Interference Of The Tumor Supportive Effects Of BCL2 and MCL1 Sensitize Malignant Plasma Cells To The Lethal Effects Of Lenalidomide and Dexamethasone Regimen: An Important Clinical Path For BCL2 Targeting Drugs

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1928-1928
Author(s):  
Aneel Paulus ◽  
Kasyapa S. Chitta ◽  
Sharoon Akhtar ◽  
Maja Kuranz ◽  
Shaji Kumar ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cell Death ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Dependent Manner ◽  
Mrna Quantification ◽  
Bcl2 Family ◽  
Dose Dependent

Abstract Background Multiple myeloma (MM) is the second most common hematologic malignancy in the U.S. and is characterized by increased immunoglobulin production and infiltration of the bone marrow by malignant plasma cells. Myeloma cell growth is supported by both the elements present in the bone marrow microenvironment as well as deregulation of internal cellular systems associated with proliferation and apoptosis. Defective programmed cell death by BCL2 or MCL1 upregulation is observed in >80% of myeloma cases and is associated with an aggressive clinical course. Remarkably, there is no approved drug with the ability to target BCL2 or MCL1. Various strategies to mitigate the effects of increased BCL2 functionality, via small molecule inhibitors or BCL2-specific antisense oligonucleotides have been previously examined in MM. These studies revealed that 1) apart from BCL2, additional anti-apoptotic members belonging to the BCL2 family (i.e. MCL1, BCL-xL) play integral roles in maintenance of the myeloma cell phenotype, 2) drugs, which target only BCL2 offer limited therapeutic advantage and 3) given that survival factors engaged in MM are multifactorial, perhaps isolated targeting of the BCL2 pathways may not be sufficient. We therefore hypothesized that the clinical development of drugs that target various antiapoptotic BCL2 family members will require a partnership with established anti-MM regimens whose activity can potentially be further enhanced with correction of the apoptotic response system. Aim Since lenalidomide and dexamethasone (LD) is an established therapy for plasma cell cancers, we investigated if the pan-BCL2 inhibitor AT-101 (BH3 mimetic), which has high binding specificity for BCL2, MCL1 and BCL-xL, can be an effective therapeutic partner to enhance anti-MM effects of LD. Methods Human MM (KMS11, U266, OPM2) and WM (BCWM.1) cell lines along with their corresponding bortezomib resistant (BR) clones, (KMS11/BR, U266/BR, OPM2/BR and BCWM.1/BR) which were developed in our laboratory were used in this study. For gene expression profiling, the Illumina HumanHT-12 v3 whole-genome gene expression array and Nanostring nCounter mRNA quantification assays (NanoString, Seattle, WA) were utilized. Statistical analysis was conducted using R-based packages and the MeV software (TIGR). Apoptosis was measured by annexin-v/PI staining, and mitochondrial membrane permeability (MOMP) was assessed using TMRM followed by flow cytometry. Protein profiles were ascertained by western blot. Results Gene expression and immunoblot analysis of six plasma cell cancer models showed upregulation of various BCL2 family members, notably MCL1 and BCL-xL. In a dose-dependent manner, AT-101 was able to downregulate BCL2 and MCL1 and induced apoptotic cell death in MM and WM cells in a dose dependent manner. Tumor cell death was associated with caspase and PARP-1 cleavage accompanied by an increase in MOMP. This cytotoxic effect and BCL2 downregulation were further potentiated when AT-101 was combined with lenalidomide/dexamethasone (LDA). Nanostring nCounter mRNA quantification and IPA analysis revealed differential changes in the CCNA, FRZB, FYN, IRF1, PTPN11 genes in LDA treated cells. IPA canonical pathway analysis demonstrated the p53 signaling and the cyclins and cell cycle regulation pathways to be the most significantly activated by LDA therapy. Summary In summary, we describe for the first time the cellular and molecular events associated with the use of AT-101 in combination with lenalidomide/dexamethasone in preclinical models of plasma cell malignancy. This study lays the rationale for engaging and handicapping the intrinsic apoptotic system through the pan-BCL2 targeting capabilities of AT-101; thus enhancing the anti-MM effects of lenalidomide/dexamethasone. Disclosures: Foran: Celgene: Research Funding.

scholarly journals THE BONE MARROW ON STERNAL ASPIRATION IN MULTIPLE MYELOMA

Blood ◽  
1948 ◽  
Vol 3 (9) ◽  
pp. 987-1018 ◽  
Author(s):  
EDWIN D. BAYRD
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Multinucleated Cells ◽  
Cytoplasmic Bodies ◽  
Plasma Cell Type ◽  
Well Differentiated ◽  
Diagnostic Pitfalls

