Poster FP-028 Aminopeptidase Gene Expression in Myeloma Nina Nupponen,1 Muntasir Majumder,2 Paul Dowling,3 Juha Lievonen,4 Despina Bazou,5 Ana Slipicevic,1 Raija Silvennoinen,4 Pekka Anttila,4 Peter O`Gorman,5 Fredrik Lehmann,1 and Caroline A. Heckman6 1Oncopeptides AB, Stockholm, Sweden; 2Institute for Molecular Medicine Finland (FIMM), Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland; 3Maynooth University, Dublin, Ireland; 4Department of Hematology, Helsinki University Hospital and Comprehensive Cancer Center, Helsinki, Finland; 5Mater Misericordiae University Hospital, Dublin, Ireland; 6Helsinki University Hospital Cancer Center, Helsinki, Finland INTRODUCTION RESULTS A hallmark of myeloma is high-level production • Aminopeptidase gene expression levels were ranked based on abundance levels in all • We also investigated whether any aminopeptidase could be linked to disease progression and • Ex vivo testing of patient cells with the • Survival analysis revealed patient samples exhibiting 2× or higher LAP3 of immunoglobulins leading to a heavy load on samples (Figure 1A) found no significant difference (Figure 2) aminopeptidase inhibitor tosedostat showed expression had poorer prognosis with a median survival of 6 months that the viability of approximately 30% of from the sampling date (P=0.0001, HR 4.5; 95% CI 1.45-14.05) ( ) protein folding and homeostasis in tumor cells. Aminopeptidases were differentially expressed compared to heathy plasma cells - Expression levels of LAP3, ERAP1, METAP2, and DPP7 (P>0.005) appeared higher in Figure 5 • relapsed myeloma samples was reduced The aminopeptidase gene family catalyze the (Figure 1B) relapsed/refractory multiple myeloma (RRMM) than in newly diagnosed multiple myeloma hydrolysis of amino acid residues from proteins (NDMM) samples (Figure 4) - The majority of the genes in patient samples showed related expression patterns or Figure 5. LAP3 Expression Is Associated With Poor Prognosis or peptides and are last in line for protein - RNA expression of LAP3, DPP3, METAP1D, were modestly overexpressed (DPP7, DPP3, METAP2 and LAP3) compared to healthy - A comparison of expression levels in 6 paired NDMM and RRMM samples showed a trend for degradation. They are thus an important group CTSV, TPP2, and XNPEP1 was found to 1.0 LAP3+ (n=18) plasma cells increased LAP3 at relapse of metalloenzymes implicated in cellular significantly correlate with tosedostat Others (n=48) functions such as differentiation, cell cycle, - Decreased expression was detected for several aminopeptidases including MMP14, response (Spearman P<0.005) 0.8 DNA repair, and apoptosis. However, there is MMP15, ANPEP, ENPEP, and CTSH Figure 2. Gene Expression Changes During Disease Progression - The figure demonstrates LAP3 and DDP3 limited information about the expression of RNA expression in relation to tosedostat aminopeptidases in myeloma and their possible LAP3 ERAP1 METAP2 DPP7 LAP3 0.6 Figure 1. Gene Expression Profiles of Aminopeptidases 400 50 100 100 100 ns treatment utilization for novel therapeutic approaches 300 60 80 including peptide conjugated drugs. A Abundant Modestly Expressed Low or No Housekeeping Genes 200 40 50 80 0.4 Aminopeptidases Aminopeptidases Expression (n=17) 