A Deptor Inhibitor Induces Its Degradation with Resulting Anti-Myeloma Cytotoxicity in Vitro and In Vivo

Multiple myeloma (MM) is a hematological disorder characterized by a proliferation of malignant monoclonal plasma cells in the bone marrow (BM) and / or in extramedullary sites. Despite recent progress in OS rates, MM remains an incurable disease and most patients will relapse and require treatment. Deptor is a component of mTOR complexes and a constitutive inhibitor of their activities. It is known that the inhibition of Deptor results in the inhibition of the proliferation and induction of apoptosis in MM cells. In addition, high levels of Deptor are predictive of a poor response to conventional therapies, indicating that Deptor expression are important as a prognostic marker for patients with myeloma and is a possible therapeutic target. Our group previously identified a drug which prevents mTOR-Deptor binding (NSC126405) and induces cellular cytotoxicity in MM (Shi Y, et al 2016). In this study, we developed a new related chemical inhibitor (43 M) capable of inducing the inhibition of the mTOR / Deptor interaction and results in the negative regulation of Deptor that leads to the inhibition of proliferation and induces apoptosis in several MM cell lines. The cytotoxic effect of 43 M is not dependent of caspase activation and induces the activation of p70 and AKT (T308). This leads to the induction of apoptosis in MM cell lines and tumor cells derived from MM patients. The degradation of Deptor induced by 43 M is dependent on the proteasome complex since it was prevented in the presence of MG132. In vivo, 43 M prevents the expression of Deptor in a xenograft tumor, and delayed tumor growth and interestingly, induces the eradication of tumors in 40% of mice in a murine model of MM, without significant toxic implications. Recent studies show that Deptor expression protects MM cells against Bortezomib treatment, suggesting that anti-Deptor drugs can synergize with proteasome inhibitors (PIs). However, the combination of 43 M + Bortezomib was not synergistic, and was antagonistic in vitro. These results are probably due to the prevention of the proteasomal degradation of Deptor, suggesting a possible use of the 43 M inhibitor in MM in the absence of the current PIs. This study describes for the first time the possible role of Deptor as a therapeutic target using a chemical inhibitor capable of degrading and inducing a cytotoxic effect in MM cell lines. In addition, Deptor is reported as an important therapeutic target in an in vivo MM model. Shi Y, Daniels-Wells TR, Frost P, Lee J, Finn RS, Bardeleben C, Penichet ML, Jung ME, Gera J, Lichtenstein A. Cytotoxic Properties of a DEPTOR-mTOR Inhibitor in Multiple Myeloma Cells. Cancer Res. 2016 Oct 1;76(19):5822-5831 Disclosures No relevant conflicts of interest to declare.

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JAM-A as a Prognostic Factor and New Therapeutic Target in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.307.307 ◽

2016 ◽

Vol 128 (22) ◽

pp. 307-307 ◽

Cited By ~ 1

Author(s):

Antonio Solimando ◽

Andreas Brandl ◽

Mattenheimer Katharina ◽

Carolin Graf ◽

Miriram Ritz ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Therapeutic Target ◽

Healthy Subjects ◽

Plasma Cells ◽

Cell Membrane Protein ◽

And Migration ◽

Rpmi 8226

Abstract Cell adhesion in the multiple myeloma (MM) microenvironment is a mechanism by which MM plasma cells escape the effects of therapy and survive. To improve clinical strategies and overcome drug resistance, approaches directed to both MMPCs and bone marrow microenvironment are under investigation. Here, we examined the cell membrane protein Junctional adhesion molecule-A (JAM-A) as a clinical biomarker and novel therapeutic target for MM. We evaluated JAM-A expression by real time PCR (RT-PCR), flow cytometry and immunofluorescence microscopy in 132 MM patients at different stages and various MM cell lines. Next, we measured the concentrations of soluble JAM-A from MM and healthy subjects sera by enzyme linked immune assay (ELISA). We investigated JAM-A functionally in vitro and in vivo by transient gene silencing (siRNA) and with blocking antibodies. Patient-derived plasma cells (MMPCs) expressed increased JAM-A expression levels when compared to control PC from healthy individuals. Elevated JAM-A expression correlated with poor prognosis (Figure 1A,B). Furthermore, soluble JAM-A was significantly increased in MM patient sera when compared to healthy subjects. Additionally, MM cell lines showed high expression of both membrane and cytoplasmic JAM-A. Consequently, inhibition of JAM-A using specific siRNA treatment resulted in diminished tumorigenic potential, including decreased colony formation, chemotaxis and migration. Importantly, treatment of luciferase+RPMI-8226 MM bearing NSG with a JAM-A blocking monoclonal antibody reduced significantly MM progression and dissemination in vivo when compared to MM bearing mice that received an non-specific isotype control antibody (Figure 1C). Conclusively, our data suggest that JAM-A can serve as a biomarker of malignancy in MM patients. Soluble plasma JAM-A could contribute to serum-based clinical stratification. Furthermore, therapeutic targeting of JAM-A appears attractive for clinical translation. Figure 1 Figure 1. Disclosures Einsele: Celgene: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Amgen: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria.

