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. 2012 Apr 15;18(8):2210-9.
doi: 10.1158/1078-0432.CCR-11-2413. Epub 2012 Feb 28.

Aurora A inhibitor (MLN8237) plus vincristine plus rituximab is synthetic lethal and a potential curative therapy in aggressive B-cell non-Hodgkin lymphoma

Daruka Mahadevan  1 , Amy Stejskal, Laurence S Cooke, Ann Manziello, Carla Morales, Daniel O Persky, Richard I Fisher, Thomas P Miller, Wenqing Qi
Affiliations

Aurora A inhibitor (MLN8237) plus vincristine plus rituximab is synthetic lethal and a potential curative therapy in aggressive B-cell non-Hodgkin lymphoma

Daruka Mahadevan et al. Clin Cancer Res. .

Erratum in

"VSports app下载" Abstract

Purpose: Aurora A and B are oncogenic serine/threonine kinases that regulate mitosis. Overexpression of Auroras promotes resistance to microtubule-targeted agents. We investigated mechanistic synergy by inhibiting the mitotic spindle apparatus in the presence of MLN8237 [M], an Aurora A inhibitor with either vincristine [MV] or docetaxel [MD] in aggressive B-cell non-Hodgkin lymphoma (B-NHL). The addition of rituximab [R] to MV or MD was evaluated for synthetic lethality. VSports手机版.

Experimental design: Aggressive B-NHL cell subtypes were evaluated in vitro and in vivo for target modulation and anti-NHL activity with single agents, doublets, and triplets by analyzing cell proliferation, apoptosis, tumor growth, survival, and mechanisms of response/relapse by gene expression profiling with protein validation V体育安卓版. .

Results: MV is synergistic whereas MD is additive for cell proliferation inhibition in B-NHL cell culture models. Addition of rituximab to MV is superior to MD, but both significantly induce apoptosis compared with doublet therapy. Mouse xenograft models of mantle cell lymphoma showed modest single-agent activity for MLN8237, rituximab, docetaxel, and vincristine with tumor growth inhibition (TGI) of approximately 10% to 15% V体育ios版. Of the doublets, MV caused tumor regression, whereas TGI was observed with MD (approximately 55%-60%) and MR (approximately 25%-50%), respectively. Although MV caused tumor regression, mice relapsed 20 days after stopping therapy. In contrast, MVR was curative, whereas MDR led to TGI of approximately 85%. Proliferation cell nuclear antigen, Aurora B, cyclin B1, cyclin D1, and Bcl-2 proteins of harvested tumors confirmed response and resistance to therapy. .

Conclusions: Addition of rituximab to MV is a novel therapeutic strategy for aggressive B-NHL and warrants clinical trial evaluation VSports最新版本. .

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Conflict of interest statement

Conflict-of-interest disclosure

The authors declare no competing financial interests.

