ARRY-797, a Potent and Selective Inhibitor of p38 Map Kinase, Inhibits LPS-Induced IL-6 and In Vivo Growth of RPMI-8226 Human Multiple Myeloma Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3478-3478
Author(s):  
Dale Wright ◽  
Shannon L. Winski ◽  
Deborah Anderson ◽  
Patrice Lee ◽  
Mark Munson ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Small Molecule ◽  
Single Agent ◽  
Tnf Alpha ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells within the bone marrow. Their growth, survival, and migration are mediated in part via cytokines. Interleukin 6 (IL-6) is necessary for sustaining the in vitro growth of many MM cell lines and enhancing the proliferation of explanted human myeloma cells. The mitogen-activated protein kinase family member, p38, is activated by cytokines and growth factors and plays a significant role in inflammatory diseases. However, its role in the pathogenesis of multiple myeloma is poorly understood. Specific p38 inhibitors inhibit paracrine MM cell growth which is associated with IL-6 and VEGF secretion from bone marrow stromal cells (BMSCs). Furthermore, p38 inhibition blocks TNF-alpha-induced IL-6 secretion in BMSCs, thereby further inhibiting MM cell growth and survival. Although these data suggest an important role for p38 in MM, the direct effects of p38 inhibiton on MM has not been extensively explored. Therefore, we investigated the effects of p38 inhibition on in vitro and in vivo IL-6 production and MM cell growth in vivo after lipopolysaccaride (LPS) stimulation. LPS has been shown to induce various cytokines, including TNF-alpha and IL-6, via the p38 pathway. ARRY-797, an orally bioavailable, small molecule inhibitor of p38 directly inhibited LPS-induced IL-6 production from RPMI-8226 (IC50 = 100 pM) in vitro. In SCID-beige mice, LPS (3 μg/kg) induced IL-6 (7897 ± 827 pg/mL) and TNF-alpha (1922 ± 282 pg/mL) after 2 hours and these cytokines were inhibited by oral administration of ARRY-797 (30 mg/kg) by 91% and 95%, respectively. In MM xenograft models, ARRY-797 (30 mg/kg, BID, PO) inhibited RPMI 8226 tumor growth by 72% as a single agent and by 56% when LPS was administered to stimulate growth in vivo. In addition, ARRY-797 inhibited LPS-induced phosphorylation of p38 in RPMI-8226 xenografts. Together, these data support a role for p38 in IL-6-mediated growth of multiple myelomas. To our knowledge, ARRY-797 is the first small molecule p38 inhibitor to demonstrate single agent activity in a MM xenograft model and it has been advanced into preclinical development.

Gadolinium Containing Contrast Agent Promotes Multiple Myeloma Cell Growth: Implication for Clinical Use of MRI in Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1809-1809
Author(s):  
Mariateresa Fulciniti ◽  
Swaminathan Sundararaman ◽  
Puru Nanjappa ◽  
Samir B Amin ◽  
Prajwal Chevireddy ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Contrast Agent ◽  
Conflicts Of Interest ◽  
Bone Lesions ◽  
Myeloma Cells ◽  
Growth Promoting

