Expression of CRBN Signaling Pathway Proteins Assessed By Immunohistochemical Staining As Candidate Biomarkers for Outcome in Myeloma Patients Treated with IMiDs

Abstract Cereblon (CRBN), Ikaros (IKZF1), Aialos (IKZF3) and multiple myeloma oncogene 1 (MUM1) are important component of CRBN signaling pathway when treat MM cells with IMiDs. CRBN interacts with the DNA damage-binding protein-1 (DDB1), Cullin 4 (Cul4A or Cul4B) and regulator of Cullins 1 (ROC1) to form the functional E3 ubiquitin ligase complex (E3ULC). CRBN increases the interaction between E3ULC and IKZF1/3, leading to increased ubiquitination degradation of IKZF1/3, and then induce cytotoxicity in myeloma cells. Subsequently, degradation of IKZF1/3 induce depression of multiple myeloma oncogene 1 (MUM1), which is also called interferon regulatory factors (IRF4) and proved to be involved in the anti-MM activity of IMIDs in previous studies. Immunohistochemical (IHC) staining may be a convenient approach for researchers to differentiate the myeloma cells and non-myeloma cells in BM samples. In this study, we evaluated CRBN, IKZF1/3 and MUM1 expression level in bone marrow (BM) by immunohistochemical (IHC) staining and investigated the relationship between expression level and treatment outcome after IMiDs-based or bortezomib-based therapy in 123 newly diagnosed multiple myeloma (NDMM) patients. H-score method was applied according to both intensity and extent of staining. The intensity was graded from 0 to 3("0"for absent staining, "1" for weak expression, "2" for intermediate expression, and "3" for strong expression of the protein). The extent was graded from "0" to "100" to represent the percentage of MM cells with positive staining of any intensity. H-score was obtained by multiplying the intensity and extent score, ranging from 0 to 300, which reflected protein expression level in MM cells. The median H-score of CRBN, IKZF1, IKZF3 and MUM1 were 200, 0, 180 and 180, respectively. According to the median H-score, we classified the patients into high or low expression group. One hundred and twenty-three NDMM patients were enrolled in this study, including 64 males (52.0%) and 59 females (48.0%). The median age was 60 years (range 34-84). Fifty-one patients (41.5%) received IMiDs-containing regimen as the first-line therapy. The median follow-up time was 24.0 months (range, 10-76 months). After treated with IMiDs, patients with high level of CRBN and MUM1 achieved better overall response rate (ORR) than those expressed low level (CRBN, 88.0% vs. 42.3%, P=0.001; MUM1, 83.3% vs. 48.1%, P=0.009). Besides, patients with CRBN and MUM1 overexpression also had better overall survival (median OS, CRBN, not reached vs. 21.0 months, P=0.004; MUM1, not reached vs. not reached, P=0.021) and progression free survival (median PFS, CRBN, 28.0 vs. 12.0 months, P=0.002; MUM1, 32.0 vs. 12.0 months, P<0.001) than patients with low level, as well as 2-year OS rate (CRBN, 86% vs. 44%, P=0.005; MUM1, 81% vs. 51%, P=0.003) and PFS rate (CRBN, 66% vs. 17%, P=0.001; MUM1, 63% vs. 20%, P<0.001). In addition, in high CRBN and MUM1 expression group, patients treated with IMiDs had a higher 2-year PFS rate than Bortezomib presentation (CRBN, 66% vs. 45%, P=0.004; MUM1, 63% vs. 36%, P=0.003). In low CRBN and MUM1 expression group, patients treated with IMiDs had an inferior OS (median OS, CRBN, 21.0 vs. 57.0 months, P=0.001; MUM1, not reached vs. 57.0 months, P<0.001) and PFS (median PFS, CRBN, 12.0 vs. 31.0 months, P=0.003; MUM1, 12.0 vs. 32.0 months, P<0.001) than patients with Bortezomib, as well as 2-year OS rate (CRBN, 44% vs. 91%, P=0.002; MUM1, 51% vs. 100%, P<0.001) and PFS rate (CRBN, 17% vs. 59%, P=0.010; MUM1, 20% vs. 68%, P<0.001). This study identified high levels of CRBN pathway proteins CRBN and MUM1 were correlated with favorable ORR and survival in NDMM patients with IMiDs therapy, also suggested that expression levels of CRBN signaling pathway proteins in plasma cells assessed by IHC staining could better direct clinic individualized therapy. Disclosures No relevant conflicts of interest to declare.

