High Trip13 and Low P31 Comet Cause Drug Resistance and Poor Prognosis In Multiple Myeloma

Abstract Background We have recently established that increased chromosomal instability (CIN) signature is linked to drug resistance and poor outcome in multiple myeloma (MM) and other cancers. Thyroid Hormone Receptor Interactor 13 (Trip13), one of the 56 drug-resistant genes, plays a key role in chromosomal recombination and structure development during meiosis and has been reported to be increased in some malignancies including lung cancer, prostate cancer and breast cancer. In this study, we investigated how important Trip13 is in myelomagenesis and progression. Materials and Methods Gene expression profiling (GEP) was analyzed on plasma cells from 22 healthy donors, 44 patients with monoclonal gammopathy of undetermined significance (MGUS), 351 patients with newly diagnosed multiple myeloma, and 9 human myeloma cell lines, as well as on 36 sequential samples at diagnosis, pre-1st, pre-2nd and post-2nd autologous stem cell transplantation (ASCT). Over-expression and knock-down experiments of Trip13 were performed on myeloma cell lines by lentivirus transfection. Cell viability was assessed by trypan exclusion assay. Western blots were used to detect the expression of Trip13, P31 comet, caspase-8, caspase-9, caspase-3 and PARP, and checkpoint related proteins MAD2 and CDC20 in Trip13 overexpressed or Trip13 shRNA-transfected myeloma cells. Results Sequential GEP samples showed that Trip13 expression increased in 8 of 9 patients after chemotherapy and ASCT compared to the samples at diagnosis strongly suggesting that increased Trip13 is associated with drug resistance. Trip13 was already significantly increased in MGUS patients, newly diagnosed MM patients and MM cell lines compared with normal plasma cells. Furthermore, Trip13 was significantly higher in high-risk MMs than in low-risk MMs and increased Trip13 was linked to an inferior event-free survival (EFS) (p<0.01) and overall survival (OS) (p<0.01) in 351 newly diagnosed MMs. In contrast, the Trip13-interacting gene P31 comet was down-regulated in high-risk MMs and high expression of P31 was associated with good outcome. Interestingly, patients with high Trip13 and low P31 comet have the worst outcome compared to patients with only one of these, suggesting the interaction of Trip 13 and p31 has a synergistic effect on MM progression. Transfection of Trip13 into ARP1 and OCI-My5 cells significantly increased cell proliferation, while knock-down Trip13 in OCI-My5, H929, RPMI8226 cells inhibited cell growth and induced MM cell apoptosis with increases of cleaved caspase-8, caspase-9, caspase-3 and PARP. Mechanistic studies showed that Trip13 over-expression decreased P31comet and MAD2 expression by western blotting, but increased CDC20. Conclusions The association of increased Trip13 and decreased p31 is a good biomarker for MM drug resistance and poor prognosis. Our results also show Trip13 and P31 comet could be potential targets to overcome drug resistance in MM. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of apoptosis may lead to the development of bortezomib resistance in multiple myeloma cancer cells

Turkish Journal of Biochemistry ◽

10.1515/tjb-2019-0521 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Emine Öksüzoğlu ◽

Gül Kozalak

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Cell Lines ◽

Molecular Mechanisms ◽

Caspase 3 ◽

Plasma Cells ◽

Expression Levels ◽

Diphenyltetrazolium Bromide ◽

Apoptotic Pathways ◽

Resistant Cells

AbstractBackgroundMultiple myeloma (MM), a malignancy of plasma cells, is the second most prevalent hematological cancer. Bortezomib is the most effective chemotherapeutic drug used in treatment. However, drug-resistance prevents success of chemotherapy. One of the factors causing drug-resistance is dysfunction of apoptotic-pathways. This study aimed to evaluate the relationship between expression levels of Bcl-2, Bax, caspase-3 and p-53 genes involved in apoptosis and the development of bortezomib-resistance in MM cell lines.Materials and methodsMultiple myeloma KMS20 (bortezomib-resistant) and KMS28 (bortezomib-sensitive) cell lines were used. 3-[4,5-Dimethylthiazol-2-yl] 1-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine IC50 values of bortezomib. RNAs were isolated from bortezomib-treated cell lines, followed by cDNA synthesis. Expression levels of the genes were analyzed by using q-Realtime-PCR.ResultsAs a result, Bcl-2/Bax ratio was higher in KMS20 (resistant) cells than in KMS28 (sensitive) cells. Expression of caspase-3 decreased in KMS20-cells, whereas increased in KMS28-cells. The results indicate that apoptosis was suppressed in resistant cells.ConclusionThese findings will enable us to understand the molecular mechanisms leading to drug-resistance in MM cells and to develop new methods to prevent the resistance. Consequently, preventing the development of bortezomib resistance by eliminating the factors which suppress apoptosis may be a new hope for MM treatment.

