Targeting Glycolysis in Multiple Myeloma: Novel Strategies in the Treatment of Proteasome Inhibitor Resistant in Hypoxic Conditions

Introduction: Multiple myeloma (MM) is one of the hematological malignancy and characterized by the clonal expansion of plasma cells in the bone marrow. The treatment of MM patients has been dramatically changed by new agents such as proteasome inhibitors and immunomodulatory drugs, however, many patients will relapse even if new agents provide therapeutic advantages. Therefore, a new strategy is still needed to increase MM patient survival. Hypoxia is an important component of the bone marrow microenvironment. Hypoxia may increase myeloma cell survival. Because cells shift primarily to a glycolytic mode for generation of energy in hypoxic conditions, glycolytic activities can be targeted therapeutically in MM patients. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) is responsible for maintaining the cellular levels of fructose-2,6-bisphosphate which is a regulator of glycolysis. Materials and Methods: In this study, we investigated whether PFKFB was involved in myeloma cells in hypoxia condition. We also investigated whether PFKFB inhibitors could suppress myeloma cells and enhance the sensitivity of myeloma cells to proteasome inhibition. Results: We first investigated the expression of PFKBP in the myeloma cell lines in hypoxia condition. PFKFB family contains four tissue-specific isoenzymes encoded by four different genes. We found expression of PFKBP3 and PFKBP4 were increased in hypoxia condition. We found gene expression of PFKBP3 and PFKBP4 were involved in myeloma cell lines and myeloma patient samples in hypoxia condition from the public microarray datasets (GSE80140 and GSE80545). In hypoxia condition, expression of hypoxia-inducible factor 1α (HIF1α) was increased and phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was activated in myeloma cell lines. Expression of PFKBP3 and PFKBP4 were inhibited by HIF1α inhibitor and p38 MAPK inhibitor treatment. In the hypoxia condition, activity of proteasome inhibitors were reduced compared to normoxia condition. We next investigated whether PFKBP3 inhibitor, PFK158 and PFKBP4 inhibitor, 5MPN could inhibit the proliferation of myeloma cells. We found PFK158 and 5MPN treatment inhibited the growth of myeloma cells in a dose dependent manner in hypoxia condition. Combined treatment of myeloma cells with carfilzomib and PFK158 or 5MPN caused more cytotoxicity than each drug alone. Caspase 3/7 activity and cellular cytotoxicity was also increased. We found proteasomal activity was also reduced by carfilzomib and PFK158 or 5MPN treatment. Adenosine triphosphate (ATP) is the most important source of energy for intracellular reactions. Intracellular ATP levels drastically decreased after carfilzomib and PFK158 or 5MPN treatment. Because mitochondria generate ATP and participate in signal transduction and cellular pathology and cell death. The quantitative analysis of JC-1 stained cells changed mitochondrial membrane potential in cell death, which were induced by carfilzomib and PFK158 or 5MPN on myeloma cells. In the hypoxia condition and inhibitor treatment, glycolytic activities (e.g. glucose and lactate) were changed in myeloma cells. Conclusion: The PFKBP3 and PFKBP4 are enhanced in hypoxia condition and involved in proteasome inhibitor sensitivity. Our data also suggested that administration of PFKBP3 and PFKBP4 inhibitors may be a powerful strategy against myeloma cells and enhance cytotoxic effects of proteasome inhibitors in hypoxia condition. Disclosures No relevant conflicts of interest to declare.

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Therapeutic targeting of PFKFB3 and PFKFB4 in multiple myeloma cells under hypoxic conditions

10.21203/rs.3.rs-1091276/v1 ◽

2021 ◽

Author(s):

Seiichi Okabe ◽

Yuko Tanaka ◽

Akihiko Gotoh

Keyword(s):

