A Monoclonal IgM Antibody With Specificity To Heat Shock Protein GRP78/BIP Shows Anti- Myeloma Activity In Vitro and In Vivo, Synergy In Combination With Lenalidomide and Safety In a Pilot Phase I Study

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3213-3213
Author(s):  
Leo Rasche ◽  
Stephanie Braendlein ◽  
Johannes Duell ◽  
Stefan Knop ◽  
Valentina Dubljevic ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Phase I ◽  
Plasma Cells ◽  
Pilot Phase ◽  
Myeloma Cells ◽  
Refractory Multiple Myeloma ◽  
Igm Antibody

Abstract Monoclonal antibodies have entered the therapy of multiple myeloma (MM) and are currently being evaluated in phase I-III trials. PAT-SM6 is a fully human IgM antibody that specifically binds to a cancer-specific cell surface variant of the chaperone molecule glucose regulated protein 78 (GRP78). Finding a GRP78 cancer-specific form on the surface of cancer cells, but not normal cells in vivo, presents an opportunity for cancer-specific targeting. This antibody is able to specifically target primary myeloma cells without showing cross-reactivity to healthy tissues (including plasma cells from healthy donors). Moreover, antibody treatment in vitro led to apoptosis in primary myeloma cells (Rasche L; PLOS One 2013). In vitro,PAT-SM6 was combined with Lenalidomide and/or Bortezomib and Dexamethasone in double and triple combinations on myeloma cell lines. Synergistic and additive cytotoxic effects were analyzed using the Chou-Talalay method. All double and triple combinations showed synergistic effect with a combination index (CI) <1. In all double combinations, low doses of agents appear more effective than high doses. In triple, PAT-SM6 + Dexamethasone + Lenalidomide seem to be the most efficient combination (CI from 0.005 to 0.011). In vitro data is further supported by positive in vivodata using PAT-SM6 in a 5T33 multiple myeloma mouse model. Upon injection of 5T33 cells mice developed multiple myeloma disease with clinical, biological and genetic characteristics similar to those of the human disease. A total of 6 doses PAT-SM6 were given i.p. followed by the collection of serum and bone marrow samples. Doses >10mg/kg resulted in a significant reduction of plasma cells in the bone marrow (up to 54%) and a reduction of blood levels (up to 48%) of M protein. No cytotoxicity was observed. Based on these results we performed a Phase I clinical trial to examine the tolerability and safety of the PAT-SM6 antibody in patients with relapsed / refractory multiple myeloma. A pilot Phase I dose-escalating study was initiated (NCT01727778). Relapsed myeloma patients according to IMWG criteria were treated in different dose cohorts (0.3, 1,3 and 6mg/kg/dose) with at least four doses of PAT-SM6 as single agent in a two week cycle. A serological staging was performed on day 36. At the date of the abstract submission 9/12 subjects were treated. PAT-SM6 therapy was very well tolerated. No dose limiting toxicity (DLT), no related SAE and no related adverse events greater than grade 3 were observed. Mild leucopenia seemed to be a specific side effect. At date of submission 8 patients are evaluable for response. Two out of 8 patients showed stable disease according to IMWG criteria. In summary, PAT-SM6 provides a very promising approach for the immune therapy of patients with relapsed and refractory multiple myeloma. Disclosures: Braendlein: Patrys Ltd: Consultancy. Dubljevic:Patrys Ltd: Employment. Einsele:Celgene GmbH: Consultancy, Honoraria, Research Funding. Topp:Patrys Ltd: Honoraria.

