scholarly journals Overcoming the Supportive Stroma-Induced Proliferation in Waldenstrom's Macroglobulinemia By Selective Inhibition of the FGF/FGF-Receptor Axis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1822-1822
Author(s):  
Cinzia Federico ◽  
Antonio Sacco ◽  
Katia Todoerti ◽  
Arianna Giacomini ◽  
Gaia C Ghedini ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Growth ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Research Funding ◽  
Significant Inhibition ◽  
Advisory Committees ◽  
Dose Dependent

The human fibroblast growth factor receptor (FGF-R) family plays an essential role in a wide range of cellular processes, such as cell growth, proliferation, differentiation, migration and survival. It has been reported that FGF-Rs are expressed in hematopoietic cells; and FGF/FGFR signaling deregulation is largely involved in hematologic malignancies, including Waldenström macroglobulinemia (WM). WM is still an incurable disease, and patients succumb due to disease progression. Therefore, novel therapeutics designed to specifically target deregulated signaling pathways in WM are required. We aimed to investigate the role of FGF/FGF-R system in FGF-dependent WM cell lines by using an anti-pan FGF trap molecule (NSC12), responsible for FGF/FGF-R blocking. We first interrogated the GSE9656 dataset in order to confirm the expression of FGFs and FGF-Rs in WM cells, demonstrating an enrichment of several FGF- and FGF-R-isoforms in primary WM patients' derived tumor cells compared to the normal cellular counterpart (P<0.05); and demonstrated the ability of NSC12 to inhibit FGF-secretion within the conditioned media of NCS12-treated WM cells, as shown by ELISA. Wide-transcriptome profiling of NSC12-treated WM cells (BCWM.1; MWCL1) revealed a significant inhibition of Myc-target related genes, coupled with silencing of genes involved in cell cycle progression, cell proliferation, PI3K-AKT-mTOR signaling, oxidative phosphorylation (Hallmark; FDR<0.25; P<0.05). This prompted us to evaluate the anti-tumor functional sequelae exerted by NSC12 in WM cells: NSC12 induced significant inhibition of WM cell growth (BCWM1 and WMCL1) in a dose-dependent fashion (0.1-10μM; IC50 ~3μM), even in the presence of bone marrow microenvironment. In addition, a significant effect was also observed in primary tumor cells from WM patients; while no effect was observed on healthy donor-derived peripheral blood mononuclear cells. The growth inhibitory effect was associated with induction of apoptotic cell death, caspase activation and PARP cleavage, as demonstrated by flow cytometry and western blot, respectively. Moreover, we also observed a NSC12 dose-dependent increase of mitochondrial reactive oxigen species (ROS), at protein level. Cell cycle analysis revealed a reduction of the S-phase and increase of G0/G1 phase. Mechanistically, NSC12 targeted WM cells by inhibiting MAPK, JAK/STAT3 and PI3K-Akt pathways known to be FGFRs-activated signaling cascades. Importantly, the same effect was maintained in WM cells even in the presence of the supporting BM microenvironment. Functional studies demonstrated the ability of NSC12 to impair the adhesion of both cell lines to the supportive primary bone marrow stromal cells, in vitro. NCS12-dependnet anti-WM activity was also tested in combination with bortezomib, carfilzomib, everolimus and ibrutinib: the combinatory treatment (48h) resulted in a more significant dose-dependent inhibition of WM cell survival and proliferation (P<0.05), thus suggesting the rational for combining FGF-blockade with proteasome-, mTOR-, or BTK-inhibitors. In vivo studies are being performed, in order to further corroborate the anti-WM activity of NSC12 using WM animal models. Disclosures Ronca: Associazione Italiana per la Ricerca sul Canctro (AIRC): Research Funding. Rossi:Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Roccaro:AstraZeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; European Hematology Association: Research Funding; Associazione Italiana per al Ricerca sul Cancro (AIRC): Research Funding; Transcan2-ERANET: Research Funding; AstraZeneca: Research Funding; European Hematology Association: Research Funding; Transcan2-ERANET: Research Funding.

Role of TORC1 and TORC2 in Multiple Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1815-1815
Author(s):  
Patricia Maiso ◽  
Yi Liu ◽  
Abdel Kareem Azab ◽  
Brittany Morgan ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Stromal Cells ◽  
Research Funding ◽  
Plasma Cells ◽  
Advisory Committees ◽  
Cycle Arrest

Abstract Abstract 1815 Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 (Raptor) and TORC2 (Rictor). TORC1 leads to the phosphorylation of p70S6 kinase and 4E- BP1, while TORC2 regulates phosphorylation of Akt and other kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin and its analogues have not shown significant activity in MM, likely due to the lack of inhibition of TORC2. In this study, we dissected the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. Methods: Eight different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, immunochemistry, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. In vivo homing was checked by in vivo flow. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: Raptor (TORC1) and Rictor (TORC2) knockdowns led to significant inhibition of proliferation of MM cells even in the presence of bone marrow stromal cells, this effect was also accompanied by inactivation of p-Akt, p-rS6 and p-4EBP1. We used INK128, a dual and selective TORC1/2 kinase inhibitor with similar effects to Raptor plus Rictor knockdown. We examined the protein expression levels of both mTOR complex and their downstream effectors in MM plasma cells from patients and cell lines. mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all samples. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin) even in the presence of cytokines or stromal cells. INK128 induced cell cycle arrest, autophagy and apoptosis in cell lines and primary plasma cells even in the presence of bone marrow stromal cells (BMSCs). INK128 also showed a significant effect inhibiting cell adhesion in our in vivo homing model. Oral daily treatment with INK128 highly decreased the percentage of CD138+ tumor plasma cells in mice implanted with MM cells and reduced the levels of p-Akt and p-4EBP. These results suggest that potent and complete blockade of mTOR as part of TORC1 and TORC2 is potential therapeutic strategy to induce cell cycle arrest, apoptosis and disruption of MM cells interaction with the BM microenvironment. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Liu: Intellikine: Employment. Roccaro:Roche: Research Funding. Rommel:Intellikine: Employment. Ghobrial:Celgene: Consultancy; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals The KDM3A-KLF2-IRF4 Axis Maintains Myeloma Cell Survival