Bone Marrow ◽

Plasma Cells ◽

Combined Treatment ◽

Proteasome Inhibitors ◽

Myeloma Cell ◽

Mitogen Activated Protein Kinase ◽

Gene Expressions ◽

New Agents ◽

Myeloma Cells ◽

Hypoxic Conditions

Abstract BackgroundMultiple myeloma (MM) is a hematological malignancy characterized by the clonal expansion of plasma cells in the bone marrow. The treatment of MM patients has been dramatically changed by new agents, such as proteasome inhibitors and immunomodulatory drugs; however, many patients will relapse, even if the new agents provide therapeutic advantages. Hypoxia is an important component of the bone-marrow microenvironment. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) is responsible for maintaining cellular levels of fructose-2,6-bisphosphate, which regulates glycolysis.MethodsIn this study, we investigated the PFKFB functions in myeloma cells under hypoxic conditions. We also investigated whether PFKFB inhibitors could suppress myeloma cells and enhance their sensitivity to proteasome inhibition.ResultsWe first investigated the expression of PFKFBs in the myeloma cell lines under hypoxic conditions. Using public microarray datasets (GSE80140 and GSE80545), we found that the gene expressions of PFKFB3 and PFKFB4 were elevated under hypoxic conditions. Hypoxia-inducible factor 1α (HIF1α) was increased, and the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was activated. Under hypoxia, the activity of proteasome inhibitors was reduced. The PFKFB3 inhibitor, PFK158 and PFKFB4 inhibitor, 5MPN treatment were found to inhibit the growth of myeloma cells. The combined treatment of myeloma cells with carfilzomib and PFK158 or 5MPN was more cytotoxic than each drug alone. Caspase 3/7 activity and cellular cytotoxicity were also increased. In addition, we found that proteasomal activity was reduced by carfilzomib and PFK158 or 5MPN treatment. Intracellular adenosine triphosphate (ATP) levels drastically decreased after combined treatment. The combined treatment also changed the mitochondrial membrane potential in cell death and was effective on the bortezomib-resistant cell line.ConclusionPFKFB3 and PFKFB4 are enhanced in hypoxic conditions and are involved in proteasome-inhibitor sensitivity. Our data also suggested that administration of PFKFB3 and PFKFB4 inhibitors may be a powerful strategy against myeloma cells and may enhance the cytotoxic effects of proteasome inhibitors in hypoxic conditions.

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A Role for Syntenin-1 in Multiple Myeloma Cell Survival

Blood ◽

10.1182/blood-2018-99-113594 ◽

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.

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

Blood ◽

10.1182/blood.v104.11.1419.1419 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1419-1419

Author(s):

Soraya Wuilleme-Toumi ◽

Nelly Robillard ◽

Patricia Gomez-Bougie ◽

Philippe Moreau ◽

Steven Le Gouill ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Disease Severity ◽

Cell Lines ◽

Plasma Cells ◽

Myeloma Cell ◽

Lymphocytic Leukemia ◽

Myeloma Cells

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

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Upregulation of MMP-2 & 9 by Co-Culture of Human Myeloma Cell Lines and Endothelial Cells May Be Responsible for Protection Against Cytotoxic Drugs.

Blood ◽

10.1182/blood.v108.11.5080.5080 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5080-5080

Author(s):

Shankaranarayana Paneesha ◽

Raghu Adya ◽

Hemali Khanji ◽

Ed Leung ◽

C. Vijayasekar ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Endothelial Cells ◽

Mrna Expression ◽

Cell Lines ◽

Myeloma Cell ◽

Myeloma Cells ◽

Human Myeloma Cell

Abstract Multiple myeloma is a clonal lymphoproliferative disorder characterised by the proliferation of plasma cells in the bone marrow. Inspite of good initial response, it is associated with universal relapse. We hypothesise this is due to sanctuary provided to myeloma cells by the endothelium. Matrix metalloproteinases (MMPs) are shown play a role in cell growth, invasion, angiogenesis, metastasis and bone degradation. We show here the protection offered by endothelial cells to human myeloma cell lines in in-vitro co-culture with upregulation of MMP-2 & 9 and the role of GM6001 MMP inhibitor (Ilomastat) in overcoming this protection. Human myeloma cell lines (H929, RPMI 8226, U266 & JJN3) with or without endothelial cells (human umbilical vein endothelial cells and EaHy 926 cell line) in-vitro co-culture were treated with melphalan, dexamethasone, arsenic trioxide and Ilomastat. Cytotoxicity/proliferation were assessed by the alamarBlue™ assay (Serotec) and validated by Annexin V-FITC apoptosis detection Kit (Calbiochem) and BrDU proliferation assay (BD Pharmingen™). Gelatin Zymography was used to demonstrate activity of MMP-2 & 9 in the supernatant. MMP-2 and 9 mRNA expression was quantified by Real Time Quantitative PCR (ROCHE). Co-culture of human myeloma cell lines with endothelial cells lead to increase in the proliferation of myeloma cell lines and also protected them from the cytotoxicity of chemotherapeutic agents. MMP-2 & 9 activity was upregulated by the co-culture. MMP-2 mRNA expression in human myeloma cell lines increased following 4 hr co-culture. Treatments with Ilomastat lead to the suppression of proliferation in co-culture in a dose dependent manner, associated with a reduction of MMP-2 and 9 activity. Our study shows endothelial cells offer protection to human myeloma cell lines in the presence of cytotoxic agents. This may result in the sanctuary of myeloma cells in bone marrow leading to ultimate relapse of disease. Our study also demonstrates the upregulation of MMP-2 and 9 by co-culture and increased cytotoxicity achieved by the inhibition of MMPs. Further studies are needed to determine the exact role of MMPs in myeloma biology as MMP inhibition may be an interesting therapeutic target and help in averting relapse in multiple myeloma.