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Bioactive Compounds from Herbal Medicine Targeting Multiple Myeloma

Applied Sciences ◽

10.3390/app11104451 ◽

2021 ◽

Vol 11 (10) ◽

pp. 4451

Author(s):

Coralia Cotoraci ◽

Alina Ciceu ◽

Alciona Sasu ◽

Eftimie Miutescu ◽

Anca Hermenean

Keyword(s):

Multiple Myeloma ◽

Survival Rate ◽

Plasma Cells ◽

Inhibition Of Proliferation ◽

In Vivo Experiments ◽

Clonal Proliferation ◽

Hematological Cancers ◽

Monoclonal Proteins

Multiple myeloma (MM) is one of the most widespread hematological cancers. It is characterized by a clonal proliferation of malignant plasma cells in the bone marrow and by the overproduction of monoclonal proteins. In recent years, the survival rate of patients with multiple myeloma has increased significantly due to the use of transplanted stem cells and of the new therapeutic agents that have significantly increased the survival rate, but it still cannot be completely cured and therefore the development of new therapeutic products is needed. Moreover, many patients have various side effects and face the development of drug resistance to current therapies. The purpose of this review is to highlight the bioactive active compounds (flavonoids) and herbal extracts which target dysregulated signaling pathway in MM, assessed by in vitro and in vivo experiments or clinical studies, in order to explore their healing potential targeting multiple myeloma. Mechanistically, they demonstrated the ability to promote cell cycle blockage and apoptosis or autophagy in cancer cells, as well as inhibition of proliferation/migration/tumor progression, inhibition of angiogenesis in the tumor vascular network. Current research provides valuable new information about the ability of flavonoids to enhance the apoptotic effects of antineoplastic drugs, thus providing viable therapeutic options based on combining conventional and non-conventional therapies in MM therapeutic protocols.

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Global Genomic Analysis of Newly Diagnosed t(4 ;14) Multiple Myeloma Reveals a Specific Mutational Spectrum and Identifies PKD2 As a Potential Therapeutic Target

Blood ◽

10.1182/blood.v128.22.4462.4462 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4462-4462

Author(s):

Xiu Ly Song ◽

Raphaël Szalat ◽

Alexis Talbot ◽

HaiVu Nguyen ◽

Mehmet K. Samur ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Therapeutic Target ◽