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Tetraspanin Overexpression Induces Multiple Myeloma Cell Death.

Blood ◽

10.1182/blood.v106.11.5067.5067 ◽

2005 ◽

Vol 106 (11) ◽

pp. 5067-5067

Author(s):

Tali Tohami ◽

Liat Drucker ◽

Judith Radnay ◽

Hava Shapiro ◽

Michael Lishner

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Cell Death ◽

Cell Lines ◽

Caspase 3 ◽

Annexin V ◽

Myeloma Cell ◽

Transfected Cells ◽

Over Expression ◽

Rpmi 8226

Abstract Background: Medullary and extra-medullary dissemination of multiple myeloma (MM) cells involves cell-cell and cell-extracellular matrix (ECM) interactions. Proteins coordinating these intricate networks regulate the signaling cascades in a spatial and time dependent manner. Tetraspanins facilitate multiprotein complexing in defined membranal microdomains and select family members have been identified as metastasis suppressors. In preliminary studies, we observed that tetraspanins CD82, frequently down regulated or lost at the advanced clinical stages of various cancers, was absent in MM (8 BM samples, 5 cell lines) and CD81, characteristically expressed in leukocytes plasma membranes, was under-expressed (4/8 BM samples, 4/5 cell lines). We aimed to investigate the consequences of CD81 and CD82 over-expression in myeloma cell lines. Methods: CAG and RPMI 8226 were transfected with pEGFP-N1/C1 fusion vectors of CD81 and CD82. Transfected cells were assessed for - cell morphology (light and fluorescent microscope); cell survival (eGFP+/PI- cells); cell death (Annexin V/7AAD, pre-G1, activated caspase-3 (IC), caspase dependence with pan caspase inhibitor z-VAD-fmk); cell cycle (PI staining). Results: CD82 induced cell death was determined by morphologic characteristics in stably transfected CAG cells (50%) compared to their mock-transfected counterparts (8%) (p<0.05). Activated caspase-3 was also detected (40% of the CD82 transfected cells) (p<0.05). In CD82 transiently transfected MM cell lines a reduced fraction of surviving cells was observed compared to mocks (~60%) (p<0.05) yet, no increases in pre-G1 or Annexin V+/7AAD- subgroups were observed. Moreover, CD82 induced cell death could not be inhibited by the use of z-VAD-fmk. CD82 transfection did not affect the cell cycle of CAG and RPMI 8226 lines. CD81 stably transfected cell lines (CAG and RPMI 8226) could not be established. Indeed, in transiently transfected cells we determined a massive rate of CD81 induced cell death. This is demonstrated in a surviving fraction of only 10% CAG cells and 30% RPMI 8226 (compared to mock) (p<0.05). The CD81 transfected cells were negative for PS exposure, pre-G1 sub-population, or inhibition of death with z-VAD-fmk. The death inducing effect of both tetraspanins in the two cell lines was evident with the pEGFP-N1 orientation vector only. Conclusions: CD81 and CD82 over-expression in MM cell lines causes cell death. Based on the restriction of the killing effect to the pEGFP-N1 clone it may be speculated that its implementation is either dependent on the interactions of the N1 tetraspanin terminus or the proteins’ conformation. It is of interest that CD81 though normally expressed in RPMI 8226 still induced cell death when over-expressed, possibly indicative of ’negative signaling’. Tetraspanins’ suppressive effects on adhesion, motility, and metastasis in solid tumors combined with its capacity to induce myeloma cell death underscore the significance of its absence in MM cell lines and patients. We suspect that a better understanding of CD81/82 mediated signaling pathways will promote future treatment of myeloma cell in their microenvironment. Current studies designed to assess the involvement of oxidative stress in CD81/CD82 induced death are underway.