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Survival Signals, Growth Cytokines, Immunosuppressive Factors and Their Cellular Perpetrators in the Myeloma Tumor Microenvironment.

Blood ◽

10.1182/blood.v112.11.1664.1664 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1664-1664

Author(s):

Jayakumar R Nair ◽

Louise M Carlson ◽

Noreen Ersing ◽

Asher Alban Chanan-Khan ◽

Kelvin P. Lee

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Tumor Microenvironment ◽

Cell Lines ◽

Plasma Cells ◽

Immune Escape ◽

Human Monocyte ◽

Fold Increase ◽

Myeloma Cell ◽

Myeloma Cells

Abstract Multiple myeloma (MM) is an incurable neoplasia of terminally differentiated plasma cells in the bone marrow. Essential interactions of MM cells with host bone marrow stromal cells (BMSC) induce growth factors essential for MM progression and pathogenesis, as well as induce an immunosuppressive environment that inhibits endogenous and therapeutically-induced immune responses against the MM cells. However, despite their importance, little is known about the identity of these BMSC cells or the molecular basis of their interaction with myeloma cells. A potential MM surface protein that could be involved in these interactions is CD28, based on its known pro-survival role in T cells. Clinical studies have shown that expression of CD28 in multiple myeloma highly correlates (p=0.006) with myeloma disease progression. Moreover, CD28+ MM cells invariably express the CD28 ligand CD86. A survival role for MM-CD28 might involve interactions with cellular partners that express the B7 (CD80/CD86) ligands. Potential candidates would include CD86+ myeloma cells themselves or B7+ dendritic cells (DC) that are known to be closely associated with myeloma cells in the patient bone marrow. When myeloma-myeloma interactions were disrupted by using the high affinity CD80/CD86 blocker CTLA4Ig (Abatacept®), increased sensitivity to arsenic trioxide (ATO) and melphalan (MEL) was observed in all the three MM cell lines U266, RPMI8226 and MM1S. For U266 viability was 93% in media alone, 84% with CTLA4Ig (100 μg/ml) alone, 86% with 2 μM ATO alone and was significantly reduced to 36% with CTLA4Ig + ATO. Similar drops in viability were observed with 25 μM MEL in combination with CTLA4Ig (33% as opposed to 71–74 % with CTLA4Ig or MEL alone). Our data suggests that this does not involve the downregulation of anti-apoptotic proteins Bcl-2, Bcl-xL or Mcl-1, commonly associated with drug resistance in myeloma. In the second part of the study, we demonstrate that myeloma cell lines or primary CD138+ myeloma cells can enhance via direct contact the ability of human monocyte derived immature DC to produce the immunosuppressive tryptophan depleting enzyme indoleamine 2,3 dioxygenase (IDO, as estimated by kynurenine (Kyn) (a tryptophan catabolite) levels in the supernatant) and also the pro-plasma cell survival cytokine IL-6. In co-cultures of IFNg treated immature DCs with either MM cell lines or with primary CD138+ myeloma cells from patient BM aspirates, the activity of IDO was enhanced ~ 2–8 fold (81 mM kyn with U266 and 20–43mM with primary cells) over that observed in control IFNg-treated DCs (9.7 mM Kyn). Western analysis also demonstrated increased IDO expression relative to IFNg activated DC controls. Blocking MM-CD28 with (Fab)2 fragments of anti-hCD28 mAb 9.3 downregulated IDO activity (9.3 mM) close to that of control, demonstrating the involvement of MM-CD28 in these interactions. We also demonstrated a significant up-regulation of the pro-myeloma survival cytokine IL-6 when immature DCs were co-cultured with CD28+ MM1S (90–300 pg/ml), a 4–9 fold increase over that of DC only control (25 – 35 pg/ml). This was further enhanced when immature DCs cultured with IL-10 (+ GM-CSF + IL-4) was used in co-cultures with MM-1S (800 – 1300 pg/ml), or with primary CD138+ myeloma cells from patient bone marrow aspirates (128–1142 pg/ml). In conclusion, our data demonstrates that blocking myeloma-CD28 - myeloma-CD86 “autocrine” interaction can enhance drug cytotoxicity, while interactions with DCs produce the essential growth cytokines IL-6 and immunosuppressive enzyme IDO with potential implications in MM survival and immune escape. Use of clinically approved agents (e.g. Abatacept®) to block myeloma-CD28 binding to its B7 ligands (increase chemotherapeutic efficacy), 1-MT to inhibit IDO and targeting DCs in the microenvironment to disrupt the tumor microenvironment could be viable therapeutic strategies for the future treatment of multiple myeloma.