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.

scholarly journals A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo

Leukemia ◽  
2016 ◽  
Vol 31 (8) ◽  
pp. 1743-1751 ◽  
Author(s):  
S Hipp ◽  
Y-T Tai ◽  
D Blanset ◽  
P Deegen ◽  
J Wahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cell ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Selective Lysis ◽  
Bispecific T Cell Engager

Abstract B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein that is expressed on malignant plasma cells of multiple myeloma (MM) patients and therefore is an ideal target for T-cell redirecting therapies. We developed a bispecific T-cell engager (BiTE) targeting BCMA and CD3ɛ (BI 836909) and studied its therapeutic impacts on MM. BI 836909 induced selective lysis of BCMA-positive MM cells, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA-negative cells were not affected. Activity of BI 836909 was not influenced by the presence of bone marrow stromal cells, soluble BCMA or a proliferation-inducing ligand (APRIL). In ex vivo assays, BI 836909 induced potent autologous MM cell lysis in both, newly diagnosed and relapsed/refractory patient samples. In mouse xenograft studies, BI 836909 induced tumor cell depletion in a subcutaneous NCI-H929 xenograft model and prolonged survival in an orthotopic L-363 xenograft model. In a cynomolgus monkey study, administration of BI 836909 led to depletion of BCMA-positive plasma cells in the bone marrow. Taken together, these results show that BI 836909 is a highly potent and efficacious approach to selectively deplete BCMA-positive MM cells and represents a novel immunotherapeutic for the treatment of MM.

CD38-Targeted Immunochemotherapy Of Multiple Myeloma: Preclinical Evidence For Its Combinatorial Use In Lenalidomide and Bortezomib Refractory/Intolerant MM Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 277-277 ◽  
Author(s):  
Inger S. Nijhof ◽  
Willy A. Noort ◽  
Jeroen Lammerts van Bueren ◽  
Berris van Kessel ◽  
Joost M. Bakker ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Research Funding ◽  
Plasma Cells ◽  
Cell Lysis ◽  
In Vitro Assays ◽  
Human Antibody ◽  
Myeloma Cells ◽  
Clinical Phase

Abstract Multiple myeloma (MM) remains an incurable malignancy of clonal plasma cells. Although the new generation of immunomodulatory agents, such as lenalidomide (LEN), and the potent proteasome inhibitor bortezomib (BORT) have significantly improved the overall survival of MM patients, all chemotherapy strategies are eventually hampered by the development of drug-resistance. The outcome of patients who are refractory to thalidomide, lenalidomide (LEN) and bortezomib (BORT) is very poor. Set out with the idea that targeted immunotherapy with human antibodies may offer new perspectives for MM patients, we have recently developed daratumumab (DARA), a CD38 human antibody with broad-spectrum killing activity, mainly via ADCC (antibody dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity). In our previous preclinical studies and in current clinical phase I/II trials, DARA induces marked anti-MM activity. Based on these encouraging results, we now explored the potential activity of DARA for patients who are refractory to LEN- and/or BORT. In a recently developed human-mouse hybrid model that allows the in vivo engraftment and outgrowth of patient-derived primary myeloma cells in immune deficient Rag2-/-gc-/- mice, single dose DARA treatment appeared to effectively inhibit the malignant expansion of primary MM cells derived from a LEN- and BORT-refractory patient, indicating the potential efficacy of DARA even in LEN/BORT refractory patients. To substantiate the conclusions of these in vivo data, we conducted in vitro assays, in which full BM-MNCs from LEN (n=11) and LEN/BORT (n=8) refractory patients were treated with DARA alone or the combination of DARA with LEN or BORT to induce MM cell lysis. As expected, LEN alone induced no or little lysis of MM cells in the LEN-refractory patients and also BORT was not able to induce any lysis in the BORT-refractory patients. On the contrary, DARA induced substantial levels of MM cell lysis in all LEN and LEN/BORT-refractory patients. This lysis was significantly enhanced by combination with LEN or BORT. The combination of DARA and BORT improved MM lysis by additive mechanisms. However, LEN improved DARA-mediated lysis of MM cells in a synergistic manner through the activation of effector cells involved in DARA-mediated ADCC. In conclusion, our results demonstrate that DARA is also effective against multiple myeloma cells derived from LEN- and BORT-refractory patients. Especially LEN seems to improve responses in a synergistic manner. Our results provide a rationale for clinical evaluation of DARA in combination with LEN to achieve more effective results in LEN- and BORT-refractory patients. Disclosures: Lammerts van Bueren: Genmab: Employment. Bakker:Genmab: Employment. Parren:Genmab: Employment. van de Donk:Celgene: Research Funding. Lokhorst:Genmab A/S: Consultancy, Research Funding; Celgene: Honoraria; Johnson-Cilag: Honoraria; Mudipharma: Honoraria.