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3633-3633
Author(s):  
Hiroto Ohguchi ◽  
Teru Hideshima ◽  
Manoj Bhasin ◽  
Gullu Gorgun ◽  
Loredana Santo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Growth ◽  
Cell Survival ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Plasma Cells ◽  
Chip Assay ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Histone methylations are tightly regulated by a balance between methyltransferases and demethylases that mediate the addition and removal of these modifications. Importantly, dysregulation of histone methylation is implicated in pathogenesis of cancers, including multiple myeloma (MM). For example, the t(4;14) (p16;q32) is present in 15 - 20% of MM patients and results in overexpression of WHSC1, a histone H3 lysine 36 (H3K36) methyltransferase. On the other hand, approximately 10% of MM patients without the t(4;14) have inactivating mutations in KDM6A, a H3K27 demethylase. KDM3A is a Jumonji C-domain-containing histone demethylase which catalyzes removal of H3K9 mono- and dimethylation (H3K9me1 and H3K9me2). KDM3A is implicated in pathogenesis of different types of cancers. Here we investigated the biological impact of KDM3A in MM. KDM3A expression was significantly elevated in MM patient samples compared to normal plasma cells in publicly available dataset (GSE5900, GSE6691). To evaluate the functional role of KDM3A, shRNAs targeting KDM3A were transduced into MM cell lines: knockdown of KDM3A significantly inhibited MM cell growth (RPMI8226, MM.1S, U266, H929) in vitro and in xenograft model (MM.1S). Apo2.7 staining showed that apoptotic cells were significantly increased after knockdown of KDM3A. We next examined gene expression profiles after knockdown of KDM3A in RPMI8226 cells. With a cutoff of > 1.5-fold downregulation, a total of 305 probe sets were downregulated in KDM3A-knockdown cells relative to control cells. Among putative KDM3A targets, a gene of particular interest is KLF2 which plays a key role in maintenance of B cell and plasma cell phenotype, and function. Another intriguing gene is IRF4, given its known crucial role in MM cell survival. We confirmed that expression of KLF2 and IRF4 was downregulated after knockdown of KDM3A by quantitative realtime PCR and immunoblots in RPMI82226, MM.1S, and U266 cells. KDM3A binding to KLF2 and IRF4 core promoters was demonstrated by chromatin immunoprecipitation (ChIP) assay in RPMI8226 cells. Moreover, knockdown of KDM3A increased H3K9me1 and me2 levels at both promoter regions, indicating that KDM3A directly regulates KLF2 and IRF4 expression by removing H3K9 methylation marks at their promoters in MM cells. shRNAs targeting KLF2 were next transduced into MM cell lines: silencing of KLF2 significantly reduced cell growth of MM cell lines, associated with decreased IRF4. Promoter reporter assays using human IRF4 promoter showed that KLF2 significantly increased luciferase expression in a dose-dependent manner. Moreover, ChIP assay showed that KLF2 bound to IRF4 promoter in RPMI8226 cells. Since transcription factors could form an autoregulatory feedback loop, we hypothesized that IRF4 might regulate KLF2 expression. As expected, knockdown of IRF4 downregulated KLF2 expression at both the mRNA and protein levels in 3 MM cell lines. In addition, ChIP assays demonstrated that IRF4 bound to KLF2 second intron that contains tandem IRF4 motifs in RPMI8226 cells. Collectively, these results suggest that KLF2 activates IRF4 expression and vice versa, forming an autoregulatory loop in MM cells. KLF2 has been reported to control homing of plasma cells to the bone marrow; we therefore hypothesized that KDM3A-KLF2-IRF4 axis might regulate adhesion and homing of MM cells to the bone marrow. Importantly, knockdown of KDM3A, KLF2, or IRF4 decreased adhesion of 3 MM cell lines to bone marrow stromal cells. Furthermore, bone marrow homing of MM.1S cells was significantly reduced after knockdown of KDM3A, KLF2, or IRF4 in a murine xenograft MM model, indicating that KDM3A-KLF2-IRF4 axis regulates, at least in part, MM cell adhesion and homing to the bone marrow. In conclusion, our study demonstrated that KDM3A is a crucial epigenetic regulator of MM cell survival, and that inhibition of KDM3A represents a novel therapeutic strategy in MM. Disclosures Raje: Amgen: Consultancy; Takeda: Consultancy; Novartis: Consultancy; Celgene Corporation: Consultancy; BMS: Consultancy; Acetylon: Research Funding; Eli Lilly: Research Funding; Onyx: Consultancy; AstraZeneca: Research Funding; Millenium: Consultancy. Richardson:Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees. Harigae:Chugai Pharmaceutical Co., Ltd.: Research Funding. Anderson:Oncopep: Equity Ownership; Gilead: Consultancy; BMS: Consultancy; Millennium: Consultancy; Celgene: Consultancy; Acetylon: Equity Ownership.