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A Novel Mouse Model of Multiple Myeloma Representative of Human Disease and Its Use in Preclinical Therapeutic Assessment

Blood ◽

10.1182/blood.v118.21.2907.2907 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2907-2907

Author(s):

Rosemary A Fryer ◽

Timothy J Graham ◽

Emma M Smith ◽

Brian A Walker ◽

Gareth J Morgan ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Flow Cytometry ◽

Cell Lines ◽

Myeloma Cell ◽

Human Condition ◽

Myeloma Cells ◽

Treatment Groups ◽

Hyperintense Signal

Abstract Abstract 2907 In order to aid the pre-clinical development of novel therapeutics for multiple myeloma, an in vivo model which recapitulates the human condition in particular tumor growth patterns and response to treatment is required. An important feature of such a model is the interaction of the myeloma cells with the bone marrow microenvironment as this is known to modulate tumor activity and protect against drug-induced apoptosis. We have developed a model with myeloma restricted to the bone marrow, which proceeds rapidly from initial inoculation to disease progression, and possesses a range of chemo-sensitive markers with which to monitor anti-tumor response. Female NOD/SCID γcnull mice were injected inta-osseously with luciferase-tagged myeloma cell lines. Disease progression was monitored weekly by bioluminescent imaging (BLI) and measurement of paraprotein levels (ELISA). These methods were compared to histological assessment of tumor infiltration and MRI which provided a quantitative measurement of progression. On T2-weighted images tumor was identified as a hyperintense signal enclosed within cortical bone. Tumor burden was quantified from regions of interest drawn on the periphery of the hyperintense signal. Luciferase-tagged cells engrafted by 3 weeks at the injection site and progressed to the femurs, spine and pelvis from week 4. BLI showed a significant increase in radiance from 5.6×105 to 43.0×105p/s/cm2/sr between weeks 5 and 7 (p<0.05). Quantification of tumor volume by MRI showed a significant increase from 6.4mm3 to 27.6mm3 between weeks 4 and 8 (p<0.05) and μCT demonstrated lytic disease. Serum levels of Igλ increased from 860ng/ml to 4325ng/ml during this period (p<0.05), which mirrored the changes seen with BLI and MRI. Flow cytometry and histology confirmed the confinement of CD138 positive myeloma cells within the bone. These results indicate successful engraftment of human myeloma cell lines with induction of myeloma in a pattern similar to the human condition. We have adapted this model to study primary patient material. 10 mice were implanted with samples from 3 cases of plasma cell leukemia with complex cytogenetics. 5 of these developed myeloma confined to the bone marrow, 2 with additional plasmacytoma localized at the injection site, over a period of 1–5months. We have characterized the original patient cells with gene expression, SNP based gene mapping and have characterized the nature of the engrafted cells using similar technology. We have also shown the model is suitable for preclinical assessment of anti-myeloma agents using bortezomib and a novel aminopeptidase inhibitor, tosedostat (CHR-2797). Non-treated mice displayed a significant increase in radiance from 16.13×105 to 69.00×105p/s/cm2/sr (p<0.01). In comparison, in the bortezomib and tosedostat treated groups no significant increase in radiance was seen (bortezomib: 5.22×105 to 1.12×105 p/s/cm2/sr; tosedostat: 9.92×105 to 13.78×105p/s/cm2/sr). Paraprotein levels mimicked these changes in BLI. At the end of treatment Igλ levels in control, bortezomib and tosedostat treated mice were 2473.7, 132.5 and 923.0ng/ml, respectively. Igλ levels in both treatment groups were significantly different from control (p<0.001). Average tumor volumes derived from MRI were significantly different in bortezomib (14.7mm3) and tosedostat treated (23.4mm3) groups compared to non-treatment (33.0mm3). The volumes for the bortezomib treated group showed no significant difference from control mice. In addition, there was a decrease in CD138 expression by flow cytometry in bone aspirates from treatment groups compared to control which was mirrored in histological samples. In conclusion using both myeloma cell lines and primary patient cells, we have developed a model which recapitulates human myeloma with secretion of paraprotein, disease confined to the bone marrow, lytic bone lesions and spinal compression. In addition, this model is suitable for assessing the efficacy of novel therapeutics in vivo, using a number of non-invasive tumor markers such as BLI and MRI. Disclosures: Morgan: J&J: Honoraria, Speakers Bureau. Davies:J&J: Honoraria, Speakers Bureau.

