NVP-LAQ824 is a potent novel histone deacetylase inhibitor with significant activity against multiple myeloma

Abstract Histone deacetylase (HDAC) inhibitors are emerging as a promising new treatment strategy in hematologic malignancies. Here we show that NVP-LAQ824, a novel hydroxamic acid derivative, induces apoptosis at physiologically achievable concentrations (median inhibitory concentration [IC50] of 100 nM at 24 hours) in multiple myeloma (MM) cell lines resistant to conventional therapies. MM.1S myeloma cell proliferation was also inhibited when cocultured with bone marrow stromal cells, demonstrating ability to overcome the stimulatory effects of the bone marrow microenvironment. Importantly, NVP-LAQ824 also inhibited patient MM cell growth in a dose- and time-dependent manner. NVP-LAQ824-induced apoptotic signaling includes up-regulation of p21, caspase cascade activation, and poly (adenosine diphosphate [ADP]) ribose (PARP) cleavage. Apoptosis was confirmed with cell cycle analysis and annexin-propidium iodide staining. Interestingly, treatment of MM cells with NVPLAQ824 also led to proteasome inhibition, as determined by reduced proteasome chymotrypsin-like activity and increased levels of cellular polyubiquitin conjugates. Finally, a study using NVP-LAQ824 in a preclinical murine myeloma model provides in vivo relevance to our in vitro studies. Taken together, these findings provide the framework for NVP-LAQ824 as a novel therapeutic in MM. (Blood. 2003;102:2615-2622)

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1′-Acetoxychavicol Acetate (ACA) Is a Novel NF-κB Inhibitor with Significant Activity Against Multiple Myeloma in Vitro and in Vivo.

Blood ◽

10.1182/blood.v104.11.765.765 ◽

2004 ◽

Vol 104 (11) ◽

pp. 765-765 ◽

Cited By ~ 2

Author(s):

Keisuke Ito ◽

Tomonori Nakazato ◽

Yoshitaka Miyakawa ◽

Ming Ji Xian ◽

Taketo Yamada ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Myeloma Cell ◽

Serine Phosphorylation ◽

Significant Activity ◽

Dependent Manner ◽

Myeloma Cells ◽

Induced Apoptosis

Abstract 1′-acetoxychavicol acetate (ACA) is a component of traditional Asian condiment, obtained from rhizomes of the commonly used ethno-medicinal plant Languas galanga (Zingiberacetate). Recent extensive studies revealed that ACA has potent chemopreventive effects against various tumors. More recently, we have reported that ACA induces apoptosis of myeloid leukemic cells via mitochondrial- and Fas-mediated dual pathway. The transcription factor NF-κB confers significant survival potential in myeloma cells; therefore, it has emerged as a therapeutic target for the treatment of multiple myeloma. Multiple myeloma is an incurable hematological disorders, which has been fatal outcome despite of high dose chemotherapy with stem cell transplantation; therefore, a novel biologically based therapeutic approach is desired. In this study, we investigated the effects of ACA on myeloma cells in vitro and in vivo, and further examined the molecular mechanisms of ACA-induced apoptosis in myeloma cells. ACA dramatically inhibited cellular growth of various human myeloma cell lines (RPMI8226, U266, IM9, and HS-Sultan) as well as freshly isolated myeloma cells from patients, but not normal bone marrow cells, in a dose (0-20 μM)- and time (0-24 h)-dependent manner. Cultivation with 10 μM ACA rapidly increased the population of cells in the G0/G1 phase with a reduction of cells in the S phase, and a strong induction of apoptosis was shown by the appearance of a hypodiploid DNA peak with sub-G1 DNA content 3 h after treatment. Treatment with ACA induced both caspase-3, -9, and caspase-8 activities, suggesting that ACA-induced apoptosis in myeloma cells mediates both mitochondrial- and Fas-dependent pathways. Furthermore, we investigated the effects of ACA on NF-κB activity in myeloma cells, and were able to demonstrate that ACA significantly inhibited serine phosphorylation and degradation of IκBα in a time-dependent manner. ACA rapidly decreased the nuclear expression of NF-κB, but increased the accumulation of cytosol NF-κB in RPMI8226 cells, indicating that ACA inhibits translocation of NF-κB from the cytosol to the nucleus. In addition, we also confirmed the inhibitory effects of ACA on NF-κB activation by ELISA in myeloma cell lines and fresh samples. ACA had a synergistic proapoptotic effect with another NF-κB inhibitor, MG-132 and TLCK. In contrast, NF-κB activator, PMA, dramatically abrogated ACA-induced apoptosis in myeloma cells. These in vitro studies prompted us to examine whether the effects of ACA are equally valid in vivo. To evaluate the effects of ACA in vivo, RPMI8226-transplanted NOD/SCID mice were treated with ACA. Tumor weight decreased in the mice that were injected ACA (mean weight: 0.04±0.06 g in the ACA-treated group vs. 0.63±0.29 g in the control group; p<0.01). During the treatment, ACA-treated mice appeared healthy, and pathological analysis at autopsy revealed no ACA-induced tissue changes in any of the organ, indicating that ACA might be developed as a new potent anti-cancer agent for the management of multiple myeloma. In conclusion, ACA has an inhibitory activity of NF-κB, and induces apoptosis of myeloma cells in vitro and in vivo. Therefore, ACA provides the new biologically based therapy for the treatment of multiple myeloma patients as a novel NF-κB inhibitor.