Phase I study of TH-302, an investigational hypoxia-targeted drug, and dexamethasone in patients with relapsed/refractory multiple myeloma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 8602-8602
Author(s):  
Irene M. Ghobrial ◽  
Jacob Laubach ◽  
Philippe Armand ◽  
Erica Boswell ◽  
Courtney Hanlon ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Phase I ◽  
Clinical Activity ◽  
Heavily Pretreated ◽  
Hepatorenal Function ◽  
Definition Of ◽  
Ecog Ps

8602 Background: TH-302 is an investigational 2-nitroimidazole prodrug of the DNA alkylator Br-IPM designed to be selectively activated in hypoxia. In multiple myeloma (MM) mouse models, diseased animals demonstrate a marked expansion of areas of hypoxia in the bone marrow. TH-302 exhibited anti-tumor activity against MM in vitro and in vivo and synergism was seen when combined with bortezomib (Hu et al, Blood 2010; Chesi et al, Blood 2012). Based on these findings, a phase I/II study of TH-302 plus dexamethasone (dex) was initiated for patients (pts) with relapsed/refractory MM. Methods: Eligible pts in the study (NCT01522872) had ECOG PS ≤ 2, receipt of at least two prior therapies, and acceptable hepatorenal function and hematologic status. A standard 3+3 dose escalation design was used with a fixed oral 40 mg dose of dex and 40% dose increments of TH-302. TH-302 was administered IV with dex on days 1, 4, 8, and 11 of a 21-day cycle. The objectives were to determine DLTs and the MTD; assess the safety, tolerability and preliminary clinical activity of TH-302 plus dex; and study the relationship between hypoxia within the bone marrow and response to TH-302. Results: Eleven pts have been treated: 7M/4F with a median age 61 years (range: 53 – 86) and 6 prior therapies (range: 3 – 10). All received both bortezomib and lenalidomide/thalidomide containing regimens. TH-302 was dosed at 240 (n=5), 340 (n=4), and 480 (n=2) mg/m² for a median of 5 cycles. No DLTs were reported at 240 or 340 mg/m². Two pts treated at 480 mg/m² had DLTs of grade 3 mucositis, exceeding the definition of MTD. A dose expansion is thus ongoing at 340 mg/m2. Two patients had SAEs related to TH-302 (pneumonia). Five pts continue on study after a median of 7 cycles (range: 2–11). Nine pts have had efficacy evaluations: 2 pts with partial responses, 2 pts with minimal responses, and 5 pts with stable disease, for an overall response rate (of MR or better) of 44%. Conclusions: TH-302 can be administered at 340 mg/m2 biweekly + dex, with dose limiting mucositis seen at higher doses. Initial clinical activity has been noted with an ORR of 44% in heavily pretreated MM pts who are relapsed/refractory to both bortezomib and lenalidomide. Clinical trial information: NCT01522872.

MCL1 Inhibitors in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. SCI-12-SCI-12
Author(s):  
Karin Vanderkerken ◽  
Kim De Veirman ◽  
Ken Maes ◽  
Eline Menu ◽  
Elke De Bruyne
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Plasma Cells ◽  
Conflicts Of Interest ◽  
Therapeutic Option ◽  
Therapy Resistance ◽  
Bone Marrow Microenvironment ◽  
Molecular Heterogeneity