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Serum/Glucocorticoid-Regulated Kinase 1 (SGK1) Is a Prominent Target Gene of the Transcriptional Response to Cytokines In Multiple Myeloma and Supports the Growth of Myeloma Cells

Blood ◽

10.1182/blood.v116.21.1915.1915 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1915-1915

Author(s):

Unn-Merete Fagerli ◽

Thorsten Stühmer ◽

Toril Holien ◽

Randi Utne Holt ◽

Ove Bruland ◽

...

Keyword(s):

Multiple Myeloma ◽

Growth Factors ◽

Cell Lines ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Transcriptional Response ◽

Myeloma Cell ◽

Myeloma Cells ◽

Growth And Survival ◽

Stat3 Phosphorylation

Abstract Abstract 1915 Multiple myeloma is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We hypothesized that the intracellular signals evoked by cytokines converge and regulate transcription of a set of genes that are common targets for several growth factors and therefore constitute pivotal mediators of the tumor-promoting effects of autocrine or paracrine stimuli. To identify such targets, we determined the changes in gene expression induced by IL-6, TNFalpha, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase SGK1, which is a down-stream effector of PI3-kinase and highly homologous to AKT. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the JAK/STAT pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, shRNA-mediated knock-down of STAT3 reduced basal and induced SGK1 levels, demonstrating the involvement of the JAK/STAT3 signaling pathway in SGK1 induction. Furthermore, down-regulation of SGK1 by shRNAs resulted in decreased proliferation and viability of myeloma cell lines. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their growth and survival and represents an attractive candidate for further evaluation as a therapeutic target. Disclosures: No relevant conflicts of interest to declare.

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The Expression Of CD200 As a Prognostic Factor In Newly Diagnosed Multiple Myeloma

Blood ◽

10.1182/blood.v122.21.3082.3082 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3082-3082

Author(s):

Yi Li ◽

Wenjun Wu ◽

Jingsong He ◽

Xiaoyan Han ◽

Gaofeng Zheng ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Conflicts Of Interest ◽

Progression Free Survival ◽

Staging System ◽

Newly Diagnosed ◽

New Approach ◽

Myeloma Cells ◽

Targeting Therapy ◽

Molecular Therapeutic

Abstract Introduction multiple myeloma (MM) is currently an incurable hematological malignancy. Discovering molecular therapeutic targets is a new approach to improve the outcome in the treatment of the malignant disease. As CD200 is a type Ⅰmembrane glycoprotein expressed on myeloma cells, we asked if the expression of CD200 could serve as a prognostic marker for MM patients. Our data indicated that the expression level of CD200 is indeed correlated with the prognosis of the MM patients. Methods bone marrow samples from 96 newly diagnosed MM patients from April 2011 to July 2013 were evaluated by flow cytometry, using PE-conjugated anti-CD200 mAb, FITC-conjugated anti-CD138 mAb, and PE-Cy7-conjugated anti-CD45 mAb. PE-or FITC-conjugated normal mouse IgG was used as isotype-matched controls. Results 96 MM patients were investigated in the present study, including 60 men and 36 women, with a median age of 63 years (range 34–86 years). 81/96(84%) MM patients were CD200 positive with a median Mean Fluorescence Intensity (MFI) of 127 as analyzed by flow cytometry, which was consistent with the previous studies. While in 15 of 96 patients, CD200 expression was undetectable. Among the CD200 positive ones 7.40% patients were classified as stage Ⅰ, 12.35% were stage Ⅱ, and 80.25% were stage Ⅲ according to Durie–Salmon staging criteria. 40.74% patients were stageⅠ, 22.22% were stage Ⅱ, and 37.04% were stage Ⅲ, according to the International Staging System (ISS). Analysis of the CD200 positive patients revealed the MFI<127 group had a better progression free survival (PFS) (p=0.046) (Fig 1A) and overall survival (OS) (p=0.069) compared to those with MFI≥127. In the patients with age ≥65 years old, PFS (p=0.023) (Fig 1B) and OS (p=0.044) (Fig 1C) were much shorter in the MFI≥127 group, compared to the MFI<127 ones. Conclusions Our study demonstrated that the expression and MFI of CD200 on primary multiple myeloma cells is correlated with the prognosis of the MM patients. The better PFS and OS were observed in the MFI<127 group compared to the patients with MFI≥127, especially in the patients with age≥65 years old. Improved PFS in CD200 positive ones is likely due to the immune suppression mediated by CD200. Our study suggests that targeting therapy against CD200 may become a new approach to the treatment of MM in clinical practice. Disclosures: No relevant conflicts of interest to declare.

