Preclinical studies in support of defibrotide for the treatment of multiple myeloma and other neoplasias

Abstract

Purpose of the study: Defibrotide, an orally bioavailable polydisperse oligonucleotide, has promising activity in hepatic veno-occlusive disease, a stem cell transplantation-related toxicity characterized by microangiopathy. The antithrombotic properties of defibrotide and its minimal hemorrhagic risk could serve for treatment of cancer-associated thrombotic complications. Given its cytoprotective effect on endothelium, we investigated whether defibrotide protects tumor cells from cytotoxic antitumor agents. Further, given its antiadhesive properties, we evaluated whether defibrotide modulates the protection conferred to multiple myeloma cells by bone marrow stromal cells.

Methods-results: Defibrotide lacks significant single-agent in vitro cytotoxicity on multiple myeloma or solid tumor cells and does not attenuate their in vitro response to dexamethasone, bortezomib, immunomodulatory thalidomide derivatives, and conventional chemotherapeutics, including melphalan and cyclophosphamide. Importantly, defibrotide enhances in vivo chemosensitivity of multiple myeloma and mammary carcinoma xenografts in animal models. In cocultures of multiple myeloma cells with bone marrow stromal cells in vitro, defibrotide enhances the multiple myeloma cell sensitivity to melphalan and dexamethasone, and decreases multiple myeloma-bone marrow stromal cell adhesion and its sequelae, including nuclear factor-kappaB activation in multiple myeloma and bone marrow stromal cells, and associated cytokine production. Moreover, defibrotide inhibits expression and/or function of key mediators of multiple myeloma interaction with bone marrow stromal cell and endothelium, including heparanase, angiogenic cytokines, and adhesion molecules.

Conclusion: Defibrotide's in vivo chemosensitizing properties and lack of direct in vitro activity against tumor cells suggest that it favorably modulates antitumor interactions between bone marrow stromal cells and endothelia in the tumor microenvironment. These data support clinical studies of defibrotide in combination with conventional and novel therapies to potentially improve patient outcome in multiple myeloma and other malignancies.

Figure 1. In vitro viability of MM cell lines treated with defibrotide (DF) alone or its combination or novel anti-MM agents

Panel A: The results of in vitro DF treatment of MM cell lines, including MM-1S, its dexamethasone-resistant subline MM-1R, the chemo-sensitive RPMI-8226/S and its Doxorubucin-resistant subline Dox40, as well as the OPM-1 cells. Panel B: WST-1 assays were performed to assess the putative impact of DF on the in vitro response of MM cells to diverse agents, including melphalan; doxorubicin, bortezomib (PS341), the IMID pomalidomide (CC-4047), and dexamethasone. These results represent the combination of two separate independent experiments.

Figure 2. In vivo studies of DF in combination with other anti-tumor agents

Changes in tumor volume over time in mice xenografted with human MM-1S cells (subcutaneous xenograft model in panels A and B, and model of diffuse MM lesions in panel C) and treated with DF either alone or in combination with melphalan (panels A and C) or cyclophosphamide (CTX, panel B).

Figure 3. Defibrotide (DF) treatment enhances the anti-MM activity of conventional therapeutics in the context of MM-BMSCs interactions

Illustrative examples of results from treatment of primary MM cells with dexamethasone (Dex, panel A) or melphalan (Mel, panel B), in the presence or absence of bone marrow stromal cells (BMSCs), with or without treatment with DF. Among MM samples which exhibit constitutive responsiveness to these agents in the absence of stroma, but significantly less pronounced response in MM-BMSC co-cultures, DF is able to lead to variable degrees of sensitization to these agents in the presence of stromal cells. In each panel, primary samples 3 to 6 are examples of how DF treatment can increase the presence of stromal cells higher response to treatment with Dex or Mel, respectively.

Figure 4. Defibrotide (DF) modulates multiple myeloma (MM) cell adhesion to bone marrow stromal cells (BMSCs or SCs) and its sequelae

In vitro adhesion assays (with CFSE labeling of MM cells and quantification of adhesion with fluorescence plate reader) show that DF treatment decreases the adhesion of primary MM tumor cells to BMSCs (panel A). Furthermore, DF treatment suppresses the increase, triggered by MM-BMSCs interaction, in secretion of IL-6 (panel B) or VEGF (panel C). Cytokine levels in panels B and C are expressed, in each experiment, as % of levels in cultures of stromal cells alone.

Figure 5. Defibrotide (DF) treatment suppresses the expression and function of heparanase

RT-PCR was performed as described in “Material and methods”. Results are expressed as mean mRNA heparanase level normalized by β-actin housekeeping gene. DF treatment suppresses the levels of heparanase transcript in RPMI-8226 (panel A) MM cells. Furthermore, DF suppresses the intracellular protein expression of heparanase (measured by flow cytometric analysis) (panel B); as well as the heparanase activity in cellular extracts of RPMI-8226 (panel C) MM cells. Student test: ^*p< 0.05 and ^**p<0.01.

Figure 6. Defibrotide (DF) modulates the invasive potential of MM cells and the molecular sequelae of their interactions with endothelial cells

The invasive potential of MM cells was assessed in vitro on the basis of their ability to invade from the top to the bottom chamber of a dual-chamber in vitro culture system when these 2 chambers are separated by matrigel and a PET membrane. The invasive potential of the MM cells was enhanced by either exogenous addition of heparanase (HSPE) in the culture (panel A) or MM cell transfection with heparanase construct (panel B), but was suppressed, in both cases, by DF treatment. The interaction with MM cells triggers in human microvascular endothelial cells (HMECs) upregulation of transcripts for heparanase (panel C); VEGF and FGF-2 (panel D), but these events are suppressed by DF treatment. Student test: ^§,*p< 0.05 and ^§§,**p<0.01.