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High GRP78 (78-kDa Glucose-Regulated Protein) Expression Predicts for a Favorable Clinical Outcome in Patients with Multiple Myeloma and May be a Potentially Useful Therapeutic Target in the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.4206.4206 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4206-4206 ◽

Cited By ~ 2

Author(s):

Hang Quach ◽

Daniel North ◽

Susanna Freddi ◽

Shuh Y Tan ◽

Lenny Straszkowski ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Clinical Outcome ◽

Cell Lines ◽

Plasma Cells ◽

Myeloma Cell ◽

Newly Diagnosed ◽

Favorable Clinical Outcome ◽

Glucose Regulated Protein

Abstract Background: GRP78 (78-kDa glucose-regulated protein) is a molecular chaperone that is upregulated during cellular stress. It has been well demonstrated that GRP78 upregulation is associated with chemoresistance and metastasis in solid tumours. GRP78 has not been widely explored in multiple myeloma (MM), however, we and others have shown that GRP78 is much more overexpressed in myeloma cell lines compared to other cell lines. To assess the clinical relevance of GRP78 overexpression in MM, we investigated the association of plasma cell GRP78 expression on primary bone marrow (BM) trephines to clinical outcome in patients with MM, and correlate this finding to concurrent in vitro studies to investigate the potential usefulness of targeting GRP78 for the treatment of MM. Method: The degree of GRP78 expression within CD138+ plasma cells was assessed by immunohistochemistry (IHC) on archived bone marrow trephines of patients with newly diagnosed MM, who underwent autologous stem cell transplant (ASCT) at St.Vincent's Hospital Melbourne. Independent assessment of GRP78 was performed by 3 hematopathologists, who underwent initial calibration. The degree of GRP78 expression within plasma cells was assigned as low, medium or high. Clinical data was abstracted from medical records of the corresponding patients with respect to baseline demographics, treatment-response, progression free survival (PFS), time to next treatment (TTNT) and overall survival (OS). The association GRP78 expression to each of these clinical parameters was assessed using Kaplan-Meier product limit method and the Mantel-Cox logrank test. In vitro, GRP78 expression was also quantified in various myeloma cell lines by RT-PCR and western blot. The association of GRP78 expression to MM-cell survival and drug resistance was assessed in vitro. The impact of GRP78 inhibition on reversal of drug resistance and myeloma-cell viability was investigated. Result: Between the years 2000 to 2014, a total of 243 patients with newly diagnosed MM underwent ASCT as part of initial therapy, and were included in the study. Baseline bone marrow trephine was available for CD138 and GRP78 staining for 91 patients. Of these, 20, 42 and 34% of patients had low, medium and high expression of GRP78 within BM plasma cells, respectively. Low GRP78 expression was associated with a shorter PFS (HR 2.4, p=0.0006) and shorter TTNT (HR 2.5, p=0.008) compared to intermediate or high GRP78 expression. No significant difference was seen in OS. High GRP78 correlated with a higher probability of achieving CR (p=0.03). In vitro, inhibition of GRP78 resulted in decreased myeloma cell viability, and sensitized myeloma cells to various antimyeloma agents. As a result, synergistic anti-myeloma activity was seen when GRP78 inhibition was combined with melphalan (synergy quotient (SQ) 1.2), dexamethasone (SQ 1.97) and especially bortezomib (SQ 2.06). Conclusion: In contrast to what is reported for solid tumours in the literature, higher GRP78 expression appeared to predict for a more favorable clinical outcome in patients with MM. In vitro, GRP78 inhibition resulted in significant anti-myeloma effects and increased the antimyeloma activity of various agents especially bortezomib. Together, these findings suggest that GRP78 is potentially a useful biomarker and therapeutic target that warrants further investigation in patients with MM. Disclosures Quach: Celgene Corp, ONYX, Janssen, Takeda, Novartis, BMS: Honoraria, Research Funding.

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Loss of Spleen Visualization on Whole-Body Diffusion-Weighted Imaging in Patients with Multiple Myeloma Is Associated with Poor Prognosis and High Tumor Burden

Blood ◽

10.1182/blood-2021-147250 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3797-3797

Author(s):

Toshiki Terao ◽

Youichi Machida ◽

Ukihide Tateishi ◽

Takafumi Tsushima ◽

Kentaro Narita ◽

...