Abstract Generalizing, it can be said that the pathologic cells seen in smears of the bone marrow in multiple myeloma resemble the plasma cell and vary from the very anaplastic and immature cell to the well-differentiated and almost characteristic plasma cell. The feature which the "myeloma" cell shares with the plasma cell is the abundant, granular, basophilic cytoplasm which tends to be fragile and undergo the same degenerative changes in each; namely, the formation of Russell bodies and vacuolization. Fairly frequently a perinuclear clear area or Hof is present and the nucleus tends to be eccentrically placed. Cytoplasmic extensions or pseudopodia may also be seen in either case, but they occur more often and more dramatically in instances of multiple myeloma. Multinucleated cells are commonly seen. In addition, myeloma-plasma cells will often have a large clear nucleolus and a leptochromatic nucleus and will exhibit a tendency to the formation of isolated areas of condensed chromatin. Cytoplasmic extrusions, free cytoplasmic bodies, occasionally complete with Russell bodies and vacuoles are almost universally present. All cases were of the plasma cell type; there was no exception. In these cases, the myeloma-plasma cell constituted from 2.5 to 96 per cent of the leukocytic elements present. The opinion was expressed that all so-called types of multiple myeloma are merely variations in differentiation of this same cell. It was noted that anaplasia, hypernucleation and lack of plasma cell predominance in certain cases were diagnostic pitfalls. Additional evidence was adduced to confirm the reticulo-endothelial origin of the myeloma-plasma cell. It was further observed that certain prognostically valuable information could be gleaned from a careful review of the cytologic characteristics in these cases.

Aminopeptidase Inhibition as a Targeted Treatment Strategy in Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2505-2505 ◽  
Author(s):  
Faith E. Davies ◽  
Hannah E. Moore ◽  
Emma L. Davenport ◽  
Alan S. Dunlop ◽  
Srikanth Muralikrishnan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cell Death ◽  
Protein Turnover ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Unfolded Protein ◽  
Marrow Stroma ◽  
The Unfolded Protein Response ◽  
Protein Response

Abstract Myeloma cells are highly dependent on the unfolded protein response to assemble folded immunoglobulins correctly. Therefore targeting protein handling within a myeloma cell by inhibiting the aminopeptidase enzyme system that catalyses the hydrolysis of amino acids from the N terminus of proteins may be a novel therapeutic approach. The effect of the aminopeptidase inhibitor CHR-2797 on myeloma cell proliferation and survival, gene expression, protein turnover, cell migration and myeloma-bone marrow stromal cell interactions was determined on a panel of myeloma cell lines and patient cells. CHR-2797 is able to inhibit the proliferation of myeloma cell lines and primary patient cells, whereas its derivative CHR-79888, an acid metabolite with low cell membrane permeability fails to induce myeloma cell death. This occurs though apoptosis as demonstrated by trypan blue exclusion and annexin V/PI staining, and is proceeded by G1 growth arrest. Western blot analysis demonstrates apoptosis occurs via a non-caspase dependant mechanism. Importantly CHR-2797 is able to induce apoptosis in cells known to be resistant to conventional chemotherapeutic agents. Analysis of the pathways involved using Affymetrix gene expression arrays demonstrates CHR-2797 causes an upregulation of many genes involved in the proteasome/ubiquitin pathway, as well as amino acid deprivation response genes and some aminopeptidases. A further mechanism contributing to cell death is activation of the unfolded protein response with activation of all three UPR pathways demonstrated by splicing of XBP1 to its active from XBP1s, an increase in CHOP with activation of the PERK pathway and cleavage of ATF6. Cytoplasmic inclusions are also present on light microscopy suggestive of the build up of misfolded proteins within the cytoplasm. CHR-2797 causes minimal inhibition of the proliferation of bone marrow stroma, but is able to overcome the protective effects of the micro-environment on myeloma cells, as the drug is still able to inhibit the proliferation of myeloma cells when they are bound to bone marrow stromal cells. Aminopeptidase inhibition is also able to inhibit the increase VEGF that occurs when myeloma cells and bone marrow stroma are bound together. Combination experiments of CHR-2797 with dexamethasone demonstrate synergy, in keeping with the different mechanisms of action the two drugs. CHR-2797 in combination with the proteasome inhibitor bortezomib demonstrates an additive effect. Although both drugs target intracellular protein turnover, gene expression studies of cells treated with CHR-2797 or bortezomib show deregulation of a number of genes specific to aminopeptidase inhibition, as well as a series of genes characteristic of protein turnover. These differences in the mechanism of action of the two drugs are also reflected in the Western blot analysis that demonstrates a predominately non-caspase mediated cell death in CHR2797 compared to a caspase mediated cell death with bortezomib. In summary inhibiting intracellular protein turnover using the aminopeptidase inhibitor CHR-2797 results in myeloma cell death and represents a novel therapeutic approach for the treatment of myeloma. A phase 1 clinical trial has been initiated in haematological malignancies and the results will also be presented at this meeting.