Abstract Abstract 1809 Poster Board I-835 Bone marrow infiltration by myeloma cells and osteolytic bone lesions are the major features of Multiple Myeloma. Magnetic Resonance Imaging (MRI) has been used in MM not only to image bone marrow (BM) and to identify lytic bone disease but to also evaluate therapeutic response and prognosis. Gadolinium (Gd)-based contrast agents are frequently used to enhance MRI resolution. We evaluated effect of the most common Gd-containing agent, Omniscan, on myeloma cells. We observed that Omniscan induced both time and dose dependent MM cell growth in vitro (8-20 fold increase relative to control). Importantly, the presence of BMSC enhanced the effect of Omniscan on growth of both MM cell lines and primary MM cells. However, Omniscan was not able to overcome cytotoxic effects of conventional and novel agents in MM. This growth promoting effects were not observed on normal BM stromal cells. Evaluating the molecular mechanism of action of Omniscan on MM cells, we observed time dependent ERK1/2 phosphorylation as well as reversal of growth promoting effects of Omniscan by specific inhibition of ERK signaling; however, Omniscan had no effect on STAT3 and AKT signaling pathways. Next, we investigated in vivo effect of Omniscan in a murine xenograft model of MM. Following detection of tumor, mice were treated with either iv Omniscan or PBS. Treatment with Omniscan significantly induced MM tumor growth compared to control mice (1042 ±243 mm3 vs 502 ±137 mm3 respectively; p=0.0001). Finally in autopsies in 8 MM patients with repeated exposure to Omniscan, we quantified gadolinium in various tissues using Inductively-coupled mass spectrometry. We observed massive quantities of gadolinium accumulation in tissues of these MM patients regardless of their renal function. These results, confirming both in vitro and in vivo growth promoting effects of Gd-containing contrast agent on MM, suggest the need for further analysis of the mechanism of its action on myeloma cells and careful analysis of its clinical impact in MM patients undergoing MRI evaluation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Pharmacodynamic, pharmacokinetic, and phase 1a study of bisthianostat, a novel histone deacetylase inhibitor, for the treatment of relapsed or refractory multiple myeloma

2021 ◽  
Author(s):  
Yu-bo Zhou ◽  
Yang-ming Zhang ◽  
Hong-hui Huang ◽  
Li-jing Shen ◽  
Xiao-feng Han ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Targets ◽  
Single Agent ◽  
Hdac Inhibitors ◽  
Preclinical Study ◽  
Parp Cleavage ◽  
Rpmi 8226 ◽  
Ongoing Phase

AbstractHDAC inhibitors (HDACis) have been intensively studied for their roles and potential as drug targets in T-cell lymphomas and other hematologic malignancies. Bisthianostat is a novel bisthiazole-based pan-HDACi evolved from natural HDACi largazole. Here, we report the preclinical study of bisthianostat alone and in combination with bortezomib in the treatment of multiple myeloma (MM), as well as preliminary first-in-human findings from an ongoing phase 1a study. Bisthianostat dose dependently induced acetylation of tubulin and H3 and increased PARP cleavage and apoptosis in RPMI-8226 cells. In RPMI-8226 and MM.1S cell xenograft mouse models, oral administration of bisthianostat (50, 75, 100 mg·kg-1·d-1, bid) for 18 days dose dependently inhibited tumor growth. Furthermore, bisthianostat in combination with bortezomib displayed synergistic antitumor effect against RPMI-8226 and MM.1S cell in vitro and in vivo. Preclinical pharmacokinetic study showed bisthianostat was quickly absorbed with moderate oral bioavailability (F% = 16.9%–35.5%). Bisthianostat tended to distribute in blood with Vss value of 0.31 L/kg. This distribution parameter might be beneficial to treat hematologic neoplasms such as MM with few side effects. In an ongoing phase 1a study, bisthianostat treatment was well tolerated and no grade 3/4 nonhematological adverse events (AEs) had occurred together with good pharmacokinetics profiles in eight patients with relapsed or refractory MM (R/R MM). The overall single-agent efficacy was modest, stable disease (SD) was identified in four (50%) patients at the end of first dosing cycle (day 28). These preliminary in-patient results suggest that bisthianostat is a promising HDACi drug with a comparable safety window in R/R MM, supporting for its further phase 1b clinical trial in combination with traditional MM therapies.

scholarly journals Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 11-13 ◽  
Author(s):  
XG Zhang ◽  
B Klein ◽  
R Bataille
Keyword(s):  
Multiple Myeloma ◽  
Growth Factor ◽  
Cell Growth ◽  
Interleukin 6 ◽  
Myeloma Cell ◽  
S Phase ◽  
Myeloma Cells ◽  
Severity Of The Disease