Keyword(s):

Multiple Myeloma ◽

Diffusion Weighted Imaging ◽

Poor Prognosis ◽

Tumor Volume ◽

Plasma Cells ◽

Myeloma Cell ◽

Stage Iii ◽

Whole Body ◽

Cell Infiltration ◽

Group A

Abstract Introduction Multiple myeloma (MM) is caused by the proliferation of monoclonal malignant plasma cells in the bone marrow (BM). Imaging has played a major role in visualizing myeloma lesions, assessing tumor volume, and predicting the prognosis. Recently, we reported that the total diffusion volume (tDV), assessed using a pretreatment whole-body diffusion-weighted imaging (WB-DWI), was associated with a high BM plasma cells (BMPCs) and a poor prognosis in patients with MM (Terao. et al., Eur Radiol 2021). During that study, we unexpectedly found the frequent absence of a spleen signal in patients with MM and its reappearance after treatment. Therefore, this study aimed to investigate the association between spleen visualization changes on WB-DWI and myeloma tumor load and prognosis in patients with MM. Methods The data of 96 consecutive patients with symptomatic newly-diagnosed MM (NDMM) at Kameda Medical Center from January 2016 to December 2020, 15 consecutive patients with smoldering MM (sMM), and two autopsied spleens of patients with PC dysplasia were retrospectively reviewed. All patients underwent at least one WB-DWI prior to treatment. The detail of WB-DWI was previously reported (Terao. et al., Eur Radiol 2021). "Loss of spleen visualization" (LSV) was defined as a visual loss of the spleen in maximum intensity projection on the WB-DWI (Fig1). The spleen-to-spinal cord (SC) ratio (SSR) was used in each regions-of-interest (ROI) to compare the signal intensity. The spleen ROIs were defined as non-overlapping ROIs of 30-50 pixels. The SC ROI was the largest ROI without overhanging in the image depicting the maximum size of the spleen. This study was approved by the institutional review board and conducted in accordance with the Declaration of Helsinki. All participants provided informed consent. Results The median patient age was 75.5 years and 81 patients (84.4%) were 65 years or older. Almost all patients (n=91) received proteasome inhibitors (PIs) as remission induction therapy and 33 patients received autologous stem-cell transplantation (ASCT). LSV was observed on the WB-DWI of 56/96 (58.3%) patients with NDMM and in one patient with sMM (1/15, 6.7%). Patients with NDMM and LSV had a higher median BMPC infiltration as assessed by CD138-immunohistochemistry (80.0% vs. 50.0%, p<0.001), a higher median tDV (540.2 mL vs. 137.0 mL, p=0.003), higher rate of ISS stage III (p<0.01), a lower SSR (0.36 vs. 0.96; p<0.001), and lower tDV (540.2 mL vs. 137.0 mL; p=0.003) than those without LSV. The three-year PFS (p=0.27) and three-year OS (p=0.021) were lower in patients with NDMM with LSV (PFS: 51.2% and OS: 72.5%) than in patients without LSV (PFS: 63.4% and OS: 100%). Next, we investigated the spleen signal change of patients who underwent WB-DWI twice or more during treatment (n=74). Of 42 out of the 74 patients with LSV at diagnosis, the spleen during treatment became visible on 31/42 (73.8%) patients. Representative patients with various spleen signal changes during treatment are shown in Figure 3 as group A (n=32; patients without LSV at diagnosis and during treatment), group B (n=31; patients who had LSV at diagnosis but the spleen reappeared after treatment), and group C (n=11; patients who had LSV at diagnosis, and despite treatment response, did not regain the spleen signal). Patients in group C showed significantly worse three-year PFS and OS (not available due to early events) than those in group A and B, even after excluding patients who did not achieve partial response or worse (n=11) (Fig1). In the multivariate analysis, the group C retained its prognostic significance for both PFS (hazard ratio [HR], 1.98, 95% confidence interval [CI] 1.00-3.90, p = 0.049) and OS (HR, 5.16, 95% CI 1.27-21.0, p = 0.022) even after adjustment for age over 70 years and the revised-ISS stage III. At last, to investigate the pathological cause of LSV, we reviewed two patients who underwent autopsies, who had both received WB-DWI within 3 months before their deaths (Fig1). One patient showed diffuse myeloma cell infiltration in the spleen and the other showed the amyloid deposition without myeloma cell infiltration. Conclusion This study showed that LSV and a low SSR on pretreatment WB-DWI are correlated with a high tumor volume and poor prognosis. As patients with LSV during treatment had very poor prognosis, the relationships between LSV and other variables should be investigated. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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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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Expression of functional interleukin-15 receptor and autocrine production of interleukin-15 as mechanisms of tumor propagation in multiple myeloma