scholarly journals The Lysine-Specific Demethylase KDM4A/JMJD2A Acts As a Tumor Suppressor in Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 191-191
Author(s):  
Fengyan Jin ◽  
Shaji K. Kumar ◽  
Yun Dai
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Tumor Suppressor ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Committees ◽  
Apoptotic Genes

Abstract Introduction: Histone lysine methylation, a reversible event dynamically and reciprocally regulated by lysine methyltransferases (KMTs) and demethylases (KDMs), represents one of the major epigenetic mechanisms for regulation of chromatin remodeling and gene expression re-programming. The KDM4 family, which belongs to the Jumonji C (JmjC)-domain-containing proteins (JMJDs), consists of five members, including KDM4A-E that demethylate H3K9me2/3 and/or H3K36me2/3 in a Fe2+- and α-ketoglutarate-dependent manner. KDM4 proteins are involved in various cellular processes such as gene transcription and translation, DNA replication, DNA repair, apoptosis, and stem cell renewal. Notably, increasing evidence implicates KDM4 dysregulation in promoting genomic instabilities and oncogenesis, thereby which is considered as a potential target for emerging cancer epigenetic therapy. Although KDM4A, a member of the KDM4 family, has been widely studied in many solid tumors including breast, prostate, bladder cancer, its role in hematopoietic malignancies, including multiple myeloma (MM), remains unknown. Materials and Methods: Human MM cell lines (U266, RPMI8226, H929, OPM-2) were employed. After exposed to hypoxia (or the chemical hypoxia mimetic lactic acid) and anti-MM agents (e.g., bortezomib/Btz), cells were analyzed by flow cytometry, qPCR, Western blot to monitor apoptosis, cell cycle, proliferation (Ki67), DNA double-strand break/DSB (γH2A.X), expression of 1q21 and anti-apoptotic genes, as well as activation of the NF-κB and HIF pathways. The shRNA approach was used to knock down KDM4A for functional evaluation. The findings from in vitro experiments involving cell lines were then validated in primary MM samples to link KDM4A expression to disease progression and therapeutic response. Results: Analysis of the MM genome-wide GEP databases revealed that KDM4A mRNA was significantly up-regulated in MGUS and MM, but not SMM, compared to normal control, as well as in relapsed MM, compared to newly-diagnosed MM. To our surprise, KDM4A expression rather favored overall survival of MM patients, including those carrying 1q21 gain in whom KDM4A expression was indeed lower than those who did not have this high risk cytogenetic abnormality. Moreover, KDM4A expression correlated adversely with expression of 1q21 genes (e.g., CKS1B, MCL1, PSMD4, ARNT). Whereas basal KDM4A protein level was moderately but clearly higher in MM cell lines carrying 1q21 gain or acquired drug resistance than their counterparts, exposure to hypoxia or lactic acid (but not cobalt chloride) resulted in marked KDM4A up-regulation, accompanied by NF-κB and HIF pathway activation. However, while NF-κB inhibition and to a lesser extent ARNT/HIF-1β knockdown led to a robust increase in hypoxia-induced KDM4A expression, shRNA knockdown or pharmacological inhibition of KDM4A triggered NF-κB activation and HIF expression, as well as up-regulated anti-apoptotic proteins (e.g., Mcl-1, TNFAIP3/A20, CKS1B), in association with increased H3K36me3 rather than H3K9me3. Furthermore, KDM4A knockdown or inhibition sharply diminished Btz lethality and overrode hypoxia-mediated cytoprotection. Interestingly, KDM4A knockdown also increased MM cell proliferation, promoted S phase entry, and attenuated Btz-induced DSB. Last, IHC of sequential bone marrow biopsies revealed that while KDM4A protein was relatively low at diagnosis, its level was markedly increased when patients achieved CR and then fell to the baseline low level at relapse. Conclusion: KDM4A/JMJD2A, a lysine demethylase that has been recognized as an pro-oncogenic protein via its epigenetic and/or non-epigenetic properties, is identified for the first time as a potential tumor suppressor in MM, particularly in a high risk subtype carrying 1q21 gain. Whereas KDM4A is expressed in MM and can be further induced by hypoxia that naturally exists in bone marrow niche, it seems to play multiple inhibitory roles in cell growth, cell cycle, DNA repair, and drug resistance by suppressing expression of oncogenic and anti-apoptotic genes (especially 1q21 genes), likely via H3K36me3 demethylation, and antagonizing NF-κB and HIF activation. These findings suggest that in contrast to its pro-oncogenic role in certain solid tumors, KDM4A might instead act as a tumor suppressor in MM. This work was supported by NNSFC (81471165, 81670189, and 81670190). Disclosures Kumar: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Inhibition of the Proteasome in Bone Marrow-Derived CD138+ Tumor Cells Following Carfilzomib Administration in Relapsed or Refractory Myeloma Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1845-1845
Author(s):  
Suzanne Trudel ◽  
Susan Lee ◽  
Christopher J. Kirk ◽  
Nashat Gabrail ◽  
Sagar Lonial ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Whole Blood ◽  
Research Funding ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Advisory Committees ◽  
Post Dose ◽  
Blood Samples