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Phase II Randomized Trial of Bevacizumab Versus Bevacizumab and Thalidomide for Relapsed/Refractory Multiple Myeloma.

Blood ◽

10.1182/blood.v106.11.2571.2571 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2571-2571 ◽

Cited By ~ 3

Author(s):

George Somlo ◽

William Bellamy ◽

Todd M. Zimmerman ◽

Paul Frankel ◽

Joe Tuscano ◽

...

Keyword(s):

Multiple Myeloma ◽

Stem Cell ◽

Stem Cell Transplant ◽

Plasma Cells ◽

Progression Free Survival ◽

Staining Intensity ◽

Cell Transplant ◽

Myeloma Cells ◽

Anti Vegf ◽

Grade 3

Abstract Vascular endothelial growth factor (VEGF) plays a seminal role in neo-angiogenesis. VEGF is present on myeloma cells, and its receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR) are detectable on the surface of neighboring myeloid and monocytic elements. Hence, VEGF is implicated in the pathogenesis of multiple myeloma (MM). Thalidomide, an important agent in the treatment of MM, among its many postulated mechanisms of actions also inhibits VEGF-mediated neo-angiogenesis. We set out to test the feasibility and explore the efficacy of combining an anti-VEGF agent with thalidomide. With the availability of the anti-VEGF antibody rhuMAB bevacizumab, a trial of bevacizumab 10 mg/kg given intravenously every 2 weeks alone (in thalidomide-exposed patients) versus a randomized comparison of bevacizumab +/− thalidomide 50–400 mg/day (in thalidomide naive patients) was initiated by the California Cancer Consortium. Twelve patients (median age:58 years; range:50–75) with initial stages of I (n:2), II (n:2) and III (n: 8), all with refractory MM have been enrolled. Patients received a median of 1 prior regimen (range:0–5). Six patients had failed an autologous stem cell transplant prior to enrollment. In patients who have received bevacizumab alone, grade 3 toxicities included fatigue and neutropenia (1), hypertension (1), and hyponatremia (1). In the group receiving bevacizumab and thalidomide, grade 3 lymphopenia was observed in 1 patient during cycle 3, and one patient was taken off study due to exacerbation of pre-exisiting (diet pill induced) pulmonary hypertension and was considered inevaluable. Median time to progression for the 6 patients treated with bevacizumab alone was 2 (range 1–4) months. Progression-free survival for the 5 evaluable patients treated with bevacizumab and thalidomide is 6 +, 7, 8 +, 10, and 30 + months, with 2 patients still on study and in response. Two of these patients did not progress but were taken off study (one for patient’s choice, and one due to the physician’s choice to pursue a stem cell transplant at 7.5 months, this patient is listed above as in response at 30 + months). Immunohistochemical staining (IHC) revealed 2 + to 4 + expression of VEGF on myeloma cells in 7 cases of the available 8 pre-treatment bone marrow samples. Weak staining (1+) of VEGFR1 was observed on the surface of myeloma cells in 5 cases. VEGFR2 expression was also observed on plasma cells by IHC (1+ to 2+) in 5 cases. Myeloma cells from a patient treated with bevacizumab alone for a duration of 4 months, and from a patient receiving bevacizumab and thalidomide for 7.5 months before going on to transplant, demonstrated the strongest staining intensity for VEGF. Due to slow accrual the study had been closed to accrual, although 2 patients continue on the bevacizumab and thalidomide arm. However, in light of our findings further testing of bevacizumab, preferably in combination with other active agents is warranted. Supported by NO1 CM 17101.

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Autocrine Sonic Hedgehog Protects Multiple Myeloma Cells From Apoptosis and Enhances Drug Resistance

Blood ◽

10.1182/blood.v118.21.2904.2904 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2904-2904

Author(s):

Zhiqiang Liu ◽

Yuhuan Zheng ◽

Haiyan Li ◽

Yong Lu ◽

Donna M. Weber ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Tumor Growth ◽

