Acquired Resistance to Bortezomib in Human RPMI-8226 Multiple Myeloma Cells: Molecular Characterization, Cross-Resistance with Other Proteasome Inhibitors but Marked Sensitization to Glucocorticoids

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2640-2640
Author(s):  
Niels E Franke ◽  
Gertjan L Kaspers ◽  
Nynke van den Berg ◽  
Ina van Zantwijk ◽  
Katarina Vojtekova ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Molecular Mechanism ◽  
Plasma Cell ◽  
Clinical Sample ◽  
Acquired Resistance ◽  
Proteasome Inhibitors ◽  
Gene Set Enrichment Analysis ◽  
Cross Resistance ◽  
Phenotypic Characterization ◽  
Rpmi 8226

Abstract The proteasome inhibitor bortezomib (BTZ, PS-341, Velcade®) has proven to be an effective drug in the treatment of multiple myeloma (MM) patients, both in first line as in refractory disease. However, emergence of drug resistance, for which the molecular mechanism(s) remain elusive, can hamper its clinical efficacy. Recently, we showed (Oerlemans & Franke, Blood 2008) that resistance to BTZ in human THP1 acute myeloid leukemia cells was conferred by an acquired mutation in the PSMB5 gene. This mutation introduced an amino acid substitution in the highly conserved substrate binding pocket of the β5 subunit of the proteasome, which is the primary target of BTZ. In the present study we investigated whether chronic exposure to BTZ could provoke acquired resistance to BTZ in the human RPMI-8226 MM cell line model and whether a PSMB5 gene mutation would be responsible. To this end, 8226 cells were exposed in vitro to stepwise increasing concentrations of BTZ from 0.1 nM BTZ up to 7nM (8226/BTZ7) over a period of 6 months and up to 30nM (8226/BTZ30) over a period of 12 months. In parallel to these selective concentrations, RPMI-8226 cells were cultured without BTZ (8226/WT). Characterization of 8226/BTZ7 vs 8226/WT cells revealed: 2.6-fold resistance to BTZ along with cross-resistance to other peptide-based proteasome inhibitors; MG132 (2.2-fold), MG262 (1.9-fold) and 4A6 (4.9 fold), unchanged sensitivity to other anti-MM drugs, including doxorubicin, melphalan and thalidomide, a marked gain in sensitivity for the glucocorticoids dexamethasone (IC50: 25 nM vs >10 uM, respectively) and prednisolone (IC50: 2 uM vs > 1000 uM, respectively), other than 8226/WT cells, no G2M cell cycle arrest after BTZ exposure in 8226/BTZ7 cells, no significant changes in mRNA and functional activity of the three dominant catalytic subunits of the proteasome; β1, β2 and β5, although at a protein level β5 expression was markedly increased (5–10 fold), and sequencing of the PSMB5 gene in 8226/BTZ7 and 8226/BTZ30 cells, and one clinical sample of plasma cells of a BTZ-resistant plasma cell leukemia patient, showed no evidence for PSMB5 mutations. Microarray experiments were initiated to further elicit possible molecular mechanism(s) of BTZ resistance in 8226/BTZ7 cells. Initial analysis showed upregulation of several genes involved in microenvironment interaction and plasma cell differentiation. In this context, Gene Set Enrichment Analysis (GSEA) showed enrichment in the 8226/BTZ7 for the gene sets related to CD138 upregulation and MYC upregulation, indicative for a more differentiated phenotype of 8226/BTZ7 cells. Consistently, immune phenotypic characterization showed a shift to a dominant CD138dim/CD45+/CD20dim/CD27dim cell population for 8226/WT cells grown in parallel with 8226/BTZ7, which retained a CD138+/CD45−/CD20−/CD27− marker profile. Collectively, these data indicate that, other than in acute leukemia, the onset of acquired resistance to BTZ in 8226 MM cells proceeds very slowly, is associated with the upregulation of a (non-mutated) β5 catalytic proteasome subunit, and goes along with a shift towards a more mature immune phenotype in combination with an enrichment of genes that support cell growth. Notwithstanding this fact, the marked sensitization of BTZ-resistant 8226 MM cells for glucocorticoids may further corroborate the use of these drugs in combination with BTZ.