Abstract Abstract 1845 Poster Board I-871 Background: Proteasome inhibition is an effective strategy for the treatment of multiple myeloma. In patients, proteasome inhibition has primarily been measured in peripheral blood samples (whole blood or mononuclear cells). However, it is unknown whether myeloma cells in the bone marrow (BM) are equally sensitive to proteasome inhibitors such as bortezomib (BTZ) and carfilzomib (CFZ). Aim: To measure proteasome inhibition in purified tumor cells from BM samples taken from patients enrolled in two ongoing Phase 2 trials of single agent CFZ in relapsed or refractory myeloma: PX-171-003 (003) and PX-171-004 (004). Methods: CFZ was administered as an IV bolus of 20 mg/m2 on Days 1, 2, 8, 9, 15 and 16 of a 28-day cycle on both trials. Bone marrow samples, from an optional sub-study of both trials, were taken during screening and Day 2 (post-treatment) and sorted into CD138+ and CD138− cells. Proteasome activity was measured by an enzymatic assay using a fluorogenic substrate (LLVY-AMC) for the chymotrypsin-like (CT-L) activity and an active site ELISA (ProCISE) to quantitate levels of the CT-L subunits of the constitutive proteasome (Beta5) and immunoproteasome (LMP7) and the immunoproteasome subunit MECL1. Results: Whole blood samples from patients treated with CFZ showed inhibition of CT-L activity of ∼80+, similar to values obtained in Phase 1 studies. A total of 10 CD138+ screening samples, 6 from 004 and 4 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed for proteasome levels and activity. In addition, 15 CD138−screening samples, 7 from 004 and 8 from 003, and 9 post-dose samples, 5 from 004 and 4 from 003, were analyzed. When compared to the average base-line activity, CFZ treatment resulted in 88% CT-L inhibition in CD-138+tumor cells from 004 patients (P = 0.0212 by unpaired t-test) and 59% CT-L inhibition in CD-138+ tumor cells from 003 patients (P = 0.25). Baseline CT-L activity in CD138+ tumor cells was 3-fold higher in 004 than 003, which includes a more heavily pre-treated patient population with greater prior exposure to BTZ. Higher specific enzymatic activity was due to increased levels of both constitutive and immunoproteasomes in tumor cells, where immunoproteasomes account for >75% of total cellular proteasomes. No differences between trials were seen in baseline CT-L activity from non-tumor (CD138−) cells. Inhibition in CD138− cells was 84% (P = 0.0380 and 42% (P = 0.38) in 004 and 003, respectively. Using ProCISE, we measured inhibition of LMP7 (66%), beta5 (48%) and MECL1 (64%) in CD138+ tumor cells from 004 patients. Three patients from 004 and one from 003 had both a screening and post-dose tumor cell samples available for analysis. Inhibition of CT-L activity was >80% in two of the 3 patients on 004; the third patient showed no proteasome inhibition by ProCISE and was unavailable for analysis by CT-L. CT-L activity in the CD138+ tumor cells in the 003 patient was not inhibited, however, inhibition was seen in non-tumor cells. Conclusions: CFZ inhibits the proteasome activity of myeloma cells in the bone marrow of relapsed and refractory myeloma patients. The levels of inhibition were similar to those measured in whole blood samples, supporting the use of the blood-based assay as a surrogate marker for proteasome inhibition in tumor cells. CFZ treatment resulted in inhibition of both CT-L subunits as well as additional subunits of the immunoproteasome in tumor cells. Reduced baseline activity in the more heavily pretreated 003 patients may reflect reduced tumor-dependency on the proteasome and may be related to prior treatment with BTZ in these patients. More samples are needed in order to make correlations between levels of proteasome inhibition in bone marrow tumor cells and prior therapies or response. These observations support further evaluation of proteasome activity and the effects of this promising new agent in primary tumors cells from myeloma patients. Disclosures: Trudel: Celgene: Honoraria, Speakers Bureau; Ortho Biotech: Honoraria. Lee:Proteolix, Inc.: Employment. Kirk:Proteolix, Inc.: Employment. Lonial:Celgene: Consultancy; Millennium: Consultancy, Research Funding; BMS: Consultancy; Novartis: Consultancy; Gloucester: Research Funding. Wang:Proteolix, Inc.: Research Funding. Kukreti:Celgene: Honoraria. Stewart:Genzyme, Celgene, Millenium, Proteolix: Honoraria; Takeda, Millenium: Research Funding; Takeda-Millenium, Celgene, Novartis, Amgen: Consultancy. Jagannath:Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. McDonagh:Proteolix: Research Funding. Zonder:Celgene: Speakers Bureau; Pfizer: Consultancy; Seattle Genetics, Inc.: Research Funding; Amgen: Consultancy; Millennium: Research Funding. Bennett:Proteolix: Employment.

