Multiple Myeloma Inhibitory Activity of Plant Natural Products

A literature search on plant natural products with antimyeloma activity until the end of 2020 resulted in 92 compounds with effects on at least one human myeloma cell line. Compounds were divided in different compound classes and both their structure–activity-relationships as well as eventual correlations with the pathways described for Multiple Myeloma were discussed. Each of the major compound classes in this review (alkaloids, phenolics, terpenes) revealed interesting candidates, such as dioncophyllines, a group of naphtylisoquinoline alkaloids, which showed pronounced and selective induction of apoptosis when substituted in position 7 of the isoquinoline moiety. Interestingly, out of the phenolic compound class, two of the most noteworthy constituents belong to the relatively small subclass of xanthones, rendering this group a good starting point for possible further drug development. The class of terpenoids also provides noteworthy constituents, such as the highly oxygenated diterpenoid oridonin, which exhibited antiproliferative effects equal to those of bortezomib on RPMI8226 cells. Moreover, triterpenoids containing a lactone ring and/or quinone-like substructures, e.g., bruceantin, whitaferin A, withanolide F, celastrol, and pristimerin, displayed remarkable activity, with the latter two compounds acting as inhibitors of both NF-κB and proteasome chymotrypsin-like activity.

Expression of the chemokine receptor CCR1 promotes the dissemination of multiple myeloma plasma cells in vivo

Multiple myeloma (MM) disease progression is dependent on the ability of MM plasma cells (PCs) to egress from the bone marrow (BM), enter the circulation and disseminate to distal BM sites. Expression of the chemokine CXCL12 by BM stromal cells is crucial for MM PC retention within the BM. However, the mechanisms which overcome CXCL12-mediated retention to enable dissemination are poorly understood. We have previously identified that treatment with the CCR1 ligand CCL3 inhibits the response to CXCL12 in MM cell lines, suggesting that CCL3/CCR1 signalling may enable egress of MM PC from the BM. Here, we demonstrated that CCR1 expression was an independent prognostic indicator in newly diagnosed MM patients. Furthermore, we showed that CCR1 is a crucial driver of dissemination in vivo, with CCR1 expression in the murine MM cell line 5TGM1 being associated with an increased incidence of bone and splenic disseminated tumours in C57BL/KaLwRij mice. Furthermore, we demonstrated that CCR1 knockout in the human myeloma cell line OPM2 resulted in a >95% reduction in circulating MM PC numbers and BM and splenic tumour dissemination following intratibial injection in NSG mice. Therapeutic targeting of CCR1 with the inhibitor CCX9588 significantly reduced OPM2 or RPMI-8226 dissemination in intratibial xenograft models. Collectively, our findings suggest a novel role for CCR1 as a critical driver of BM egress of MM PCs during tumour dissemination. Furthermore, these data suggest that CCR1 may represent a potential therapeutic target for the prevention of MM tumour dissemination.

Identification of Potential Glucocorticoid Receptor Therapeutic Targets in Multiple Myeloma

Glucocorticoids (GC) are a cornerstone of combination therapies for multiple myeloma. However, patients ultimately develop resistance to GCs frequently based on decreased glucocorticoid receptor (GR) expression. An understanding of the direct targets of GC actions, which induce cell death, is expected to culminate in potential therapeutic strategies for inducing cell death by regulating downstream targets in the absence of a functional GR. The specific goal of our research is to identify primary GR targets that contribute to GC-induced cell death, with the ultimate goal of developing novel therapeutics around these targets that can be used to overcome resistance to GCs in the absence of GR. Using the MM.1S glucocorticoid-sensitive human myeloma cell line, we began with the broad platform of gene expression profiling to identify glucocorticoid-regulated genes further refined by combination treatment with phosphatidylinositol-3'-kinase inhibition (PI3Ki). To further refine the search to distinguish direct and indirect targets of GR that respond to the combination GC and PI3Ki treatment of MM.1S cells, we integrated 1) gene expression profiles of combination GC treatment with PI3Ki, which induces synergistic cell death; 2) negative correlation between genes inhibited by combination treatment in MM.1S cells and genes over-expressed in myeloma patients to establish clinical relevance and 3) GR chromatin immunoprecipitation with massively parallel sequencing (ChIP-Seq) in myeloma cells to identify global chromatin binding for the glucocorticoid receptor (GR). Using established bioinformatics platforms, we have integrated these data sets to identify a subset of candidate genes that may form the basis for a comprehensive picture of glucocorticoid actions in multiple myeloma. As a proof of principle, we have verified two targets, namely RRM2 and BCL2L1, as primary functional targets of GR involved in GC-induced cell death..

