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Multiple myeloma (MM) is a hematological malignancy whose curability is greatly challenged by recurrent patient relapses and therapy resistance. We have previously proposed the high expression of ADAM8 , ADAM9 and ADAM15 (A Disintegrin And Metalloproteinase 8/9/15) as adverse prognostic markers in MM. This study focused on the so far scarcely researched role of ADAM8/9/15 in MM using two patient cohorts and seven human MM cell lines (HMCL). High ADAM8 / 9 / 15 expression was associated with high-risk cytogenetic abnormalities and extramedullary disease. Furthermore, ADAM8 / 15 expression increased with MM progression and in relapsed/refractory MM compared to untreated patient samples. RNA sequencing and gene set enrichment analysis comparing ADAM8 / 9 / 15 high/low patient samples revealed an upregulation of proliferation markers and proliferation-associated gene sets in ADAM8 / 9 / 15 high patient samples. High ADAM8 / 9 / 15 expression correlated with high Ki67 and high ADAM8 / 15 expression with high MYC protein expression in immunohistochemical stainings of patient tissue. Conversely, siRNA-mediated knockdown of ADAM8 / 9 / 15 in HMCL downregulated proliferation-related gene sets. Western blotting revealed that ADAM8 knockdown regulated IGF1R/AKT signaling and ADAM9 knockdown decreased mTOR activation. Lastly, high ADAM8 / 9 / 15 expression levels were verified as prognostic markers independent of Ki67/MYC expression and/or high-risk abnormalities. Overall, these findings suggest that ADAM8/9/15 play a role in MM progression and proliferation signaling.

Cell lines represent the everyday workhorses for in vitro research on multiple myeloma (MM) and are regularly employed in all aspects of molecular and pharmacological investigations. Although loss-of-function studies using RNA interference in MM cell lines depend on successful knockdown, no well-established and widely applied protocol for efficient transient transfection has so far emerged. Here, we provide an appraisal of electroporation as a means to introduce either short-hairpin RNA expression vectors or synthesised siRNAs into MM cells. We found that electroporation using siRNAs was much more efficient than previously anticipated on the basis of transfection efficiencies deduced from EGFP-expression off protein expression vectors. Such knowledge can even confidently be exploited in \"hard-to-transfect\" MM cell lines to generate large numbers of transient knockdown phenotype MM cells. In addition, special attention was given to developing a protocol that provides easy implementation, good reproducibility and manageable experimental costs.

Background Proteasome inhibitors (PIs) are indispensable for the treatment of multiple myeloma (MM), the second most common hematologic malignancy. Although primary resistance to PIs is rare, most patients eventually relapse and develop acquired resistance, with underlying mechanisms that remain incompletely understood and appear to be drug-specific. In the case of bortezomib, resistance is often associated with PSMB5 mutations. In contrast, resistance to carfilzomib (CFZ) is mediated by overexpression of the drug efflux transporter ABCB1. However, the regulatory mechanisms driving ABCB1 upregulation in CFZ-resistant MM remain unclear. Methods An integrative multi-omics analysis was conducted using paired samples from a CFZ-sensitive and -resistant MM patient, alongside resistant cell line models. Whole-genome sequencing (WGS), whole-genome bisulfite sequencing (WGBS), and RNA sequencing (RNA-seq) were used to assess the genotype (structural variants, single nucleotide variants, and copy number variations), methylation status, and the expression of the ABCB1 locus. ABCB1 promoter methylation levels and expression levels in an independent MM subcohort were analyzed to determine clinical relevance. Functional validation was performed using dual-luciferase reporter assays, DNMT1 knockdown, and treatment with DNA methyltransferase inhibitors (DNMTis) to evaluate methylation-dependent regulation of ABCB1 expression. Results Significant hypomethylation of the ABCB1 downstream promoter region was identified (GH07J087598) in a CFZ-resistant patient sample, which correlated with elevated ABCB1 expression. Consistent with the paired CFZ-resistant case, the independent MM subcohort showed a significant inverse association between ABCB1 promoter methylation and ABCB1 expression. These findings align with results obtained from CFZ-resistant MM cell line models, which demonstrated reduced promoter methylation and elevated ABCB1 expression compared to their wild-type counterparts. Furthermore, treatment with DNA methyltransferase inhibitors as well as DNMT1 knockdown enhanced ABCB1 expression while demethylating the promoter, thereby validating the functional significance of promoter hypomethylation in ABCB1 overexpression. Conclusions Our findings highlight ABCB1 promoter hypomethylation as a potential epigenetic driver of CFZ resistance in MM. These results underscore the clinical relevance of epigenetic regulation in drug resistance and the potential of targeting DNA methylation as a therapeutic strategy to overcome resistance in MM.

Approximately 20% of multiple myeloma (MM) cases harbor a point mutation in KRAS. However, there is still no final consent on whether KRAS-mutations are associated with disease outcome. Specifically, no data exist on whether KRAS-mutations have an impact on survival of MM patients at diagnosis in the era of novel agents. Direct blockade of KRAS for therapeutic purposes is mostly impossible, but recently a mutation-specific covalent inhibitor targeting KRASp.G12C entered into clinical trials. However, other KRAS hotspot-mutations exist in MM patients, including the less common exon-4 mutations. For the current study, the coding regions of KRAS were deep-sequenced in 80 newly diagnosed MM patients, uniformely treated with three cycles of bortezomib plus dexamethasone and cyclophosphamide (VCD)-induction, followed by high-dose chemotherapy and autologous stem cell transplantation. Moreover, the functional impact of KRASp.G12A and the exon-4 mutations p.A146T and p.A146V on different survival pathways was investigated. Specifically, KRASWT, KRASp.G12A, KRASp.A146T, and KRASp.A146V were overexpressed in HEK293 cells and the KRASWT MM cell lines JJN3 and OPM2 using lentiviral transduction and the Sleeping Beauty vector system. Even though KRAS-mutations were not correlated with survival, all KRAS-mutants were found capable of potentially activating MEK/ERK- and sustaining PI3K/AKT-signaling in MM cells.