Plasma Cells ◽

Genomic Analysis ◽

Sequencing Analysis ◽

Newly Diagnosed ◽

Potential Therapeutic Target ◽

Mutational Landscape

Abstract In Multiple Myeloma (MM), the t(4;14) translocation is associated with a poor outcome. However, beside this translocation, the genetic events which determine the adverse evolution of the disease and the resistance to treatments remain elusive. In this study we performed whole exome or RNA sequencing analysis of samples from 65 newly diagnosed t(4;14) MM. We found that NRAS, KRAS, MAPK and FGFR3 are frequently mutated (12%, 9%, 13.8%, and 20% respectively). Overall, the FGFR3/RAS/BRAF/MAPK genes were mutated in 36 cases (54%). There was a negative correlation between mutations in FGFR3 and those occurring in NRAS, KRAS and BRAF as expected from the mutually exclusive occurrence of mutations in these genes. In addition to alterations in TP53 and DIS3, we found marked elevated frequency of mutations in PRKD2 (10.7%), ATM/ATR (10.7%) and MYCBP2 (7.6%), reduced frequency in FAM46C (1.5%) and no mutation in TRAF3 and CCND1. Mutations in ATM/ATR were strongly associated with the MB4-2 breakpoint (Bp) (p = 1.62 10-4) and significantly correlated with mutations affecting genes coding for members of the MAPK family. We observed a positive correlation between non-silent mutations in PRKD2 and the MB4-1 or MB4-3 Bp (p = 1.3 10-2). Of note, PRKD2 mutations are exclusively found in 3 t(4;14) MM cell lines and among the 84 MM sequenced by Bolli et al. (1), none of the non t(4;14) patient were mutated in PRKD2, indicating that this genetic lesion is associated with t(4;14) MM. In the NCI-H929 t(4;14) MM cell line, which is mutated for PRKD2, encoding the PKD2 serine/threonine kinase, we observed elevated levels of phosphorylated PKD2. Furthermore, inhibition of PKD, decreased PKD2 phosphorylation and triggered reduced proliferation and apoptosis of MM cell lines and fresh plasma cells from patients in vitro. These results define a specific mutational landscape for t(4;14) MM and identify PKD2 as a potential therapeutic target in MM patients. Altogether, these results define a specific mutational landscape for t(4;14) MM and identify PKD2 as a potential therapeutic target in MM patients. Reference 1. Bolli, N., Avet-Loiseau, H., Wedge, D.C., Van Loo, P., Alexandrov, L.B., Martincorena, I., Dawson, K.J., Iorio, F., Nik-Zainal, S., Bignell, G.R., et al. (2014). Heterogeneity of genomic evolution and mutational profiles in multiple myeloma. Nat Commun 5, 2997. Disclosures Munshi: Janssen: Consultancy; Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Merck: Consultancy; Pfizer: Consultancy; Oncopep: Patents & Royalties.

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Anti-CD138-IFNα Fusion Proteins Are Effective in Treating Multiple Myeloma

Blood ◽

10.1182/blood.v120.21.939.939 ◽

2012 ◽

Vol 120 (21) ◽

pp. 939-939

Author(s):

Esther Yoo ◽

Alex Vasuthasawat ◽

Danh Tran ◽

Alan Lichtenstein ◽

Sherie Morrison

Keyword(s):

