Purpose: Prior studies indicate that bone marrow resident plasma cells (BMRPCs) demonstrate varying sensitivity to proteasome inhibition (PI). Effects of carfilzomib (CFZ) monotherapy desensitization on CD138⁺ BMRPC gene expression were analyzed.

Methods: HLA-sensitized transplant candidates received escalating CFZ doses (20, 27, 36 mg/m²⁾ with BM aspirates performed before and after CFZ. CD138⁺ BMRPCs were isolated to >98% purity;total RNA was analyzed on Ilumina TruSeq NGS platform (30 million reads/sample with paired end 75 bp sequencing). Strongly and differentially expressed gene sets were clustered and evaluated using enrichment and prior knowledge-based network and interactions analyses using ToppGene Suite (toppgene.cchmc.org/).

Results: CFZ therapy reduced CD138⁺ BMRPC by a mean of 72%.RNAseq analysis of CD138+ BMRPC identified 2,024 differentially expressed transcripts corresponding to 1755 genes (1273 upregulated and 482 down regulated) following CFZ. Network analysis revealed strong shifts in B-cell and plasma cell (PC) genes; PC and B cell development genes were exclusively upregulated whereas genes associated with mature B cells were both up-regulated and down-regulated. Upregulated genes including many associated with 1) ubiquitin+proteasome system (UPS), 2) apoptosis negative regulation, and 3) negative regulation of cell-cycle control. Genes encoding proteolysis in the immunoproteasome (IP) (PSMB8, PSMB9, PSM10) were upregulated more than those in the constitutive proteasome (CP), indicating potential changes in PI sensitivity. In vitro assessment of chymotrypsin-like activity in BMRPCs after CFZ treatment revealed reduced chymotryptic-like inhibition by CFZ, bortezomib and ixazomib, but increased inhibition with an IP-specific inhibitor. These results indicate a shift toward IP-specific proteolysis may mediate relative CFZ resistance in surviving BMRPCs.

Conclusions: This first RNAseq expression profile analysis of untransformed, human CD138⁺ BMRPCs revealed wide-ranging PI-induced effects on genes controlling B cell differentiation into PCs and high connectivity and dependence on genes associated with UPS and autophagy. Additional in vitro studies indicated changes from CP to IP proteolytic capacity post-CFZ. These studies suggest several new strategies to personalize and enhance PI-based PC depletional therapy, such as sequential therapy with CP and IP-selective inhibitors.

CITATION INFORMATION: Driscoll J, Aronow B, Tremblay S, Alloway R, Woodle E. RNAseq Transcriptome Analysis of Bone Marrow CD138⁺ Plasma Cell Responses to Carfilzomib Monotherapy Desensitization. Am J Transplant. 2016;16 (suppl 3).

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