"VSports" Figures

Figure 1
Figure 1. Cell proliferation (MTS) assays of Granta-519 treated with MLN8237 or Vincristine or Both
(A). The IC50 for MLN8237 was determined to be 109.8 nM and when combined with vincristine (V), decreased to 2.65 nM. (B). IC50 for V was determined to be 2.74 nM and when combined decreased to 0.067 nM with a CI value for ED50 to be 0.048.
Figure 2
Figure 2. Rituximab and microtubule targeting agents enhance MLN8237-induced apoptosis in B-cell NHL cells
(A) RL, Granta-519 and SUDHL-4 cells were treated with docetaxel at 5 nM, rituximab at 10 μg/ml and MLN8237 at 5 nM alone or the combinations as indicated at same doses for 72 hr. (B) Granta-519 cells were treated as same as (A) except docetaxel was substituted for vincristine at the dose of 0.1 nM. Apoptosis was analyzed by flow cytometry after annexin V and PI staining. The graph represents the mean percentage of apoptosis ± S.D. (n=3). * p<0.05 and ** p<0.001. (C) Granta-519 cells treated with AT9283 (5nM) (pan-Aurora inhibitor) and vincristine (0.1nM) ± rituximab (10 μg/ml). Apoptosis was analyzed by flow cytometry after annexin V and PI staining. The graph represents the mean percentage of apoptosis ± S.D. (n=3).
Figure 2
Figure 2. Rituximab and microtubule targeting agents enhance MLN8237-induced apoptosis in B-cell NHL cells
(A) RL, Granta-519 and SUDHL-4 cells were treated with docetaxel at 5 nM, rituximab at 10 μg/ml and MLN8237 at 5 nM alone or the combinations as indicated at same doses for 72 hr. (B) Granta-519 cells were treated as same as (A) except docetaxel was substituted for vincristine at the dose of 0.1 nM. Apoptosis was analyzed by flow cytometry after annexin V and PI staining. The graph represents the mean percentage of apoptosis ± S.D. (n=3). * p<0.05 and ** p<0.001. (C) Granta-519 cells treated with AT9283 (5nM) (pan-Aurora inhibitor) and vincristine (0.1nM) ± rituximab (10 μg/ml). Apoptosis was analyzed by flow cytometry after annexin V and PI staining. The graph represents the mean percentage of apoptosis ± S.D. (n=3).
Figure 2
Figure 2. Rituximab and microtubule targeting agents enhance MLN8237-induced apoptosis in B-cell NHL cells
(A) RL, Granta-519 and SUDHL-4 cells were treated with docetaxel at 5 nM, rituximab at 10 μg/ml and MLN8237 at 5 nM alone or the combinations as indicated at same doses for 72 hr. (B) Granta-519 cells were treated as same as (A) except docetaxel was substituted for vincristine at the dose of 0.1 nM. Apoptosis was analyzed by flow cytometry after annexin V and PI staining. The graph represents the mean percentage of apoptosis ± S.D. (n=3). * p<0.05 and ** p<0.001. (C) Granta-519 cells treated with AT9283 (5nM) (pan-Aurora inhibitor) and vincristine (0.1nM) ± rituximab (10 μg/ml). Apoptosis was analyzed by flow cytometry after annexin V and PI staining. The graph represents the mean percentage of apoptosis ± S.D. (n=3).
Figure 3
Figure 3. Rituximab increases in vivo anti-NHL activity of MLN8237 and MLN8237 plus docetaxel in a Granta-519 MCL xenograft mouse model
(A). Evaluation of in vivo therapeutic activity of MLN8237 [M], rituximab [R] and MR in a MCL xenograft mouse model. SCID mice bearing Granta-519 tumors (n = 12) were treated with saline (control), M at 10 mg/kg or 30 mg/kg, R at 10 mg/kg, M 10 mg/kg + R 10 mg/kg and M 30 mg/kg + R 10 mg/kg. Tumor burden were measured three times a week and graphed. All values are presented as mean ± S.E.M. (B). Evaluation of tumor growth inhibition of M + R versus M + docetaxel [D] versus M + D + R. Granta-519 xenograft mice were treated with M 30 mg/kg plus R 10 mg/kg, M 30 mg/kg plus D 10 mg/kg and M 30 mg/kg + D 10 mg/kg + R 10 mg/kg. Tumor volume versus time is presented as mean ± S.E.M. The arrows represent the start and the end of treatment.
Figure 3
Figure 3. Rituximab increases in vivo anti-NHL activity of MLN8237 and MLN8237 plus docetaxel in a Granta-519 MCL xenograft mouse model
(A). Evaluation of in vivo therapeutic activity of MLN8237 [M], rituximab [R] and MR in a MCL xenograft mouse model. SCID mice bearing Granta-519 tumors (n = 12) were treated with saline (control), M at 10 mg/kg or 30 mg/kg, R at 10 mg/kg, M 10 mg/kg + R 10 mg/kg and M 30 mg/kg + R 10 mg/kg. Tumor burden were measured three times a week and graphed. All values are presented as mean ± S.E.M. (B). Evaluation of tumor growth inhibition of M + R versus M + docetaxel [D] versus M + D + R. Granta-519 xenograft mice were treated with M 30 mg/kg plus R 10 mg/kg, M 30 mg/kg plus D 10 mg/kg and M 30 mg/kg + D 10 mg/kg + R 10 mg/kg. Tumor volume versus time is presented as mean ± S.E.M. The arrows represent the start and the end of treatment.
Figure 4
Figure 4. MLN8237 plus Vincristine plus Rituximab is synthetic lethal and curative in a mouse xenograft model of MCL
