Abstract
Dysregulated proteostasis is the hallmark of clonal plasma cell disorders (PCD), including amyloidosis (AL), monoclonal gammopathy of undetermined significance (MGUS), and multiple myeloma (MM). Plasma cell clones can also be amyloidogenic, producing misfolded light chains that form conglomerates of amyloid fibrils penetrating and damaging different organs. No clinical test or biomarker is available to determine plasma cell clone pathogenicity or to assess the potential to progress into MM, produce amyloid, or both. Currently, in the absence of early diagnostic biomarkers, AL is often diagnosed at a late stage leading to significant organ failure, high rates of hospitalization, and excess mortality. There is an urgent need to discover novel biomarkers and identify potential therapeutic targets through an understanding of molecular mechanisms conferring amyloidogenicity and malignancy to clonal plasma cells.
We used next-generation RNA sequencing with viably preserved bone marrow aspirates from 84 treatment-naïve PCD patients, of which 46 yielded good quality of DNA, RNA and proteins and passed quality control comprised of AL (14), MGUS (8), smoldering MM (SMM) (1), AL/MM overlapping (2), and MM (21). Clinical annotations were obtained for clonal burden, treatment history, and response. The RNA-seq data (100bp paired end reads) were aligned to the GRCh38 reference genome using STAR, and transcriptome was quantified using Salmon. Differential expression analysis was done with tximport/edgeR workflow. Variant calling in mRNA was performed using Strelka disabling high-depth filters. After annotation with ANNOVAR, Polymorphism phenotyping (Polyphen) and sorting intolerant from tolerant (SIFT) scores were obtained from the Ensembl variant effect predictor. Only variants present over 100X depth, 0.1 VAF with damaging Polyphen (>0.85) and SIFT (<0.05) were considered. Mutation and expression data were analyzed to identify a novel and unique AL profile compared to MM and MGUS.
The plasma cell burden varied from 6-50% in AL, 0-7% in MGUS, and 3-95% in MM. Consistent with previous reports (Hasipek et al., Cancers 2021), expression analysis demonstrated dysregulated protein quality control pathways included endoplasmic reticulum (ER) stress, UPR (Unfolded Protein Response), and proteosome degradation pathways across different PCD. Variant calling at the mRNA level identified disproportionally increasing mutational burden in these pathways from AL to MM. As expected, most mutations were associated with the UPR and ERAD (ER-associated degradation) pathways, including ring finger protein (RNF) families, early in the disease pathophysiology. The expression of ER resident chaperone PDIA1 (Protein Disulfide Isomerase A1) is positively correlated with increasing plasma cell burden (R=0.256, p=0.04).
MM shows more diverse and frequent mutations compared to AL and MGUS in the UPR and RNF families. AL patients had unique mutational signatures, with more mutations in RNF31 encoding an E3 ubiquitin ligase in ubiquitin complex – affecting degradation of abnormal proteins – and PPP1R15A (Protein Phosphatase 1 Regulatory Subunit 15A) in the UPR pathway regulating ER stress response. There are unique gene mutations found only in the AL group, which may serve as early-stage predictive biomarkers. More than 20% of AL patients show mutations in JKAMP (JNK Pathway-Associated Membrane Protein) modifying ER stress response through JNK-signaling pathway, and around 15% of AL patients have a mutation in NPLOC4 (Ubiquitin Recognition Factor), GRINA (Glutamate Receptor Ionotropic), or DDIT3 (DNA Damage Inducible Transcript 3) involved in ERAD and UPR. Thus, suggesting a distinct maladaptive mutation pattern in AL. These mutations create a dysregulated ER proteostasis network resulting in misfolded light chain production and secretion, providing potential early-stage diagnostic biomarkers and therapeutic targets in ER proteostasis-related pathways presenting a selective vulnerability in AL.
In summary, we report that AL amyloidosis is genomically less complex than MM and typically more akin to MGUS, except in overlap syndromes. Dysregulated proteostasis can be one of the first key steps in PCD. PCD pathogenicity and our data suggest gain in ER function while loss in proteasomal proteostasis may leading to survival of aberrant plasma cell clones. Further studies are warranted to investigate the malignancy and amyloidogenicity of individual plasma clones.