Multiple Myeloma ◽

Fusion Protein ◽

Cell Lines ◽

Dna Content ◽

Fusion Proteins ◽

Conflicts Of Interest ◽

Myeloma Cell ◽

Inhibition Of Proliferation ◽

Discontinue Therapy

Abstract Abstract 939 Although IFNα has shown some efficacy in the treatment of multiple myeloma (MM), this efficacy has been limited in large part because systemic toxicity makes it difficult if not impossible to reach therapeutically effective doses at the site of the tumor. The short half-life of IFN also makes it difficult to sustain high levels during treatment, and because of the side effects, the patients often discontinue therapy. To address these issues, we have genetically fused IFNα2 to a chimeric IgG1 antibody specific for the antigen CD138 expressed on the surface of MM cells, yielding anti-CD138-IFNα. We have also produced a fusion protein (anti-CD138-mutIFNα) using a mutant IFNα that binds the IFN receptor (IFNAR) more tightly. The fusion proteins continued to bind CD138 and retained IFN activity and showed anti-proliferative activity against a broad panel of myeloma cell lines (HMCL) representing MM with different characteristic. To investigate the events responsible for the inhibition of proliferation, 8226/S, ANBL-6, MM1-144, H929, OCI-My5 and U266 cells were incubated with 500 pM anti-CD138-IFNα for 72 h and their DNA content analyzed by FLOW cytometry following permeabilization and staining with PI. The different cell lines exhibited different responses. All of the cell lines except OCI-My5 underwent apoptosis. For 8226/S, OCI-My5 and U266 there was little change in DNA content following treatment. ANBL-6 showed a slight increase in the number of cells in S. However, MM1-144 and H929 showed a marked accumulation in G2 with H929 also showing accumulation of cells with sub-G0content of DNA. Therefore, there is heterogeneity in the response of different HMCL to treatment with targeted IFNα2. For many but not all of the cell lines, anti-CD138-mutIFNα was more effective than anti-CD138-IFNα in inhibiting proliferation and causing DNA fragmentation. Anti-CD138-mutIFNα was more effective than anti-CD138-IFNα in inducing senescence-associated β-galactosidase and STAT1 activation in OCI-My5 cells. Treatment with anti-CD138-IFNα or anti-CD138-mutIFNα resulted in a decrease in the amount of IRF4 present in U266, suggesting that this may be responsible for the efficacy of the fusion proteins in this cell line. Treatment of the other cell lines did not alter the level of IRF4 present, but anti-CD138-IFNα and anti-CD138-mutIFNα treatment caused a decrease in the amount of ppRB present in 8226/S, OCI-My5 and MM1-144, and to a lesser extent in H929. To determine the in vivo efficacy of fusion protein treatment, SCID mice were injected subcutaneously with OCI-My5 cells and treated intravenously on days 14, 16 and 18 with 100 μg of the indicated proteins and monitored for tumor growth (Figure 1). Mice were sacrificed when tumors exceeded 1.5 cm in diameter. Treatment with anti-CD138-IFNα provided some protection (p ≤ 0.0001 compared to PBS). However, treatment with anti-CD138-mutIFNα was even more effective (p = 0.0004 compared to anti-CD138-IFNα). Anti-CD138-mutIFNα was also found to be more effective than anti-CD138-IFNα against primary MM cells. Patients with active myeloma were biopsied while off therapy and the marrow cells isolated by a negative antibody selection to >95% purity. After 72 h incubation with 25 nM of protein, anti-CD138 was found to have little effect. In contrast treatment with anti-CD138-IFNα caused a decrease in viability with anti-CD138-mutIFNα treatment leading to an even greater decrease in cell viability. Following 72 h of treatment, 25 nM of anti-CD138-mutIFNα was found to have more potent cytoreductive effects than 100 nM of anti-CD138-IFNα. Disclosures: No relevant conflicts of interest to declare.

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MCL1 Inhibitors in Multiple Myeloma

Blood ◽

10.1182/blood-2019-121104 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. SCI-12-SCI-12

Author(s):

Karin Vanderkerken ◽

Kim De Veirman ◽

Ken Maes ◽

Eline Menu ◽

Elke De Bruyne

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Therapeutic Option ◽

Therapy Resistance ◽

Bone Marrow Microenvironment ◽

Molecular Heterogeneity

Apoptosis plays a key role, not only in normal homeostasis but also in protection against genomic instability. Protection against apoptosis is a hallmark of cancer and is mainly regulated by the overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-Xl or Mcl-1. This results in increased survival of the tumor cells and resistance to therapy. This presentation will focus on MCL-1 (myeloid cell leukemia 1), its expression and its role as potential target in multiple myeloma (MM). MCL1 gene regions are one the most amplified gene regions in several human cancers and Mcl-1 activity is often associated with therapy resistance and relapse. Mcl-1 binds to and sequesters the pro-apoptotic BH3 proteins, thereby preventing apoptosis. Mcl-1 is overexpressed on MM cells from newly diagnosed patients compared to normal plasma cells and in MM cells at relapse. This overexpression is furthermore associated with a shorter survival of these patients. Increased Mcl-1 expression can result either from genetic lesions or by induction through interaction with the bone marrow microenvironment. Its expression is correlated with the molecular heterogeneity of the myeloma patients; while the CCDN1 group has high BCL2 and low MCL-1 expression; the MMSET and MAF group has high MCL-1 and low BCL2 expression. Unlike Bcl-2 and Bcl-Xl, Mcl-1 has a large unstructured aminoterminus and its activity is mainly dependent on posttranslational modifications. The bone marrow microenvironment, by producing high levels of interleukin 6, also induces the upregulation of Mcl-1. Furthermore, our group recently demonstrated that not only stromal cells in the bone marrow microenvironment, but also MDSC (myeloid derived suppressor cells) induce survival of MM cells by increasing Mcl-1 levels through the AMPK pathway. As such, these data suggest the potential therapeutic benefit of targeting Mcl-1 in MM patients. Developing the first-generation inhibitors appeared to be challenging, especially in view of the occurrence of unwanted off target effects. Recent preclinical data with new, selective Mcl-1 inhibitors show promising anti-tumor effects both in vitro and in in vivo myeloma models, either alone or in combination with the Bcl-2 selective inhibitor, venetoclax, especially as it was demonstrated that high levels of MCL-1 are associated with venetoclax resistance in MM. In addition, it was also shown that proteasome inhibition can trigger Mcl-1 accumulation, further pointing to the importance of Mcl-1 inhibition. Induction of NOXA, as an inhibitor of Mcl-1, is also suggested as a therapeutic option, especially in combinations with other drugs. Clinically, following preclinical results, several new Mcl-1 inhibitors have entered phase I trials. Most of them are still recruiting patients, and as such too early to have results. Disclosures No relevant conflicts of interest to declare.