Dual Targeting of -TORC1 and -TORC2 as a New Strategy In the Treatment of Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 133-133 ◽  
Author(s):  
Patricia Maiso ◽  
AbdelKareem Azab ◽  
Yang Liu ◽  
Yong Zhang ◽  
Feda Azab ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Board Of Directors ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Plasma Cells ◽  
Bone Marrow Microenvironment ◽  
Advisory Committees

Abstract Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

CYC065, a Potent Derivative of Seliciclib Is Active In Multiple Myeloma In Preclinical Studies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2999-2999 ◽  
Author(s):  
Samantha Pozzi ◽  
Diana Cirstea ◽  
Loredana Santo ◽  
Doris M Nabikejje ◽  
Kishan Patel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Preliminary Data ◽  
Research Funding ◽  
Preclinical Studies ◽  
Dependent Manner ◽  
Advisory Committees

Abstract Abstract 2999 Multiple myeloma (MM) is a treatable but incurable hematological malignancy and novel targeted therapies are under investigation. MM is characterized by dysregulation of the cell cycle, consequent to the overexpression of cyclins and their related kinases, the cyclins dependent kinases (CDK), a group of Ser/Thr proteine kinases. CDKs represent a promising therapeutic target, and inhibitors have been developed for anticancer treatment. We have previously studied seliciclib in the context of MM. CYC065, a second generation CDK inhibitor is the more potent derivative of seliciclib. It is mainly active on CDK 2, 5 and 9, involved in progression of the cell cycle and protein transcription. It has already shown promising results in preclinical studies in breast cancer and acute leukemia. We tested CYC065 in in vitro experiments in MM. Our preliminary data in 7 MM cell lines showed cytotoxicity of CYC065, both in MM cell lines sensitive as well as resistant to conventional chemotherapy, with an IC50 ranging between 0.06 and 2μ M, at 24 and 48h. Tritiated thymidine uptake assay confirmed the antiproliferative effects of CYC065 in MM, and its ability to overcome the growth advantage conferred by co-culture with bone marrow stromal cells derived from MM patients, and cytokines like interleukin 6 (10ng/ml) and insulin like growth factor-1 (50ng/ml). The anti-proliferative effect was evident both at 24 and 48h, starting at concentrations as low as 0.015μ M. The AnnexinV/PI assay in the MM1.s cell line confirmed CYC065's ability to induce apoptosis in a time dependent manner starting at 9 hours of treatment, at a concentration of 0.125 μ M, inducing 82% of apoptosis after 48h of exposure. Cell cycle analysis in the same MM1.s cell line showed an increase of subG1 phase, starting at 9 hours of treatment, at 0.125 μ M of CYC065. Preliminary results of western blot analysis confirmed the apoptotic effect of CYC065 in the MM1s cell line, highlighted by the cleavage of caspase 3, 8, 9 and PARP. The compound was tested in primary CD138+ cells isolated from three refractory MM patients, confirming its efficacy at 0.125 μ M, both at 24 and 48h. Comparative analysis in PBMCs from normal donors, for the evaluation of the drug toxicity is ongoing and will be presented. In conclusion our preliminary data confirm the efficacy of CYC065 in MM cell lines and primary MM cells, at nanomolar concentrations. Ongoing mechanistic and in vivo studies will delineate its role in the now increasing spectrum of CDK inhibitors in MM and better define its potential for clinical development in MM. Disclosures: Green: Cyclacel: Employment. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Scadden:Fate Therapeutics: Consultancy, Equity Ownership, Patents & Royalties. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Acetylon: Research Funding.

A Novel Acanthoic Acid Analog NPI-1342 Blocks IκB Kinase-α and Trigger In Vitro and In Vivo cytotoxicity in Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1841-1841
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Arghya Ray ◽  
Teru Hideshima ◽  
Paul G. Richardson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Growth ◽  
Board Of Directors ◽  
Tumor Cells ◽  
Cell Lines ◽  
Inhibitory Effect ◽  
Advisory Committees ◽  
Acid Analog