scholarly journals A Role for Syntenin-1 in Multiple Myeloma Cell Survival

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1008-1008
Author(s):  
Tyler Moser-Katz ◽  
Catherine M. Gavile ◽  
Benjamin G Barwick ◽  
Sagar Lonial ◽  
Lawrence H. Boise
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Cell Survival ◽  
Cell Lines ◽  
Plasma Cells ◽  
Surface Expression ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract Multiple myeloma is the second most common hematological malignancy in the U.S. with an estimated 30,700 new diagnoses in 2018. It is a clonal disease of plasma cells that, despite recent therapeutic advances, remains incurable. Myeloma cells retain numerous characteristics of normal plasma cells including reliance on survival signals in the bone marrow for long term viability. However, malignant transformation of plasma cells imparts the ability to proliferate, causing harmful bone lesions in patients, and in advanced stages independence of the bone-marrow microenvironment. Therefore, we are investigating the molecular mechanisms of myeloma cell survival that allow them to become extramedullary. We identified syntenin-1 (SDCBP) as a protein involved in myeloma cell survival and a potential therapeutic target. Syntenin-1 is an adapter protein that has been shown to regulate surface expression of several transmembrane proteins by binding with membrane phospholipids and mediating vesicular trafficking of proteins throughout the cell. Syntenin-1 regulates the surface expression of CD138, a plasma/myeloma cell marker. Syntenin-1 has been shown to regulate apoptosis in numerous cancer cell lines including breast cancer, glioma, and pancreatic cancer but its role in multiple myeloma survival has not been studied. To determine if syntenin-1 expression has an effect on myeloma cell survival, we utilized the CoMMpass dataset (IA12), a longitudinal study of myeloma patients that includes transcriptomic analysis throughout treatment. We found that patients with the highest expression of syntenin-1 mRNA (top quartile) had significantly worse overall survival, progression-free survival, and a shorter response duration than those in the bottom quartile of expression. To determine if syntenin-1 has a role in myeloma cell survival, we used short hairpin RNA to knock down syntenin-1 (shsyn) in RPMI 8226 and MM1.s myeloma cell lines. We then determined the amount of cell death using Annexin-V staining flow cytometry four days following lentiviral infection. We found increased cell death in syntenin-1-silenced cells compared to our empty vector control in both RPMI 8226 (control=42.17%, shsyn=71.53%, p=0.04) and MM1.s cell lines (control=8.57%, shsyn=29.9%, p=0.04) suggesting that syntenin-1 is important for myeloma cell survival. Syntenin-1 contains two PDZ domains that allow it to bind to receptor proteins via their corresponding PDZ-binding motifs. We therefore wanted to look at correlation of syntenin-1 expression with CD138 and CD86, two PDZ-binding domain containing proteins expressed on the surface of myeloma cells. Using the CoMMpass dataset, we found patients with high expression of syntenin-1 had a median expression of CD86 that was twice as high as the total population (P<0.0001) while syntenin-1-low patients expressed CD86 at levels that were half as much as the population (P<0.0001). In contrast, there was no clear relationship between syntenin-1 and CD138 mRNA expression. Indeed if one takes into account all patients, there is a positive correlation between CD86 and syntenin-1 expression (r=0.228, P<0.0001) while there is a negative correlation between CD138 and syntenin-1 (r=-0.1923, P<0.0001). The correlation with CD86 but not CD138 suggests a previously undescribed role for syntenin-1 in myeloma cells. Our lab has previously shown that expression of CD86 is necessary for myeloma cell survival, and signals via its cytoplasmic domain to confer drug resistance. Silencing syntenin-1 results in a decrease in CD86 surface expression. However, there is no change in CD86 transcript or total cellular CD86 protein levels in our shsyn treated cells. Moreover, knockdown of CD86 resulted in increased protein expression and transcript levels of syntenin-1. Taken together, these data suggest that syntenin-1 may regulate CD86 expression on the cell surface. Our data supports a novel role for syntenin-1 in myeloma cell viability and as a potential regulator of CD86 surface expression. The role of syntenin-1 has not previously been explored in multiple myeloma and determining its molecular function is warranted as it may be an attractive target for therapeutic treatment of the disease. Disclosures Lonial: Amgen: Research Funding. Boise:AstraZeneca: Honoraria; Abbvie: Consultancy.

Preferential Inhibition of Malignant Cell Growth by CDDO in Waldenström Macroglobulinemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2528-2528
Author(s):  
Sherine F. Elsawa ◽  
Anne J. Novak ◽  
Marina Konopleva ◽  
Michael Andreeff ◽  
Thomas E. Witzig ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Cell Death ◽  
B Cells ◽  
Cell Growth ◽  
Peripheral Blood ◽  
Malignant Cell ◽  
Dependent Manner ◽  
Malignant Cells ◽  
Dose Dependent