Factors Influencing the Abundance of the Side Population in a Human Myeloma Cell Line

Side population (SP) refers to a group of cells, which is capable to efflux Hoechst 33342, a DNA-binding dye. SP cells exist both in normal and tumor tissues. Although SP abundance has been used as an indicator for disease prognostic and drug screening in many research projects, few studies have systematically examined the factors influencing SP analysis. In this study we aim to develop a more thorough understanding of the multiple factors involved in SP analysis including Hoechst 33342 staining and cell culture. RPMI-8226, a high SP percentage (SP%) human myeloma cell line was employed here. The results showed that SP% was subject to staining conditions including: viable cell proportion, dye concentration, staining cell density, incubation duration, staining volume, and mix interval. In addition, SP% was highest in day one after passage, while dropped steadily over time. This study shows that both staining conditions and culture duration can significantly affect SP%. In this case, any conclusions based on SP% should be interpreted cautiously. The relation between culture duration and SP% suggests that the incidence of SP cells may be related to cell proliferation and cell cycle phase. Maintaining these technical variables consistently is essential in SP research.

Establishment and Characterization of a Novel Human Myeloma Cell Line, AMU-MM1, From a Multiple Myeloma Patient Involving Central Nerve System After Treatment with Bortezomib

Abstract 4991 Bortezomib improved the survival in patients with multiple myeloma (MM), however, cannot cure this disease even when it is combined with autologous stem cell transplant(s) and other new drugs. Myeloma cells cannot be excluded completely from a patient due to drug refractoriness and/or inefficiency of drug delivery in extramedullary sites such as cerebrospinal fluid (CSF). Therefore, it is important to clarify the mechanisms of bortezomib resistance and invasion to central nerve system (CNS) of myeloma for the progress of myeloma therapy. In this study, we established a novel human myeloma cell line from a myeloma patient involved with CNS and analyzed its characters including sensitivity to bortezomib. The patient had been treated with bortezomib for 1.5 years when her CNS was involved with myeloma. The cells from patient's CSF were cultured in RPMI 1640 supplemented with 20% heat-inactivated fetal bovine serum (FBS) and rhIL-6. After 3 months of culture, cell proliferation became continuous with 10% FBS and without rhIL-6. The cell line, named AMU-MM1 was established and negative for EBV. A doubling time of AMU-MM1 cells was about 48 hours. AMU-MM1 cells were positive for CD38, CD54, CD138 and cytoplasmic kappa chain and negative for CD19, CD20, CD33 and CD56 by flow cytometry analysis while those in patient's bone marrow were positive for CD56. AMU-MM1 showed hypo- and pseudodiploid karyotypes with t(4;14), t(8;13), t(1;19), del13q, amp1q21 and others but without del17p by cytogenetic analyses including FISH. The G322A mutation in the proteosome beta 5 subunit (PSMB5) gene, which is reported as a mutation found in bortezomib-resistant cell lines induced via repeated drug selection, was not detected in AMU-MM1 by direct sequencing. Apoptosis analysis using Annexin V/PI assay indicated that AMU-MM1 was sensitive to bortezomib. Our data suggest that AMU-MM1 was derived from the cells that invaded to CSF in which bortezomib concentration was very low and not resistant to bortezomib, and the downregulation of CD56 might play a role in the pathogenesis of CNS involvement as reported before. In addition, we are now focusing on new chimera or dysregulated genes in t(8;13), t(1;19) and other sites as well. In conclusion AMU-MM1 is a useful cell line for analysis of mechanisms of CNS involvement and also possibly the acquired resistance to bortezomib. Disclosures: No relevant conflicts of interest to declare.