Multiple myeloma (MM) is a genetically heterogeneous disease and the management of relapses is one of the biggest clinical challenges. TP53 alterations are established high‐risk markers and are included in the current disease staging criteria. KRAS is the most frequently mutated gene affecting around 20% of MM patients. Applying Clonal Competition Assays (CCA) by co‐culturing color‐labeled genetically modified cell models, we recently showed that mono‐ and biallelic alterations in TP53 transmit a fitness advantage to the cells. Here, we report a similar dynamic for two mutations in KRAS (G12A and A146T), providing a biological rationale for the high frequency of KRAS and TP53 alterations at MM relapse. Resistance mutations, on the other hand, did not endow MM cells with a general fitness advantage but rather presented a disadvantage compared to the wild‐type. CUL4B KO and IKZF1 A152T transmit resistance against immunomodulatory agents, PSMB5 A20T to proteasome inhibition. However, MM cells harboring such lesions only outcompete the culture in the presence of the respective drug. To better prevent the selection of clones with the potential of inducing relapse, these results argue in favor of treatment‐free breaks or a switch of the drug class given as maintenance therapy. In summary, the fitness benefit of TP53 and KRAS mutations was not treatment‐related, unlike patient‐derived drug resistance alterations that may only induce an advantage under treatment. CCAs are suitable models for the study of clonal evolution and competitive (dis)advantages conveyed by a specific genetic lesion of interest, and their dependence on external factors such as the treatment.

Growing evidence points to multiple myeloma (MM) and its stromal microenvironment using several mechanisms to subvert effective immune and anti-tumor responses. Recent advances have uncovered the tumor-stromal cell influence in regulating the immune-microenvironment and have envisioned targeting these suppressive pathways to improve therapeutic outcomes. Nevertheless, some subgroups of patients include those with particularly unfavorable prognoses. Biological stratification can be used to categorize patient-, disease- or therapy-related factors, or alternatively, these biological determinants can be included in a dynamic model that customizes a given treatment to a specific patient. Genetic heterogeneity and current knowledge enforce a systematic and comprehensive bench-to-bedside approach. Given the increasing role of cancer stem cells (CSCs) in better characterizing the pathogenesis of solid and hematological malignancies, disease relapse, and drug resistance, identifying and describing CSCs is of paramount importance in the management of MM. Even though the function of CSCs is well-known in other cancer types, their role in MM remains elusive. With this review, we aim to provide an update on MM homing and resilience in the bone marrow micro milieu. These data are particularly interesting for clinicians facing unmet medical needs while designing novel treatment approaches for MM.

High expression of the receptor tyrosine kinase (RTK) insulin-like growth factor-1 receptor (IGF1R) and RTK mutations are associated with high-risk/worse prognosis in multiple myeloma (MM). Combining the pIGF1R/pINSR inhibitor linsitinib with the proteasome inhibitor (PI) bortezomib seemed promising in a clinical trial, but IGF1R expression was not associated with therapy response. Because the oncogenic impact of IGF1R mutations is so far unknown, we investigated the functional impact of IGF1R mutations on survival signaling, viability/proliferation and survival response to therapy. We transfected four human myeloma cell lines (HMCLs) with IGF1RWT, IGF1RD1146N and IGF1RN1129S (Sleeping Beauty), generated CRISPR-Cas9 IGF1R knockouts in the HMCLs U-266 (IGF1RWT) and L-363 (IGF1RD1146N) and tested the anti-MM activity of linsitinib alone and in combination with the second-generation PI carfilzomib in seven HMCLs. IGF1R knockout entailed reduced proliferation. Upon IGF1R overexpression, survival signaling was moderately increased in all HCMLs and slightly affected by IGF1RN1129S in one HMCL, whereby the viability remained unaffected. Expression of IGF1RD1146N reduced pIGF1R-Y1135, especially under serum reduction, but did not impact downstream signaling. Linsitinib and carfilzomib showed enhanced anti-myeloma activity in six out of seven HMCL irrespective of the IGF1R mutation status. In conclusion, IGF1R mutations can impact IGF1R activation and/or downstream signaling, and a combination of linsitinib with carfilzomib might be a suitable therapeutic approach for MM patients potentially responsive to IGF1R blockade.

Das multiple Myelom ist eine altersassoziierte maligne Plasmazellerkrankung, die vorrangig im Knochenmark auftritt und zur Verdrängung der normalen Hämatopoese führt. Fortschritte in der molekulargenetischen Diagnostik haben das Verständnis der biologischen Grundlage der Erkrankung entscheidend erweitert. Die schrittweise Einführung von neuen Medikamenten wie Proteasominhibitoren, Immunmodulatoren und monoklonalen Antikörpern sowie der Einsatz wirksamer Kombinationstherapien haben die Prognose auch mehrfach vorbehandelter Patienten deutlich gebessert. Dennoch ist eine Heilung weiterhin nicht möglich. Neue Therapiestrategien vor allem mit immunologischen und zielgerichteten Ansätzen werden die Behandlungsmöglichkeiten in Zukunft bereichern und die Prognose für Patienten mit multiplem Myelom weiter verbessern.