Waldenström macroglobulinemia (WM) is a B cell disorder with a highly variable clinical outcome, where some patients remain asymptomatic, while others have significant symptoms and require therapeutic intervention. Clinical symptoms include infiltration of lymphoplasmacytic cells into the bone marrow, production of a monoclonal IgM protein, anemia, lymphadenopathy, and serum hyperviscosity. Despite the introduction of multiple chemotherapeutic regimens over the past several decades, WM remains an incurable disease. 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) and its methyl ester derivative (CDDO-Me) and imidazolide derivative (CDDO-Im) are synthetic triterpenoids derived from oleanolic acid. These compounds have been shown to induce apoptosis of several tumor cell types including breast cancer, lung cancer, ovarian cancer, melanoma, osteosarcoma, leukemia, and multiple myeloma cells. The goal of this study was to evaluate the potential role of synthetic triterpenoids in WM. Preliminary studies on malignant B cells indicated that CDDO-Im induced the greatest amount of cell death and we therefore used this derivative of CDDO for our studies. CD19+ CD138+ cells from bone marrow biopsy specimens obtained from WM patients were isolated by positive selection and were treated with varying concentrations of CDDO-Im (62.5 nM to 750 nM ) and cell viability was determined after 24 hours (n=3). Compared to the nil control 47% of the malignant cells remained viable at a CDDO-Im concentration of 62.5 nM and only 11% remained viable at 125 nM CDDO-Im. To determine if CDDO-Im had specific toxic effects on non-malignant cells, we cultured CD19- CD138- cells from WM patient bone marrows with CDDO-Im and found that non-malignant cells were less sensitive to the drug, 80% being viable at 62.5 nM and 65% being viable at 125 nM. Similarly, we found that normal peripheral blood B cells and CD19+ CD138+ bone marrow B cells from healthy donors were less sensitive to CDDO-Im. Compared to the nil control 93% of the CD19+ CD138+ bone marrow B cells and 70% of the peripheral blood B cells remained viable at a CDDO-Im concentration of 62.5 nM and 95% and 68% remained viable at 125 nM CDDO-Im respectively. We next examined the effect of CDDO-Im on WM cell proliferation and found that CDDO-Im inhibited cell proliferation in a dose-dependent manner. Similar to the viability assays, there was a differential effect of CDDO-Im on malignant and non-malignant cells. Compared to the nil control, at 125 nM, there was a complete inhibition of malignant cell growth, while approximately 40% of the non-malignant cells remained proliferative. To determine the mechanism of cell death, CD19+ CD138+ cells were cultured in the presence or absence of various doses of CDDO-Im for 6 hours and cell lysates were examined for cleavage of PARP. There was evidence of PARP cleavage in a dose-dependent manner, suggesting that CDDO-Im induced malignant cell death occurs through a caspase-dependent mechanism. In summary, the synthetic triterpenoid CDDO-Im decreased the viability of WM B cells in a dose-dependent manner, and CDDO-Im had a greater effect on the viability of the malignant cells compared to non-malignant cells from the same WM patients. CDDO-Im also inhibited malignant cell growth in a dose-dependent manner and the mechanism of CDDO-Im mediated cell death appears to be a caspase-mediated event. Overall, our data indicate that CDDO-Im may have potential efficacy in WM patients.

A Novel Role For Histone Deacetylase 11 (HDAC11) In Plasma Cell Differentation and Survival

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1907-1907
Author(s):  
Eva Sahakian ◽  
Jason B. Brayer ◽  
John Powers ◽  
Mark Meads ◽  
Allison Distler ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
B Cells ◽  
Plasma Cell ◽  
Research Funding ◽  
Plasma Cells ◽  
Myeloma Cells ◽  
Peripheral Blood Plasma

Abstract The role of HDACs in cellular biology, initially limited to their effects upon histones, is now appreciated to encompass more complex regulatory functions that are dependent on their tissue expression, cellular compartment distribution, and the stage of cellular differentiation. Recently, our group has demonstrated that the newest member of the HDAC family of enzymes, HDAC11, is an important regulator of IL-10 gene expression in myeloid cells (Villagra A Nat Immunol. 2009). The role of this specific HDAC in B-cell development and differentiation is however unknown. To answer this question, we have utilized a HDAC11 promoter-driven eGFP reporter transgenic mice (TgHDAC11-eGFP) which allows the monitoring of the dynamic changes in HDAC11 gene expression/promoter activity in B-cells at different maturation stages (Heinz, N Nat. Rev. Neuroscience 2001). First, common lymphoid progenitors are devoid of HDAC11 transcriptional activation as indicated by eGFP expression. In the bone marrow, expression of eGFP moderately increases in Pro-B-cells and transitions to the Pre- and Immature B-cells respectively. Expression of eGFP doubles in the B-1 stage of differentiation in the periphery. Of note, examination of both the bone marrow and peripheral blood plasma cell compartment demonstrated increased expression of eGFP/HDAC11 mRNA at the steady-state. These results were confirmed in plasma cells isolated from normal human subjects in which HDAC11 mRNA expression was demonstrated. Strikingly, analysis of primary human multiple myeloma cells demonstrated a significantly higher HDAC11 mRNA expression in malignant cells as compared to normal plasma cells. Similar results were observed in 4/5 myeloma cell lines suggesting that perhaps HDAC11 expression might provide survival advantage to malignant plasma cells. Support to this hypothesis was further provided by studies in HDAC11KO mice in which we observed a 50% decrease in plasma cells in both the bone marrow and peripheral blood plasma cell compartments relative to wild-type mice. Taken together, we have unveiled a previously unknown role for HDAC11 in plasma cell differentiation and survival. The additional demonstration that HDAC11 is overexpressed in primary human myeloma cells provide the framework for specifically targeting this HDAC in multiple myeloma. Disclosures: Alsina: Millennium: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Baz:Celgene Corporation: Research Funding; Millenium: Research Funding; Bristol Myers Squibb: Research Funding; Novartis: Research Funding; Karyopharm: Research Funding; Sanofi: Research Funding.