Blood ◽

10.1182/blood.v95.2.610 ◽

2000 ◽

Vol 95 (2) ◽

pp. 610-618 ◽

Cited By ~ 87

Author(s):

Inge Tinhofer ◽

Ingrid Marschitz ◽

Traudl Henn ◽

Alexander Egle ◽

Richard Greil

Keyword(s):

Multiple Myeloma ◽

Cell Survival ◽

Cell Lines ◽

Plasma Cells ◽

Constitutive Expression ◽

Myeloma Cell ◽

Myeloma Cells ◽

Cytotoxic Treatment ◽

Interleukin 15 ◽

Induced Apoptosis

Interleukin-15 (IL-15) induces proliferation and promotes cell survival of human T and B lymphocytes, natural killer cells, and neutrophils. Here we report the constitutive expression of a functional IL-15 receptor (IL-15R) in 6 of 6 myeloma cell lines and in CD38high/CD45low plasma cells belonging to 14 of 14 patients with multiple myeloma. Furthermore, we detected IL-15 transcripts in all 6 myeloma cell lines, and IL-15 protein in 4/6 cell lines and also in the primary plasma cells of 8/14 multiple myeloma patients. Our observations confirm the existence of an autocrine IL-15 loop and point to the potential paracrine stimulation of myeloma cells by IL-15 released from the cellular microenvironment. Blocking autocrine IL-15 in cell lines increased the rate of spontaneous apoptosis, and the degree of this effect was comparable to the pro-apoptotic effect of depleting autocrine IL-6 by antibody targeting. IL-15 was also capable of substituting for autocrine IL-6 in order to promote cell survival and vice versa. In short-term cultures of primary myeloma cells, the addition of IL-15 reduced the percentage of tumor cells spontaneously undergoing apoptosis. Furthermore, IL-15 lowered the responsiveness to Fas-induced apoptosis and to cytotoxic treatment with vincristine and doxorubicin but not with dexamethasone. These data add IL-15 to the list of important factors promoting survival of multiple myeloma cells and demonstrate that it can be produced and be functionally active in an autocrine manner.

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ABCG2 expression, function, and promoter methylation in human multiple myeloma

Blood ◽

10.1182/blood-2005-10-009084 ◽

2006 ◽

Vol 108 (12) ◽

pp. 3881-3889 ◽

Cited By ~ 106

Author(s):

Joel G. Turner ◽

Jana L. Gump ◽

Chunchun Zhang ◽

James M. Cook ◽

Douglas Marchion ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Drug Resistance ◽

Protein Expression ◽

Cell Lines ◽

Promoter Methylation ◽

Plasma Cells ◽

Cancer Resistance ◽

Response To Chemotherapy ◽

Mrna And Protein Expression

AbstractWe investigated the role of the breast cancer resistance protein (BCRP/ABCG2) in drug resistance in multiple myeloma (MM). Human MM cell lines, and MM patient plasma cells isolated from bone marrow, were evaluated for ABCG2 mRNA expression by quantitative polymerase chain reaction (PCR) and ABCG2 protein, by Western blot analysis, immunofluorescence microscopy, and flow cytometry. ABCG2 function was determined by measuring topotecan and doxorubicin efflux using flow cytometry, in the presence and absence of the specific ABCG2 inhibitor, tryprostatin A. The methylation of the ABCG2 promoter was determined using bisulfite sequencing. We found that ABCG2 expression in myeloma cell lines increased after exposure to topotecan and doxorubicin, and was greater in logphase cells when compared with quiescent cells. Myeloma patients treated with topotecan had an increase in ABCG2 mRNA and protein expression after treatment with topotecan, and at relapse. Expression of ABCG2 is regulated, at least in part, by promoter methylation both in cell lines and in patient plasma cells. Demethylation of the promoter increased ABCG2 mRNA and protein expression. These findings suggest that ABCG2 is expressed and functional in human myeloma cells, regulated by promoter methylation, affected by cell density, up-regulated in response to chemotherapy, and may contribute to intrinsic drug resistance.