Abstract It has recently been demonstrated that interleukin-6 (IL-6) is a potent myeloma-cell growth factor in the majority of patients with multiple myeloma (MM). Using an anti-bromodeoxyuridine monoclonal antibody (MoAb) to specifically count myeloma cells in the S-phase (ie, labeling index, LI), we demonstrate that the IL-6 responsiveness of myeloma cells in vitro is directly correlated with their LI in vivo. Myeloma cells from all 13 patients with high LIs in vivo (greater than or equal to 1%) responded in vitro to IL-6, the strongest response occurring in cells from five patients with plasma-cell leukemia. In contrast, the cells of only two of eight patients with low myeloma-cell LIs in vivo (less than 1%) responded to IL-6 in vitro. After seven days of culturing with 1,000 U/mL recombinant IL-6 (rIL-6), the median LI value in the first group of patients (in vivo LI greater than or equal to 1%) was 11%, ie 11 times higher (P less than .01) than the median LI value (1%) in the second group of patients (in vivo LI less than 1%). Thus, the in vitro IL-6 responsiveness of myeloma cells is directly related to their in vivo proliferative status, and hence to the severity of the disease.

Anti-Tumor Activity of IPI-504, a Novel Hsp90 Inhibitor in Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4922-4922 ◽  
Author(s):  
Vito J. Palombella ◽  
Emmanuel Normant ◽  
Janid Ali ◽  
John Barrett ◽  
Michael Foley ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tumor Cells ◽  
Aqueous Solubility ◽  
Hsp90 Inhibitor ◽  
Tumor Xenograft ◽  
Active Metabolites ◽  
Myeloma Cells ◽  
Rpmi 8226

Abstract IPI-504 is a novel inhibitor of Hsp90 based on the geldanamycin pharmacophore. When placed in rat, monkey, and human blood, IPI-504 rapidly converts to the known and well-studied compound 17-allylamino-17-demethoxy-geldanamycin (17-AAG). 17-AAG is the subject of multiple clinical trials for the treatment of hematologic and solid tumors. However, 17-AAG suffers from poor aqueous solubility necessitating the use of sub-optimal formulations to deliver this agent to patients. IPI-504 is over 1000-fold more soluble than 17-AAG in aqueous solution. In vitro, both 17-AAG and IPI-504 bind tightly to, and selectively inhibit Hsp90 derived from cancer cells. The cytotoxic effect of IPI-504, as well as its ability to stimulate the degradation of Hsp90 client proteins and increase the intracellular levels Hsp70, were monitored in two human multiple myeloma cells lines (RPMI-8226 and MM1.S). The effects of IPI-504 were compared to 17-AAG. We demonstrate that the actions of IPI-504 are bioequivalent to 17-AAG and that both compounds induce apoptosis in these cells and stimulate the degradation of HER2 and c-Raf. In addition, both agents stimulate Hsp70 protein levels. In all cases the EC50s are virtually the same for both molecules (~200–400 nM). Furthermore, IPI-504 inhibits the secretion of immunoglobulin light chain from the RPMI-8226 multiple myeloma cells (EC50 ~300 nM). Importantly, IPI-504 is active in tumor xenograft models of multiple myeloma. The data indicate that active metabolites of IPI-504 accumulate in these xenografts long after these metabolites are cleared from the plasma compartment, suggesting that they preferentially accumulate in tumor cells based on their increased affinity to Hsp90 derived from tumor cells. In conclusion, we have developed IPI-504 as a novel, potent inhibitor of Hsp90 with greatly increased solubility over 17-AAG, and that IPI-504 is an active anti-tumor agent in vitro and in vivo.