Signaling Pathway ◽

Sonic Hedgehog ◽

Hedgehog Signaling ◽

Conflicts Of Interest ◽

Myeloma Cells ◽

Apoptosis Rate ◽

Chemotherapy Drugs

Abstract Abstract 2904 The secreted protein sonic hedgehog (SHH) and the hedgehog signaling are of great importance in proliferation and differentiation of cells in the hematopoietic system, and also play a vital role in oncogenesis of B cell malignance. However, the functions and mechanism of SHH signaling in multiple myeloma (MM) is mostly unknown. Thus far, aberrant activation of the hedgehog signaling in tumor growth promoting and/or survival capabilities as well as a paracrine model of SHH secretion have been demonstrated in MM. In the current study, we demonstrated a new autocrine SHH functioning manner in MM cells. The Shh mRNA and the SHH protein were highly expressed both in the MM cell lines and in purified CD138+ MM cells from patients using real-time PCR, Western Blot and immunohistochemistry analyses, respectively; and the SHH protein was also detected in the culture medium. Accordingly, the Hh ligand receptor PTCH1 and PTCH2 as well as the transcriptional factor GLI1 were all overexpressed in MM cells, indicating the activation of Hh signaling pathway. Autocrine SHH played a role in MM cells survival and protected MM cells from apoptosis in vitro, and autocrine SHH accelerated xenograft tumor growth in myeloma-SCID mouse model in vivo. Moreover, autocrine SHH enhanced drug resistance of MM cells, as SHH overexpressed CAG cells (SHH+CAG) had a significantly low apoptosis rate when treated with chemotherapy drugs dexamethasone or bortezomib, as compared with wild type cells (wt-CAG). On the contrary, SHH knockdown cells (SHH-CAG) had a dramatically higher apoptosis rate. Blocking autocrine SHH ligand and treating cells with dexamethasone or bortezomib significantly improved the drug killing effect. Finally, we found that upregulated BLC2 via SHH-Gli1signaling is the signaling pathway by which MM cells enhanced the drug resistance. Our study provides a new insight into the biologic function of the autocrine SHH in proliferation, survival and the drug resistance in the myeloma cells. Disclosures: No relevant conflicts of interest to declare.

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The Epigenetic Repression of Mir-375 Is the Dominant Mechanism for the Constitutive Activation of PDPK1/RSK2 Signaling Axis in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.4182.4182 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4182-4182

Author(s):