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

Acta Pharmacologica Sinica ◽

10.1038/s41401-021-00728-y ◽

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.

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CXCR4 inhibitor AMD3100 disrupts the interaction of multiple myeloma cells with the bone marrow microenvironment and enhances their sensitivity to therapy

Blood ◽

10.1182/blood-2008-10-186668 ◽

2009 ◽

Vol 113 (18) ◽

pp. 4341-4351 ◽

Cited By ~ 278

Author(s):

Abdel Kareem Azab ◽

Judith M. Runnels ◽

Costas Pitsillides ◽

Anne-Sophie Moreau ◽

Feda Azab ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Therapeutic Agents ◽

Cytotoxic Agents ◽

Parp Cleavage ◽

Hematopoietic Stem ◽

Akt Phosphorylation ◽

Enhanced Sensitivity

Abstract The interaction of multiple myeloma (MM) cells with their microenvironment in the bone marrow (BM) provides a protective environment and resistance to therapeutic agents. We hypothesized that disruption of the interaction of MM cells with their BM milieu would lead to their sensitization to therapeutic agents such as bortezomib, melphalan, doxorubicin, and dexamethasone. We report that the CXCR4 inhibitor AMD3100 induces disruption of the interaction of MM cells with the BM reflected by mobilization of MM cells into the circulation in vivo, with kinetics that differed from that of hematopoietic stem cells. AMD3100 enhanced sensitivity of MM cell to multiple therapeutic agents in vitro by disrupting adhesion of MM cells to bone marrow stromal cells (BMSCs). Moreover, AMD3100 increased mobilization of MM cells to the circulation in vivo, increased the ratio of apoptotic circulating MM cells, and enhanced the tumor reduction induced by bortezomib. Mechanistically, AMD3100 significantly inhibited Akt phosphorylation and enhanced poly(ADP-ribose) polymerase (PARP) cleavage as a result of bortezomib, in the presence of BMSCs in coculture. These experiments provide a proof of concept for the use of agents that disrupt interaction with the microenvironment for enhancement of efficacy of cytotoxic agents in cancer therapy.

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Phenyhexyl Isothiocyanate, a Novel Inhibitor of Histone Deacetylation and Methylation, Induce Growth Arrest and Apoptosis of Myeloma Cells.

Blood ◽

10.1182/blood.v108.11.5033.5033 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5033-5033

Author(s):

Quanyi Lu ◽

Jane Feng ◽

Karen Seiter ◽

Tauseef Ahmed ◽

Dicky J.W. Chiao ◽

...

Keyword(s):

Histone Deacetylase ◽

Hdac Inhibitors ◽

Hematologic Malignancies ◽

Myeloma Cell ◽

Histone Deacetylation ◽

Dependent Manner ◽

Myeloma Cells ◽

Human Myeloma Cell Line ◽

Endothelial Growth Factor

Abstract Histone deacetylase (HDAC) inhibitors are emerging as a promising family of new agents in the therapy of hematologic malignancies. We have shown that phenyhexyl isothiocyanate(PHI) is a novel histone deacetylase inhibitor and can also modulate histone methylation in leukemia cells. In this study we investigated the effect of PHI on human myeloma cell line RPMI8226 in vitro. We have observed that cell proliferation was inhibited by PHI in a dose and time dependent manner. Cell cycle analysis indicated an arrest in G0/G1 phase, and significant apoptosis was detected in PHI treated RPMI8226 cells. The accumulation of P21 and reduced Levels of PCNA were detected. We also examined the effect of PHI on vascular endothelial growth factor (VEGF) production by the myeloma cells. Treatment with 20μM PHI for 24hrs led to a decrease of VEGF concentration by 50% of that secreted by the control cells. VEGF production further decreased to 25% when the cells were exposed to PHI for 48hrs. These data suggest that myeloma cells are sensitive to the novel HDAC inhibitor, and PHI may become a novel agent in multiple myeloma therapy. PHI Inhibit VEGF production by Myeloma Cells PHI Inhibit VEGF production by Myeloma Cells

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

Blood ◽

10.1182/blood.v104.11.1419.1419 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1419-1419

Author(s):

Soraya Wuilleme-Toumi ◽

Nelly Robillard ◽

Patricia Gomez-Bougie ◽

Philippe Moreau ◽

Steven Le Gouill ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Disease Severity ◽

Cell Lines ◽

Plasma Cells ◽

Myeloma Cell ◽

Lymphocytic Leukemia ◽

Myeloma Cells

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

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The Tyrophostin Adaphostin (NSC680410) Inhibits Multiple Myeloma Bone Marrow Angiogenesis In Vitro and In Vivo.