A Novel Role for Histone Deacetylase 11 (HDAC11) in B Cell Lymphopoiesis and Plasma Cell Survival in Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4715-4715
Author(s):  
Jason B. Brayer ◽  
Eva Sahakian ◽  
John Powers ◽  
Mark B Meads ◽  
Susan Deng ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Proteasome Inhibitors ◽  
Myeloma Cell ◽  
Resistant Cell ◽  
Rpmi 8226 ◽  
Human Myeloma Cell

Abstract While multiple myeloma (MM) remains incurable presently, expanded therapeutic options over the past decade have improved patient survival markedly. Proteasome inhibitors have redefined the treatment paradigm for myeloma, often serving as the backbone of front-line treatment. Histone deacetylase (HDAC) inhibitors (HDI), although only marginally active as single agent therapy in hematological malignancies, have demonstrated an ability to salvage bortezomib responsiveness in refractory patients, prompting heightened interest in this class of targeted therapeutics in myeloma. HDAC’s represent a family of enzymes, currently with 11 known members in the classical HDAC family, and subdivided into 4 sub-classes. HDAC11 is currently the only member of the sub-class IV and, as the newest member of the HDAC family, its impact on B cell lymphopoiesis and myeloma development is only starting to be unveiled. Intriguingly, we show that mice with germ-line silencing of HDAC11 (HDAC11KO mice) exhibit a 50% decrease in plasma cells in both the bone marrow and peripheral blood plasma cell compartments relative to wild-type mice. Consistent with this, Tg-HDAC11-eGFP mice, a transgenic strain engineered to express GFP under control of the HDAC11 promoter (Heinz, N Nat. Rev. Neuroscience 2001) reveals that HDAC11 expression is increased in the plasma cell population and to a lesser extent B1 B cells, as compared to earlier lineage stages. Similar observations based on measurements of HDAC11 mRNA were seen in normal human plasma cells. Significant increases in HDAC11 mRNA expression were observed in 7 of 11 primary human multiple myeloma samples and 11 of 12 human myeloma cell lines as compared to normal plasma cells, further emphasizing the potential relevance of HDAC11 to the underlying pathologic processes driving myeloma development and/or survival. Targeted silencing of HDAC11 in RPMI-8226 cells lines using siRNA results in a modest decrease in cell viability as measured by Annexin/PI staining and detection of activated caspase-3. Quisinostat, a second generation pan-HDI, has previously demonstrated activity against human myeloma cell lines in vitro (Stuhmer, Brit J Haematol, 2010), and suppressed bone destruction in an in vivo murine myeloma model (Deleu, Cancer Res, 2009). We similarly observe dose-dependent survival impairment in 10 human myeloma cell lines when cultured in the presence of quisinostat, with EC50’s consistently in the 1-10nM range. Importantly, quisinostat acts synergistically with proteasome inhibitiors (bortezomib and carfilzomib) in RPMI-8226 cells; more importantly, the degree of synergism is amplified in the RPMI-6226-B25 bortezomib-resistant cell line. Although a clear mechanism of action remains to be elucidated, preliminary data suggests that RPMI-8226 cells exposed to quisinostat appear to exhibit a decrease nuclear, but not cytosolic HDAC11. Collectively, these data illustrate a previously unknown role for HDAC11 in plasma cell differentiation and survival. Increased HDAC11 expression seen in myeloma patient specimens and primary myeloma cell lines highlights the potential of HDAC11 as a therapeutic target. Furthermore, we show that quisinostat, a pan-HDI with selectivity towards HDAC11 at lower dosing, acts synergistically with proteasome inhibitors in vitro in proteasome inhibitor sensitive and resistant cell lines. Future work will focus on further elucidating the role of HDAC11 in myeloma survival and drug response, with particular emphasis on proteasome inhibitors. Disclosures No relevant conflicts of interest to declare.