Shotaro Tatekawa ◽

Junya Kuroda ◽

Yoshiaki Chinen ◽

Yuji Shimura ◽

Hisao Nagoshi ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Copy Number ◽

Plasma Cells ◽

Expression Level ◽

Chromosome 16 ◽

Myeloma Cells ◽

U6 Snrna ◽

Normal Plasma ◽

Signaling Axis

Abstract [Introduction] Multiple myeloma (MM) is a cytogenetically/molecularly heterogeneous hematologic malignancy that remains mostly incurable, and the identification of a universal and relevant therapeutic target molecule is essential for the further development of therapeutic strategy. We have recently identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), a serine threonine kinase, and its major downstream substrate RSK2, a member of the 90 kDa ribosomal S6 kinase family of serine threonine kinases, were universally active in eleven human MM-derived cell lines (HMCLs) examined regardless of the type of cytogenetic abnormality, the mutation state of RAS, RAF and FGFR3 genes and myeloma cells of approximately 90% of symptomatic patients at diagnosis. Our study also disclosed that PDPK1/RSK2 signaling axis played pivotal roles in myeloma pathophysiology by regulating series of downstream molecules, such as c-MYC, IRF4, D-type cyclins, or PLK1, while the inactivation of either PDPK1 or N-terminal domain of RSK2 resulted in the induction of apoptosis in myeloma cells which was accompanied by the activation of BH3-only proteins BIM and BAD (Shimura Y, Mol Caner Ther 2012; Chinen Y, Cancer Res 2014). Here we assessed the underlying mechanism for PDPK1 overexpression in MM. [Methods] The miR-375 expression level was analyzed by the quantitative RT-PCR in 11 HMCLs and 92 patient-derived myeloma cells isolated by CD138-positive cell sorting (normal plasma cells (N=10), MGUS (N=21), newly diagnosed MM (NDMM) (N=27), relapsed/refractory (RRMM) (N=34). The pre-miR-375 precursor molecule (miR-375 mimics), the siRNA targeted against PDPK1, or a negative control RNA-oligonucleotides was transfected into 8 cell lines by utilizing Hemagglutinating Virus of Japan (HVJ)-envelope vector. The copy number abnormality of PDPK1 gene was assessed by double-color FISH for PDPK1 gene and the centromere of chromosome 16. The methylation status of miR-375 promoter site was analyzed by methylation-specific PCR (MSP). This study was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Boards. Patient-derived samples were obtained with informed consent, and normal bone marrow plasma cells were obtained from volunteers who were not affected by hematologic disease. [Result] The level of miR-375 expression was calculated with 2-ΔCt methods. Human U6 snRNA was examined as the reference. The median log102-ΔCt ± SD of normal plasma cells, MGUS, NDMM, RRMM and HMCLs were -2.46 ± 0.67, -3.64 ± 0.68, -4.23 ± 0.95, -3.92 ± 1.24 and -3.69 ± 0.29 respectively. When compared to normal plasma cells, the miR-375 expression was significantly decreased in NDMM and RRMM (p<0.01, respectively), and tended to be decreased in MGUS (p=0.083) and HMCLs (p=0.097). As the causative of miR-375 repression, our study disclosed that the promoter sites of miR-375 gene were hypermethylated in 8/8 of HMCLs when examined by MSP. The interphase FISH for PDPK1 with centromere chromosome 16 indicated the copy number of PDPK1 gene was increased in 11/11 HMCLss, however, this was never the case with patient-derived myeloma cells (0/7). Importantly, the miR-375 gene transfection resulted in the reduction of PDPK1 expression in 7 of 8 HMCLs, and it simultaneously caused the reduction of the expression levels of IGF1 receptor and JAK2, the known targets of miR-375. Furthermore, when treated with 5-Azacitidine and/or Trichostatin A, miR-375 was markedly upregulated, suggesting that the overlapping epigenetic deregulations, such as DNA hypermethylation or histone deacetylation, are involved in the silencing of miR-375. [Conclusion and Discussion] PDPK1 is activated by autophosphorylation and, therefore, its expression level is the crucial determinant for its activity, Because our study revealed the miR-375 expression as the major regulator of PDPK1 expression, it is suggested that the abnormally repressed miR-375 is the major causative for the constitutive hyperactivation of the PDPK1/RSK2 signaling axis in MM. Moreover, since the decreased miR-375 expression was observed in plasma cells of MGUS and was more pronounced in MM, miR-375 repression by epigenetic deregulation may be involved in both disease development and progression of MM. Disclosures No relevant conflicts of interest to declare.

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Expression and Clinical Significance of CD137L in Multiple Myeloma Cells

Blood ◽

10.1182/blood.v128.22.5616.5616 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5616-5616

Author(s):

Chengcheng Fu ◽

Shuang Yan ◽

Depei Wu

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Clinical Significance ◽

Cell Lines ◽

Myeloma Cell ◽

Expression Level ◽

Newly Diagnosed ◽

Myeloma Cells ◽

Low Level ◽

Rpmi 8226

Abstract 【 Background 】 Human CD137L molecule, a member of the TNF superfamily, was found to be expressed in a variety of malignant tumors, such as acute myeloid leukemia, non Hodgkin's lymphoma, associated with complete remission. Our previous experiments showed that high level of CD137L expressed on the surface of myeloma cell line RPMI-8226, U266, LP1, MY5 and KMS-11, as well as MM primary cell. However, we have no idea about the level of CD137L on MGUS (monoclonal gammapathy of undetermined significance) and the relation between the expression level and tumor stage, bionomics, prognosis of multiple myeloma. 【 Objective 】 (1) To determine expression and clinical significance of CD137L molecular in patients with MGUS and multiple myeloma cells; (2)To explore function of CD137L in multiple myeloma cell lines. 【 Methods 】 (1) The expression of CD137L molecule on myeloma cells/normal plasma cell surface was detected by flow cytometry; (2) Clinical significance of CD137L molecule expressed by multiple myeloma cells was accessed via rank sum test; expression level of high/low of CD137L on overall survival was evaluated through survival analysis and Log-Rank test; (3) SiRNA, the customization of SiRNA for CD137L gene, transfected myeloma cell lines U266, RPMI-8226, KMS-11 by Lipo3000.Then the expression of CD137L was detected by RT-PCR; cell cycle distribution after inhibition of CD137L signal was detected by PI; cell proliferation was detected by CCK8. 【 Results 】 (1) Fresh bone marrow specimens of 28 patients with newly diagnosed multiple myeloma patients were collected. The expression of CD137L molecule on CD45-/CD38+/CD138+ cell group in bone marrow was detected, and the median expression level was 29 (7-94)%; the expression of CD137L molecule on 9 patients with MGUS was 7 (2-57)%; (2) That the expression level of CD137L between patients with MGUS and newly diagnosed MM showed statistic difference indicated that it could be as a marker for differential diagnosis; the different expression level by rank sum test between those with newly diagnosed MM and post-treated MM, post-treated MM and RRMM indicated that it could be a marker for MRD; (3) The follow-up of patients found that after the treatment CD137L level of 11/13 patients decreased, and these patients at least achieved PR; (4) there is no related with the level of CD137L and type, ISS stage, DS stage, white blood cell count, hemoglobin concentration, platelet count, serum beta 2- microglobulin, lactate dehydrogenase, serum albumin, calcium concentration, the ratio of bone marrow plasma cell by correlation analysis; (5) According to median values of CD137L expression level, all the newly diagnosed patients were divided into two groups, low level expression and high level and survival analysis showed no significant difference by Log-Rank test. The 2 years survival rate of low level group and the high one was 84.7%, 74.1%; (6) KMS-11, RPMI 8226,U266 cells were transfected using Lipo3000 and only U266 cell line was inhibited obviously. Inhibition of CD137L induced cell proliferation by CCK8 test and a distribution change of G1 and S phase on cell cycle. 【 Conclusions 】 Multiple myeloma cell lines and primary myeloma cells had a high expression level of CD137L, MGUS cells had a low level while normal plasma cells surface without CD137L expression; CD137L can be a marker for diagnosis of MM and MGUS and for minimal residual disease; CD137L expression of MM patients had no correlation with clinical and biological features; In vitro the inhibition of CD137L signaling on U266 cells can induce cells proliferation. Disclosures No relevant conflicts of interest to declare.

