P-020: Altered mRNA splicing identifies novel biomarkers and therapeutic targets in AL (Amyloid light-chain) Amyloidosis

Abstract

<h3>Background</h3> Amyloid light-chain Amyloidosis (ALA) is a plasma cell (PC) dyscrasia characterized by the accumulation of amyloid fibrils formed by clonal PCs in the bone marrow (BM). While multiple myeloma (MM) is present at the time of diagnosis in 10% of patients with ALA, nearly 30% of MM patients have subclinical amyloid deposits in the BM and/or in vital organs. Differential diagnoses of ALA, monoclonal gammopathy of undetermined significance (MGUS), and MM are challenging because these malignancies share genetic similarities. Unrecognized ALA in an MM setting can be life-threatening due to the side effects of some MM treatment regimens. Thus, a molecular signature selectively associated with ALA is needed to improve patient outcomes. <h3>Methods</h3> To identify a selective gene-signature linked to ALA, we evaluated altered mRNA splicing (AltS) events in 8 ALA, 24 MGUS, 33 smoldering MM, 40 MM patient samples and 10 normal donor plasma cells (NPC). We used the custom pipeline to identify AltS events present only in ALA cells and absent in other PC dyscrasias. Gene and transcript level analyses showed that 1609 genes are aberrantly spliced (894 genes are upregulated and 615 downregulated) in ALA patients compared to NPC (P< 001). <h3>Results</h3> Focusing on upregulated gene-splicing events occurring in the mRNA coding region, we observed significant upregulation of CD200, CDKN2B, and B7H3 splice variants in ALA PCs. Since splicing of these genes was also detected in other PC dyscrasias, custom transcript level analyses were used to identify ALA-specific AltS events. These analyses indicated that ~19% of the mRNAs were aberrantly spliced in each subgroup of patient samples, and ~17% retained introns, a marker of malignant transformation. In ALA patients we identified 1607 unique AltS events on 624 genes, 132 of which retained introns, 429 showed exon skipping alterations, and 250 were subjected to nonsense-mediated decay and degraded. These analyses showed significant upregulation of certain splice variants of CD200 and B7H3 in ALA PCs. These variants are attractive targets for therapy; CD200 and B7H3 are immune checkpoint proteins and targeting them may overcome checkpoint-induced drug resistance. In patient's samples harboring upregulated spliced genes, we evaluated the expression of cis-/trans-splicing molecules. Samples with upregulated AltS variants overexpressed SNRPN70 and RBM8A genes, both of which are part of the U1 spliceosome. Alteration of this complex assembly leads to AltS at the 5'sites of exons, causing intron retention and/or uncontrolled exon skipping. Therefore, targeting these splicing factors has the potential to control AltS in ALA. <h3>Conclusion</h3> Our study (i) identifies splice variants that are selectively expressed in ALA PCs, (ii) provides potential ALA molecular biomarkers to aid the differential diagnoses of ALA and other PC dyscrasias, and (iii) identifies potential therapeutic strategies targeting altered splicing in ALA.