acquisition of a senescence-associated secretory phenotype, associated to accumulation of DNA damage, micronuclei and heterochromatin.

Ribosome profiling provided that U266 cells, but not NCI-H929 cells, preferentially translated shorter mRNA fragments, with ribosome footprint lengths centered around 27 nt and 28 nt after 48h of arg deprivation. The ribosome translation frame analysis revealed a preference for the first codon in 28 nt fragments and the second codon in 27 nt fragments, confirming alterations in translation. Applying AS-Quant we identified novel alternative splicing events, common in both cell lines, occurring in the skipped exons (leading to a significant increase in the expression of ASNS, ATF5, Caspase 4-6-8) and in mutually exclusive exons (leading to increased translation of CD74, a transmembrane glycoprotein involved in MHC II protein formation and transport, responsible of downstream translation of genes involved in apoptosis, immune response, and cell migration).

Taken together, our findings suggest that arg deprivation in TME conveys a complex transcriptional and translational response, leading to peptides translated from short-coding or noncoding regions of the genome, providing a basis for subsequent further functional studies, including the evaluation of potential new ne-antigens for further immunotherapy development.

No relevant conflicts of interest to declare.