Prognostic significance of esterase gene expression in multiple myeloma

Background Esterase enzymes differ in substrate specificity and biological function and may display dysregulated expression in cancer. This study evaluated the biological significance of esterase expression in multiple myeloma (MM). Methods For gene expression profiling and evaluation of genomic variants in the Institute for Molecular Medicine Finland (FIMM) cohort, bone marrow aspirates were obtained from patients with newly diagnosed MM (NDMM) or relapsed/refractory MM (RRMM). CD138+ plasma cells were enriched and used for RNA sequencing and analysis, and to evaluate genomic variation. The Multiple Myeloma Research Foundation (MMRF) Relating Clinical Outcomes in MM to Personal Assessment of Genetic Profile (CoMMpass) dataset was used for validation of the findings from FIMM. Results MM patients (NDMM, n = 56; RRMM, n = 78) provided 171 bone marrow aspirates (NDMM, n = 56; RRMM, n = 115). Specific esterases exhibited relatively high or low expression in MM, and expression of specific esterases (UCHL5, SIAE, ESD, PAFAH1B3, PNPLA4 and PON1) was significantly altered on progression from NDMM to RRMM. High expression of OVCA2, PAFAH1B3, SIAE and USP4, and low expression of PCED1B, were identified as poor prognostic markers (P <0.05). The MMRF CoMMpass dataset provided validation that higher expression of PAFAH1B3 and SIAE, and lower expression of PCED1B, were associated with poor prognosis. Conclusions Esterase gene expression levels change as patients progress from NDMM to RRMM. High expression of OVCA2, PAFAH1B3, USP4 and SIAE, and low expression of PCED1B, are poor prognostic markers in MM, suggesting a role for these esterases in myeloma biology.Peer reviewe

Growth Response and Differentiation of Bone Marrow-Derived Mesenchymal Stem/Stromal Cells in the Presence of Novel Multiple Myeloma Drug Melflufen

Mesenchymal stem/stromal cells (MSCs) are self-renewing and multipotent progenitors, which constitute the main cellular compartment of the bone marrow stroma. Because MSCs have an important role in the pathogenesis of multiple myeloma, it is essential to know if novel drugs target MSCs. Melflufen is a novel anticancer peptide–drug conjugate compound for patients with relapsed refractory multiple myeloma. Here, we studied the cytotoxicity of melflufen, melphalan and doxorubicin in healthy human bone marrow-derived MSCs (BMSCs) and how these drugs affect BMSC proliferation. We established co-cultures of BMSCs with MM.1S myeloma cells to see if BMSCs increase or decrease the cytotoxicity of melflufen, melphalan, bortezomib and doxorubicin. We evaluated how the drugs affect BMSC differentiation into adipocytes and osteoblasts and the BMSC-supported formation of vascular networks. Our results showed that BMSCs were more sensitive to melflufen than to melphalan. The cytotoxicity of melflufen in myeloma cells was not affected by the co-culture with BMSCs, as was the case for melphalan, bortezomib and doxorubicin. Adipogenesis, osteogenesis and BMSC-mediated angiogenesis were all affected by melflufen. Melphalan and doxorubicin affected BMSC differentiation in similar ways. The effects on adipogenesis and osteogenesis were not solely because of effects on proliferation, seen from the differential expression of differentiation markers normalized by cell number. Overall, our results indicate that melflufen has a significant impact on BMSCs, which could possibly affect therapy outcome

Growth Response and Differentiation of Bone Marrow-Derived Mesenchymal Stem/Stromal Cells in the Presence of Novel Multiple Myeloma Drug Melflufen

Mesenchymal stem/stromal cells (MSCs) are self-renewing and multipotent progenitors, which constitute the main cellular compartment of the bone marrow stroma. Because MSCs have an important role in the pathogenesis of multiple myeloma, it is essential to know if novel drugs target MSCs. Melflufen is a novel anticancer peptide–drug conjugate compound for patients with relapsed refractory multiple myeloma. Here, we studied the cytotoxicity of melflufen, melphalan and doxorubicin in healthy human bone marrow-derived MSCs (BMSCs) and how these drugs affect BMSC proliferation. We established co-cultures of BMSCs with MM.1S myeloma cells to see if BMSCs increase or decrease the cytotoxicity of melflufen, melphalan, bortezomib and doxorubicin. We evaluated how the drugs affect BMSC differentiation into adipocytes and osteoblasts and the BMSC-supported formation of vascular networks. Our results showed that BMSCs were more sensitive to melflufen than to melphalan. The cytotoxicity of melflufen in myeloma cells was not affected by the co-culture with BMSCs, as was the case for melphalan, bortezomib and doxorubicin. Adipogenesis, osteogenesis and BMSC-mediated angiogenesis were all affected by melflufen. Melphalan and doxorubicin affected BMSC differentiation in similar ways. The effects on adipogenesis and osteogenesis were not solely because of effects on proliferation, seen from the differential expression of differentiation markers normalized by cell number. Overall, our results indicate that melflufen has a significant impact on BMSCs, which could possibly affect therapy outcome