The Location of Multiple Myeloma Adhesion Variants in Diverse Niches within the Bone Marrow Affects Plasma Cells Enumeration.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5063-5063
Author(s):  
Liat Nadav ◽  
Ben-Zion Katz ◽  
Shoshana Baron ◽  
Lydia Lydia ◽  
Aaron Polliack ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Gene Expression Profile ◽  
Plasma Cell ◽  
Expression Profile ◽  
Plasma Cells ◽  
Response To Therapy ◽  
Morphological Evaluation

Abstract Background - The diagnosis of multiple myeloma (MM) is based on clinical and laboratory criteria combined with bone marrow (BM) plasmocytosis, estimated by inspection of bone marrow aspirates. Recent advances in flow-cytometry (FCM) have provided an additional tool for the diagnosis of MM and for monitoring response to therapy. However, significant discrepancy has been reported regarding the enumeration of plasma cells in marrow samples of MM patients using these two methods. Aims - In this study we compared the bone marrow plasmocytosis by microscopic examination of BM aspirates, to the flow cytometry results in samples obtained form MM patients. We tested whether the noted discrepancy between these two methods applies only to MM, or represents a trend in other hematopoietic malignancies as well. We defined this discrepancy and explained it. Methods - The number of plasma cells or blasts from BM aspirates of 41 MM or seven acute myeloid leukemia (AML) patients respectively were analyzed simultaneously by morphological evaluation and by FCM. Each sample was assessed independently by two qualified laboratory specialists and/or hemato-pathologist. In MM we found plasma cell fractions that were characterized by FCM and gene expression profile. Results - In MM it was evident that FCM under-estimated the number of BM plasma cells samples by an average of 60%, compared with conventional morphological evaluation. On the other hand in AML there was a good correlation between the morphological and FCM assessments of the blast cell population, indicating that the discrepancy observed in the MM BM samples may be related to unique characteristics of the malignant plasma cells. Since flow cytometry is performed on the bone marrow fluid which is depleted of fat tissue-adhesive plasma cells, we disrupted spicules from MM BM samples (by repeated passages through 21g needle) and found a 40% increase in plasma cell compared with the fluid of the same BM samples. In order to determine the FCM profile of the cells in these two fractions, we isolated BM derived spicules from aspirates of MM patients and treated them with extracellular matrix (ECM) degrading enzymes followed by mechanical shearing. This combination released the highly adhesive plasma cells from the spicules. The released myeloma cells displayed a different FCM profile and in particular had a higher level of CD138 expression. Gene expression profile, which was performed on similar adhesion variants of cultured MM cells, demonstrated distinct oncogenic and transcriptional programs. Summary - We have shown a major discrepancy between the percentage of MM cells obtained by routine BM morphology and flow cytometry counts. It is possible that this discrepancy is partially attributable to the two distinct microenvironmental components occupied by MM cells in the BM sample - the lipid spicules, and the fluid phase. MM cells located in different niches of the BM also differ in their FCM and gene expression profile. This study indicates that multiple myeloma patients contain heterogeneous populations of malignant plasma cells. These sub-populations may play distinct roles in the biological and clinical manifestations of the disease and differ in their response to anti-myeloma therapy.

BP-2-047, a Novel Small-Molecule Inhibitor of Stat3 Is Active in Myeloma Pre-Clinical Models: Rationale for Treatment of Stat3-Dependent Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 576-576
Author(s):  
Victor Hugo Jimenez-Zepeda ◽  
Brent DG Page ◽  
Li Zhia Hua ◽  
Ellen Wei ◽  
Leif Bergsagel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cell Lines ◽  
Myeloma Cell ◽  
Luciferase Reporter ◽  
Stat3 Phosphorylation ◽  
Induced Apoptosis ◽  
Dose Dependent ◽  
Important Therapeutic Target