Apoptosis plays a key role, not only in normal homeostasis but also in protection against genomic instability. Protection against apoptosis is a hallmark of cancer and is mainly regulated by the overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-Xl or Mcl-1. This results in increased survival of the tumor cells and resistance to therapy. This presentation will focus on MCL-1 (myeloid cell leukemia 1), its expression and its role as potential target in multiple myeloma (MM). MCL1 gene regions are one the most amplified gene regions in several human cancers and Mcl-1 activity is often associated with therapy resistance and relapse. Mcl-1 binds to and sequesters the pro-apoptotic BH3 proteins, thereby preventing apoptosis. Mcl-1 is overexpressed on MM cells from newly diagnosed patients compared to normal plasma cells and in MM cells at relapse. This overexpression is furthermore associated with a shorter survival of these patients. Increased Mcl-1 expression can result either from genetic lesions or by induction through interaction with the bone marrow microenvironment. Its expression is correlated with the molecular heterogeneity of the myeloma patients; while the CCDN1 group has high BCL2 and low MCL-1 expression; the MMSET and MAF group has high MCL-1 and low BCL2 expression. Unlike Bcl-2 and Bcl-Xl, Mcl-1 has a large unstructured aminoterminus and its activity is mainly dependent on posttranslational modifications. The bone marrow microenvironment, by producing high levels of interleukin 6, also induces the upregulation of Mcl-1. Furthermore, our group recently demonstrated that not only stromal cells in the bone marrow microenvironment, but also MDSC (myeloid derived suppressor cells) induce survival of MM cells by increasing Mcl-1 levels through the AMPK pathway. As such, these data suggest the potential therapeutic benefit of targeting Mcl-1 in MM patients. Developing the first-generation inhibitors appeared to be challenging, especially in view of the occurrence of unwanted off target effects. Recent preclinical data with new, selective Mcl-1 inhibitors show promising anti-tumor effects both in vitro and in in vivo myeloma models, either alone or in combination with the Bcl-2 selective inhibitor, venetoclax, especially as it was demonstrated that high levels of MCL-1 are associated with venetoclax resistance in MM. In addition, it was also shown that proteasome inhibition can trigger Mcl-1 accumulation, further pointing to the importance of Mcl-1 inhibition. Induction of NOXA, as an inhibitor of Mcl-1, is also suggested as a therapeutic option, especially in combinations with other drugs. Clinically, following preclinical results, several new Mcl-1 inhibitors have entered phase I trials. Most of them are still recruiting patients, and as such too early to have results. Disclosures No relevant conflicts of interest to declare.

Dual Src/Abl Kinase Targeted Inhibition in Myeloma Microenvironment Promotes Myeloma Cell Apoptosis Both in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2813-2813
Author(s):  
Karthik Ramasamy ◽  
Lee Macpherson ◽  
Ghulam J Mufti ◽  
Stephen Schey ◽  
Yolanda Calle
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Stromal Cells ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Myeloma Cells ◽  
Abl Kinase ◽  
Osteoclast Function