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Mcl-1 Is Overexpressed in Multiple Myeloma and Correlates with Disease Severity.

Blood ◽

10.1182/blood.v104.11.1419.1419 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1419-1419

Author(s):

Soraya Wuilleme-Toumi ◽

Nelly Robillard ◽

Patricia Gomez-Bougie ◽

Philippe Moreau ◽

Steven Le Gouill ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Disease Severity ◽

Cell Lines ◽

Plasma Cells ◽

Myeloma Cell ◽

Lymphocytic Leukemia ◽

Myeloma Cells

Abstract Multiple Myeloma (MM) is a fatal malignancy of B-cell origin characterized by the accumulation of plasma cells within the bone marrow. The expression of the pro-survival members of the Bcl-2 family has been shown to be a key process in the survival of myeloma cells. More particularly, Mcl-1 expression turned out to be critical for their survival. Indeed, knockdown of Mcl-1 by antisenses induces apoptosis in myeloma cells. Finally, Mcl-1 was found to be the only anti-apoptotic Bcl-2 family member which level of expression was modified by cytokine treatment of myeloma cells. For these reasons, we have evaluated the expression of Mcl-1 in vivo in normal, reactive and malignant plasma cells (PC) i.e., myeloma cells from 55 patients with MM and 20 human myeloma cell lines using flow cytometry. We show that Mcl-1 is overexpressed in MM in comparison with normal bone marrow PC. Forty-seven percent of patients with MM at diagnosis (p=.017) and 80% at relapse (p=.014 for comparison with diagnosis) overexpress Mcl-1. Of note, only myeloma cell lines but not reactive plasmocytoses have abnormal Mcl-1 expression, although both plasmocyte expansion entities share similar high proliferation rates (>20%). Of interest, Bcl-2 as opposed to Mcl-1, does not discriminate malignant from normal PC. This shows that the overexpression of Mcl-1 is clearly related to malignancy rather than to proliferation. It will be important to know whether the overexpression of Mcl-1 is related to an abnormal response to cytokines like Interleukin-6 or to mutations of the promoter of the Mcl-1 gene as already described in B chronic lymphocytic leukemia. Finally, level of Mcl-1 expression is related to disease severity, the highest values being correlated with the shortest event-free survival (p=.01). In conclusion, Mcl-1 which has been shown to be essential for the survival of human myeloma cells in vitro is overexpressed in vivo in MM and correlates with disease severity. Mcl-1 represents a major therapeutical target in MM.

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In vivo antitumor effects of the mTOR inhibitor CCI-779 against human multiple myeloma cells in a xenograft model

Blood ◽

10.1182/blood-2004-03-1153 ◽

2004 ◽

Vol 104 (13) ◽

pp. 4181-4187 ◽

Cited By ~ 137

Author(s):

Patrick Frost ◽

Farhad Moatamed ◽

Bao Hoang ◽

Yijiang Shi ◽

Joseph Gera ◽

...

