Understanding the Molecular Mechanism of Plasma Cell Clonality and Disease Evolution in Light Chain Amyloidosis

Amyloidosis, also known as light chain amyloidosis (AL), is one of the most common types of systemic amyloidosis affecting patients of older ages with a median age of 64 years. Nearly 4000 new cases are diagnosed annually in the US alone (Dima et. al. JCO Oncology Practice 2023). AL is a proteostasis disorder stemming from misfolded immunoglobulin light chains caused by abnormal protein production by clonal plasma cells and in rare cases clonal B cells. These misfolded proteins can be deposited in the organs, tissues, and nerves and lead to permanent and even lethal damage. This dysregulated feature of AL resembles findings in multiple myeloma (MM) but there are several differences in the pathogenesis (Madan et. al. Mayo Clinic Proceedings 2010). Due to the lack of molecular biomarkers for detection at early stages, AL is usually diagnosed at a late stage when significant organ failure is already present. There is a high rate of hospitalization due to heart failure, renal failure, and other debilitating symptoms (Dima et. al. JCO Oncology Practice 2023). Thus, there is an urgent need to understand the molecular pathogenesis of AL and to develop biomarkers and therapeutic strategies to prevent clonal evolution.

Although AL and MM are clinically distinct, there are several shared clinical features with overlapping pathogenesis (Madan et. al. Mayo Clinic Proceedings 2010). The mechanisms that confer amyloidogenicity on clonal plasma cells have yet to be fully elucidated. Our aim is to achieve an improved and novel understanding of the distinct transcriptional programs that confer amyloidogenicity to plasma cells. For this purpose, we have adopted a unique approach utilizing next-generation sequencing of the CD138+ and CD138- fraction of a large cohort of AL patients. We will report the transcriptional profile of AL amyloid plasma cell clones and the cellular microenvironment they interact with to identify molecular biomarkers and potential therapeutic targets.

In this study, we used treatment-naïve human frozen banked plasma cells and sorted bone marrow aspirate samples of AL patients. The 67 out of 84 patient samples yielded a good quality of RNA, DNA, and proteins. Among 67 samples, 22 were AL, 31 confirmed multiple myeloma (MM), 3 overlapped with MM/AL, 2 were smoldering myeloma (sMM), and 9 were monoclonal gammopathy of undetermined significance (MGUS). Clinical annotation was obtained regarding bone marrow clonal burden, treatment history, and response. We used analytical flow cytometry coupled with the purification of plasma cells using antibodies against CD138 (MicroBeads Kit). The percentages of CD138+ fraction ranged from 0.04% to 14.1%. We utilized samples with 5% or more (n=10) to purify CD138+ and CD138- cells. These ten samples include AL (5 cases), MGUS (3 cases), MM (1 case), and sMM (1 case). RNA from CD138+ cells as well as CD138- cells of these samples along with bulk RNA from all 67 samples were extracted and next-generation RNA sequencing was performed. The RNASeq data were analyzed to identify the novel and unique clonal AL amyloidosis profile compared to MM and normal cells.

In addition, to compare the proteostasis pathways of AL and MM, we utilized four distinct cellular modes of MM (MM1.S, RPMI8226, KMS12, and U266). Our preliminary data demonstrated gain in endoplasmic reticulum (ER) function as one of the common mechanisms in AL and MM. Consistent with our previous report about MM, (Hasipek et. al. Cancers 2021) we observed that therapeutic resistance and persistence of plasma cell clones emerge as a result of gain in the function of ER, unfolded protein response, and ER-dependent protein degradation. To further understand the mechanism of AL, the expression level of PDIA1 (protein disulfide isomerase A1) was investigated. PDIA1, a resident ER chaperon essential for light chain exit from plasma cells, is a major member of the protein disulphide isomerase family involved in the protein folding process. Inhibition of PDIA1 positively correlates with the survival rate of patients with relapsed myeloma (Hasipek et. al. Cancers 2021). The levels of PDIA1 in 67 samples were compared to identify PDIA1 as a potential therapeutic target in both AL and MM.

In summary, this study establishes the basis of the novel predictive model for AL in both molecular pathogenesis and transcriptional profile with the potential to develop novel biomarkers and therapeutic targets.

Khouri:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Consultant; Prothena: Honoraria; Legend: Membership on an entity's Board of Directors or advisory committees; GPCR Therapeutics, Inc.: Honoraria. Williams:Bristol Myers Squibb: Honoraria; Janssen: Honoraria; Abbvie Inc.: Research Funding.