Altered Expression of Epigenetic Modifiers Identifies Novel Biomarkers and Therapeutic Targets in AL Amyloidosis

Background: Immunoglobulin light chain amyloidosis (ALA) is a plasma cell dyscrasia characterized by the accumulation of amyloid fibrils formed by clonal plasma cells (PC) in the bone marrow (BM). While MM (multiple myeloma) is present at the time of diagnosis in 10% of ALA patients, 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 can be life-threatening due to the side effects of some MM treatment regimens. Thus, identification of epigenetic alterations detected at the transcriptome level selectively associated with ALA is needed to improve patient outcomes.

Alternative splicing (AS) is a normal epigenetic phenomenon and a key regulator of gene expression. Although splicing is a normal process, defects in this process are inevitable. Highly recurrent alterations in genes encoding components of the spliceosome, splicing factors (SF), are among the most unexpected and dramatic findings in the cancer genome. Abnormal splicing events are associated with malignant transformations, particularly in genes associated with susceptibility and/or progression of cancer. Here, we proposed to identify aberrantly spliced transcripts and define splicing molecules associated with those altered splicing events in patients with ALA.

Methods: To identify aberrantly spliced transcripts selectively linked to ALA, we evaluated altered mRNA splicing (AltS) events in 8 ALA, 24 MGUS, 33 smoldering MM (sMM), and 40 MM patient samples, as well as in 10 normal donor plasma cells (NPCs). We developed a custom pipeline and identified AltS events present only in ALA cells and absent in other PC dyscrasias or NPCs. These analyses showed that 1609 genes encoding functional proteins are aberrantly spliced (894 genes are upregulated and 615 downregulated) in ALA patients compared to NPC (P<0.001). 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.

Results: These analyses indicated that ~19% mRNAs were aberrantly spliced in samples from each patient subgroup, and ~17% retained introns, a marker of malignant transformation. In ALA patient samples, we identified 1607 unique AltS events on 624 genes, 132 of which retained introns (RI), 429 showed exon skipping alterations, and 250 were subjected to nonsense-mediated decay and degraded. These analyses showed significant upregulation of CD200 and B7H3 splice variants in ALA PCs; these variants are attractive targets for therapy since CD200 and B7H3 are immune checkpoint proteins and targeting them may overcome checkpoint inhibitor-induced drug resistance. CDKN2B is a tumor suppressor which prevents the activation of the CDKs; p15 splice variant upregulation leads to loss of p15-induced growth suppression and therefore represents another novel target in ALA patients.

In patient 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. Studies show that 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.

We determined the expression signature of epigenetic modifiers (miRNAs) in ALA samples and detected significant upregulation of onco-miRNAs including miR16 and Let7f-2 in these samples. Targeting these miRs may be beneficial for ALA patients since miR-16 regulates BCL-2 and Let-7f modulates many oncogenic pathways.

Conclusion: Our study (1) identifies splice variants that are selectively expressed in ALA PCs, (2) provides potential ALA molecular biomarkers to aid in the differential diagnoses of ALA and other plasma cell dyscrasias, and (3) identifies potential therapeutic strategies targeting altered splicing in ALA.