Keyword(s):

Multiple Myeloma ◽

Therapeutic Potential ◽

Xenograft Model ◽

Mtor Inhibitors ◽

Antitumor Effects ◽

Mtor Inhibition ◽

Inhibition Of Proliferation ◽

Induction Of Apoptosis

Abstract In vitro studies indicate the therapeutic potential of mTOR inhibitors in treating multiple myeloma. To provide further support for this potential, we used the rapamycin analog CCI-779 in a myeloma xenograft model. CCI-779, given as 10 intraperitoneal injections, induced significant dose-dependent, antitumor responses against subcutaneous growth of 8226, OPM-2, and U266 cell lines. Effective doses of CCI-779 were associated with modest toxicity, inducing only transient thrombocytopenia and leukopenia. Immunohistochemical studies demonstrated the antitumor responses were associated with inhibited proliferation and angiogenesis, induction of apoptosis, and reduction in tumor cell size. Although CCI-779-mediated inhibition of the p70 mTOR substrate was equal in 8226 and OPM-2 tumor nodules, OPM-2 tumor growth was considerably more sensitive to inhibition of proliferation, angiogenesis, and induction of apoptosis. Furthermore, the OPM-2 tumors from treated mice were more likely to show down-regulated expression of cyclin D1 and c-myc and up-regulated p27 expression. Because earlier work suggested heightened AKT activity in OPM-2 tumors might induce hypersensitivity to mTOR inhibition, we directly tested this by stably transfecting a constitutively active AKT allele into U266 cells. The in vivo growth of the latter cells was remarkably more sensitive to CCI-779 than the growth of control U266 cells.

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The Role of PHF19 As a Promoter of Tumorigenicity and Therapeutic Target in Multiple Myeloma

Blood ◽

10.1182/blood-2019-130595 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 508-508

Author(s):

Carolina D. Schinke ◽

Pingping Qu ◽

Shmuel Yaccoby ◽

Valeriy V Lyzogubov ◽

Veronica MacLeod ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Candidate Gene ◽

Cell Lines ◽

Crucial Role ◽

Plasma Cells ◽

Total Therapy

Introduction - Multiple Myeloma (MM) is a hematologic malignancy characterized by clonal growth of differentiated plasma cells (PCs). Despite improvement in MM therapy, the disease remains mostly incurable and is characterized by recurrent relapses with development of resistant clones that eventually lead to patient death. The pathways that lead to resistant and aggressive MM are not fully understood highlighting the need to improve our understanding of MM biology to identify potential new pathways and therapeutical targets. PHD Finger Protein 19 (PHF19) is a regulator of Polycomb Repressive Complex 2 (PRC2), the sole methyltransferase complex capable of catalyzing H3K27me3 to induce and enforce gene repression. PRC2 employs enhancer of zeste homolog 1 and 2 (EZH1/EZH2) as enzymatic subunits to hypermethylate H3K27. While overexpression and gain of function mutations of EZH1/2 have been observed in many cancers the role of this particular pathway in MM remains poorly understood. In the present study, we report on PHF19 as a new candidate gene to play a potential crucial role in MM oncogenesis. Methods- Gene expression profiling (GEP; Affymetrix U133 Plus 2.0) was performed on 739 MM patients (from total therapy trials [TT] 3-5; low risk MM n=636, high risk MM n=103), 42 patients with monoclonal gammopathy of undetermined significance (MGUS), 73 smoldering MM patients, 42 patients with