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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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Expression Of Truncated Protein Tyrosine Phosphatase, Receptor Type, O (PTPROt) Influences Multiple Myeloma Sensitivity To Bortezomib Through Akt Signaling, and Is a Favorable Clinical Prognostic Factor

Blood ◽

10.1182/blood.v122.21.2520.2520 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2520-2520

Author(s):

Hua Wang ◽

Veerabhadran Baladandayuthapani ◽

Zhiqiang Wang ◽

Jiexin Zhang ◽

Heather Yan Lin ◽

...

Keyword(s):

Multiple Myeloma ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Myeloma Cell ◽

Advisory Committees ◽

Over Expression ◽

Resistant Disease ◽

Drug Naïve ◽

Rpmi 8226

Abstract Background Proteasome inhibitors such as bortezomib and carfilzomib are an important part of our current chemotherapeutic armamentarium against multiple myeloma, and have improved outcomes in the up-front, relapsed, and relapsed/refractory settings. Their efficacy has been demonstrated both as single agents, and as part of rationally designed combination regimens, but they are at this time used empirically, since biomarkers to identify patients who would most or least benefit from their application have not been clinically validated. Moreover, the vast majority of patients eventually develop drug-resistant disease which precludes further proteasome inhibitor use through mechanisms that have not been fully elucidated. Methods We compared gene expression profiles (GEPs) of a panel of bortezomib-resistant myeloma cell lines and their vehicle-treated, drug-naïve counterparts to identify significant changes associated with drug resistance. The list of genes whose expression was changed by at least 2-fold was compared with independent RNA interference studies whose goal was to identify genes whose suppression conferred drug resistance. Further validation of genes of interest was pursued in a panel of myeloma cell lines, and in clinically annotated GEP databases. Results Suppression of PTPROt expression was noted in bortezomib-resistant RPMI 8226 and ANBL-6 myeloma cells compared to isogenic, drug-naïve controls, and this was confirmed by quantitative PCR. Overexpression of PTRPOt in RPMI 8226, ANBL-6 and other myeloma cell lines was by itself sufficient to increase the level of apoptotic, sub-G0/G1 cells compared to vector controls, or cells expressing a phosphatase-dead PTPROt mutant. Moreover, PTPROt enhanced the ability of bortezomib to reduce myeloma cell viability, in association with increased activation of caspases 8 and 9. Exogenous over-expression of PTPROt was found to reduce the activation status of Akt, a known anti-apoptotic pathway that reduces bortezomib activity, based on Western blotting with antibodies to phospho-Akt (Ser473), and Akt kinase activity assays. Notably, we also found that exogenous over-expression of PTPROt resulted in increased expression levels of p27Kip1. Interestingly, array CGH data from studies of myeloma cell lines and primary cells showed that the PTPROt gene was located in a genomic region with a high propensity for loss. Analysis of the Total Therapy databases of GEP and patient outcomes available on the Multiple Myeloma Genomics Portal showed that higher than median expression of PTPROt was associated with better long-term survival (P=0.0175). Finally, analysis of the Millennium Pharmaceuticals database of studies of bortezomib in the relapsed and relapsed/refractory setting showed high PTRPOt expression was more frequently seen in patients who achieved complete remission (P<0.01), and was associated with a better median overall survival (P=0.0003). Conclusions Taken together, the data support the possibility that high expression of PTPROt is a good prognostic factor for response to bortezomib-containing therapies, and that this may occur through modulation by PTPROt of the Akt pathway. Moreover, they suggest that strategies to enhance the expression of PTPROt should be investigated to restore bortezomib sensitivity in patients with proteasome inhibitor-resistant disease. Disclosures: Orlowski: Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees.