Abstract Abstract 1841 Introduction: The dimeric Nuclear Factor-kappa B (NF-κB) transcription factor plays a key role during multiple myeloma (MM) cell adhesion-induced cytokine secretion in bone marrow stromal cells, which in turn triggers MM cell growth in a paracrine manner. NF-κB signaling pathway is mediated via canonical (IKK-α/IKK-β/NEMO-P50/65 or NF-κB1) and non-canonical (IKK-α/IKK-α/NIK-p52/RelB or NF-κB2) components. Prior studies have also linked constitutive activation of non-canonical NF-κB pathway to genetic abnormalities/mutation, allowing for an autocrine growth of MM cells. Other recent studies showed that constitutive NF-κB activity in tumor cells from MM patients renders these cells refractory to inhibition by bortezomib; and in fact, that bortezomib induces canonical NF-κB activity. These reports provided the impetus for the development of an agent with ability to modulate canonical and/or non-canonical NF-κB axis, allowing for a more robust and specific inhibition of NF-κB. Recent research and development efforts at Nereus Pharmaceuticals, Inc., have identified a novel small molecule acanthoic acid analog NPI-1342 as a potent NF-κB inhibitor. Here, we examined the effects of NPI-1342 on canonical versus non-canonical NF-κB signaling pathways, as well as its anti-tumor activity against MM cells using both in vitro and in vivo model systems. Methods: We utilized MM.1S, MM.1R, RPMI-8226, U266, KMS12PE, NCI-H929, OCI-MY5, LR5, Dox-40, OPM1, and OPM2 human MM cell lines, as well as purified tumor cells from patients with MM. Cell viability assays were performed using MTT and Trypan blue exclusion assays. Signal transduction pathways were evaluated using immunoblot analysis, ELISA, and enzymology assays. Animal model studies were performed using the SCID-hu model, which recapitulates the human BM milieu in vivo. Results: We first examined the effects of NPI-1342 on lipopolysaccharides (LPS)-induced NF-κB activity. Results showed that NPI-1342 inhibits LPS-stimulated NF-κB activity in vitro, as measured by phosphorylation of IkBa. To determine whether NPI-1342 triggers a differential inhibitory effect on IKKβ versus IKKα, MM.1S MM cells were treated with NPI-1342 for 48 hours, and protein lysates were subjected to kinase activity assays. NPI-1342 blocked IKKα, but not IKKβ or IKKγ phosphorylation. We next assessed whether the inhibitory effect of NPI-1342 on NF-κB activity is associated with cytotoxicity in MM cells. We utilized a panel of MM cell lines: at least five of these have mutations of TRAF3 (MM.1S, MM.1R, DOX40 and U266); one has no known NF-κB mutations (OPM2), and one has amplification of NF-κB1 (OCI-MY5). Treatment of MM cell lines and primary patient (CD138 positive) MM cells for 48 hours significantly decreased their viability (IC50 range 15–20 μM) (P < 0.001; n=3) without affecting the viability of normal peripheral blood mononuclear cells, suggesting selective anti-MM activity and a favorable therapeutic index for NPI-1342. NPI-1342-induced a marked increase in Annexin V+ and PI- apoptotic cell population (P < 0.001, n=3). Mechanistic studies showed that NPI-1342-triggered apoptosis in MM cells is associated with activation of caspase-8, caspase-9, caspase-3, and PARP cleavage. We next examined the in vivo effects of NPI-1342 in human MM xenograft models. For these studies, we utilized the SCID-hu MM model, which recapitulates the human BM milieu in vivo. In this model, MM cells are injected directly into human bone chips implanted subcutaneously in SCID mice, and MM cell growth is assessed by serial measurements of circulating levels of soluble human IL-6R in mouse serum. Treatment of tumor-bearing mice with NPI-1342 (20 mg/kg intraperitoneally, QD1-5 for 2 weeks), but not vehicle alone, significantly inhibits MM tumor growth in these mice (10 mice each group; P = 0.004). The doses of NPI-1342 were well tolerated by the mice, without significant weight loss. Finally, immunostaining of implanted human bone showed robust apoptosis and blockade of NF-κB in mice treated with NPI-1342 versus vehicle alone. Conclusions: We demonstrate the efficacy of a novel small molecule inhibitor of NF-κB NPI-1342 in MM using both in vitro and in vivo models. NPI-1342 blocks NF-κB activity with a preferential inhibitory activity against IKK-α component of NF-κB signaling. Our preclinical studies support evaluation of NPI-1342 as a potential MM therapy. Disclosures: Hideshima: Acetylon: Consultancy. Richardson:Millennium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Palladino:Nereus Pharmaceuticals, Inc: Employment, Equity Ownership. Anderson:Celgene: Consultancy; Millennium: Consultancy; Onyx: Consultancy; Merck: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acetylon:; Nereus Pharmaceuticals, Inc: Consultancy.

scholarly journals Dissecting Signaling Network Responses to PAK4 Allosteric Modulators (PAMs) in Cell Subsets within Primary Human Acute Myeloid Leukemia Samples

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3686-3686
Author(s):  
Paul Brent Ferrell ◽  
William Senapedis ◽  
Alexander Cook ◽  
Erkan Baloglu ◽  
Yosef Landesman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Board Of Directors ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Leukemia Stem Cells ◽  
Allosteric Modulators ◽  
Advisory Committees ◽  
Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults and has a poor outcome with limited treatment options in patients with relapsed or resistant disease. Therapy resistance in AML is likely related to the inadequacy of therapy within leukemia cell subsets, including leukemia stem cells (LSCs). The p21-activated kinase (PAK) family of proteins was shown to be overexpressed in cancer cells and to play a key role in proliferation, survival, and maintenance of cellular structure. The series of orally bioavailable PAK4 allosteric modulators (PAM) have previously been shown to have activity in hematological cancer cell lines, including those derived from acute myeloid leukemia (AML) (Senapedis et al. Blood124, 2208-2208). Understanding how therapies target cellular subsets within primary patient samples could aid drug development by revealing any subset specific drug effects. In this project, we studied the effects of p21-activated kinase 4 (PAK4) modulation in AML samples. PAK4 modulation has been shown to have significant effects on many intracellular signaling pathways, including PI3K/AKT, MAPK/ERK and WNT/β-catenin pathways (Senapedis et al. Blood124, 2208-2208). It is unknown whether PAMs will have similar activity in primary leukemia cells. Likewise, it is currently unclear to what extent PAMs will differentially impact primary cell subsets including leukemia stem cells and non-malignant cell subsets that may be critical to recovery of bone marrow functions. We have previously shown that the single cell biology platform of flow cytometry is well-suited for dissecting clinically relevant signaling network mechanisms in primary human AML (Irish et al. Cell, 118(2):217-28). Methods: Flow cytometry was used to dissect the impact of an orally bioavailable PAM in AML cell lines and primary patient tissue. Cell lines chosen for this study included NRAS mutant KG-1 and Kasumi-1, which carry t(8;21) and express the AML1:ETO fusion protein. Primary AML biopsies were acquired from bone marrow or blood prior to any treatment and patients were identified and consented for this study according to a local Institutional Review Board-approved protocol. AML tissue samples were viably cryopreserved and then assayed ex vivo. Established protocols were used for phospho-specific flow cytometry, fluorescent cell barcoding, and data analysis in Cytobank (Irish et al. Cell, 118(2):217-28, Doxie and Irish, Curr Top Microbiol Immunol. 377:1-21). Results: Differential effects of PAK4 inhibition were observed between cell lines and among cell subsets from AML patient bone marrow. In leukemia cell lines and patient samples, p-ERK and p-S6 showed marked inhibition via PAM, though degree of inhibition varied. In AML patient samples, PAMs blocked signaling responses in p-ERK specifically in AML blasts, but spared normal CD45hi mononuclear cells (0.88 vs. 0.29-fold reduction (arcsinh scale) in p-ERK at 10 nM). Within the AML blast population, CD34+ CD38- and CD34+ CD38+ AML subsets showed similar PAM dose response via p-ERK. Conclusions: Single cell analysis effectively distinguishes effects of PAK4 inhibition via a series of allosteric modulators of PAK4 (PAMs) on leukemia and non-leukemia subsets in the same sample. PAM reduced immediate p-ERK and p-S6 levels in primary leukemia and cell lines. Notably, inhibition in various subsets within human AML was successfully measured by phospho-flow cytometry. Signaling changes in p-ERK were minimal within non-leukemic mature CD45+ mononuclear cells found in primary patient biopsies. Analysis of CD34+ CD38- cells indicates that PAMs could have activity within leukemia stem cells, and, at least, effect the AML progenitors. These findings support further investigation into the mechanism of action and treatment potential of PAMs in AML. Disclosures Senapedis: Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Baloglu:Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Landesman:Karyopharm: Employment. Irish:Novartis: Honoraria; Cytobank, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; InCyte: Research Funding. Savona:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Activity of Carfilzomib (CFZ) in Acute Myeloid Leukemia (AML) As a Single Agent and in Novel Combinations