Growth Response and Differentiation of Bone Marrow-Derived Mesenchymal Stem/Stromal Cells in the Presence of Novel Multiple Myeloma Drug Melflufen

Mesenchymal stem/stromal cells (MSCs) are self-renewing and multipotent progenitors, which constitute the main cellular compartment of the bone marrow stroma. Because MSCs have an important role in the pathogenesis of multiple myeloma, it is essential to know if novel drugs target MSCs. Melflufen is a novel anticancer peptide-drug conjugate compound for patients with relapsed refractory multiple myeloma. Here, we studied the cytotoxicity of melflufen, melphalan and doxorubicin in healthy human bone marrow-derived MSCs (BMSCs) and how these drugs affect BMSC proliferation. We established co-cultures of BMSCs with MM.1S myeloma cells to see if BMSCs increase or decrease the cytotoxicity of melflufen, melphalan, bortezomib and doxorubicin. We evaluated how the drugs affect BMSC differentiation into adipocytes and osteoblasts and the BMSC-supported formation of vascular networks. Our results showed that BMSCs were more sensitive to melflufen than to melphalan. The cytotoxicity of melflufen in myeloma cells was not affected by the co-culture with BMSCs, as was the case for melphalan, bortezomib and doxorubicin. Adipogenesis, osteogenesis and BMSC-mediated angiogenesis were all affected by melflufen. Melphalan and doxorubicin affected BMSC differentiation in similar ways. The effects on adipogenesis and osteogenesis were not solely because of effects on proliferation, seen from the differential expression of differentiation markers normalized by cell number. Overall, our results indicate that melflufen has a significant impact on BMSCs, which could possibly affect therapy outcome.Peer reviewe

The Peptide–Drug Conjugate Melflufen Modulates the Unfolded Protein Response of Multiple Myeloma and Amyloidogenic Plasma Cells and Induces Cell Death

Immunoglobulin light-chain (AL) amyloidosis is a rare disease caused by clonal plasma cell secretion of misfolded light chains that assemble as toxic amyloid fibrils, depositing in vital organs including the heart and kidneys, causing organ dysfunction. Plasma cell–directed therapeutics are expected to reduce production of toxic light chain by eliminating amyloidogenic cells in bone marrow, thereby diminishing amyloid fibril deposition and providing the potential for organ recovery. Melphalan flufenamide (melflufen) is a first-in-class peptide–drug conjugate that targets aminopeptidases and rapidly releases alkylating agents inside tumor cells. Melflufen is highly lipophilic, permitting rapid uptake by cells, where it is enzymatically hydrolyzed by aminopeptidases, resulting in intracellular accumulation of the alkylating agents, including melphalan. Previous data demonstrating sensitivity of myeloma cells to melflufen suggest that the drug might be useful in AL amyloidosis. We describe the effects of melflufen on amyloidogenic plasma cells in vitro and ex vivo, demonstrating enhanced cytotoxic effects in comparison to melphalan, as well as novel mechanisms of action through the unfolded protein response (UPR) pathway. These findings provide evidence that melflufen-mediated cytotoxicity extends to amyloidogenic plasma cells, and support the rationale for the evaluation of melflufen in patients with AL amyloidosis.Peer reviewe

Aminopeptidase Expression in Multiple Myeloma Associates with Disease Progression and Sensitivity to Melflufen

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

Aminopeptidase Expression in Multiple Myeloma Associates with Disease Progression and Sensitivity to Melflufen

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

Melflufen, a peptide‐conjugated alkylator, is an efficient anti‐neo‐plastic drug in breast cancer cell lines