Abstract Abstract 576 Stat3 appears to play a critical role in sustaining and generating memory B-cells and long-lived plasma cells (PCs) the normal counterparts of myeloma cells. In contrast to the transient nature of Stat3 activation in normal cells, many hematological tumors including multiple myeloma (MM) harbor constitutive Stat3 activity. Aberrant STAT signaling is recognized as a master regulator of direct and indirect tumor processes including proliferation, apoptosis, invasion, angiogenesis and cancer inflammation and thus Stat3 represents an important therapeutic target. Using structure-based design, targeting the phosphotyrosine (pY)-SH2 domain interactions that stabilizes active Stat3 dimers, phosphopeptides have been identified that block Stat3 dimerization and DNA binding activity. Peptidic agents however suffer from limited cell permeability and metabolic lability, properties that have restricted their practical application in vivo at therapeutic doses. Here we describe the dependence of myeloma tumors on Stat3 and characterize the anti-tumor activity of BP-2-047, a novel, non-phosphorylated small molecule with reduced peptidic character. We analyzed the activity of Stat3 using a Stat3 gene expression index based on Stat3 target genes in 60 human myeloma cell lines (HMCLs). Several available myeloma cell lines (XG6, XG7, JJN3 and UTMC2) demonstrated elevated STAT3 gene expression signatures (2.3 to 14.8) with confirmed constitutive Stat3 phosphorylation by Western blot analysis. RNA interference revealed that Stat3 is essential for XG6, JJN3, and UTMC2 survival. We similarly analyzed the activity of Stat3 in publicly available gene expression profile data sets derived from newly diagnosed MM patients. Approximately a third of patients demonstrated a functionally significant level of Stat3 signature, comparable to those in the Stat3-dependent cell lines. We next evaluated the anti-myeloma activity of the BP-2-047 in cell-based assays against a panel of HMCLs with high and low STAT3 activity. BP-2-047 delivered a desirable activity profile for targeted therapy. Consistent with its predicted mechanism of action, we observed dose-dependent inhibition of Tyr705Stat3 phosphorylation in XG6, XG7, JJN3 and UTMC2 cells, presumably through the blockade of Stat3 binding to the pTyr motifs of gp130 and the prevention of de novo phosphorylation by JAKs. Using a luciferase reporter assay we confirmed inhibition of Stat3 transcriptional activity in MM cells stably expressing the Stat3-dependent luciferase reporter (pLucTKS3). Inhibition of transcription was associated with decreased expression of short half-life proteins, c-Myc and Mcl-1. BP-2-047 inhibited viability of myeloma tumors with IC50 values of 3.1–7.4 microM, in cell lines with constitutive Stat3 activity. Inhibition of Stat3 phosphorylation resulted in induction of dose-dependent apoptosis as determined by PI/annexin V staining and PARP cleavage. However, cells were resistant to BP-2-047-induced apoptosis when co-cultured with bone marrow stroma cells. Identification of factors that confer survival advantage in the presence of stroma is in progress. Exposure of MM patient derived bone marrow mononuclear cells to < 15 microM BP-2-047 preferentially induced apoptosis of CD138 + MM cells. In contrast, BP-2-047 at concentrations of 30 microM failed to inhibit normal bone marrow-derived CD34 colony formation. In vitro combination studies in cell lines demonstrate synergy with dexamethasone, bortezomib and lenalidomide. Studies assessing in vivo activity of BP-2-047 as a single agent and in combination with bortezomib against Stat3 activated myeloma tumors in xenograft mice are ongoing and will be reported. These data suggest that Stat3 may represent an important therapeutic target in a subset of myeloma patients and supports innovative drug development with a focus on Stat3. Disclosures: No relevant conflicts of interest to declare.

CD28-Mediated Pro-Survival Signaling in Multiple Myeloma

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 941-941
Author(s):  
Megan E Murray ◽  
Jayakumar R Nair ◽  
Kelvin P Lee
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Death ◽  
Plasma Cells ◽  
Cell Interactions ◽  
Complex Cell ◽  
Dose Dependent Fashion ◽  
Survival Signaling ◽  
Dose Dependent ◽  
Cell Cell