Abstract Abstract 2813 Poster Board II-789 Osteoclast, in addition to eroding the bone resulting in lytic lesions, enhances plasma cell proliferation and survival via direct cell to cell contact. Src family protein tyrosine kinases (SFKs) and c-Abl kinase play important role downstream of integrin adhesion receptors, and regulate the cytoskeletal organisation, cell motility and gene expression in response to cell adhesion. We hypothesised targeting SFKs and Abl kinase with the small molecule tyrosine kinase inhibitor Dasatinib has potential to reduce adhesion of plasma cells to ECM proteins in the bone marrow and modify the microenvironment by inhibiting osteoclast function, specifically bone resorption. As a result, myeloma cells could be sensitised to drugs with cytotoxic properties such as dexamethasone. Osteoclasts were generated from primary bone marrow mononuclear cells of myeloma and MGUS patients (n=10). Using Immunofluorescence, we found that Dasatinib 100nM but not dexamethasone inhibited osteoclastogenesis and disrupted the actin cytoskeletal organisation with actin clusters formed in the periphery of the cell. There was absence of actin ring formation at sealing zones which is essential for bone resorption. This effect consistently led to impaired osteoclast function, evidenced by fewer resorption pits formed on rabbit dentine slices on toluidine blue staining. Experiments were repeated ≥ 3 times. In plasma cells, the combination of dexamethasone and Dasatinib synergistically (Calcusyn software) inhibited cell proliferation at clinically relevant concentrations and induced apoptosis of human and murine myeloma cell lines alone and in cocultures with human stromal cells ( p<.001). Dasatinib alone at 200 nM concentration does not inhibit plasma cell proliferation with maximal serum concentration achieved in Phase I CML trials being 180nM. Additionally, Dasatinib and Dexamethasone in combination inhibited secretion of IL-6 but not MIL -1 alpha in stromal cell cocultures. Dasatinib but not dexamethasone significantly inhibited adhesion of myeloma cell lines on Fibronectin despite integrin activation with Magnesium EGTA. This effect was mediated through down regulation of both Src and Abl phosphorylation. Both Dasatinib and Dexamethasone inhibited adhesion of PC on stromal cells and osteoclasts. Taken together, our in vitro results suggest that Dasatinib and dexamethasone could be an effective therapeutic combination with Dasatinib impairing adhesion of plasma cells to the bone marrow microenvironment as well as osteoclast function and resultant bone disease thereby sensitising myeloma cells to the cytotoxic effect of dexamethasone. We have also established that the combination of Dasatinib 75mg/kg and dexamethasone 1mg/kg is not toxic to C57BL/KaLwRij mice. The anti-myeloma efficacy of these drugs alone and in combination is being currently studied. The combination of Dasatinib 100 mg OD days 1-28 and Dexamethasone 20mg OD on Day 1-4, 15-18 has resulted in a partial response (EBMT criteria) in 2 multiply relapsed and steroid refractory myeloma patients without significant toxicity. Serum calcium levels fell commensurate with disease response and we are currently performing experiments to analyse the effect of the drug combination on osteoclast function in vivo. These findings warrant exploring this drug combination in steroid resistant myeloma and patients with extensive skeletal disease prospectively in a phase I/II trial. Disclosures: Off Label Use: Dasatinib is not licensed for Myeloma.

scholarly journals Altered Iron Metabolism Is a New Targetable Hallmark for Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3059-3059
Author(s):  
Can Li ◽  
Xuelian Tan ◽  
Qierra Brockman ◽  
Yogesh Jethava ◽  
Marta Chesi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Western Blot ◽  
Plasma Cells ◽  
Macrophage Polarization ◽  
Mouse Bone Marrow ◽  
Iron Dextran