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Inhibition of p-glycoprotein does not increase the efficacy of proteasome inhibitors in multiple myeloma cells

10.1101/2020.07.16.206102 ◽

2020 ◽

Author(s):

Rachel L. Mynott ◽

Craig T. Wallington-Beddoe

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Proteasome Inhibitor ◽

Proteasome Inhibitors ◽

Specific Inhibitor ◽

Myeloma Cell ◽

Drug Transporter ◽

Myeloma Cells ◽

Multiple Myeloma Cell ◽

P Glycoprotein

AbstractThe aim of this study is to determine whether manipulation of the drug transporter P-glycoprotein improves the efficacy of proteasome inhibitors in multiple myeloma cells. P-glycoprotein is a well-known drug transporter that is associated with chemotherapy resistance in a number of cancers but its role in modulating the efficacy of proteasome inhibitors in multiple myeloma is not well understood. Research has shown that the second generation proteasome inhibitor carfilzomib is a substrate of P-glycoprotein and as such its efficacy may correlate with P-glycoprotein activity. In contrast to carfilzomib, research concerning the first-in-class proteasome inhibitor bortezomib is inconsistent with some reports suggesting that inhibition of P-glycoprotein increases bortezomib cytotoxicity in multiple myeloma cells whereas others have shown no effect. Through the mining of publicly available gene expression microarrays of patient bone marrow, we show that P-glycoprotein gene expression increases with the disease stages leading to multiple myeloma. However, RNA-seq on LP-1 cells treated with bortezomib or carfilzomib demonstrated minimal basal P-glycoprotein expression which did not increase with treatment. Moreover, only one (KMS-18) of nine multiple myeloma cell lines expressed P-glycoprotein, including RPMI-8226 cells that are resistant to bortezomib or carfilzomib. We hypothesised that by inhibiting P-glycoprotein, multiple myeloma cell sensitivity to proteasome inhibitors would increase, thus providing a potential approach to improving responses and reversing resistance to these agents. However, the sensitivity of multiple myeloma cells lines to proteasome inhibition was not enhanced by inhibition of P-glycoprotein with the specific inhibitor tariquidar. In addition, targeting glucosylceramide synthase with eliglustat did not inhibit P-glycoprotein activity and also did not improve proteasome inhibitor efficacy except at a high concentration. We conclude that P-glycoprotein is poorly expressed in multiple myeloma cells, its inhibition does not enhance the efficacy of proteasome inhibitors, and it is unlikely to be a useful avenue for further translational research.

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Induction of Apoptosis in Multiple Myeloma by Ligands of Peroxisome Proliferator-Activated Receptor γ (PPAR-γ).