(A). Granta-519 xenograft mice (n=12 per cohort) were treated with saline (control), M 30 mg/kg + R 10 mg/kg, M 30 mg/kg + V 0.375 mg/kg and M 30 mg/kg + V 0.375 mg/kg + R 10 mg/kg. MLN8237 was given by PO Q1D × 3 weeks, vincristine and rituximab by IV Q1W × 4 weeks. MV1 represents the phase of tumor regression while MV2 represents the phase of lymphoma relapse. Tumor burdens were measured, graphed and represented as mean ± S.E.M. (B). Kaplan-Meier survival curves show overall survival differences between MVR in comparison to control, MR and MV.
Figure 4
Figure 4. MLN8237 plus Vincristine plus Rituximab is synthetic lethal and curative in a mouse xenograft model of MCL
(A). Granta-519 xenograft mice (n=12 per cohort) were treated with saline (control), M 30 mg/kg + R 10 mg/kg, M 30 mg/kg + V 0.375 mg/kg and M 30 mg/kg + V 0.375 mg/kg + R 10 mg/kg. MLN8237 was given by PO Q1D × 3 weeks, vincristine and rituximab by IV Q1W × 4 weeks. MV1 represents the phase of tumor regression while MV2 represents the phase of lymphoma relapse. Tumor burdens were measured, graphed and represented as mean ± S.E.M. (B). Kaplan-Meier survival curves show overall survival differences between MVR in comparison to control, MR and MV.
Figure 5
Figure 5. Re-activation of cell cycle regulators and anti-apoptosis is a mechanism of resistance to MVabrogated by MVR therapy
(A). Tumor sizes of 3 mice per cohort sacrificed 3h after end of the last treatment are shown for control, MR, MV and MVR therapy. (B). Gene expression profiling identified unique and common gene ontologies repressed by individual drug treatments compared to control and each other (Venn diagram and list of repressed functionalities). (C). Protein was isolated from the tumors 3h after the end of last treatment as indicated and at relapse (MV2). Western blotting analysis demonstrated that Proliferating cell nuclear antigen (PCNA), Aurora B, cyclin B1 cyclin D1 and Bcl-2 were inhibited by MV1 (tumor regression phase) and MVR but not MR and MV2 (tumor relapse phase). (D). PCNA was evaluated by immunohistochemistry from tumors 3h after the end of last treatment for control, MR, MV1, MVR and MV2 (at relapse).
Figure 5
Figure 5. Re-activation of cell cycle regulators and anti-apoptosis is a mechanism of resistance to MVabrogated by MVR therapy
(A). Tumor sizes of 3 mice per cohort sacrificed 3h after end of the last treatment are shown for control, MR, MV and MVR therapy. (B). Gene expression profiling identified unique and common gene ontologies repressed by individual drug treatments compared to control and each other (Venn diagram and list of repressed functionalities). (C). Protein was isolated from the tumors 3h after the end of last treatment as indicated and at relapse (MV2). Western blotting analysis demonstrated that Proliferating cell nuclear antigen (PCNA), Aurora B, cyclin B1 cyclin D1 and Bcl-2 were inhibited by MV1 (tumor regression phase) and MVR but not MR and MV2 (tumor relapse phase). (D). PCNA was evaluated by immunohistochemistry from tumors 3h after the end of last treatment for control, MR, MV1, MVR and MV2 (at relapse).
Figure 5
Figure 5. Re-activation of cell cycle regulators and anti-apoptosis is a mechanism of resistance to MVabrogated by MVR therapy
(A). Tumor sizes of 3 mice per cohort sacrificed 3h after end of the last treatment are shown for control, MR, MV and MVR therapy. (B). Gene expression profiling identified unique and common gene ontologies repressed by individual drug treatments compared to control and each other (Venn diagram and list of repressed functionalities). (C). Protein was isolated from the tumors 3h after the end of last treatment as indicated and at relapse (MV2). Western blotting analysis demonstrated that Proliferating cell nuclear antigen (PCNA), Aurora B, cyclin B1 cyclin D1 and Bcl-2 were inhibited by MV1 (tumor regression phase) and MVR but not MR and MV2 (tumor relapse phase). (D). PCNA was evaluated by immunohistochemistry from tumors 3h after the end of last treatment for control, MR, MV1, MVR and MV2 (at relapse).
Figure 5
Figure 5. Re-activation of cell cycle regulators and anti-apoptosis is a mechanism of resistance to MVabrogated by MVR therapy
(A). Tumor sizes of 3 mice per cohort sacrificed 3h after end of the last treatment are shown for control, MR, MV and MVR therapy. (B). Gene expression profiling identified unique and common gene ontologies repressed by individual drug treatments compared to control and each other (Venn diagram and list of repressed functionalities). (C). Protein was isolated from the tumors 3h after the end of last treatment as indicated and at relapse (MV2). Western blotting analysis demonstrated that Proliferating cell nuclear antigen (PCNA), Aurora B, cyclin B1 cyclin D1 and Bcl-2 were inhibited by MV1 (tumor regression phase) and MVR but not MR and MV2 (tumor relapse phase). (D). PCNA was evaluated by immunohistochemistry from tumors 3h after the end of last treatment for control, MR, MV1, MVR and MV2 (at relapse).
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