Conventional therapies to multiple myeloma (MM) are not aimed at specific molecular targets leading ultimately to treatment resistance. Recent reports have shown that iron is instrumental in cancer development and progression and that high intracellular iron levels are associated with poor prognosis. We have demonstrated that MM cells exhibit dysregulated iron homeostasis and that limitation of cytosolic iron inhibits MM cell growth both in vitro and in vivo. The potential therapeutic role of iron should be further investigated to better understand how targeting high-iron MM cells could prevent or delay MM development and recurrence. Our study will provide crucial insights into the iron biology of MM pathogenesis and may lead to novel MM therapy. In this study, two mouse models, young Vk*MYC and old KaLwRij mice, were injected with iron dextran (1.25 mg/kg, IP, once a week). Tumor burden was monitored by serial Serum Protein Electrophoresis (SPEP) tests, flow cytometry, and immunohistochemistry. In vitro co-culturing of ARP1 MM cells with macrophages was employed to determine iron transfer. To determine iron's roles in MM evolution, we injected iron dextran into Vk*MYC mice at 8-week age. Vk*MYC mice develop MGUS around 40-50 weeks with plasma cell (PC) bone marrow infiltration and kidney damage etc. Iron-dextran was used because it is primarily taken up by macrophages. After 14-16 weeks of iron injection, M spike was detected in the injected Vk*MYC mice. The percentage of bone marrow plasma cells (CD138+) were significantly increased to 9% in the Vk*MYC mice injected with iron compared to control mice injected with vehicle by flow cytometry and immunohistochemistry. The acceleration of disease progression via iron injection was also tested in KaLwRij mice, which also spontaneously develops MGUS in old age. M protein was detected in 12 of 15 mice (80%) injected with iron dextran for 10 weeks and 1 of 5 KaLwRij (20%) control mice at 18-months of age. CD138+ B220- plasma cells were determined by flow cytometry. A significant increase of CD138+B220- plasma cells in iron treated mice (4% versus 2%) was observed compared to vehicle control mice. Deparaffined sections of bone marrow from the above mice were stained with Prussian blue and confirmed positive staining of macrophages from iron administrated mice. These results indicate that iron accelerates MGUS development in vivo. We next evaluated whether MM cells accumulate iron from the microenvironment. ARP1 MM cells were co-cultured with primary macrophages derived from mouse bone marrow to mimic disease environment in vitro. Under these conditions, MM cells induced macrophage polarization from M0 to M1 and M2. Furthermore, increased macrophage polarization was confirmed in vivo from the KaLwRij mice injected with 5TGM1 MM cells. To confirm that MM cells uptake iron from macrophages, increased intracellular ferritin levels were observed by western blot in ARP1 MM cells following co-culture with iron-loaded macrophages. We observed that this increase in intracellular ferritin was mediated via the transferrin receptor. This iron mobilization was prevented by iron chelation. Additionally, we confirmed that ferritin levels were higher in CD138+ primary human MM cells compared to CD138- non-MM cells by western blot. Our data indicate that MM cells promote macrophage polarization resulting in the transferring of iron into MM cells. The blockade of iron trafficking between MM cells and macrophages might hold a promise for the prevention and therapy in MM. Disclosures Bergsagel: Celgene: Consultancy; Ionis Pharmaceuticals: Consultancy; Janssen Pharmaceuticals: Consultancy. Zhan:BIPHARM LLC: Consultancy, Other: % Allocation of Profit.

Targeting Cyclin D1 in the Treatment of Multiple Myeloma: Preclinical Validation of a Novel Specific Small Molecule Cyclin D1 Inhibitor, P276-00.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3454-3454
Author(s):  
Noopur Raje ◽  
Teru Hideshima ◽  
Sonia Vallet ◽  
Shweta Chhetri ◽  
Constantine Mitsiades ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multiple Myeloma ◽  
Bone Marrow ◽  
Phase I ◽  
Cyclin D1 ◽  
Therapeutic Target ◽  
Growth Arrest

