Gene Expression Profiling of Kinetic Response to Proteasome Inhibitors in Innate and Acquired Resistant Multiple Myeloma

Extensive inter-individual variation in response to chemotherapy is a serious cause of concern in the treatment of multiple myeloma (MM). Drug resistance may be categorized into innate resistance already present in drug-naive refractory patients or emerging (acquired) resistance where a patient's tumor ultimately undergoes relapse in course of treatment despite initial response.

Using a panel of human myeloma cell lines (HMCLs), we have earlier presented a gene expression signature that could not only distinguish good and poor Proteasome inhibitor (PI)-response in the HMCL panel, but was also successful in stratifying exceptional response to PI-based chemotherapy in MM clinical datasets.

In this study, we used six (6) most PI-sensitive and six (6) most PI-resistant HMCLs representing innate PI response and clonally-derived PI-resistant (R) cell lines and PI-sensitive (P) HMCLs representing acquired PI-response to identify kinetic changes in gene expression patterns following test dosing with the 2^nd generation PI drug Ixazomib. High quality RNA was extracted from untreated/baseline/0Hr and treated/24hrs post-treatment myeloma cell lines and RNA sequencing was performed on llumina's HiSeq 2000 using 50bp paired-end protocol with depth of >20million reads-per-sample. Transcriptome profiles were then compared to characterize differences in kinetic response to PIs.

Our analysis revealed, at FDR<0.05, 74 genes changed significantly post-treatment in Ix-sensitive lines, with a |fold-change|> 1.5 while no gene showed any significant kinetic change in Ix-resistant cell lines. When our kinetic innate Ix-response profile was compared to Shaughnessy's 80-gene kinetic signature derived from Bz test-dosing, 16 genes were found similar, including PSMD4 that was identified earlier as novel high-risk feature in MM patients on Bz-based clinical trial. On the other hand, kinetic expression analysis of the clonally-related PI-sensitive parental and PI-resistant cell line pairs representing acquired PI-resistance revealed 651 genes were significantly differentially expressed in the sensitive/parental cell lines compared to 21 in clonally-derived resistant cell lines. Further, we observed that the gene signatures of kinetic innate PI-response were different from the signature of acquired PI-response. We also observed strikingly lower number of genes that significantly changed post-treatment in both innate and acquired PI-resistant cell lines when compared to the PI-sensitive cell lines. Ingenuity Pathway Analysis (IPA) identified the proteasome ubiquitination pathway, NRF2-mediated oxidative stress response pathway, oxidative stress response and Glutathione depletion/ Glutathione redox reactions as top canonical pathways associated with the most differentially regulated kinetic response genes. Several of these genes, including CCND1 (cyclin D1) and E2F, have been earlier shown involved in proliferative activity as well as susceptibility to chemotherapy.

Our results thus provide key insights into the phenomenon of kinetic PI response in MM and highlights the notable differences between innate and acquired drug response. We are currently validating our in vitro findings using ex vivo kinetic transcriptome analysis in primary myeloma cells derived from drug-naive newly diagnosed MM patients representing variation in PI-response.