CHIR258, a Novel Multi-Targeted Tyrosine Kinase Inhibitor, for the Treatment of t(4;14) Multiple Myeloma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 641-641 ◽  
Author(s):  
Suzanne Trudel ◽  
Zhi Hua Li ◽  
Ellen Wei ◽  
Marion Wiesmann ◽  
Katherine Rendahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Mouse Model ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
Wild Type ◽  
Myeloma Cells

Abstract The t(4;14) translocation that occurs uniquely in a subset (15%) of multiple myeloma (MM) patients results in the ectopic expression of the receptor tyrosine kinase, Fibroblast Growth Factor Receptor3 (FGFR3). Wild-type FGFR3 induces proliferative signals in myeloma cells and appears to be weakly transforming in a hematopoeitic mouse model. The subsequent acquisition of FGFR3 activating mutations in some MM is associated with disease progression and is strongly transforming in several experimental models. The clinical impact of t(4;14) translocations has been demonstrated in several retrospective studies each reporting a marked reduction in overall survival. We have previously shown that inhibition of activated FGFR3 causes morphologic differentiation followed by apoptosis of FGFR3 expressing MM cell lines, validating activated FGFR3 as a therapeutic target in t(4;14) MM and encouraging the clinical development of FGFR3 inhibitors for the treatment of these poor-prognosis patients. CHIR258 is a small molecule kinase inhibitor that targets Class III–V RTKs and inhibits FGFR3 with an IC50 of 5 nM in an in vitro kinase assay. Potent anti-tumor and anti-angiogenic activity has been demonstrated in vitro and in vivo. We employed the IL-6 dependent cell line, B9 that has been engineered to express wild-type FGFR3 or active mutants of FGFR3 (Y373C, K650E, G384D and 807C), to screen CHIR258 for activity against FGFR3. CHIR258 differentially inhibited FGF-mediated growth of B9 expressing wild-type and mutant receptors found in MM, with an IC50 of 25 nM and 80 nM respectively as determined by MTT proliferation assay. Growth of these cells could be rescued by IL-6 demonstrating selectivity of CHIR258 for FGFR3. We then confirmed the activity of CHIR258 against FGFR3 expressing myeloma cells. CHIR258 inhibited the viability of FGFR3 expressing KMS11 (Y373C), KMS18 (G384D) and OPM-2 (K650E) cell lines with an IC50 of 100 nM, 250 nM and 80 nM, respectively. Importantly, inhibition with CHIR258 was still observed in the presence of IL-6, a potent growth factors for MM cells. U266 cells, which lack FGFR3 expression, displayed minimal growth inhibition demonstrating that at effective concentrations, CHIR258 exhibits minimal nonspecific cytotoxicity on MM cells. Further characterization of this finding demonstrated that inhibition of cell growth corresponded to G0/G1 cell cycle arrest and dose-dependent inhibition of downstream ERK phosphorylation. In responsive cell lines, CHIR258 induced apoptosis via caspase 3. In vitro combination analysis of CHIR258 and dexamethasone applied simultaneously to KMS11 cells indicated a synergistic interaction. In vivo studies demonstrated that CHIR258 induced tumor regression and inhibited growth of FGFR3 tumors in a plasmacytoma xenograft mouse model. Finally, CHIR258 produced cytotoxic responses in 4/5 primary myeloma samples derived from patients harboring a t(4;14) translocation. These data indicate that the small molecule inhibitor, CHIR258 potently inhibits FGFR3 and has activity against human MM cells setting the stage for a Phase I clinical trial of this compound in t(4;14) myeloma.

Novel Murine Model To Study Modulation of Genes and Molecular Pathways Induced Following In Vivo Interaction between Multiple Myeloma Cells and Human BM Milieu.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3409-3409
Author(s):  
Paola Neri ◽  
Pierfrancesco Tassone ◽  
Masood Shammas ◽  
Mariateresa Fulciniti ◽  
Yu-Tzu Tai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Growth ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Murine Model ◽  
Cell Signalling