Blood ◽

10.1182/blood.v104.11.4848.4848 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4848-4848

Author(s):

Jan Eucker ◽

Katharina Baengeroth ◽

Ivana Zavrski ◽

Holger Krebbel ◽

Chuanbing Zang ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Cell Lines ◽

Myeloma Cell ◽

Peroxisome Proliferator ◽

Peroxisome Proliferator Activated Receptor ◽

Myeloma Cells ◽

Ppar Γ ◽

Induction Of Apoptosis ◽

Rpmi 8226

Abstract Peroxisome proliferator-activated receptor γ (PPAR-g) is a member of a nuclear receptor superfamily, which is expressed in different tumor tissues. Activation of PPAR-γ by its ligands has been shown to reduce tumor growth, interfere with tumor cell differentiation, and induce apoptosis in a variety of human malignancies including solid tumors like colon, breast, lung, liver, prostate cancer, as well as hematological malignancies like myeloid leukemia. Recently, it has been shown that both human B-lymphocytes and B-lymphomas express PPAR-γ and induce apoptosis. 15-deoxy-delta-12,14-prostaglandin J2 (15d-PGJ2) is a natural activator of PPAR-γ. Thiazolidinediones, including troglitazone, rosiglitazone (RGZ), and pioglitazone (PGZ), comprise a group of synthetic PPAR-γ agonists that are currently in use for the treatment of type 2 diabetes mellitus, and have revealed anti-tumor activity in vitro. We investigated in five human multiple myeloma cell lines (LP-1, U-266, RPMI-8226, OPM-2 and IM-9) and sorted human bone marrow myeloma cells whether treatment with PGZ, RGZ or 15d-PGJ2 inhibited tumor cell growth. Expression of PPAR-γ protein was demonstrated by western blot analysis in these cell lines. All 5 cell lines were sensitive to the PPAR-γ agonists. MTT assays revealed growth arrest induced by the natural activator of PPAR-γ 15d-PGJ2 and a lower antiproliferative effect with PGZ and RGZ in a dose dependent manner. At a dose of 50 μM 15d-PGJ2 cell proliferation was reduced to values between 0% and 26% in all multiple myeloma cell lines tested. In most cell lines the anti-proliferative effect was already detectable at 10 μM. At a dose level of 50 μM PGZ cell proliferation was reduced in MTT assay after 48 hours of incubation to 48% in LP-1, 52% in IM-9, 56% in OPM-2, 72% in U-266 and 77% in RPMI-8226. Comparable results were obtained with RGZ. Induction of apoptosis was indicated by annexin V staining. Cell lines were incubated with 50 μM of PPAR-γ agonists, a concentration which had been proven to be effective for growth inhibition in MTT assay before. Again, 15-dPGJ2 was more effective than PGZ and RGZ. All of the 15d-PGJ2 treated cell lines revealed specific apoptosis ranging between 60% and 92%. Apoptosis induced by PGZ in U-266, RPMI-8226-S, IM-9, and OPM-2 cell lines ranged between 17% and 43%, for RGZ it ranged between 20% and 50%. Furthermore, in sorted bone marrow plasma cells from myeloma patients induction of apoptosis was detected. Bone marrow multiple myeloma cells from five different patients were tested. The specific apoptosis rate induced by 15-dPGJ2 lay between 29% and 96%. Apoptosis induced by PGZ showed interindividual differences. In the myeloma cells from four patients the rate of specific apoptosis ranged between 9% and 28%, but in one patient induction of apoptosis was observed neither with PGZ nor with RGZ. For RGZ, the rate of apoptosis induced in the myeloma cells from the other four patients ranged between 7% and 26%. The rate of specific apoptosis induced by 15D-PGJ2 was not statistically different for sorted human bone marrow myeloma cells sensitive versus refractory to conventional chemotherapy with anthracyclines and alkylating agents (p = 0.8). This is one of the first studies evaluating PPAR-γ expression and its therapeutical implications in human multiple myeloma cells. Thiazolidinediones comprise anti-myeloma activity and should be explored further for the treatment of multiple myeloma.