scholarly journals Immunotherapy of Multiple Myeloma With a Monoclonal Antibody Directed Against a Plasma Cell-Specific Antigen, HM1.24

Blood ◽  
1997 ◽  
Vol 90 (8) ◽  
pp. 3179-3186 ◽  
Author(s):  
Shuji Ozaki ◽  
Masaaki Kosaka ◽  
Shingo Wakatsuki ◽  
Masahiro Abe ◽  
Yasuo Koishihara ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Plasma Cell ◽  
Severe Combined Immunodeficiency ◽  
Specific Antigen ◽  
Chemotherapeutic Agents ◽  
Scid Mice ◽  
Dependent Manner ◽  
Rpmi 8226

Abstract Multiple myeloma remains an incurable malignancy because of marked resistance of tumor cells to conventional chemotherapeutic agents. Alternative strategies are needed to solve these problems. To develop a new strategy, we have generated a monoclonal antibody (MoAb), which detects a human plasma cell-specific antigen, HM1.24. In this report, we evaluated the in vivo antitumor effect of unconjugated anti-HM1.24 MoAb on human myeloma xenografts implanted into severe combined immunodeficiency (SCID) mice. Two models of disseminated or localized tumors were established in SCID mice by either intravenous or subcutaneous injection of human myeloma cell lines, ARH-77 and RPMI 8226. When mice were treated with a single intraperitoneal injection of anti-HM1.24 MoAb 1 day after tumor inoculation, the development of disseminated myeloma was completely inhibited. In mice bearing advanced tumors, multiple injections of anti-HM1.24 MoAb reduced the tumor size and significantly prolonged survival, including tumor cure, in a dose-dependent manner. The proliferation of cultured human myeloma cells was inhibited in vitro by anti-HM1.24 IgG-mediated complement-dependent cytotoxicity, but not by the antibody alone. Moreover, spleen cells from SCID mice mediated antibody-dependent cell cytotoxicity against RPMI 8226 cells. These results indicate that anti-HM1.24 MoAb can be used for immunotherapy of multiple myeloma and related plasma cell dyscrasias.

The sitotoxic effect of gemcitabine on multiple myeloma (RPMI-8226) and Ig G plasma cell leukemia (ARH 77) cell lines

2012 ◽  
Vol 54 (4) ◽  
pp. 263 ◽  
Author(s):  
Zehra Coban ◽  
Ferit Avcu ◽  
Ali Ural ◽  
Okan Kuzhan ◽  
Sefik Guran
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cell Leukemia ◽  
Cell Leukemia ◽  
Rpmi 8226 ◽  
Ig G

Characterization of Bortezomib (Velcade ™) Resistance in a New Set of Human Cell Lines Obtained by Stepwise Selection.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2741-2741
Author(s):  
Jie Cai ◽  
Xian Jin Lian ◽  
Christopher von Roretz ◽  
Chaim Shustik ◽  
Imed Gallouzi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Stress Responses ◽  
Proteasome Inhibitors ◽  
Proteasome Activity ◽  
26S Proteasome ◽  
Cross Resistance ◽  
Stepwise Selection ◽  
Drug Concentrations