Abstract Interaction between multiple myeloma (MM) cells and the bone marrow (BM) microenvironment plays a critical role in promoting MM cell growth, survival, migration and development of drug resistance. This interaction within the bone marrow milieu is unique and its understanding is important in evaluating effects of novel agents in vitro and in vivo. We here describe a novel murine model that allows us to study the expression changes in vivo in MM cells within the human BM milieu. In this model, the green fluorescent protein (INA-6 GFP+) transduced IL-6-dependent human MM cell line, INA-6, was injected in human bone chip implanted into SCID mice. At different time points the bone chip was retrieved, cells flushed out and GFP+ MM cells were purified by CD138 MACS microbeads. Similar isolation process was used on INA-6 GFP+ cells cultured in vitro and used as control. Total RNA was isolated from these cells and gene expression profile analyzed using the HG-U133 array chip (Affymetrix) and DChip analyzer program. We have identified significant changes in expression of several genes following in vivo interaction between INA-6 and the BM microenvironment. Specifically, we observed up-regulation of genes associated with cytokines (IL-4, IL-8, IGFB 2–5) and chemokines (CCL2, 5, 6, 18, 24, CCR1, 2, 4), implicated in cell-cell signalling. Moreover genes implicated in DNA transcription (V-Fos, V-Jun, V-kit), adhesion (Integrin alpha 2b, 7, cadherin 1 and 11) and cell growth (CDC14, Cyclin G2, ADRA1A) were also up-regulated and genes involved in apoptosis and cell death (p-57, BCL2, TNF1a) were down-regulated. Using the Ingenuity Pathway Analysis the most relevant pathways modulated by the in vivo interaction between MM cells and BMSCs were IL-6, IGF1, TGF-beta and ERK/MAPK-mediated pathways as well as cell-cycle regulation and chemokine signalling. These results are consistent with previously observed in vitro cell signalling studies. Taken together these results highlight the ability of BM microenvironment to modulate the gene expression profile of the MM cells and our ability to in vivo monitor the changes. This model thus provides us with an ability to study in vivo effects of novel agents on expression profile of MM cells in BM milieu, to pre-clinically characterize their activity.

Silencing The Sialyltransferase Gene ST3GAL6 Inhibits Adhesion and Migration Of Myeloma Cells In Vitro and Reduces The Homing and Proliferation Of Tumor Cells In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 275-275
Author(s):  
Siobhan Glavey ◽  
Salomon Manier ◽  
Antonio Sacco ◽  
Michaela R Reagan ◽  
Yuji Mishima ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Research Funding ◽  
Advisory Board ◽  
Myeloma Cells ◽  
Sialyl Lewis ◽  
And Migration ◽  
Rpmi 8226