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Rational Drug Design: Proteasome Inhibitor Mediated Down-Regulation of the FA/BRCA Pathway Is Synergistic with PARP Inhibition in Myeloma Cell Lines

Blood ◽

10.1182/blood.v118.21.2922.2922 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2922-2922

Author(s):

Liang Nong ◽

Linda Mathews ◽

Mark B Meads ◽

William Dalton ◽

Kenneth H. Shain

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Dna Repair ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Myeloma Cell ◽

Sequential Treatment ◽

Parp Inhibition ◽

Repair Pathway ◽

Myeloma Cells

Abstract Abstract 2922 The development of new and biologically-based therapeutic regimens is critical for the successful control, if not cure, of multiple myeloma. Incorporation of the novel agents, including the proteasome inhibitor bortezomib, harbored large strides in disease modification. However, even with the success of bortezomib containing regimens, drug resistance and disease relapse remain inevitable. As such, it is critical that we use preclinical models to not only develop drugs, but also to consider strategies for co-development of novel drug combinations capitalizing on complementary biological activities. Our investigations in drug resistance recently revealed that increased homologous recombination (HR) potential, via over-expression of the FA/BRCA DNA repair pathway (FA/BRCA pathway), contributed to acquired melphalan-resistance in myeloma cell lines.(Yarde et al 2009) Drug resistance was causally linked to a novel transcriptional regulation of the FA/BRCA by NF-κB. Further examination demonstrated that bortezomib attenuated this component of the HR repair pathway and reversed melphalan resistance. To this end, we anticipated that bortezomib treatment may sensitize cells to inhibitors of complementary DNA repair pathways in a manner similar to the synthetic lethality elicited in by PARP1/2 inhibition in BRCA1 or FANCD1/ BRCA2 mutant cancers.(Farmer 2005, Bryant 2005) Consistent with this rationale, treatment of myeloma cells with bortezomib and the PARP inhibitor AZD2281/olaparib demonstrated synergism in specific myeloma cell lines. Pre-treatment of RPMI8226 myeloma cells with bortezomib for 6 hours greatly enhanced myeloma cell sensitivity to PARP inhibition with AZD2281/olaparib. The inhibitory concentration(IC)-50 was decreased by 17.7-fold (n=3; IC50 AZD2281 alone: 62.7 microM (39.0–84.0) and pretreated with bortezomib 3.54 microM (2.4–4.6)). Combination Index (CI) demonstrated a mean of 0.41 in 8226 and 0.43 in U266 myeloma cells, consistent with a synergistic relationship. Further analysis confirmed that synergism correlated with decreased expression of FANCD2 mRNA and protein by 6 hours. In contrast to sequential treatment, concomitant treatment with these agents did not elicit the synergistic phenotype. Interestingly, sequential treatment of NCIH929 myeloma cells did not demonstrate the same synergistic response (CI :0.89, slight synergism). Consistent with this, treatment of NCIH929 cells with bortezomib did not negatively regulate FANCD2 mRNA or protein expression, suggesting that FA/BRCA pathway can be differentially regulated in myeloma cells. To more specifically determine if FANCD2 was a key factor regulated by bortezomib, we targeted FANCD2 with siRNA. Pretreatment of myeloma cells with FANCD2 siRNA also sensitized cells to AZD2281/olaparib relative to siRNA control (IC50: 19.0 microM vs 35.0 microM n=4; p<0.05). These results show that bortezomib (or other proteosome inhibitors) and AZD2281/olaparib (or other PARP inhibitors) may represent an exciting new combination therapy for myeloma. We are currently examining the applicability of these studies to other proteosome inhibitors and the clinical relevance with ex vivo studies with myeloma patient samples. We believe that data presented here are innovative as they introduce a novel biological rationale, the abrogation complementary pathways in DNA damage repair, for the preclinical development of novel targeted drug combinations in myeloma. Further, we anticipate that although this study has focused on multiple myeloma, the results of the proposed research will be applicable to a wide range of hematologic and solid tumors. Disclosures: No relevant conflicts of interest to declare.