Blood ◽

10.1182/blood.v110.11.2507.2507 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2507-2507 ◽

Cited By ~ 1

Author(s):

Klaus Podar ◽

Jing Zhang ◽

Marc S. Raab ◽

Sonia Vallet ◽

Mariateresa Fulciniti ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Endothelial Cells ◽

Endothelial Cell ◽

Dependent Manner ◽

Antiangiogenic Effect ◽

Myelocytic Leukemia ◽

Endothelial Cell Growth

Abstract Our own and other previous studies demonstrate marked anti-proliferative activity of the tyrophostin adaphostin (NSC680410) in a variety of hematologic malignancies including chronic myelocytic leukemia (CML), chronic lymphcytic leukemia (CLL), acute myelocytic leukemia (AML), and Multiple Myeloma. Here we show that adaphostin (NSC680410), similar to bortezomib, additionally inhibits tumor angiogenesis within the MM bone marrow (BM) microenvironment. This effect is elicited both indirectly by inhibition of VEGF production and secretion in MM cells, as well as directly by abrogation of endothelial cell growth. Specifically, adaphostin triggers marked downregulation of nuclear c-Myc expression in MM cells. Both adaphostin, as well as specific downregulation of c-Myc using siRNA, lead to a decrease in cobalt chloride- induced Hif-1alpha- expression and Hif-1alpha activity, as evidenced by western blot analysis and expression of Hif-1alpha- driven luciferase, respectively. Indeed secretion of the Hif-1alpha target gene VEGF is markedly inhibited in a dose- and time- dependent manner. Importantly, neither knockdown of c-Abl expression nor exogenous overexpression of caspase- cleavage- induced c-Abl fragment abrogates drug- induced Hif-1alpha downregulation or inhibition of its activity. Taken together, these results indicate the existence of a c-Myc/ Hif-1alpha- dependent, but c-Abl- independent, pathway modulating MM cell production and secretion of VEGF. In contrast, we demonstrate a direct antiangiogenic effect of adaphostin on endothelial cells, similar to H2O2, is mediated via c-Jun upregulation, inhibition of cell proliferation, and the induction of cell apoptosis. Moreover, our data further demonstrate activity of adaphostin within the BM microenvironment. Adaphostin, similar to bortezomib, significantly inhibits VEGF secretion triggered by adhesion of MM cells to BMSCs and endothelial cells. Consequently, conditioned medium derived from adaphostin- treated co-cultures markedly inhibits endothelial cell growth and tubule formation in a dose- dependent manner. Finally, we confirmed these in vitro results using an in vivo xenograft mouse model of human MM. Specifically, western blot analysis, as well as immunohistochemistry, demonstrate marked downregulation of both Hif-1alpha and CD31 in tumors isolated from adaphostin- treated animals versus control animals, confirming the in vivo antiangiogenic effect of adaphostin. Similar effects were obtained using a SCIDhu mouse model as well as a significant decrease of MM- related bone disease, due to anti- VEGF activity of adaphostin. Taken together, these data provide the rationale for the clinical evaluation of adaphostin to target both MM cells and the BM milieu to improve patient outcome in Multiple Myeloma.

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OSU-HDAC42, a Novel Histone Deacetylase Inhibitor, Induces Apoptosis in a Caspase-Dependent Manner and Induces p21WAF1/CIP1 and p16 Expression in Multiple Myeloma Cell Lines.

Blood ◽

10.1182/blood.v108.11.5078.5078 ◽

2006 ◽

Vol 108 (11) ◽

pp. 5078-5078

Author(s):