primary plasma cell leukemia and 34 healthy donors. Myeloma risk was determined by the GEP 70 signature as previously defined. To test the implications of functional PHF19 knock down (KD) we used TRIPZ inducible PHF19 shRNA vs. scrambled control (Dharmacon) in two MM cell lines (JJN3 and ARP1). Real time PCR as well as western blotting was used to confirm PHF19 KD as well as to elucidate the effect on H3K27me3 (Cell Signaling). Functional in vitro studies included proliferation (Promega), clonogenic assays (StemCell), cell cycle and apoptosis assays (both Invitrogen). In vivo studies were performed using SCID mice that were subjected to tail vain injection with PHF19 KD JJN3 cells (n=10) or scrambled shRNA control (n=10). Weekly ELISA (Bethyl) and in vivo imaging (Xenogen) were performed and survival was recorded. Results- GEP of the previously mentioned patient populations and healthy controls identified PHF19 (chr9q33.2) as a candidate gene that was consistently dysregulated in MM patients. Mean expression levels at different MM stages correlated with disease aggressiveness (ANOVA, p<0.0001), Figure 1. High expression of PHF19 (log2>10.46) at diagnosis correlated significantly with adverse clinical parameters, including ISS III, anemia and elevated LDH, as well as worse overall survival (5 yr OS = 29% for patients with high PHF19 expression vs 77% for patients with low PHF19 expression [log2<10.46], p< 0.0001). These results led us to test the implications of functional PHF19 KD using TRIPZ inducible PHF19 shRNA vs. scrambled control in the JJN3 and ARP1 MM cell lines. PHF19 KD led to a drastic reduction of H3K27me3 thereby resulting in significantly reduced proliferation via cell cycle arrest, while apoptosis was not substantially altered. Clonogenic assays showed a significant reduction in colony numbers and size of MM cells with PHF19 KD compared to the control (>75% reduction in both cell lines, p<0.05). Xenograft studies showed consistently less tumor burden in the mice injected with PHF19 KD cells compared to scrambled control, evident through ELISA testing for IgG Kappa (Median =180 mg/ml for scrambled control vs 80 mg/ml for PHF19 KD at week 8, p=0.07) and bioimaging (Median bioilumisence 2.1x108 p/s for scrambled control vs. 0.8x108 p/s for PHF19 KD at week 8, non-significant). Median OS in mice injected with PHF19 KD cell was substantially longer (66 days) compared to mice subjected to scrambled control cells (54 days), p=0.052. Conclusion- In summary we show that PHF19 is upregulated in malignant plasma cells of MM patients and that PHF19 expression levels increase with advanced MM stages. High PHF19 expression was a marker of adverse prognosis in our total therapy (TT 3-5) cohort. Most importantly, in-vitro and in-vivo functional studies showed that PHF19 has important biological functions in MM. These results suggest that epigenetic regulation through histone methylation, in particular, H3K27 trimethylation, plays a crucial role in MM and the affected downstream pathways should be further elucidated. Disclosures Boyle: Janssen: Honoraria, Other: Travel; Abbvie: Honoraria; Amgen: Honoraria, Other: travel; Takeda: Honoraria, Other: travel; Celgene Corporation: Honoraria, Other: Travel. van Rhee:Kite Pharma: Consultancy; Adicet Bio: Consultancy; Karyopharm Therapeutics: Consultancy; Takeda: Consultancy; Sanofi Genzyme: Consultancy; Castleman Disease Collaborative Network: Consultancy; EUSA: Consultancy. Walker:Celgene: Research Funding.