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AMPK Expressed By Multiple Myeloma Cells Inhibited Metformin-Induced Myeloma Cells Proliferation

Blood ◽

10.1182/blood.v128.22.5703.5703 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5703-5703

Author(s):

Fuming Zi ◽

Jingsong He ◽

Enfan Zhang ◽

Yang Yang ◽

He Huang ◽

...

Keyword(s):

Multiple Myeloma ◽

Western Blotting ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Annexin V ◽

Bone Lesions ◽

Blood Calcium ◽

Myeloma Cells ◽

Ampk Signaling ◽

Therapy Target

Abstract Background Multiple myeloma(MM) is a malignant plasma cells proliferative disease which is characterized by increased blood calcium level, renal insufficiency, anemia, and bone lesions(CRAB). MM is still uncured disease. To further explore the mechanisms of MM was an urgent problem needs to be solved. AMPK is an "energy sensor". Studies had demonstrated that metformin could activate AMPK and exert anti-tumor effect in many tumors. However, a paradoxical phenomenon was found that knockdown AMPK could enhance the anti-tumor effect of metformin. Recent studies had revealed that AMPK maybe a double-edged sword in tumors. Our previous study had discovered that metformin exert anti-myeloma effect without activating AMPK. In this study, we mainly focus on the expression of AMPK in MM cells and to explore whether the expression of AMPK in MM cells could block the anti-myeloma effect of metformin. Methods The expression of AMPK in MM cells was detected by Western blotting. Small interfering RNA(siRNA) was used to knockdown the expression of AMPK. Apoptotic cells of MM cells were detected by Annexin V-FITC/PI using flow cytometry analysis. Western blotting was used to elucidate the apoptotic protein of MM cells induced by metformin with or without knockdown AMPK. Results Our study revealed that AMPK was obviously expressed by MM cells(Fig.1). Metformin could not activate AMPK signaling pathway. Knockdown AMPK with siRNA could enhance the anti-myeloma effect of metformin(Fig.2 and Fig.3). Conclusions Taken together, our data indicated that AMPK was expressed by MM cells. Inhibit AMPK could enhance the anti-myeloma effect of metformin. AMPK maybe a new therapy target. Disclosures No relevant conflicts of interest to declare.

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Pretreatment Serum Levels of IL-1 Receptor Antagonist and IL-4 are Predictors of Overall Survival in Multiple Myeloma Patients Treated with Bortezomib

10.20944/preprints202111.0498.v1 ◽

2021 ◽

Author(s):

Damian Mikulski ◽

Paweł Robak ◽

Ewelina Perdas ◽

Edyta Węgłowska ◽

Aleksandra Łosiewicz ◽

...