Valerie L. White ◽

Shuhong Zhang ◽

David Lucas ◽

Ching-Shih Chen ◽

Sherif S. Farag

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Histone Deacetylase ◽

Histone Deacetylase Inhibitor ◽

Myeloma Cell ◽

Dependent Manner ◽

In Vitro Activity ◽

Myeloma Cells ◽

Deacetylase Inhibitor

Abstract Multiple myeloma (MM) is a neoplastic disorder characterized by accumulation of slowly-proliferating clonal plasma cells. OSU-HDAC42 [a.k.a. (S)-HDAC-42] is a novel histone deacetylase inhibitor that induces apoptosis in various types of cancer cells and is being developed as an anti-cancer therapy in the NCI Rapid Access to Intervention Therapy (RAID) program. In this study, we tested the in vitro activity of OSU-HDAC42 against human MM cells. OSU-HDAC42 induced myeloma cell death, with an LC50 of less than 1.6μM after 48 hours in the four cell lines tested - U266, IM-9, RPMI 8226 and ARH-77 using the MTT assay. OSU-HDAC42 induced cleavage of caspases 3, 8 and 9, as well as polyADP-ribose polymerase (PARP). Addition of the pan-caspase inhibitor Q-VD-OPH before exposure to the drug prevented apoptosis at 48 hours, as determined by Annexin V/propidium iodide staining. These results indicate that OSU-HDAC42 induced apoptosis by a mainly caspase-dependent manner. Bax expression was up-regulated at 24 and 48 hours, while Bcl-2 remains relatively constant. Mcl-1 showed increasing cleavage at increasing doses of OSU-HDAC42. These findings support a mitochondrial pathway of apoptosis. Cell cycle suppressor proteins p21WAF1/CIP1 and p16 were also significantly induced after treatment with the drug, suggesting that OSU-HDAC42 may also acts on pathways to halt cell cycle progression. In addition, the gp130 (signal-transducing) subunit of the IL-6 receptor was down-regulated by OSU-HDAC42 exposure. The tyrosine-phosphorylated form of STAT3, which is phosphorylated by dimerized gp130, was also dramatically reduced following incubation with OSU-HDAC42, supporting the finding that gp130 expression is diminished. As IL-6 is an important growth and survival factor for MM cells, down-regulation of gp130 may be an important mechanism for the activity of OSU-HDAC42 against MM cells. TRAIL, FasL, XIAP, and p53 expression were not affected by OSU-HDAC42. While other HDAC inhibitors have been shown to activate the death receptor pathway or down-regulate XIAP, this was not observed with OSU-HDAC42 in myeloma cells. In conclusion, OSU-HDAC42 has in vitro activity against myeloma cells and acts via activation of caspases, inducing the cell cycle suppressors p21WAF1/CIP1 and p16, as well as interfering with the IL-6 signal transduction pathway.

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Regulation of Histone Deacetylase in Waldenström Macroglobulinemia

Blood ◽

10.1182/blood.v112.11.2617.2617 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2617-2617

Author(s):

Xiaoying Jia ◽

Aldo M. Roccaro ◽

Abdel Kareem Azab ◽

Hai T. Ngo ◽

Antonio Sacco ◽

...

Keyword(s):

Bone Marrow ◽

Western Blot ◽

Histone Deacetylase ◽

Cell Lines ◽

Parp Cleavage ◽

Low Grade ◽

Dependent Manner ◽

Apoptotic Pathways ◽

Dose Dependent

Abstract Background: Waldenström’s Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Histone deacetylase (HDAC) inhibitors represent promising new treatment strategy in B cell malignancies. We therefore investigated the in vitro effect of the novel hydroxamic acid derivative HDAC inhibitor LBH589 in WM. Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. Cytotoxicity and DNA synthesis were measured by MTS assay and thymidine uptake assay. Cell signaling and apoptotic pathways were determined by Western Blot and immunofluorescence. Results: LBH589 induced a significant decrease of proliferation and triggered cytotoxicity in all cell lines tested and primary CD19+ WM cells (IC50 of 20–40nM), even in the presence of BMSC, IL-6 and IGF-1, which induce resistance to conventional therapies. Importantly, LBH589 did not induce cytotoxicity in healthy donor peripheral blood mononuclear cells. LBH589 induced both intrinsic and extrinsic apoptotic pathways, with caspase-9, caspase-8, caspase-3, and PARP cleavage in a dose-dependent manner. We also demonstrated significant upregulation of the proapoptotic transcription factor p53 and down-regulation of the anti-apoptotic proteins BclxL, Mcl-1 and c-myc. We then demonstrated that LBH589 induced apoptosis in WM cells in a caspase-independent manner through induction of autophagy, as shown by upregulation of LC3B and Rab7 expression. We further determined the mechanism of action of LBH589 in WM, investigating the effect of LBH589 on histone acetylation and NF-kB pathways. We found that LBH589 induced a dose-dependent increase in histone H3-H4 acetylation; and inhibited both canonical and non-canonical pathways of NF-κB, as shown by western blot and immunofluorescence. In addition, LBH589 augmented rituximab, fludarabine, bortezomib, and perifosine-induced cyotoxicity in WM cells. Conclusion: LBH589 has significant antitumor activity in WM in vitro, providing the framework for clinical trials evaluating LBH589 as a new therapeutic agent in patients with WM.

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

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.8591 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 8591-8591 ◽

Cited By ~ 1

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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