Cory Mavis

Ofatumumab demonstrates activity against rituximab-sensitive and -resistant cell lines, lymphoma xenografts and primary tumour cells from patients with B-cell lymphoma

Ofatumumab is a new monoclonal antibody (mAb) targeting a novel membrane‐proximal epitope on CD20. To better define ofatumumab’s activity, we conducted pre‐clinical studies in rituximab‐sensitive cell lines (RSCL), rituximab‐resistant cell lines (RRCL), ofatumumab‐exposed cell lines (OECLs), primary lymphoma cells, and a lymphoma xenograft model. RRCL and OECL were generated by repeated exposure of sensitive cells to escalating doses of rituximab or ofatumumab ± human serum. Antibody‐dependent cellular cytotoxicity (ADCC) and complement‐mediated cytotoxicity (CMC) assays were performed to assess cellular sensitivity to rituximab or ofatumumab. Ofatumumab elicited a higher rate of CMC in RSCL, RRCL and primary tumour cells. The chronic exposure of lymphoma cells to ofatumumab resulted in rituximab resistance but less ofatumumab resistance. In an in vivo severe combined immunodeficiency mouse model of human lymphoma, ofatumumab prolonged median survival compared to rituximab. While rituximab CMC diminished with CD20 down‐regulation in RRCL passages, ofatumumab activity in vitro diminished to a lesser degree. Our data suggest that ofatumumab is more potent than rituximab in rituximab‐sensitive or rituximab‐resistant models and has the potential to decrease the development of biological resistance in patients with repeated exposure to anti‐CD20 mAbs.

Distinct cellular and therapeutic effects of obatoclax in rituximab-sensitive and -resistant lymphomas

Bcl‐2 proteins represent a rheostat that controls cellular viability. Obatoclax, a BH3‐mimetic, has been designed to specifically target and counteract anti‐apoptotic Bcl‐2 proteins. We evaluated the biological effects of obatoclax on the anti‐tumour activity of rituximab and chemotherapy agents. Obatoclax induced cell death of rituximab/chemotherapy‐sensitive (RSCL), ‐resistant cell lines (RRCL) and primary tumour‐cells derived from patients with B‐cell lymphomas (N = 39). Obatoclax also enhanced the activity of rituximab and had synergistic activity when combined with chemotherapy agents. The ability of Obatoclax to induce PARP cleavage varied between patient samples and was not observed in some RRCL. Inhibition of caspase activity did not affect obatoclax activity, suggesting the existence of caspase‐independent death pathways. Autophagy was detected by LC3 conversion and/or electron microscopy in RRCL and in patient‐derived tumour cells. Moreover, obatoclax activity was inhibited by Beclin‐1 knockdown. In summary, obatoclax is an active Bcl‐2 inhibitor that potentiates the activity of chemotherapy agents and, to a lesser degree, rituximab. Defining the molecular events triggered by obatoclax is necessary to further its clinical development and identify potential biomarkers that are predictive of response.

Expression level and DNA methylation status of glutathione-S-transferase genes in normal murine prostate and TRAMP tumors

Glutathione‐S‐transferase (Gst) genes are downregulated in human prostate cancer, and GSTP1 silencing is mediated by promoter DNA hypermethylation in this malignancy. We examined Gst gene expression and Gst promoter DNA methylation in normal murine prostates and Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) tumors.

The novel proteasome inhibitor carfilzomib induces cell cycle arrest, apoptosis and potentiates the anti-tumour activity of chemotherapy in rituximab-resistant lymphoma.

Targeting the proteasome system with bortezomib (BTZ) results in anti‐tumour activity and potentiates the effects of chemotherapy/biological agents in multiple myeloma and B‐cell lymphoma. Carfilzomib (CFZ) is a more selective proteasome inhibitor that is structurally distinct from BTZ. In an attempt to characterize its biological activity, we evaluated CFZ in several lymphoma pre‐clinical models. Rituximab‐sensitive cell lines (RSCL), rituximab‐resistant cell lines (RRCL), and primary tumour cells derived from B‐cell lymphoma patients were exposed to CFZ or BTZ. Cell viability and changes in cell cycle were determined. Western blots were performed to detect PARP‐cleavage and/or changes in Bcl‐2 (BCL2) family members. CFZ was 10 times more active than BTZ and exhibited dose‐ and time‐dependent cytotoxicity. CFZ exposure induced apoptosis by upregulation of Bak (BAK1) and subsequent PARP cleavage in RSCL and RRCL; it was also partially caspase‐dependent. CFZ induced G2/M phase cell cycle arrest in RSCL. CFZ demonstrated the ability to overcome resistance to chemotherapy in RRCL and potentiated the anti‐tumour activity of chemotherapy agents. Our data suggest that CFZ is able to overcome resistance to chemotherapeutic agents, upregulate pro‐apoptotic proteins to promote apoptosis, and induce G2/M cell cycle arrest in lymphoma cells. Our pre‐clinical data supports future clinical evaluation of CFZ in B‐cell lymphoma.