Keyword(s):

Multiple Myeloma ◽

Overall Survival ◽

Plasma Cells ◽

Progression Free Survival ◽

Serum Levels ◽

First Line Therapy ◽

Cytokine Levels ◽

Bony Lesions ◽

The Impact ◽

Pretreatment Serum

Multiple myeloma (MM) is characterized by the malignant proliferation of monoclonal plasma cells in the bone marrow with an elevation in monoclonal paraprotein, renal impairment, hypercalcemia, lytic bony lesions, and anemia. Immune cells and associated cytokines play a significant role in MM growth, progression, and dissemination. While some cytokines and their clinical significance are well described in MM biology, others remain relatively unknown. The aim of the present study was to assess the impact of pretreatment serum levels of 27 selected cytokines on progression-free survival (PFS) and overall survival (OS) in MM patients before first-line therapy with bortezomib-based regimens. Serum cytokine levels were assayed with a Bio-Rad Bio-Plex Pro Human Cytokine 27-Plex Assay on the MAGPIX Multiplex Reader and the Bio-Plex® 200 System (Bio-Rad) including IL-1β, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, Eotaxin, FGF, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1β, PDGF-BB, RANTES, TNF-α, and VEGF. A total of 61 MM patients were examined. Most patients received a bortezomib, cyclophosphamide and dexamethasone (VCD) chemotherapy regimen. In the final multivariate model, IL-13 cytokine level (HR 0.1411, 95% CI: 0.0240-0.8291, p = 0.0302), and ASCT (HR 0.3722, 95% CI: 0.1826-0.7585, p=0.0065) significantly impacted PFS. Furthermore, ASCT (HR 0.142, 95% CI: 0.046-0.438, p=0.0007), presence of bone disease at diagnosis (HR 3.826, 95% CI: 1.471-9.949, p=0.0059) and two cytokine levels- IL-1Ra (HR 1.017, 95% CI: 1.004-1.030, p= 0.0091) and IL-4 (HR 0.161, 95% CI: 0.037-0.698, p = 0.0147) were independent predictors of OS. Three clusters of MM patients were identified with different cytokine profiles. In conclusion, serum pretreatment levels of IL-13 and IL-4 are predictors of better PFS and OS, respectively, whereas IL-1Ra pretreatment levels negatively impact OS in MM patients treated with bortezomib-based chemotherapy. Cytokine signature profile may have a potential influence on the outcome of patients treated with bortezomib.

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Identification of Atypical Plasma Cells in Patients with MGUS or Multiple Myeloma Through Expression of Immunoreceptor CD229

Blood ◽

10.1182/blood.v120.21.4969.4969 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4969-4969

Author(s):

Markus Ertmer ◽

Djordje Atanackovic ◽

Lucia Vankann ◽

Stefan Wilop ◽

Eray Gökkurt ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Plasma Cell ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Cell Phenotype ◽

Surface Markers ◽

Myeloma Cells ◽

Cell Counts ◽

Exact Function

Abstract Abstract 4969 Introduction: While multiparameter flow cytometry (MPF) is an integral part in the diagnosis, disease staging and response evaluation for hematologic malignancies such as acute leukemia or non-hodgkin-lymphoma, MPF for multiple myeloma (MM) is still mostly restricted to research studies or only performed by specialised laboratories experienced in the technique of immunophenotyping. Furthermore, the exact phenotype of malignant myeloma cells is still a matter of debate. Recently, we have identified CD229, a surface marker belonging to the family of signaling lymphocytic activation molecules (SLAM) involved in lymphocyte activation as a potential novel target for diagnosis and treatment of MM. CD229 is expressed on freshly isolated myeloma cells including their clonogenic precursors and several myeloma cell lines. In order to further validate our findings from a previous pilot study, we now analysed 151 samples from 81 patients with suspected or proven MM or monoclonal gammopathy of uncertain significance (MGUS) via MPF. Methods: Between May 2010 and May 2012, specimens (bone marrow (n=142), peripheral blood (n=10), cells from isolated plasmocytoma (n=1)) from patients (pts) with MM (n=65), plasmocytoma (n=1), MGUS (n=6), lymphoplasmacytic lymphoma (n=1) and patients with suspected MM (n=8) were simultaneously analysed via cytology and 8-colour MPF. 19 pts. were analysed at least 3 times during the course of their disease so that CD229 expression could be followed under therapy. Plasma cells were specified using surface markers CD38, CD138, CD45 and cytoplasmatic light chain restriction. Antigens analysed on plasma cells were CD19, CD28, CD33, CD56, CD81, CD117, CD200, CD221 and CD229. Results: Although plasma cell numbers determined by MPF were constantly considerably lower compared to simultaneously determined cytology results, linear regression model showed a highly significant correlation between plasma cell percentages in bone marrow measured by MPF with cytology (p<0. 0001). Plasma cell enumeration in pB also showed a strong correlative trend between cytologic and MPF results, however, due to lower numbers (n=10), this was not statistically significant (p = 0. 057). CD229 could be detected on all atypical plasma cells irrespective if they were found in MGUS or MM samples. The median of mean fluorescence intensity (MFI) of CD229 expression on plasma cells was 3, 63 (range −144. 1 – 34, 23). Median MFI on MM samples (3, 62; range −144 − 34, 23; n=131) did not differ from MFI on atypical plasma cells in pts with MGUS (3, 74, range 1. 07 – 8, 65; n=9). CD229 expression was highest on atypical plasma cells with expression of CD56 compared to CD56 negative plasma cells (p<0. 0001). This was confirmed when correlation of marker expression was evaluated. CD229 expression was clearly correlated with expression of CD56 (n=141, p = 0. 03), CD117 (n=139, p = 7E–08) and CD200 (n=140; p = 0. 03), while it was inversely correlated with expression of CD19 (n=140; p = 0. 03). Serial CD229 expression (>= twice) was determined in 39 patients. Except for three samples, where plasma cell counts became less than 1% of bone marrow cells, CD229 expression remained stable throughout the various analyses. Conclusion: While the exact function of the immunoreceptor CD229 on myeloma cells is still unknown, CD229 allows identification of atypical plasma cells by MPF. Our results show that CD229 is constantly expressed on atypical plasma cells independent of therapy and can be used in addition to other surface markers for determination of malignant plasma cell phenotype and to monitor minimal residual disease (MRD) under treatment. Disclosures: No relevant conflicts of interest to declare.