Abstract Abstract 941 Multiple myeloma (MM) is an incurable neoplasm of plasma cells which is critically dependent on the bone marrow for its survival. The microenvironment is able to support MM cells through a variety of mechanisms, including direct cell-cell interactions. Therefore, identifying and targeting these interactions or their downstream pathways would represent a novel mechanism for the treatment of MM. Unfortunately, many of these interactions remain poorly characterized. However, it stands to reason that any receptor or ligand which correlates with worse prognosis or disease progression could be a pro-survival molecule. One such receptor is CD28. Although best known as the prototypic T cell costimulatory receptor, CD28 has been shown to be expressed on normal plasma cells as well as MM cells. Additionally, we have previously shown that CD28 signaling is critical for both plasma cell and MM cell survival. To determine if CD28 signaling is important in the context of complex cell-cell interactions, we cultured MM cell line cells (MM1S) with monocyte-derived human dendritic cells (DC). We and others have previously shown that DCs infiltrate myelomatous portions of bone marrow and contribute to the clonogenicity of MM cells. Importantly, DCs also present the ligands for CD28, CD80 and CD86. To examine whether DCs can protect MM cells in a CD28-dependent fashion, we co-cultured the cells in the presence of melphalan, a clinically relevant chemotherapy. DCs were able to significantly increase the survival of MM cells (p<0.01) compared to MM cells alone; however, when CD28 was blocked (using a blocking antibody, CD28.6) or when CD80 and CD86 were blocked (using the fusion protein CTLA4-Ig), DC-mediated protection was completely abrogated (figure 1). These results demonstrate that even in complex MM-DC interactions, CD28 is the critically important pro-survival signaling molecule. In T cell biology, one of the critical signaling pathways downstream of CD28 is via PI3K and Akt. We have previously shown in MM cells that activating CD28 with an antibody causes the phosphorylation of PI3K. To determine whether or not this activation is indeed linked to survival, we cultured MM1S cells with a CD28 activating antibody ± serum and ± increasing doses of the PI3K inhibitor LY294002. As expected, when MM cells were cultured with a CD28 activating antibody, they survived the no-serum conditions. However, that survival was abrogated by LY294002 in a dose-dependent fashion, indicating that CD28 is mediating survival via PI3K activation. To determine whether Akt was the involved downstream molecule, the above experiment was repeated with an Akt inhibitor. Again, we observed that MM cells were protected by CD28 in the absence of serum, but that protection was abrogated by inhibiting Akt in a dose-dependent fashion. These experiments demonstrate that CD28 is delivering a pro-survival signal via PI3K-Akt. It has been well established in the myeloma literature that the balance of the pro-apoptotic molecule Bim determines apoptosis in MM cells. Importantly, Bim is transcriptionally regulated by the PI3K-Akt-FoxO3a axis. When FoxO3a is phosphorylated by Akt, it is excluded from the nucleus and is unable to upregulate Bim transcripts. To determine if CD28 was regulating FoxO3a, we cultured MM cells with a CD28 activating antibody and examined phospho-FoxO3a via western blot. Indeed, we observed that CD28 activation increases levels of phospho-FoxO3a. We next wanted to test whether or not CD28 activation had any effect on Bim transcript levels. We have observed via western blot and RT-PCR that CD28 activation suppresses Bim expression, and CD28 blockade increases Bim expression. To confirm that this plays a functional role in MM biology, we knocked down Bim using siRNA and cultured the cells without serum + CTLA4-Ig. Consistent with our expectations, when we culture control cells in the absence serum plus CD28 blockade, they die. However, when we knock down Bim, we see that the cells are resistant to CD28 blockade-mediated cell death. These data suggest that CD28 is able to regulate Bim via the PI3K-Akt-FoxO3a axis, and that Bim is critical for MM cell death or survival (figure 2). Taken together, these data demonstrate that CD28 is the critical pro-survival molecule even in the complex cell-cell interactions between MM cells and DCs. CD28 is mediating that prosurvival signal in a PI3K-Akt-FoxO3a-Bim dependent manner. Disclosures: No relevant conflicts of interest to declare.

GPRC5D Is a Cell Surface Plasma Cell Marker Whose Expression Is High In Myeloma Cells and Reduced Following Coculture With Osteoclasts

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3099-3099 ◽  
Author(s):  
Sathisha Upparahalli venkateshaiah ◽  
Rakesh Bam ◽  
Xin Li ◽  
Sharmin Khan ◽  
Wen Ling ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Surface ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Nuclear Fraction ◽  
Myeloma Cells ◽  
Cell Markers ◽  
Qrt Pcr

Abstract Identification of unique cell surface markers on myeloma cells is important for development of targeted therapies and detection of residual disease. GPRC5D is an orphan receptor reportedly expressed at high level in bone marrow (BM) aspirates or biopsies from myeloma patients. Other studies detected GPRC5D in skin and brain but not in other tissues. The aims of the study were to assess GPRC5D gene expression and cell surface localization in myeloma cells, the changes in expression of GPRC5D and other typical myeloma cell markers following coculture of primary myeloma cells with osteoclasts and the consequences of GPRC5D gene silencing on myeloma cell growth. Global gene expression profile (GEP), qRT-PCR and immunohistochemistry revealed exclusive high expression of GPRC5D in normal and myeloma plasma cells but not in B cells, MSCs, osteoclasts or BM mononucleated cells. GPRC5D expression was higher in myeloma plasma cells from newly diagnosed patients (n=698) compared to normal plasma cells (n=26, p<0.0001)). Among molecularly classified groups GPRC5D expression is higher in MS, MF and LB subgroups and lowest in CD2 and MM cell lines. Flow cytometry analysis and immunohistochemistry detected cell surface expression of GPRC5D in myeloma plasma cells while nuclear localization was also detected in certain myeloma cell lines (e.g. H929). Western blots analysis confirmed GPRC5D expression in whole cell lysate and nuclear fraction. We and other demonstrated phonotypical plasticity of myeloma plasma cells capable of altered expression of recognizable plasma cell markers (e.g. CD138, CD45) following coculture with stromal cells (Dezorella et al., 2009) or osteoclasts (Yaccoby, 2005). In coculture of primary CD138-selected myeloma cells with osteoclasts (n=8), CD138 (p<0,004), CD38 (p<0.001) and GPRC5D (p<0.007) were commonly and significantly downregulated, CD45 (p<0.02) was upregulated, and IRF4 expression was unaffected in cocultured myeloma cells compared to the control freshly obtained uncultured myeloma cells assessed by GEP and qRT-PCR. We also observed reduced expression of GPRC5D in MM cells purified from focal lesion compared to interstitial marrow in paired clinical samples (n=176, p<1.21E-09), suggesting that high activity of osteoclasts in osteolytic lesions mediates phenotypical alteration in myeloma cells. Stable knockdown of GPRC5D by 70% in CAG myeloma line using lentiviral particles containing shRNA had no effect on short-term growth of these cells assessed by MTT assay. These data indicate that GPRC5D is a novel cell surface marker for myeloma plasma cells and that its expression is reduced in dedifferentiated myeloma cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Targeting the Plasma Cell Specific Purine Metabolic Enzyme, AMPD1, Induces Multiple Myeloma Cell Death Accompanying NAD Depletion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3097-3097
Author(s):  
Yawara Kawano ◽  
Takayuki Sasano ◽  
Saki Kushima ◽  
Yuki Inada ◽  
Nao Nishimura ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cell Death ◽  
Cell Lines ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Annexin V ◽  
Hypoxic Condition ◽  
Metabolic Enzyme ◽  
Rt Pcr