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Microrna-138 Regulates Osteogenic Differentiation and Its Inhibition Presents a Novel Therapeutic Line to Prevent Bone Lytic Lesions in Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.4483.4483 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4483-4483

Author(s):

Shokichi Tsukamoto ◽

Karma Salem ◽

Salomon Manier ◽

Michaela R. Reagan ◽

Daisy Huynh ◽

...

Keyword(s):

Multiple Myeloma ◽

Osteogenic Differentiation ◽

Cell Lines ◽

Research Funding ◽

Plasma Cells ◽

Data Sets ◽

Normal Plasma ◽

Lytic Lesions

Abstract Introduction The bone marrow (BM) microenvironment in multiple myeloma (MM) plays a pivotal role in tumor growth and bone destructive process. Mesenchymal stromal cells (MSCs) in MM exhibit different genomic and cytokine secretion profiles that ultimately impair their osteogenic differentiation abilities compared to normal MSCs. However, the underlying molecular mechanisms are not fully understood. In the present study, we explored the role of miR-138 in MSCs derived from MM patients (MM-MSCs) and the potential for anti-miR-138 treatment to rescue impaired osteogenic differentiation in MM, both in vitro and in vivo using a human xenograft MM model. Materials and methods Primary BM aspirates were obtained from MM patients and normal healthy donors, after obtaining informed consent in accordance with the Declaration of Helsinki. MiR-138 expression in MM-MSCs was measured by quantitative real-time PCR. Publicly available microarray data sets (GSE17306 and E-TABM-508) were analyzed for miR-138 expression in MM cells compared to normal plasma cells. To test the effect of inhibiting miR-138 function, a high-affinity 15-mer locked nucleic acid (LNA)-modified anti-miR oligonucleotide and a corresponding scramble sequence control oligonucleotide were used (In collaboration with Dr. Kauppinen, Denmark). Anti-miR-138 oligonucleotides were transfected into MM-MSCs or normal MSCs co-cultured with MM cell lines and osteogenic differentiation in MSCs was assessed by alizarin red staining. For the in vivo studies, 6-week-old female SCID-beige mice (n=6, each group) were injected intravenously with anti-miR-138 or scramble control oligonucleotides (15 mg/kg) 2 times a week. 3 weeks later, GFP+Luc+ MM.1S cells (3 × 106) were injected into mice. Anti-miR-138 or control oligonucleotides were continued until day 28 after injection of myeloma cells. At day 28, the effect of anti-miR138 was assessed by the number of osteoblastic lineage cell (OBC: Lin-/CD45-/CD31-/CD51+/Sca-1-) from hematopoietic cell-depleted, collagenase-treated crushed bones of mice by flow cytometry. Results MiR-138 expression in MSCs from MM patients (n=10) was significantly higher than MSCs from normal donors (n = 4) (P<0.05). In addition, miR-138 expression was significantly higher in MM patient tumor cells compared to normal plasma cells using two independent data sets (GSE17306 and E-TABM-508), (P<0.01 and P<0.01, respectively). In three-dimensional co-culture system of MSCs from normal donors (n=6) with MM.1S cells for 2 weeks (GSE60423), miR-138 expression was increased in 4 out of 6 donors compared to MSCs cultured alone (P<0.05). MM-MSCs (n≥3) transfected in vitro with anti-miR-138 oligonucleotides showed significantly increased osteogenic differentiation after 3-4 weeks compared to MSCs with scramble control oligonucleotides (P<0.01). Under in vitro two-dimensional co-culture conditions with MM cell lines, normal MSCs transfected with anti-miR-138 oligonucleotides showed significantly increased osteogenic differentiation compared to MSCs with scramble control oligonucleotides (P<0.001). In an in vivo human xenograft MM model, treatment of anti-miR-138 significantly increased the number of OBCs in the endosteal (Lin-/CD45-) BM stromal fraction of MM bearing SCID-beige mice at day 28 compared to scramble control oligonucleotides (P<0.05). Conclusions These findings indicate that miR-138 plays an important role in impaired osteogenic differentiation in MSCs in MM. Inhibition of miR-138 promotes osteogenic differentiation of MSCs in MM and anti-miR-138 treatment holds the potential to prevent MM induced bone loss and lytic lesions. Additional studies are ongoing to further understand the connection between MM cells and MSCs mediated by miR-138. Disclosures Roccaro: Takeda Pharmaceutical Company Limited: Honoraria. Ghobrial:Novartis: Honoraria; Noxxon: Honoraria; Celgene: Honoraria, Research Funding; Takeda: Honoraria; Amgen: Honoraria; BMS: Honoraria, Research Funding.

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Characterization of clonogenic multiple myeloma cells

Blood ◽

10.1182/blood-2003-09-3064 ◽

2004 ◽

Vol 103 (6) ◽

pp. 2332-2336 ◽

Cited By ~ 534

Author(s):

William Matsui ◽

Carol Ann Huff ◽

Qiuju Wang ◽

Matthew T. Malehorn ◽

James Barber ◽

...

Keyword(s):

Multiple Myeloma ◽

B Cells ◽

Cell Lines ◽

Plasma Cells ◽

Severe Combined Immunodeficiency ◽

Clinical Samples ◽

Clonogenic Potential ◽

Anti Cd20

Abstract The identity of the cells responsible for the initiation and maintenance of multiple myeloma (MM) remains unclear largely because of the difficulty growing MM cells in vitro and in vivo. MM cell lines and clinical specimens are characterized by malignant plasma cells that express the cell surface antigen syndecan-1 (CD138); however, CD138 expression is limited to terminally differentiated plasma cells during B-cell development. Moreover, circulating B cells that are clonally related to MM plasma cells have been reported in some patients with MM. We found that human MM cell lines contained small (< 5%) subpopulations that lacked CD138 expression and had greater clonogenic potential in vitro than corresponding CD138+ plasma cells. CD138- cells from clinical MM samples were similarly clonogenic both in vitro and in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, whereas CD138+ cells were not. Furthermore, CD138- cells from both cell lines and clinical samples phenotypically resembled postgerminal center B cells, and their clonogenic growth was inhibited by the anti-CD20 monoclonal antibody rituximab. These data suggest that MM “stem cells” are CD138- B cells with the ability to replicate and subsequently differentiate into malignant CD138+ plasma cells.

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