Multiple myeloma (MM) is characterized by extensive immunoglobulin production leading to an excessive load on protein homeostasis in tumor cells. Aminopeptidases contribute to proteolysis by catalyzing the hydrolysis of amino acids from proteins or peptides and function downstream of the ubiquitin–proteasome pathway. Notably, aminopeptidases can be utilized in the delivery of antibody and peptide-conjugated drugs, such as melflufen, currently in clinical trials. We analyzed the expression of 39 aminopeptidase genes in MM samples from 122 patients treated at Finnish cancer centers and 892 patients from the CoMMpass database. Based on ranked abundance, LAP3, ERAP2, METAP2, TTP2, and DPP7 were highly expressed in MM. ERAP2, XPNPEP1, DPP3, RNPEP, and CTSV were differentially expressed between relapsed/refractory and newly diagnosed MM samples (p < 0.05). Sensitivity to melflufen was detected ex vivo in 11/15 MM patient samples, and high sensitivity was observed, especially in relapsed/refractory samples. Survival analysis revealed that high expression of XPNPEP1, RNPEP, DPP3, and BLMH (p < 0.05) was associated with shorter overall survival. Hydrolysis analysis demonstrated that melflufen is a substrate for aminopeptidases LAP3, LTA4H, RNPEP, and ANPEP. The sensitivity of MM cell lines to melflufen was reduced by aminopeptidase inhibitors. These results indicate critical roles of aminopeptidases in disease progression and the activity of melflufen in MM.

Abstract Background Tamoxifen treatment of estrogen receptor (ER)-positive breast cancer reduces mortality by 31%. However, over half of advanced ER-positive breast cancers are intrinsically resistant to tamoxifen and about 40% will acquire the resistance during the treatment. Methods In order to explore mechanisms underlying endocrine therapy resistance in breast cancer and to identify new therapeutic opportunities, we created tamoxifen-resistant breast cancer cell lines that represent the luminal A or the luminal B. Gene expression patterns revealed by RNA-sequencing in seven tamoxifen-resistant variants were compared with their isogenic parental cells. We further examined those transcriptomic alterations in a publicly available patient cohort. Results We show that tamoxifen resistance cannot simply be explained by altered expression of individual genes, common mechanism across all resistant variants, or the appearance of new fusion genes. Instead, the resistant cell lines shared altered gene expression patterns associated with cell cycle, protein modification and metabolism, especially with the cholesterol pathway. In the tamoxifen-resistant T-47D cell variants we observed a striking increase of neutral lipids in lipid droplets as well as an accumulation of free cholesterol in the lysosomes. Tamoxifen-resistant cells were also less prone to lysosomal membrane permeabilization (LMP) and not vulnerable to compounds targeting the lipid metabolism. However, the cells were sensitive to disulfiram, LCS-1, and dasatinib. Conclusion Altogether, our findings highlight a major role of LMP prevention in tamoxifen resistance, and suggest novel drug vulnerabilities associated with this phenotype.

Abstract Background Despite the well described heterogeneity in glioblastoma (GBM), treatment is standardized, and clinical trials investigate treatment effects at population level. Genomics-driven oncology for stratified treatments allow clinical decision making in only a small minority of screened patients. Addressing tumor heterogeneity, we aimed to establish a clinical translational protocol in recurrent GBM (recGBM) utilizing autologous glioblastoma stem cell (GSC) cultures and automated high-throughput drug sensitivity and resistance testing (DSRT) for individualized treatment within the time available for clinical application. Results From ten patients undergoing surgery for recGBM, we established individual cell cultures and characterized the GSCs by functional assays. 7/10 GSC cultures could be serially expanded. The individual GSCs displayed intertumoral differences in their proliferative capacity, expression of stem cell markers and variation in their in vitro and in vivo morphology. We defined a time frame of 10 weeks from surgery to complete the entire pre-clinical work-up; establish individualized GSC cultures, evaluate drug sensitivity patterns of 525 anticancer drugs, and identify options for individualized treatment. Within the time frame for clinical translation 5/7 cultures reached sufficient cell yield for complete drug screening. The DSRT revealed significant intertumoral heterogeneity to anticancer drugs (p < 0.0001). Using curated reference databases of drug sensitivity in GBM and healthy bone marrow cells, we identified individualized treatment options in all patients. Individualized treatment options could be selected from FDA-approved drugs from a variety of different drug classes in all cases. Conclusions In recGBM, GSC cultures could successfully be established in the majority of patients. The individual cultures displayed intertumoral heterogeneity in their in vitro and in vivo behavior. Within a time frame for clinical application, we could perform DSRT in 50% of recGBM patients. The DSRT revealed a remarkable intertumoral heterogeneity in sensitivity to anticancer drugs in recGBM that could allow tailored therapeutic options for functional precision medicine.