Publisher's version (útgefin grein)Melphalan flufenamide (hereinafter referred to as “melflufen”) is a peptide-conjugated drug currently in phase 3 trials for the treatment of relapsed or refractory multiple myeloma. Due to its lipophilic nature, it readily enters cells, where it is converted to the known alkylator melphalan leading to enrichment of hydrophilic alkylator payloads. Here, we have analysed in vitro and in vivo the efficacy of melflufen on normal and cancerous breast epithelial lines. D492 is a normal-derived nontumorigenic epithelial progenitor cell line whereas D492HER2 is a tumorigenic version of D492, overexpressing the HER2 oncogene. In addition we used triple negative breast cancer cell line MDA-MB231. The tumorigenic D492HER2 and MDA-MB231 cells were more sensitive than normal-derived D492 cells when treated with melflufen. Compared to the commonly used anti-cancer drug doxorubicin, melflufen was significantly more effective in reducing cell viability in vitro while it showed comparable effects in vivo. However, melflufen was more efficient in inhibiting metastasis of MDA-MB231 cells. Melflufen induced DNA damage was confirmed by the expression of the DNA damage proteins ƴH2Ax and 53BP1. The effect of melflufen on D492HER2 was attenuated if cells were pretreated with the aminopeptidase inhibitor bestatin, which is consistent with previous reports demonstrating the importance of aminopeptidase CD13 in facilitating melflufen cleavage. Moreover, analysis of CD13high and CD13low subpopulations of D492HER2 cells and knockdown of CD13 showed that melflufen efficacy is mediated at least in part by CD13. Knockdown of LAP3 and DPP7 aminopeptidases led to similar efficacy reduction, suggesting that also other aminopeptidases may facilitate melflufen conversion. In summary, we have shown that melflufen is a highly efficient anti-neoplastic agent in breast cancer cell lines and its efficacy is facilitated by aminopeptidases.We thank Inovotion for their work and support in the CAM assays.Peer Reviewe

Genetic Variability of the mTOR Pathway and Prostate Cancer Risk in the European Prospective Investigation on Cancer (EPIC)

The mTOR (mammalian target of rapamycin) signal transduction pathway integrates various signals, regulating ribosome biogenesis and protein synthesis as a function of available energy and amino acids, and assuring an appropriate coupling of cellular proliferation with increases in cell size. In addition, recent evidence has pointed to an interplay between the mTOR and p53 pathways. We investigated the genetic variability of 67 key genes in the mTOR pathway and in genes of the p53 pathway which interact with mTOR. We tested the association of 1,084 tagging SNPs with prostate cancer risk in a study of 815 prostate cancer cases and 1,266 controls nested within the European Prospective Investigation into Cancer and Nutrition (EPIC). We chose the SNPs (n = 11) with the strongest association with risk (p<0.01) and sought to replicate their association in an additional series of 838 prostate cancer cases and 943 controls from EPIC. In the joint analysis of first and second phase two SNPs of the PRKCI gene showed an association with risk of prostate cancer (ORallele = 0.85, 95% CI 0.78–0.94, p = 1.3×10−3 for rs546950 and ORallele = 0.84, 95% CI 0.76–0.93, p = 5.6×10−4 for rs4955720). We confirmed this in a meta-analysis using as replication set the data from the second phase of our study jointly with the first phase of the Cancer Genetic Markers of Susceptibility (CGEMS) project. In conclusion, we found an association with prostate cancer risk for two SNPs belonging to PRKCI, a gene which is frequently overexpressed in various neoplasms, including prostate cancer