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-7
Author(s):  
Mao Yu Peng ◽  
Yasmin Abaza ◽  
Martina Mcdermott ◽  
Monica Mead ◽  
Dennis J. Slamon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Synergistic Effect ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Fatty Acid Synthase ◽  
Single Agent ◽  
Therapeutic Strategies ◽  
Private Company ◽  
Advisory Committees

Background:Recent advances in targeted therapy have expanded the available therapeutic optionsfor patients with AML. However, many patients still have suboptimal outcomes, particularly in the relapsed/refractory setting, underscoring the need for novel therapeutic strategies. Proteasome inhibitors (PIs), such as bortezomib, exhibit antitumor activity in AML through inhibition of the nuclear factor κB pathway and induction of apoptosis. CFZ, a second-generation PI, has preferential preclinical activity in AML compared to bortezomib making it an agent of interest in AML therapy. Here we assessed the activity of CFZ as a single agent and in novel combinations with Ara-C and/or other agents targeting potential vulnerabilities in AML cell lines. Methods:20 AML cell lines were treated with a single dose of CFZ for 7 days, proliferation inhibition was measured using an IC50 cutoff for CFZ of 10 nM. 2 sensitive (ML2 and MV411) and 2 resistant (AML193 and NOMO1) cell lines were selected for further analysis. Apoptosis, cell cycle, and cell senescence analysis were performed after 72 hours of CFZ exposure at 10 nM. Combination assays using CFZ 10 nM and Ara-C 200 nM were performed to evaluate for potential interaction in the form of antagonism or potentiation. Proteomic analysis was performed at baseline using reverse phase protein assay (RPPA). Cell lines were aligned according to CFZ IC50. Several proteins involved in various physiological pathways exhibited a potential correlation with CFZ sensitivity. Combination treatments with CFZ and agents targeting these pathways were carried out in selected cell lines. Results:Single-agent CFZ induced apoptosis with apoptotic rates &gt;85% in sensitive cell lines and only 10% in resistant cell lines. Similarly, CFZ resulted in G0/G1 cell cycle arrest in sensitive, but not resistant AML cell lines. Lack of difference in cellular senescence confirmed apoptosis as the major mechanism of CFZ-induced growth inhibition in AML cell lines. No antagonism was noted when CFZ was combined with Ara-C. RPPA revealed that AML cell lines with higher expression of autophagy-related proteins (Atgs) were more resistant to CFZ treatment. Combining autophagy inhibitor hydroxychloroquine (HCQ) or ROC-325 with CFZ produced a synergistic effect to induce apoptosis in several CFZresistant cell lines. RPPA also revealed that lower basal levels of fatty acid synthase (FASN), a key enzyme involved in lipogenesis, correlated with CFZ sensitivity and CFZ resistant lines tendedto have higher basal FASN levels. The combination of CFZ with a FASN inhibitor resulted in a significant synergistic apoptosis-inducing effect that was observed in the AML lines tested. Conclusion:CFZ demonstrated single agent activity in the nanomolar range in human AML cell lines. The addition of standard-of -care chemotherapy to CFZ did not show antagonism. Combining CFZ with agents targeting autophagy or lipid-metabolism showed synergistic effect in apoptosis. These results suggest a role for CFZ in combination therapeutic strategies for AML patients. Disclosures Mcdermott: TORL Biotherapeutics:Current equity holder in private company;1200 Pharma:Current equity holder in private company.Slamon:TORL Biotherapeutics:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;1200 Pharma:Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees;Novartis:Consultancy, Research Funding;Eli Lilly:Consultancy;Bayer:Consultancy, Research Funding;Pfizer:Consultancy, Other: stock, Research Funding;Syndax:Research Funding;Aileron:Research Funding;Genetech:Research Funding;Biomarin:Membership on an entity's Board of Directors or advisory committees;Seattle Genetics:Other: Stock;Amgen:Other: Stock.Larson:BMS, Bioline, Celgene, Juno, Janssen:Research Funding;TORL Biotherapeutics:Current equity holder in private company.