Abstract Background Glycosylation is a stepwise procedure of covalent attachment of oligosaccharide chains to proteins or lipids, and alterations in this process, especially increased sialylation, have been associated with malignant transformation and metastasis. The adhesion and trafficking of multiple myeloma (MM) cells is strongly influenced by glycosylation and multiple myeloma cells express a variety of adhesion molecules, including selectin ligands and integrins, which are typically dependent on glycosylation for their function. We have previously reported that the sialyltransferase ST3GAL6 is up-regulated in plasma cells from MM patients and that increased expression is associated with inferior overall survival (OS) in MM gene expression profiling (GEP) datasets. The functional significance of increased sialylation of MM cells has not previously been reported. Methods MM cell lines MM1s and RPMI-8226 were confirmed to have high expression levels of ST3GAL6 at the gene and protein level compared to healthy controls. Knockdown of ST3GAL6 was confirmed in MM cell lines RPMI-8226 and MM1s using lentiviral shRNAs targeting different regions in the ST3GAL6 mRNA. Specific ST3GAL6 knockdown was confirmed by reduced ST3GAL6 mRNA and protein expression in comparison to a scrambled control. In a calcein-AM fluorescence based adhesion assay we next evaluated the effects of ST3GAL6 knockdown on MM-cell adhesion to bone marrow stromal cells (BMSC’s) and fibronectin coated plates. Migration to 30nM SDF1-α was assessed using transwell plates comparing ST3GAL6 knockdown cells to scrambled controls. The commercially available sialyltransferase inhibitor 3Fax-Neu5Ac was used to pre-treat MM cells in vitro prior to assessment of apoptosis by flow cytometry. shST3GAL6 MM1s cells positive for green fluorescent protein and luciferin (GFP-Luc+) were injected into tail veins of SCID-Bg mice (5x106 cells, n=5/group) and mice were followed weekly using bioluminescent imaging (BLI) for tumor development. Bone marrow homing of tumor cells was assessed using in vivoconfocal imaging of the skull vasculature (n=3/group). Results Knockdown of ST3GAL6 in MM cell lines resulted in a 50% reduction in cell surface staining with the monoclonal antibody HECA-452. This indicated reduced expression of cutaneous lymphocyte associated antigen (CLA), a carbohydrate domain shared by sialyl Lewis X (sLex) and sialyl Lewis a (sLea) antigens, confirming suppression of ST3GAL6 activity. There was a significant reduction in the ability of knockdown cells to adhere to BMSC’s and fibronectin in-vitro compared to scrambled controls (P=0.016, 0.032 respectively). Migration ability of these cells in response to SDF1-α was also reduced (P=0.01). In vivo in a xenograft SCID-Bg mouse model shST3GAL6 cells demonstrated a reduced tumor burden as assessed by weekly BLI (P=0.017 at week 4). A consolidated map of the skull bone marrow niche in mice injected with shST3GAL6 MM1s GFP-Luc+ cells revealed a reduced homing ability of these cells in comparison to mice injected with scrambled control cells. Treatment of the MM cell lines MM1s and RPMI-8226 with a sialyltransferase inhibitor 3Fax-Neu5Ac resulted in almost complete elimination of cell surface sLex and/or sLea expression as determined by HECA-452 staining. Following pre-treatment with 3Fax-Neu5Ac, MM1S cells grown in co-culture with BMSC’s cells showed increased sensitivity to Bortezomib compared to cells treated with bortezomib alone. Conclusions shRNA knockdown of ST3GAL6 in MM cells significantly inhibits adhesion and migration in vitro with reduced homing and proliferation potential in vivo. In conjunction with the results of enzymatic inhibition this indicates that sialylation may play an important role in the malignant behavior of MM cells. Studies are ongoing to address the potential role of altered glycosylation in MM. Disclosures: Ghobrial: Onyx: Advisoryboard Other; BMS: Advisory board, Advisory board Other, Research Funding; Noxxon: Research Funding; Sanofi: Research Funding.

Ex Vivo Evaluation of VLA-4 Expression in Primary Human Multiple Myeloma Bone Marrow Samples and In Vivo Mobilization of Murine Multiple Myeloma Cells with Small Molecule VLA-4 Inhibitors

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2056-2056 ◽  
Author(s):  
Chantiya Chanswangphuwana ◽  
Michael P. Rettig ◽  
Walter Akers ◽  
Deep Hathi ◽  
Matthew Holt ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Cell Lines ◽  
Small Molecule ◽  
Plasma Cells ◽  
Molecular Diagnostic ◽  
Fluorescent Intensity ◽  
Variable Expression ◽  
Rpmi 8226