100 60 Figure 4. Inhibition of Aminopeptidases 10 50 30 40 50 60 6 0.2 8 40 30 40 P=0.0001 20 40 METHODS 6 30 30 Hazard Ratio 4.5 20 4 20 20 0.0 10 20 103 bone marrow aspirates from myeloma 10 0 4 8 12 16 20 24 2 Expression (RPKM) 10 10 4 patients and 2 healthy donors were obtained 0 0 0 0 0 0 Survival (months) after written informed consent and following -2 approved protocols in compliance with -4 NDMMRRMM NDMMRRMM NDMMRRMM NDMMRRMM 2 the Declaration of Helsinki. CD138+ cells At Relapse -6 LAP3 were enriched and used for RNA or protein At Diagnosis CONCLUSIONS -8 Healthy CD138+ Healthy CD138+ Healthy CD138+ Healthy CD138+ preparation. Illumina-compatible RNA -10 sequencing libraries were prepared and Expression (Log2 RPKM) Association of prior exposure to treatments with LAP3 expression was analyzed in the patients -12 • 0 • The majority of aminopeptidase genes in myeloma sequenced. Proteomic analysis was performed (Figure 3) using Q-Exactive MS/Dionex Ultimate 3000 -14 patient samples showed similar expression patterns - Here, we compared expression between samples exposed and naïve to melphalan, TC AP2 TBP instruments. Contribution of aminopeptidase LAP3 TPP2 A4H AP1D VRN PGK1 P value 0.0007 or were modestly overexpressed compared to DPP7ERAP1AMZ2 STX16DPP3DPP8 BLMHPEPD DPP9 CTSH DPP4 PHEXL CTSV DPP6RPL19 ACTB TUBBLDHA CL SDHAGUSBALAS1 HPRT1 G6PD ERAP2 LT LNPEP RNPEP DNPEP C9orf3ANPEPENPEP MMP17TRHDE RPLP0 ABCF1 MET NPEPPS NPEPL1 MMP14 MMP16 MMP15 GAPDH POLR2APOLR1B XPNPEP1 METXPNPEP3RNPEPL1 XPNPEP2 bortezomib class, or immunomodulatory drugs (IMIDs) gene expression to survival outcome Genes -2 healthy plasma cells was estimated by Kaplan-Meier analysis. - Elevated expression of LAP3 was detected in all treated groups (melphalan P=0.01, 0 10 20 30 40 Significance of survival curves between B bortezomib P=0.04, and IMIDs P=0.04) DPP7, DPP3, METAP2, and LAP3 were most 4 3 • 2 groups (high vs. normal expression) was frequently expressed in this sample set deduced using a log rank test (Mantel-Cox). Figure 3. LAP3 Expression Underlying Disease Progression 2 2 Melphalan Bortezomib IMiDs • LAP3 was identified as a potential poor prognostic 0 4 4 4 4 marker for myeloma P=0.01 P=0.04 P=0.04 1 -2 Ex vivo treatment of myeloma patient CD138+ cells 2 2 2 2 • DPP3 with the aminopeptidase inhibitor tosedostat -4 0 results in cell death and thus strengthens the role -6 0 0 0 0 of these genes in myeloma biology LAP3 Expression -1 -8 Fold Change (Log2) P=0.05 Expression Fold Change (Log2) -2 -2 -2 -2 P value <0.0001 -10 -2 Naive AP2 AP1D LAP3 A4H TPP2 VRN DPP7DPP3 DPP9ERAP1 AMZ2 BLMHDPP8 DPP6 PEPDSTX16 CTSV L CTSHPHEX DPP4 Exposed Exposed Exposed Sensitive Alkylator 0 10 20 30 40 Copies of this poster obtained through Quick Response (QR) Code are for DNPEP RNPEP ERAP2 LT C9orf3 LNPEP MMP17 MMP16 NPEPL1MMP15 ANPEPTRHDE MMP14ENPEP Treatment XPNPEP1 MET RNPEPL1NPEPPS Refractory Refractory MET XPNPEP3 XPNPEP2 Bortezomib Bortezomib+ Not Exposed Not Exposed Not Exposed Tosedostat Drug Sensitivity Score personal use only and may not be reproduced without permission from the Len RefractoryLen Refractory Genes author of this poster. Presented at the 17th International Myeloma Workshop; September 12-15; Boston, Massachusetts..