Abstract Abstract 2741 Poster Board II-717 Bortezomib (Velcade ™, PS-341, BTZ) is a boronate dipeptide that reversibly inhibits the 26S proteasome, which is essential for the breakdown of ubiquitinated proteins and the regulation of normal cellular homeostasis. The activity of BTZ in treatment of newly diagnosed and refractory/relapsed multiple myeloma may be limited by the development of chemoresistance, the mechanisms of which are poorly understood. To investigate the molecular basis of Bortezomib resistance, BTZ-resistant (BTZr) cell lines were generated by stepwise selection procedures from HeLa, CCRF-CEM, and 4 multiple myeloma cells lines (8226S, U266, H929, and MM.1S), respectively. These BTZ-selected cell lines displayed varying degrees of elevated resistance (2 to 50 fold) to clinically relevant concentrations of BTZ. In addition, while most of the BTZr cells showed cross resistance to several other proteasome inhibitors (PIs), including MG-132 and Epoxomicin, they remained as sensitive to other chemotherapeutic drugs, such as anthracyclines, vinkalkaloids and etoposide, as their parental cells. The proteasome activity profiles are distinct among the cell lines. All parental cell lines displayed varying levels of chymotrypsin-like (CT-L) activity, which is the primary target of BTZ. Most BTZr lines showed markedly decreased CT-L activity, with a few exceptions. Moreover, the observed CT-L activity in all cell lines can be inhibited directly by BTZ and other PIs. In contrast, very low levels of caspase-like or post-glutamyl peptide hydrolyzing (PGPH) proteasome activity were detected in all cell lines. BTZ resistance in HeLa/BTZ cells was closely associated with increased resistance to PI-induced apoptosis, as shown by reduced number of Annexin V-stained cells and by delayed activation/cleavage of apoptosis proteins, such as Caspase-3 and Poly(ADP-ribose) Polymerase (PARP). Furthermore, the resistance to BTZ affected the mechanisms of cell stress responses. As for the parental HeLa cells, HeLa/BTZr cells retained the ability to form, in response to PI treatment, pro-survival foci in the cytoplasm known as stress granules (SGs). However, the drug concentrations required to induce SG formation in HeLa/BTZr cells are much higher (∼4 fold) than those for the parental HeLa, suggesting the development of stress-coping mechanisms in these BTZr cells. Gene expression profiling studies are in progress to identify transcriptomes individually or generally associated with BTZ resistance in these cell lines. Further characterization of these phenotypically similar, yet mechanistically distinct BTZr cell lines may elucidate diverse mechanisms of drug resistance to Bortezomib and other proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Functional Analysis of HDAC11 in Plasma Cell Development and Multiple Myeloma Survival

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3223-3223
Author(s):  
AGM Mostofa ◽  
Allison Distler ◽  
Mark B. Meads ◽  
Eva Sahakian ◽  
John J. Powers ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Functional Analysis ◽  
T Cells ◽  
Cell Lines ◽  
Plasma Cell ◽  
Synergistic Effects ◽  
Proteasome Inhibitors ◽  
Cell Development ◽  
Selective Inhibitor ◽  
Binding Partner