Abstract Background: The interaction of multiple myeloma (MM) cells with their microenvironment in the bone marrow (BM) affects disease progression and provides resistance to therapeutic agents. Very-late-antigen 4 (VLA-4, α4β1 integrin, CD49d/CD29) is a noncovalent, heterodimeric transmembrane receptor that is strongly implicated in the pathogenesis of MM via altering cell trafficking, proliferation and drug resistance. LLP2A is a high-affinity peptidomimetic ligand for activated VLA-4. We recently reported (Soodgupta et al. J. Nucl. Med 2016) the sensitive and specific molecular imaging of activated VLA-4 in mouse MM tumors using 64Cu-LLP2A and LLP2A-Cy5. Here we extended these studies by further characterizing VLA-4 expression in primary human MM samples and malignant plasma cells in mouse models of MM. Methods: We evaluated VLA-4 expression in 5 human MM cell lines (U266, OPM2, H929, RPMI-8226 and MM1.S), one mouse MM cell line (5TGM1) and seventeen primary human MM bone marrow samples by flow cytometry using LLP2A-Cy5, soluble VCAM-1/Fc recombinant protein and CD49d (α4) and CD29 (β1) antibodies. The relative mean fluorescence intensity (RMFI) of LLP2A-Cy5 binding was calculated by dividing the MFI of LLP2A-Cy5 binding in the absence of BIO5192 (small molecule VLA-4 inhibitor) by the MFI of LLP2A-Cy5 binding in the presence of excess BIO5192. The 5TGM1/KaLwRij immunocompetent mouse model of MM was used for in vivo study. Results: The expression of activated VLA-4 on MM cell lines as measured by LLP2A-Cy5+ mean fluorescent intensity (MFI) varied 10-fold as follows (LLP2A-Cy5 MFI in parentheses): 5TGM1 (23.7) > U266 (16.1) > OPM2 (4.6) > H929 (3.4) > RPMI-8226 (3.2) > MM1.S (2.1). We observed similar variable expression of LLP2A-Cy5 binding to primary human CD138+CD38+ MM plasma cells (PCs), with 76.47% (13/17) of MM patients exhibiting greater than 20% LLP2A-Cy5+ PCs. expressing VLA-4 on CD138+CD38+ cells. Overall, the mean percentage of positive cells and LLP2A-Cy5 relative MFI (RMFI) on malignant CD138+ PCs from these 13 patients were 78.2% (43.8-98.3%) and 4.3 (1.7-10.8), respectively. Other hematopoietic cells within the BM samples expressed less VLA-4 in descending order as follows; monocytes (58.2%, RMFI 3.0), T-lymphocytes (34.4%, RMFI 2.1) and B-lymphocytes (21.6%, RMFI 1.6). These levels of VLA-4 expression on normal cell subsets within MM patients were comparable to normal blood donors. In general, there was good correlation between LLP2A-Cy5 binding and expression of CD49d and CD29 on CD138+ PCs in MM patients. To our surprise, the four MM patients with <20% LLP2A-Cy5 binding demonstrated high expression of CD49d (92.1%) but very low percentages of CD29 positive cells (17.3%). Using BIO5192 (VLA-4 inhibitor), we found that the LLP2A-Cy5 reagent allowed more accurate detection of activated VLA-4 than the soluble VCAM-1 binding assay as determined by the magnitude of inhibition of binding in the presence of inhibitor. We next evaluated targeting VLA-4 molecule in murine MM model. Preliminary mouse mobilization studies demonstrated that VLA-4 inhibitors effectively and rapidly mobilized murine 5TGM1 MM cells from the bone marrow to the blood (2.49-fold increase in circulating GFP+CD138+ cells) within 1 hour of injection. Summary:This study is the first demonstration that activated VLA4 can be detected on primary human MM cells using LLP2A. These data support the continued development of LLP2A as a molecular diagnostic imaging reagent for MM and as a potential therapeutic target of VLA-4 in MM. Ongoing studies are testing whether small molecule VLA-4 inhibitors can sensitize MM cells to cytotoxic therapy in vivo. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inhibition of MEK and DDR Pathways Induces Synergistic Killing of Novel Mll-Af4 B-ALL Model Harboring Activated Ras Mutations

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1511-1511
Author(s):  
Evelyn J Song ◽  
S. Haihua Chu ◽  
Janna Minehart ◽  
Jonathan Chabon ◽  
Richard Koche ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Mouse Model ◽  
Small Molecule ◽  
Single Agent ◽  
Short Latency ◽  
Ras Mutations ◽  
In Vivo Treatment