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Interfering with Arginine Metabolism As a New Treatment Strategy for Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.3005.3005 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3005-3005

Author(s):

Bjoern Jacobi ◽

Lea Stroeher ◽

Nadine Leuchtner ◽

Hakim Echchannaoui ◽

Alexander Desuki ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Death ◽

Cell Lines ◽

Caspase 3 ◽

Xenograft Model ◽

Myeloma Cell ◽

Intracellular Protein ◽

Myeloma Cells ◽

Induction Of Apoptosis

Abstract Introduction Starvation of tumor cells from the amino acid arginine has recently gained particular interest because of the downregulation of the rate-limiting enzyme argininosuccinate synthethase 1 (ASS1) in various cancer entities. ASS1-deficient cells cannot resynthesize arginine from citrulline and are therefore considered arginine auxotrophic. The arginine depleting enzyme arginine deiminase (ADI-PEG20, Polaris Pharmaceuticals) is currently tested in phase I-III clinical trials for different arginine auxotrophic cancers. The natural arginine analogue canavanine can compete with arginine for arginyl-tRNA-binding sites and consequently be incorporated into nascent proteins instead of arginine. Canavanine could therefore potentially further disturb intracellular protein homeostasis, especially under arginine deprivation. The sensitivity of myeloma cells towards arginine depletion strategies has not been analyzed so far. Methods Human myeloma cell lines and CD138-sorted primary human myeloma cells from patient bone marrow were screened for ASS1 expression by western blotting (WB). The cells were cultured in arginine free medium and assessed for proliferation and metabolic activity (CFSE/MTT assays), apoptosis (caspase-3 cleavage) and cell death (annexinV/propidium iodide). Canavanine was supplied in both arginine-sufficient and -deficient conditions. The level of intracellular protein stress was determined by WB and/or flow cytometry analysis for ubiquitinated proteins, phosphorylated eukaryotic initiation factor 2α (peIF2α) and the spliced isoform of the X-Box binding protein 1 (Xbp1s). Repetitive ADI-PEG20 ± canavanine application i.p. were tested in vivo in an U266 myeloma xenograft model in NOD/SCID/IL2Rcg-/- (NSG) mice. Arginine and canavanine levels in plasma were determined by HPLC. Tumor growth was measured, mice were assessed for survival, weight and side effects. Tumor tissues were analyzed for caspase-3 cleavage and Ki67 expression by immunohistochemistry. Results 5 of 6 myeloma cell lines were negative for ASS1. Also, ASS1 was either not or only weakly expressed in the majority of primary CD138+ myeloma patient samples. Arginine starvation induced an arrest of cell proliferation and/or metabolic activity of primary myeloma cells and myeloma cell lines after 18-24 h. Addition of citrulline could only rescue ASS1 positive myeloma cells due to the intracellular resynthesis of arginine. Arginine starvation alone led to delayed induction of apoptosis (e.g. 35% cell death of NCI-H929 cells after 72 h of treatment). Addition of 100 mM canavanine strongly increased cell death specifically in the context of arginine deficiency (e.g. cell death in NCI-H929 cells: 87% after 24 h, 100 % after 48h) while it was non-toxic and had no effect on cell viability under physiological arginine conditions. Co-application of canavanine induced ubiquitination of cellular proteins and led to the prolongation of a fatal unfolded protein response (UPR) as measured by markedly elevated Xbp1s levels. Prolonged UPR ultimately led to the induction of apoptosis as reflected by annexin V binding and caspase-3 cleavage. In an U266 myeloma NSG xenograft model, systemic arginine depletion by ADI-PEG20 suppressed tumor growth in vivo and significantly prolonged median survival of mice when compared with the control group (22±3 vs. 15±3 days). Canavanine treatment alone had no influence on viability (13±0 days). However, the combination of ADI-PEG20 and canavanine demonstrated the longest median survival (27±7 days). Histological examination of explanted tumors showed the highest rates of caspase-3 cleavage in the ADI-PEG20/canavanine group. Conclusion Myeloma cells are mostly arginine auxotrophic and can be selectively targeted by arginine starvation. Combination of arginine depletion with the arginine analogue canavanine leads to a highly efficient and specific tumor cell eradication and should be further optimized in multiple myeloma preclinical models. Disclosures Bomalaski: Polaris Pharmaceuticals Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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