Abstract Background: Histone deacetylases (HDACs) are potential novel therapeutic targets for multiple myeloma (MM) treatment. A pan-HDAC inhibitor (HDI) panobinostat was approved by the FDA in 2015 to treat relapsed/refractory MM patients, and several other HDIs are currently in different phases of clinical trials. However, unfavorable side-effects of the non-selective HDIs necessitate further dissection of the roles of individual HDAC isoforms to best target plasma cell malignancies with minimal toxicity. HDAC11 was recently found to regulate function in key immune cell populations including regulatory T cells, effector T cells, neutrophils, and myeloid-derived suppressor cells (MDSC). Though HDAC11 expression is confirmed in B cells and plasma cells, its functions in these cells remain largely unknown. In this study, we attempted a functional analysis of HDAC11 in plasma cell development along with its pro-tumorigenic function in MM cells. Methods: Mouse models, including a transgenic mouse strain expressing eGFP under the regulation of the HDAC11 promoter (Tg-HDAC11-eGFP), and also an HDA11-deficient mouse (B6.HDAC11-/-) were studied to establish the importance of HDAC11 in plasma cell biology. Pharmacologic inhibition of HDAC11 in MM cell lines was accomplished by using elevenostat, a new HDAC11-selective inhibitor in comparison with pan-inhibitors quisinostat and panobinostat. Impact on viability in human-derived MM cell lines was assessed using the CCK-8 assay, while induction of cell death was measured via detection of activated Caspase-3 and annexin/propidium iodide staining by flow cytometry. Synergy studies were performed by following the Chou-Talalay method for drug combinations. Post-translational modifications and subcellular localization changes induced by HDIs exposure were assessed by western blotting of fractionated cell lysates, while immunoprecipitation and proximity ligation assays (in situ PLA) were used to identify a binding partner for HDAC11. Results: Studies in Tg-HDAC11-eGFP mice reveal that HDAC11 expression in B cell lymphopoiesis is minimally detectable prior to B cell activation but demonstrates strong induction upon maturation into a plasma cell. Consistent with this, plasma cell development is markedly impaired in the absence of HDAC11. The HDAC11-selective inhibitor elevenostat showed significant cytotoxic potential in different MM cell lines that express moderate to high level of HDAC11, with IC50 values ranging 0.6-2.0 µM. Consistently, MM cell lines expressing null/very low level of HDAC11 were insensitive to elevenostat. Moreover, combining elevenostat with proteasome inhibitors bortezomib (BTZ) and carfilzomib resulted in significant synergistic effects evident from combination index (CI) and dose-reduction index (DRI) values measured by CompuSyn software. Elevenostat was also able to re-sensitize BTZ-resistant sub-clones (e.g., RPMI-8226-B25, KAS-6-V10R, and ANBL6-V10R) to BTZ and exhibited superior synergistic effects. Furthermore, elevenostat-treated cells showed a time-dependent alteration in the subcellular localization of HDAC11. HDAC11 gradually disappeared from the nuclear fractions with simultaneous upregulation in cytoplasmic fractions; similar observations were made from pan HDIs (quisinostat and panobinostat) treatment. However, unlike pan HDIs, the elevenostat treatment caused global downregulation of HDAC11 in some MM cell lines at the later time points (72 or 96 hrs), suggesting differential effects of various HDIs. Inhibition of HDAC11 also caused downstream suppression of several pro-tumorigenic factors of MM cells including IRF4 and c-Myc. Additionally, a novel interaction between HDAC11 and IRF4, an essential regulator of PC differentiation and MM survival, was identified by using PLA. HDAC11 dynamically interacts with IRF4 which can be induced by LPS stimulation and inhibited by HDIs, indicating the involvement of HDAC11 in the IRF4-mediated regulatory circuit. Conclusions: We observe that targeted inhibition of HDAC11 can impair MM cell survival and overcome acquired resistance to proteasome inhibitors. Furthermore, we identify IRF4 as a nuclear binding partner of HDAC11 and propose this interaction as a candidate mechanism regulating PC maturation and survival. Disclosures No relevant conflicts of interest to declare.

scholarly journals Elimination of clonogenic stem cells from human multiple myeloma cell lines by a plasma cell-reactive monoclonal antibody and complement

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1482-1489 ◽  
Author(s):  
AW Tong ◽  
JC Lee ◽  
JW Fay ◽  
MJ Stone
Keyword(s):  
Stem Cells ◽  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Cell Lines ◽  
Plasma Cell ◽  
Colony Formation ◽  
Human Multiple Myeloma ◽  
Rpmi 8226 ◽  
Marrow Cells

Abstract The monoclonal antibody (MoAb) MM4 reacts with human multiple myeloma (MM) cell lines and bone marrow from patients with plasma cell dyscrasias but not with normal peripheral blood or bone marrow cells. Treatment with MM4 and rabbit complement (C') was cytotoxic to the plasma cell-derived cell lines GM 1312, RPMI 8226, and ARH-77, as demonstrated by chromium release microcytotoxicity and trypan blue exclusion assays. The same treatment eliminated greater than 99% of clonogenic myeloma stem cell colony formation of these cell lines, with less than 20% inhibition of normal human bone marrow pleuripotent progenitor colony formation in vitro. As an experimental model to explore the efficacy of MM4 + C' in purging MM-involved bone marrow, normal marrow cells were mixed with RPMI 8226 or GM 1312 cells in the ratio of 90:10 or 50:50 (marrow:myeloma cells). Colony growth assays indicated that MM4 + C' eliminated at least 2 logs of clonogenic myeloma stem cells in both 90:10 and 50:50 preparations, while sparing the majority of normal marrow progenitors (inhibition of CFU-C:10% to 13%; BFU-E:0%). The selectivity of MM4-mediated cytotoxicity may be useful for eliminating myeloma clonogenic stem cells from bone marrow of patients with multiple myeloma.