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Multiple Myeloma Cells Express IL-17A Creating An Autocrine Loop: An Attractive Therapeutic Target

Blood ◽

10.1182/blood.v122.21.3113.3113 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3113-3113

Author(s):

Rao H. Prabhala ◽

Mariateresa Fulcinitti ◽

Dheeraj Pelluru ◽

Harsha K. Prabhala ◽

Naim Rashid ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Growth ◽

Therapeutic Target ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Normal Donor ◽

Myeloma Cells ◽

Autocrine Loop ◽

Attractive Therapeutic Target

Abstract We have previously demonstrated that Th17 cells, which produce IL-17A, are significantly elevated in peripheral blood and bone marrow (BM) of patients with Multiple Myeloma (MM) and IL-17A promotes MM cell growth and survival, both in vitro and in vivo via IL-17A receptor. We have recently evaluated and observed that anti-IL-17A monoclonal antibody (mAb) significantly inhibited MM cell growth in vitro, while IL-17A induced proliferation of MM cells compared to control. We have also observed significant down-regulation of IL-6 production by anti-IL-17A mAb in MM-BMSC co-culture. Importantly, the administration of anti-IL-17A mAb weekly for 4 weeks in the SCIDhu model of human myeloma, where MM cells grow within the human microenvironment in mice led to a significant inhibition of tumor growth compared to the control mice. This remarkable activity of anti-IL17 mAb raised the question of whether the myeloma cells themselves are a possible source of IL-17. In this study, we used transcriptome sequencing (RNA-Seq) data to evaluate the expression of IL-17A in primary CD138+ myeloma cells (N=17) compared to normal plasma cells (NPC) (N=5). Whereas none of the NPCs expressed IL-17A, it was significantly over-expressed in majority of MM cells. In addition, these data also showed that the expression of other IL-17 family members (IL-7B, C, D, E & F) and Th17-associated pro-inflammatory cytokines (IL-21, IL-22 & IL-23) were not significantly elevated in primary myeloma cells compared to normal donor plasma cells. We further validated these observations by IL-17 immunoblot showing IL17 expression in all MM cell lines and 10 out of 14 primary patient MM cells; confirmed IL-17 expression in MM cells by quantitative RT-PCR, and flow cytometry and by immuno-histochemistry and confocal microscopy. We observed that IL-17 knock down by IL-17-specific siRNA inhibited MM cell growth as well as their ability to induce IL-6 production in co-cultures with BMSC. Finally, expression profile data from 172 uniformly treated patients showed that patients with lower IL-17A expression had superior overall survival compared to those with higher expression. These data confirms that MM cells express IL-17 and targeting it with a mAb will abrogate the autocrine loop making it an attractive therapeutic target. Disclosures: No relevant conflicts of interest to declare.

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