Introduction. Novel agents have improved the prognosis of multiple myeloma (MM) patients. However, MM is still an incurable disease. In order to achieve a cure, it is necessary to develop novel therapeutic agents that are highly specific to MM cells and target different pathways from the present anti-MM agents used in the clinic. In the present study, we attempt to identify a specific molecule which is specifically expressed in plasma cells and MM cells, and to examine whether it can be a novel target for MM therapy. Materials and methods. Public available gene expression websites, GenomicScape and Genevestigator were utilized to study genes specifically expressed in human plasma cells and MM cells. To examine gene expression in MM cell lines, gene expression data set from the Cancer Cell Line Encyclopedia (CCLE) was analyzed. AMPD1 (AMP deaminase 1) gene expression in normal leukocytes and hematological malignancies were analyzed by RT-PCR. AMPD1 protein expression in MM cell lines and patient derived MM cells were examined by western blot and immunohistochemistry. Genes co-expressed with AMPD1 in MM cells were identified using public available gene expression datasets. Molecular pathway analysis was conducted using Molecular Signatures Database. Cell viability of MM cell lines, peripheral blood mononuclear cells (PBMCs) and patient derived bone marrow mononuclear cells treated by AMPD1 inhibitor (compound #3) (Admyre T et al. Chemistry & Biology. 2014; 21: 1486-1496.) were analyzed by flow-cytometry after staining with 7AAD. Intracellular NAD and NADH concentrations in MM cell lines were analyzed using NAD / NADH assay kit. Detection of apoptosis in MM cell lines were examined by Annexin V and PI staining followed by flow cytometry analysis. Z-VAD-FMK (Caspase inhibitor) and Nec-1 (RIP1 kinase inhibitor) were used in combination with compound #3 to study the mechanism of AMPD1 inhibition induced MM cell death. Results. We identified several genes specifically expressed in human plasma cells and MM cells using public available gene expression websites. Among the identified genes, we focused on AMPD1, a purine metabolic enzyme that converts adenosine monophosphate (AMP) to inosine monophosphate (IMP), since this gene has not been previously studied in MM. We found that AMPD1 gene expression was limited to MM cell lines and patient derived MM cells through CCLE analysis and RT-PCR. AMPD1 protein expression was detected only in MM cell lines, bone marrow MM cells and extramedullary plasmacytomas. Genes associated with AMPD1 expression were related to hypoxic pathways. MM cell lines cultured under hypoxic condition had significantly higher AMPD1 expression compared to those cultured under normoxia, indicating that AMPD1 plays a significant role in MM cell surviving under hypoxic condition such as the bone marrow microenvironment. Compound #3 induced cell death in MM cell lines and patient derived MM cells, while toxicity against PBMCs and non-MM cells were minimal. Compound #3 was more effective against MM cell lines cultured under hypoxia compared to those under normoxia, reflecting the higher AMPD1 expression under hypoxia. Since, AMPD1 is associated with purine metabolism, we analyzed the intracellular concentration of NAD and NADH, which are major cellular metabolites, in MM cell lines post compound #3 treatment. Marked decrease of NAD concentration and NAD / NADH ratio was observed in compound #3 treated MM cell lines compared to control, demonstrating that AMPD1 inhibition depletes intracellular NAD leading to MM cell death. Compound #3 treated MM cell lines showed increase in Annexin V- PI+ and Annexin V+ PI+ fractions. Additionally, Z-VAD-FMK and Nec-1 treatment did not reverse cell death induced by compound #3, indicating a distinct mechanism of cytotoxicity by compound #3 from other anti-MM agents inducing apoptosis and necroptosis. Conclusions. This is the first report so far that AMPD1 is specifically expressed in MM cells. AMPD1 inhibitor showed specific cytotoxicity against MM cells, while toxicities against non-MM cells were minimal. Additionally, AMPD1 inhibitor was more effective under hypoxic condition, suggesting that AMPD1 inhibition works more efficiently in the bone marrow microenvironment. Considering the specificity against MM cells and its distinct mechanism of action from the present anti-MM agents, AMPD1 inhibition is a potent novel therapeutic strategy for MM. Disclosures Matsuoka: Bristol-Myers Squibb Corp.: Research Funding; Kyowa Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria.

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