scholarly journals CIC Mutation as a Molecular Mechanism of Acquired Resistance to Combined BRAF‐MEK Inhibition in Extramedullary Multiple Myeloma with Central Nervous System Involvement

2019 ◽  
Vol 25 (2) ◽  
pp. 112-118 ◽  
Author(s):  
Matteo Claudio Da Vià ◽  
Antonio Giovanni Solimando ◽  
Andoni Garitano‐Trojaola ◽  
Santiago Barrio ◽  
Umair Munawar ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Multiple Myeloma ◽  
Nervous System ◽  
Molecular Mechanism ◽  
Acquired Resistance ◽  
Central Nervous System Involvement ◽  
Mek Inhibition ◽  
System Involvement

Differential response to carfilzomib based on initial therapy with bortezomib in multiple myeloma.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e19515-e19515
Author(s):  
Sarvari Yellapragada ◽  
Chizoba Ifeorah ◽  
Luke Fletcher ◽  
Gustavo Rivero ◽  
Zahida Yasin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Medical Center ◽  
Proteasome Inhibitors ◽  
Progression Free Survival ◽  
Initial Therapy ◽  
Resistance Mechanisms ◽  
Cross Resistance ◽  
Primary Resistance ◽  
Free Survival ◽  
Depth Of Response

e19515 Background: Proteasome inhibitors (PIs) are efficacious in multiple myeloma (MM). In randomized phase 3 studies Carfilzomib doubled the progression free survival (PFS) compared to Bortezomib from 9.4 mo to 18.7 mo (ENDEAVOR study) in relapsed and refractory MM. However concern for suboptimal Carfilzomib based therapy (CBT) response in patients (pt) progressing on Bortezomib based therapy (BBT) suggests “cross-resistance”. Methods: AfterIRB approval, all pt with symptomatic MM over the last 10 years at the Michael E. DeBakey VA Medical Center with initial BBT followed by salvage CBT at progression were included. The primary aim was to evaluate ORR (PR + VGPR+CR) and and PFS by the IMWG criteria, among relapsed/refractory pt treated with salvage CBT whether or not they had an initial BBT response (i.e., primary refractory+stable vs CR+PR+VGPR). Results: In this cohort, the median overall survival was 45 mo. Pt initially treated with BBT had an ORR of 17/19 (89.4%) and median PFS of 7.7 mo. For 12 pt attaining PR, PFS was 4.3 mo, whereas for those achieving CR/VGPR was 9.1 mo, p= 0.03. Two pt were primary refractory to BBT and also to CBT. For initially treated BBT responders the ORR with CBT was 68.4% (13/19), median PFS was 5.26 mo. 6/19 pt (32%) were CBT refractory. PFS during BBT for CBT responders versus CBT refractory patients was not statistically different. Among those who responded to both CBT and BBT there was no significant correlation between the PFS or depth of response (CR vs VGPR vs PR) with initial BBT and PFS with CBT. Conclusions: In our retrospective analysis, pt who exhibited primary resistance to initial BBT did not benefit from salvage therapy with CBT, raising the possibility that hard-wired PI-resistance mechanisms exist. Among BBT responders, CBT ORR was 68%. However the PFS with CBT was significantly shorter than the published literature of CBT in RRMM naïve to PI leading us to hypothesize that uncharacterized resistance mechanisms from “priming” bortezomib leads to inferior outcome with salvage CBT. We conclude that CBT can be attempted in those who respond to BBT independent of depth of response or PFS duration. However, alternative therapies should be considered in pt with primary refractoriness to BBT.