Methyljasmonate, An Inhibitor of Glycolytic Energy Production, Displays Pre-Clinical Activity against Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1741-1741
Author(s):  
Steffen Klippel ◽  
Jana Jakubikova ◽  
Jake Delmore ◽  
Melissa G. Ooi ◽  
Douglas McMillin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Cell Death ◽  
Cancer Cells ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Energy Production ◽  
Advisory Committees ◽  
Normal Cells

Abstract Abstract 1741 Poster Board I-767 Background In contrast to most normal cells, cancer cells typically produce energy predominantly by glycolysis as demonstrated by O. Warburg more than 50 years ago. Methyljasmonate (MJ), a hormone produced by plants in response to biotic & abiotic stresses such as herbivory and wounding, has been shown to prevent the interaction of hexokinase (Hxk) and voltage dependent anion channels (VDACs), thereby significantly impacting the onset of glycolytic energy production. This may explain promising preclinical results observed with MJ against a variety of cancer cells, including myeloid leukemia and B-cell lymphoma cell lines. Methods and Results We tested the potential of MJ against Multiple Myeloma (MM) cells. We first evaluated the response of 16 different MM cell lines to 24 h of exposure to MJ concentrations of 0.5 – 3.5 mM using MTT assays. 15/16 of the MM cell lines tested displayed an IC50 of < 1.5 mM. In contrast, HS-5 stroma cells and peripheral blood mononuclear cells (PBMCs) did not respond to that MJ concentration, and even at a concentration of 2.5 mM MJ showed a maximal reduction of cell viability of 40%. Similarly to MM cell lines, purified CD138+ primary tumor cells of 3 MM patients displayed an IC50 of < 1.5 mM, suggesting that the differential sensitivity of MM vs. normal cells to MJ is not restricted to cell lines, but is also observed with primary tumor cells. Importantly, neither co-culture with HS-5 stroma nor IL-6 protected MM cells against MJ. Cell death commitment assays revealed that 1h exposure of 1.5 mM MJ induced cell death. Annexin V/PI FACS analysis of MJ-exposed MM cells showed that the cell death is mainly driven by apoptosis, evidenced by cleavage of caspases 3, 8 and 9 as well as of PARP. However, pre-incubation of MM cells with specific caspase inhibitors such as 10 mM of AC-DEVD-CHO, Z-IETD-fmk, Z-LEHD-fmk or 50 mM of Z-VAD only minimally protects the cancer cells from MJ exposure. Therefore, the impact of the MJ is not solely due to caspase triggered proteolytic cascades. Measurements of cellular ATP content by cell titer glow (CTG; Promega, Madison, WI) assay showed rapid depletion of ATP triggered by MJ action in sensitive MM cell lines. Additionally, we observed that 1 h exposure to 2 mM MJ modulated signaling pathways including IRS1/PI3K/AKT, MEK1/2, as well as Stat3 and JNK. FACS-based cell cycle analysis after propidium iodide staining did not show cell cycle arrest, but rather a rapid transition of cells to G0/G1 No correlation of sensitivity of MM cell lines and the number of mitochondria per cancer cell, as determined by Mitotracker Green (Invitrogen, Carlsbad, CA) -based flow analysis, was observed. We next examined if MJ exhibits either significant antagonism or synergy with established or novel anti-MM agents, including Bortezomib, Lenalidomide, Doxorubicin, Rapamycin or Dexamethasone, but discovered neither. However, MJ displayed synergy when combined with 2-Deoxyglucose. Finally, MJ was tested in vivo in scid/nod mice irradiated with 150 rads, injected with 1× 106 MM1S cells, and then, treated at 500 mg/kg by IP administration on a 5 days on / 2 days off schedule starting two weeks after tumor cell injection, There was an overall survival advantage of MJ-treated animals over the respective controls, with all treated mice (n=10) still alive but 6/10 control mice dead after 27 d. Conclusions Based on its rapidity of anti-MM action, favorable safety profile in preclinical models, distinct pattern of molecular sequelae, and compatibility with established anti-MM agents, MJ represents a promising investigational anti-MM agent. Disclosures Laubach: Novartis: Consultancy, Honoraria. Richardson:Millennium: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: (Speakers Bureau up to 7/1/09), Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Anderson:Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Mitsiades:Novartis Pharmaceuticals: Consultancy, Honoraria; Milllennium: Consultancy, Honoraria; Bristol-Myers Squibb : Consultancy, Honoraria; Merck &Co.: Consultancy, Honoraria; Kosan Pharmaceuticals : Consultancy, Honoraria; Pharmion: Consultancy, Honoraria; PharmaMar: Patents & Royalties; Amgen: Research Funding; AVEO Pharma: Research Funding; EMD Serono: Research Funding; Sunesis Pharmaceuticals: Research Funding.

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