Abstract Childhood B-cell acute lymphoblastic leukemia (B-ALL) that harbor a translocation of the MLL1 and AF4 genes are considered high-risk with poor prognosis (event-free survival (EFS) of 35%-50%), especially when compared to non-MLL-rearranged (MLL-R) childhood ALL (EFS >85%). An important obstacle to developing new therapeutic approaches for this patient population is the lack of models that faithfully recapitulate the short latency and aggressiveness of this disease. Recently, whole genome sequencing of patient childhood MLL-R leukemias revealed that activating mutations of the proto-oncogenes involved in signaling, most prominently, N or K-RAS were found in nearly 50% of patients. Patients with these co-occurring mutations have an even poorer overall survival rate, indicating that a model harboring both mutations is of extreme interest. Here, we report the generation of a highly aggressive, serially transplantable B-ALL by the retroviral overexpression of activating N-RasG12D mutant in bone marrow of an inducible knock-in Mll-Af4 murine model that we have previously published. Recipient mice injected with Mll-Af4/N-RasG12Dpre-leukemic bone marrow cells developed an acute B-ALL (B220+CD43+IgM-) with short latency to development of disease (median 35 days). Furthermore, the resultant primary B-ALL was serially transplantable into sub-lethally irradiated recipients with accelerated latency to secondary and tertiary disease developing at a median of 20 and 12 days, respectively. As our model includes an activating mutation in N-Ras, we wanted to see if the cells would be sensitive to small molecule inhibitors of downstream effectors of Ras. Pre-leukemic Mll-Af4/N-RasG12D cells were sensitive to two different MEK inhibitors, Trametinib or PD901, in vitro. Furthermore, in vivo treatment of tertiary B-ALL mice with Trametinib showed significant reduction in leukemia burden after 7 days of treatment, as well as increase in survival, compared to vehicle controls. However, prolonged in vivo treatment with Trametinib eventually led to loss of sensitivity and development of B-ALL in our mouse model, suggesting that Trametinib alone is insufficient to prevent leukemia progression. As single agent MEK inhibition was insufficient to generate long-term durable responses, we conducted RNA-Sequencing of primary Mll-Af4/N-RasG12D leukemias to discover pathways amenable for therapeutic intervention. Gene set enrichment analysis suggested that targeting the DNA damage response (DDR) pathway as an attractive therapeutic opportunity. We were able to demonstrate an increased basal level of replicative stress in our Mll-Af4/NrasG12D pre-leukemic cells and sensitivity to small molecule inhibition of ATR, a master regulator of the G2 to M transition of cell cycle progression, with AZ20, a selective ATR inhibitor. In vitro and in vivo treatment with AZ20 led to increased leukemia cytotoxicity. However, similar to Trametinib treatment, tertiary B-ALL mice eventually succumbed to disease with prolonged AZ20 treatment in vivo. Since neither single agent MEK nor ATR inhibition could prevent leukemic progression in vivo, we tested the combination and found increased cytotoxicity and cell cycle arrest in vitro at concentrations well below the IC50, as compared to single agent treatment. In vivocombination treatment also demonstrated decreased leukemia burden and significant prolonged survival compared to either AZ20 or Trametinib alone. Lastly, we tested out the efficacy of combination therapy in human B-ALL patient derived xenograft harboring both MLL-AF4 and activating N-RASmutations. 250,000 human primary leukemic blasts were transplanted into non-irradiated immune-compromised mice and treated with vehicle, single agent, or the combination for 14 days. Similar to the results seen in our mouse model, combination treatment with Trametinib and AZ20 led to significant reductions in leukemic burden. In summary, our model of B-ALL faithfully recapitulates the short latency and aggressiveness of this disease and was predictive of response in human patient samples harboring MLL-AF4 and activating N-RAS mutations to small molecule inhibitor therapy to MEK and DDR pathways. In the future, this model can be used as a platform to not only better understand the molecular events governing and sustaining leukemogenesis, but also as a discovery platform for novel therapeutic combinations. Disclosures Armstrong: Epizyme, Inc: Consultancy; Vitae Pharmaceuticals: Consultancy; Imago Biosciences: Consultancy; Janssen Pharmaceutical: Consultancy.

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

Blood ◽  
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 (&gt;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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