Amplification of EIF3S3 in breast and prostate cancer

Eturauhassyöpä on yleisin miehillä esiintyvä syöpä Suomessa. Eturauhassyövän hoitona käytetään hormonaalista hoitoa, joka vaikuttaa tehokkaasti hoidon alussa, mutta jonka hoitovaste yleensä lakkaa ajanmyötä. Tällä hetkellä ei ole olemassa tehokasta keinoa, millä uusiutunutta ns. hormoniresistenttiä eturauhassyöpää voitaisiin hoitaa. Tämän vuoksi olisi tärkeää tunnistaa niitä geneettisiä muutoksia, jotka aiheuttavat eturauhassyövän uusiutumisen hormonaalisen hoidon aikana. Väitostutkimuksen tavoitteena oli tunnistaa eturauhassyövän etenemiseen johtavia geneettisiä muutoksia käyttäen vertailevaa genomista hybridisaatiota (VGH). VGH:lla pystytään yhdessä hybridisaatiossa tutkimaan kaikki kasvaimessa olevat DNA-jakson kopiolukumäärän muutokset. Näin ollen VGH on erityisen hyvä molekyylisytogeneettinen menetelmä seuloa kasvaimessa esiintyviä geneettisen materiaalin häviämiä ja/tai monistumia. Tutkimuksen tulokset osoittivat, että, hormonihoidon aikana uusiutuneissa eturauhassyövissä yleisin löydös (esiintyy 80%:ssa tapauksista) oli kromosomi 8:n pitkän käsivarren monistuma. Koska 8q-monistuma on harvinainen diagnoosivaiheen eturauhassyövissä, se näyttäisi liittyvän eturauhassyövässä aggressiiviseen, hormonihoidolle resistenttiin taudinkuvaan. VGH:n avulla voitiin myös kaventaa ns. minimaalinen, yleisesti monistunut kromosomaalinen alue kromosomiraitoihin; 8q21 ja 8q23-q24. Tutkimuksen seuraavana tavoitteena oli tunnistaa kromosomi 8q-monistuman kohdegeeni tai -geenit. Uusien ehdokasgeenien etsimiseen käytettiin vaimennus-subtraktiohybridisaatio-tekniikkaa, jonka avulla voidaan valikoidusti monistaa polymeraasiketjureaktion (PCR) avulla kahdessa näytteessä erilailla ilmentyneitä geenejä. Tällä tavalla löudetty ehdokaskohdegeeni EIF3S3 paikannettiin 8q23-kromosomialueelle ja sen löydettiin olevan yli-ilmentynyt ja monistunut 20%:ssa diagnoosivaiheen rinta- ja 30%:ssa uusiutuneissa eturauhassyövissä. Seuraavaksi EIF3S3 geenin minimaalinen monistuma-alue kartoitettiin tarkemmin käyttäen fluoresenssi in situ hybridisaatiotekniikoita (FISH). Alueen kooksi määritetiin 2.5 miljoonaksi emäspariksi. Alueella sijaitsevat geenit tutkittiin ilmentymistasonsa suhteen ja näin voitiin osoittaa, että EIF3S3 on ainoa alueella sijaitseva monistunut ja yli-ilmentynyt ehdokaskohdegeeni. EIF3S3:n proteiinin toimintaa ei toistaiseksi tunneta tarkoin. Se oletetaan kuitenkin liittyvän proteiinisynteesin aloitukseen. Näin ollen EIF3S3 monistuma rinta- ja eturauhassyövässä viittaa siihen, että proteiinisynteesin säätelyn muutokset ovat tärkeitä syövän kehityksen mekanismeja. Tätä löydöstä voitaisiin mahdollisesti hyödyntää uusien entistä parempien ennustetekijöiden ja hoitojen kehittämisessä.To identify genetic alterations that underlie the progression of prostate cancer during endocrine therapy, hormone-refractory prostate carcinomas and prostate cancer cell lines were screened by comparative genomic hybridization (CGH). Recurrent hormone-refractory carcinomas showed on average 11 genetic changes per tumor, and the most common genetic aberrations were losses at 8p (73%), 1p (54%), 13q (51%) and 10q (46%) and gains at 8q (73%), 7q (43%), Xq (35%) and 18q (30%). Findings in CGH were validated by fluorescence in situ hybridization (FISH) using both pericentromeric (7, 8, 18) and locus-specific probes (CAV1, MYC, BCL2). The clonal relationship of six primary-recurrent tumor pairs was also examined. Half of these tumor pairs showed a close genetic relationship, whereas in the rest of tumors the relationship was less evident. Amplification at the long arm of chromosome 8 occurs in a large fraction of primary breast and prostate cancers. In order to clone the target genes associated with this amplification, suppression subtraction hybridization (SSH) was used to identify overexpressed genes in the breast cancer cell line SK-Br-3, which harbors amplification at 8q21 and 8q23-q24. A differentially expressed gene, the p40 subunit of eukaryotic translation initiation factor 3 (EIF3S3 alias eIF3-p40), identified by SSH, was localized to 8q23, and was found to be highly amplified and overexpressed in the breast and prostate cancer cell lines studied. High-level amplification of EIF3S3 was also found in 30% of hormone-refractory prostate tumors and in 18% of untreated primary breast tumors. In the majority of cases, EIF3S3 and MYC were amplified in equal copy numbers. Tumors with higher copy numbers of EIF3S3 than MYC were also found. Expression of EIF3S3 mRNA was analyzed by in situ hybridization. Amplification of the EIF3S3 gene was associated with overexpression of its mRNA, as expected for a functional target gene of amplification. FISH mapping data on EIF3S3 amplification narrowed the minimal highly amplified region to 2.5 Mb. All mapped ESTs within and nearby the highly amplified region were investigated as regards their expression in several cancer cell lines. Three anonymous ESTs and one known gene (EXT1) were shown to be expressed more in cancer cell lines with 8q amplification. However, these ESTs were located outside the minimal highly amplified region and EXT1 was overexpressed only in one of the cancer cell lines containing 8q amplification