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Hexokinase-2 Regulates Hypoxia-Inducible Autophagy, Leading to Enhance Anti-Apoptotic Capability of Refractory Multiple Myeloma

Blood ◽

10.1182/blood-2019-123360 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1787-1787

Author(s):

Sho Ikeda ◽

Fumito Abe ◽

Matsuda Yuka ◽

Akihiro Kitadate ◽

Takahiro Kobayashi ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Cell Lines ◽

Research Funding ◽

Pharmaceutical Research ◽

Proteasome Inhibitors ◽

Myeloma Cell ◽

Hypoxic Stress ◽

Myeloma Cells ◽

Viable Cells

(background) The drug resistance of multiple myeloma (MM) cells is thought to be induced by various factors of the bone marrow microenvironment. Of these factors, hypoxic stress may be associated with drug resistance in various hematologic malignancies, including MM. Hypoxic stress lead MM cells to induce distinct gene expressions. It has been reported that oncogenic transcription factors such as IRF4 and Myc are suppressed under hypoxia. Instead, accumulation of another transcription factor, HIF-1α upregulates anti-apoptotic proteins, increases glycolysis, and enhances neovascularization leading MM cells to represent anti-apoptotic phenotype. Autophagy is an intracellular process that encapsulates cytoplasmic components, which are directed to the lysosome for degradation. Autophagy and proteasomal degradation prevent apoptosis caused by endoplasmic reticulum (ER) stress. Although proteasome inhibitor such as bortezomib, is a key drug for MM, it may induce treatment resistance. This might be because autophagy is induced in hypoxic microenvironment. Autophagy associated molecules might be therapeutic target in MM cells adapted to hypoxia. (Aim and methods) To clarify the association of hypoxia inducible genes and autophagy, and to obtain rational basis for a new therapeutic strategy against MM, we performed following experiments in vitro using myeloma cell lines (MM.1S, KMS-12-PE, KMS-11, and H929) and primary samples (n=6) that were subjected to hypoxia (1% O2). (Results) First, we examined volcano plot analysis on our cDNA microarray data (GSE80545) of patient samples incubated in normoxia or hypoxia for 48 hours. 546 probes were significantly elevated in hypoxia (fold change > 2.0, p < 0.05). Gene ontology analysis revealed that "Glycolytic Process" contained 13 genes such as PFKFB4, ENO2, ALDOC, PFKFB3, HK2, PFKP, GPI, PGK1, LDHA, ALDOA, ENO1, PKM, and GAPDH. We focused on hexokinase-2 (HK2) because it has been reported that HK2 activates autophagy under stress conditions. Western blot analysis for patient samples revealed that HK2 expression was remarkably upregulated under hypoxia. Apoptosis assay showed that viable cells of HK2 knockdowned cell lines were significantly lower than that of control cells under hypoxia, but not under normoxia. Also, in hypoxia, we found that number of 3-bromopyruvate (3-BrPA, a HK2 inhibitor) subjected viable cells were significantly lower than that of normoxia. This suggested that HK2 contributes to anti-apoptotic phenotype of MM cells under hypoxia. Next, we examined the role of HK2 in autophagy under hypoxia. Because degradation of p62 and increase of LC3-II/LC3-I ratio is considered to be useful for autophagy detection, we examined these factors by Western blot analysis. We found that hypoxic stress decreased expression of p62 and increased the ratio of LC3-II/LC3-I in myeloma cell lines, indicating that hypoxia activates autophagy. However, under hypoxia, these changes were canceled by HK2 knockdown. We confirmed that the number of autophagosome were significantly decreased in HK2-knockdowned myeloma cells by electron microscopy analysis. These data suggested that HK2 is required for hypoxia-inducible autophagy in MM. Finally, we examined the effect of combined inhibition of HK2 and proteasome. In hypoxia, apoptosis by bortezomib was significantly increased in HK2-knockdowned myeloma cells when compared with control. Moreover, we found that the combination of 3-BrPA and bortezomib increased apoptotic cells in both normoxia and hypoxia. These results suggested that HK2-inhibition can induce apoptosis against MM cells with enhancement of sensitivity to proteasome inhibitors. (Conclusion) These results suggest that hypoxia induced HK2 promotes autophagy and inhibits apoptosis. Thus, the combination of proteasome inhibitors and HK2 inhibition may bring about a deep response against treatment resistant MM. Disclosures Ikeda: Nippon Shinyaku Research Grant: Research Funding. Takahashi:Bristol-Myers Squibb: Speakers Bureau; Eisai Pharmaceuticals: Research Funding; Pfizer: Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding; Chug Pharmaceuticals: Research Funding; Ono Pharmaceutical: Research Funding; Novartis Pharmaceuticals: Research Funding, Speakers Bureau; Astellas Pharma: Research Funding; Asahi Kasei Pharma: Research Funding.

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