Abstract Either an overexpression or dysregulation of cyclin D1, D2, or D3, has been reported in the majority of multiple myeloma (MM) tumors, suggesting a possible early unifying event in MM pathogenesis. This proposed critical role of cyclin D dysregulation in myeloma pathogenesis makes the cyclins, specifically cyclin D1, an attractive therapeutic target. We have evaluated a specific small molecule cyclin D1 inhibitor, P276-00 in MM. Its specificity has been confirmed in an in vitro kinase assay by potent inhibitory activity for Cdk4-D1 as compared to Cdk2-E. Additionally in vitro kinase assays against a broad range of other kinases have also confirmed specificity for D1 and B cyclins at nanomolar concentrations. P276-00 has been tested against a wide range of cancer cell types in both in vitro and tumor xenograft models. Based on these data, it is undergoing phase I clinical testing in North America. We have observed both time and dose dependent in vitro activity against a broad range of MM cells sensitive and resistant to conventional agents like dexamethasone, doxorubicin, and melphalan with IC50 ranging from 400–800nM. Spectral karyotyping confirmed t(11;14) (q13;q32) in KMS 12 MM cells which were sensitive to P276-00. Importantly, it has demonstrated activity in primary patient derived tumor cells. Cell cycle analysis confirmed that P276-00 induced either growth arrest or apoptosis in MM cells depending on the cell line. Apoptosis was in part caspase dependent suggested by partial reversal of cytotoxicity by Z-VAD Fmk. P276-00 inhibited Rb-1 phosphorylation as early as 6 hours in most of the MM cell lines tested associated with a decrease in cdk4 suggesting a regulatory role of P276-00 in cell cycle progression. These changes preceeded growth arrest and apoptosis of MM cells on cell cycle analysis. Ongoing studies are using SiRNA to Cyclin D1 to confirm this regulatory role of P276-00. As cyclin D1 dysregulation or overexpression can render MM cells more susceptible to proliferative stimuli such as IL-6, IGF-1, and the bone marrow microenvironment, we tested the effects of P276-00 in the presence of these cytokines and bone marrow stromal cells (BMSCs). Our data confirms that P276-00 was able to overcome these proliferative signals and induce apoptosis in MM cells. Next we evaluated in vivo efficacy of P276-00 in NOD-SCID mice bearing GFP+ MM xenografts. Animals were treated with either control PBS or P276-00 intraperitoneally at 25 mg/kg three times a week for 3 weeks. Our data confirms in vivo anti-tumor activity of P276-00 as suggested by a significant decrease in biluminesence of GFP+ MM cells (p<0.05) and a decrease in tumor volume. Immunohistochemistry on tumor tissue from P76.00 treated, and control animals validates our in vitro studies and will be presented. In vitro combination studies with bortezomib have been completed suggesting synergism. P276-00 and bortezomib combination is currently being tested in our in vivo model. These studies confirm cyclin D1 to be an important therapeutic target in MM and form the basis of a phase I/II study of P276-00 alone and in combination in the treatment of MM.

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.

Promotion of human multiple myeloma cell growth in vitro and bone marrow invasion in vivo by Notch receptors and the CXCR4/SDF1 axis.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 8591-8591 ◽  
Author(s):  
Maurizio Chiriva-Internati ◽  
Leonardo Mirandola ◽  
Elisa Lazzari ◽  
Michela Colombo ◽  
Marialuigia Lancellotti ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Growth ◽  
Plasma Cells ◽  
Myeloma Cell ◽  
Notch Receptors ◽  
Associated Lesions

8591 Background: Multiple myeloma (MM) originates from post-germinal center B cells, and is caused by malignant plasma cells accumulating in the bone marrow. Interactions of MM cells with the bone marrow stroma promote tumor growth, migration and drug resistance. The chemokine receptor CXCR4 and its ligand SDF1 are critical regulators of this process. MM cells frequently hyper-express CXCR4 and respond to SDF1,2 enhancing MM cell infiltration, proliferation and osteolysis. Notch receptors similarly promote MM cell growth, drug resistance and the associated osteolytic process. We hypothesized that the CXCR4/SDF1 axis mediates the effects of Notch signals in MM. Methods: We used real-time PCR, flow-cytometry, E.L.I.S.A. and chemotaxis assay to explore the effects of CXCR4 in cultured human MM cell lines after Notch inhibition or over-stimulation. Additionally, we validated our findings in a NOD/SCID murine model xenografted with human MM cells. Results: Our results show that Notch blocking reduced CXCR4 and SDF1 expression by MM cells. Further, Notch activation was required for MM cell chemotactic and proliferative response to SDF1 in vitro. We then investigated the outcome of anti-Notch treatment on human MM cells bone invasion in NOD/SCID mice. Interfering with Notch activity dramatically reduced xenografted MM cell ability to infiltrate the bone marrow, ultimately resulting in diminished tumor burden. Notably, such effect was associated with a decrease of CXCR4 expression. Conclusions: This was the first time that Notch receptors were reported to regulate the CXCR4/SDF1 axis and bone marrow invasion in human MM. These findings indicate that specific Notch-tailored therapies may effectively hamper CXCR4-mediated bone infiltration and associated lesions, and are expected to significantly improve treatment outcome and survival.

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