Targeting Plasticity and Stemness in Multiple Myeloma Tumor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4270-4270
Author(s):  
Stacy W. Blain ◽  
Danielle Joseph ◽  
Amitabha Mazumder
Keyword(s):  
Multiple Myeloma ◽  
Tissue Culture ◽  
Stem Cell ◽  
Tumor Cells ◽  
Plasma Cell ◽  
Cell Types ◽  
Bone Marrow Niche ◽  
Self Renewal ◽  
Accessory Cells ◽  
Rpmi 8226

Abstract Background: While there are many effective therapies that can induce remission, almost all Multiple Myeloma (MM) patients relapse. This has led to the hypothesis that a chemotherapy-resistant cancer stem cell (CSC) remains dormant and then undergoes self-renewal and differentiation to reestablish disease. To eliminate disease, novel therapies must be designed to preferentially target this CSC, either to maintain it in its dormant state, or alternatively to drive it into cycle where it will become sensitive to more traditional therapies. The MM CSC can be detected in cultured MM tissue culture models, including RPMI-8266 cells, and is defined by several parameters: 1) the lack of the plasma cell marker CD138, 2) lack of the differentiation markers Blimp1 and IRF4, 3) self-renewal as measured by the ability to form colonies in methylcellulose, 4) clinically relevant chemoresistance to Bortezomib and Lenalidomide, and 5) a G0 cell cycle state. Our finding is that the mature cancer cell (CD138+, Blimp+, IRF4+, rapidly proliferating and chemosensitive) has developmental plasticity; namely, the ability to dedifferentiate back into its own chemoresistant CSC progenitor, the CD138-, Blimp-, IRF4-, quiescent pre-plasma cell. In our 2D culture system, we observe multiple cycles of sequential differentiation and dedifferentiation in the absence of niche or supportive accessory cells, suggesting that soluble cytokines secreted by the MM cells themselves are responsible for this bidirectional interconversion and that stemness and more importantly chemoresistance are dynamic characteristics that can be acquired or lost and thus are targetable. Methods: Flow cytometric analysis of RPMI 8226, U266 and NIH-H929 demonstrates that 5-10% of these stable cells are consistently CD138-, and thus are a mixture of mature and precursor cells. Sorted >98% pure CD138- and CD138+ populations were cultured in tissue culture media, and then harvested at various times post plating and screened for CD138. Within 5 days post plating, the immature CD138- cells differentiated to produce progeny that were CD138+. When the mature CD138+ cells were plated post sorting, we found that within 5 days, a large population of CD138- cells appeared as well, suggesting that the mature CD138+ tumor cells had the capacity to dedifferentiate back into the more immature CD138- cells. While it has been shown that MM cells and other cell types are able to differentiate in the presence of a niche or accessory cells, our data suggest that the presence or absence of soluble factors secreted from the cells themselves alters their differentiation and dormancy status: the cell population has the plasticity required to maintain enough progenitor cells to provide a continuous supply of daughter cells. Using an array to examine cytokine secretion from sorted, 99% pure populations of either CD138-CSCs or CD138+ progeny from the RPMI-8226 line we identified that concomitant with interconversion, Macrophage Migration Inhibitory Factor (MIF-1) was secreted into the media. To determine if MIF-1 was responsible for interconversion, we treated pure populations of CD138+ cells with 4-IPP, a small molecule MIF-1 inhibitor. We saw that with increasing concentrations of inhibitor, the CD138+ cells rapidly accelerated dedifferentiation back into the CD138- progenitor and trapped these cells reversibly in the dormant, chemoresistant state. When we treated CD138- cells with 4-IPP, they remained CD138- and did not differentiate into CD138+ cells. This suggests that blocking MIF-1 forced and/or maintained cells in the chemoresistant CD138- state. Conclusion: MIF-1 is an inflammatory cytokine, secreted by many cell types, and shown to increase survival and proliferation of several stem cell lineages. Our data suggest that MIF1 regulates interconversion and its levels, produced from either the MM cells themselves or in vivo from the bone marrow niche, regulate the balance between chemoresistant CSCs and chemosensitive progeny. Thus, targeting MIF-1 may be a viable way to alter chemosensitivity. Disclosures No relevant conflicts of interest to declare.

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