Multi-Omics Analysis of Proteasome Inhibitor Resistance in Multiple Myeloma

Background: Proteasome inhibitors (PI) are a core component of contemporary quadruplet treatments for multiple myeloma (MM). Resistance to PI may develop due to mutations in the PSMB5 gene, which encodes for the 20S core proteasomal complex, or through the compensatory upregulation of the aggresome-autophagy pathway. Nonetheless, these mechanisms inadequately explain all cases of PI resistance. In order to identify novel resistance patterns, we here therefore aimed to analyze the proteome, methylome, and transcriptome landscape of four PI-naïve MM cell line models as well as their PI-resistant derivates.

Methods: AMO1, ARH77, L363, and RPMI8226 were continuously exposed to bortezomib (BTZ) and carfilzomib (CFZ) by gradually increasing the dosage to develop resistance. For the proteomics analysis, cells were lysed, tryptic digested, and TMT11-multiplexed. The pooled peptides of lysed cells were analyzed by Orbitrap LC-MS/MS. In parallel, the DNA and RNA were extracted from each cell line and their PI-resistant counterpart. For the methylome analysis, the DNA was subsequently bisulfite converted and analyzed using whole genome bisulfite sequencing (WGBS) with a coverage of 30x on the Illumina NovaSeq6000 platform. Finally, messenger RNA (mRNA) was separated from total RNA for transcriptome analysis using magnetic beads coupled to poly-T oligos. The libraries were subjected to paired-end sequencing on NovaSeq 6000, yielding a minimum of 20 million reads per sample.

Results: Proteomic analysis was first employed to screen for variations in protein expression profiles in parental (WTs) and BTZ-/CFZ-resistant cells. A total of nine proteins demonstrated a significantly elevated expression rate (P<0.05) in all BTZ-resistant cells (BTZres), while 14 proteins were significantly overexpressed in all CFZ-resistant models (CFZres). The most significant upregulation in BTZres was observed for NQO1, PSMB5, and FAM219A, with fold changes of 4.35, 3.65, and 3.49, respectively. In contrast, the three proteins that showed the largest increase in expression within CFZres were MAP1A (14.98-fold), CA2 (14.67-fold), and NQO1 (4.46-fold). To note, the only overlap between both PIs was seen for NQO1 (NAD(P)H dehydrogenase quinone 1), an enzyme involved in oxidative stress response that has previously been reported to stabilize TP53 and TP73 by preventing their ubiquitin-independent degradation by the 20S proteasome. BTZres exhibited a notable decrease in the expression of the two proteins DECR2 (-2.25-fold) and FUCA1 (-1.46-fold). Among the ten downregulated proteins in CFZres, CTSS and INPP1 showed notable reductions with fold changes of -3.72 and -3.25, respectively. In the subsequent WGBS analysis of CFZres models, a substantial increase in methylation levels of CTSS (30.4%) and INPP1 (17.7%) was observed. This inverse correlation between protein expression and methylation suggests that epigenetic mechanisms govern gene expression for both proteins and may contribute to the reported resistance phenotype. The evaluation of the RNA sequencing data is currently ongoing. This dataset will be associated with the WGBS data to discover genes that are regulated pre-transcriptionally. Additionally, it will be cross-referenced with the proteomics data to identify potential proteins that are affected at the post-transcriptional level.

Conclusions: By using a multi-omic, state-of-the-art screening platform, we here identified candidate proteins that may confer resistance to PI beyond PSMB5. Most notably, NQO1 upregulation was consistently associated with both CFZ and BTZ resistance. On a mechanistic level, we discovered that differential protein expression is in part driven by inverse methylation, suggesting that epigenetic mechanisms contribute to PI resistance in vitro.

Waldschmidt:Pharmamar: Honoraria; Sanofi: Consultancy; Stemline Menarini: Consultancy; Oncopeptides: Consultancy; Takeda: Consultancy; GSK: Honoraria; Pfizer: Honoraria; Beigene: Honoraria; Janssen: Consultancy. Einsele:Sanofi: Honoraria; BMS: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene/Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Rasche:Janssen: Honoraria; Skyline Dx: Research Funding; Pfizer: Honoraria; GSK: Honoraria; BMS: Honoraria; Amgen: Honoraria. Kortüm:AbbVie, BMS, GSK Janssen, Novartis, Pfizer, Sanofi, Takeda, Stemline: Consultancy; AbbVie, BMS, GSK Janssen, Novartis, Pfizer, Sanofi, Takeda, Stemline: Honoraria; University Hospital Wurzburg: Current Employment.