Entinostat, a novel histone deacetylase inhibitor is active in B‐cell lymphoma and enhances the anti‐tumour activity of rituximab and chemotherapy agents

Histone deacetylases (HDACs) inhibitors are active in T‐cell lymphoma and are undergoing pre‐clinical and clinical testing in other neoplasms. Entinostat is an orally bioavailable class I HDAC inhibitor with a long half‐life, which is under evaluation in haematological and solid tumour malignancies. To define the activity and biological effects of entinostat in B‐cell lymphoma we studied its anti‐tumour activity in several rituximab‐sensitive or ‐resistant pre‐clinical models. We demonstrated that entinostat is active in rituximab‐sensitive cell lines (RSCL), rituximab‐resistant cell lines (RRCL) and primary tumour cells isolated from lymphoma patients (n = 36). Entinostat exposure decreased Bcl‐XL (BCL2L1) levels and induced apoptosis in cells. In RSCL and RRCL, entinostat induced p21 (CDKN1A) expression leading to G1 cell cycle arrest and exhibited additive effects when combined with bortezomib or cytarabine. Caspase inhibition diminished entinostat activity in some primary tumour cells suggesting that entinostat has dual mechanisms‐of‐action. In addition, entinostat increased the expression of CD20 and adhesion molecules. Perhaps related to these effects, we observed a synergistic activity between entinostat and rituximab in a lymphoma‐bearing severe combined immunodeficiency (SCID) mouse model. Our data suggests that entinostat is an active HDAC inhibitor that potentiates rituximab activity in vivo and supports its further clinical development in B‐cell lymphoma.

Distinct molecular mechanisms responsible for bortezomib-induced death of therapy-resistant versus -sensitive B-NHL cells.

Resistance to currently available therapies is a major impediment to the successful treatment of hematological malignancies. Here, we used a model of therapy-resistant B-cell non Hodgkin lymphoma (B-NHL) developed in our laboratory along with primary B-NHL cells to study basic mechanisms of bortezomib activity. In resistant cells and a subset of primary B-NHLs, bortezomib treatment led to stabilization of Bak and subsequent Bak-dependent activation of apoptosis. In contrast to sensitive cells that die strictly by apoptosis, bortezomib was capable of killing resistant cells through activation of apoptosis or caspase-independent mechanism(s) when caspases were pharmacologically inhibited. Our data demonstrate that bortezomib is capable of killing B-NHL cells via multiple mechanisms, regardless of their basal apoptotic potential, and contributes to growing evidence that proteasome inhibitors can act via modulation of B-cell lymphoma 2 (Bcl-2) family proteins. The capacity of bortezomib to act independently of the intrinsic apoptotic threshold of a given B-NHL cell suggests that bortezomib-based therapies could potentially overcome resistance and result in relevant clinical activity in a relapsed/refractory setting.

Mitotic catastrophe and cell cycle arrest are alternative cell death pathways executed by bortezomib in rituximab resistant B-cell lymphoma cells

The ubiqutin-proteasome system (UPS) plays a role in rituximab-chemotherapy resistance and bortezomib (BTZ) possesses caspase-dependent (i.e. Bak stabilization) and a less characterized caspase–independent mechanism-of-action(s). Here, we define BTZ-induced caspase-independent cell death pathways. A panel of rituximab-sensitive (RSCL), rituximab-resistant cell lines (RRCL) and primary tumor cells derived from lymphoma patients (N = 13) were exposed to BTZ. Changes in cell viability, cell-cycle, senescence, and mitotic index were quantified. In resting conditions, RRCL exhibits a low-proliferation rate, accumulation of cells in S-phase and senescence. Exposure of RRCL to BTZ reduces cell senescence, induced G2-M phase cell-cycle arrest, and is associated with mitotic catastrophe. BTZ stabilized p21, CDC2, and cyclin B in RRCL and in primary tumor cells. Transient p21 knockdown alleviates BTZ-induced senescence inhibition, G2-M cell cycle blockade, and mitotic catastrophe. Our data suggest that BTZ can induce apoptosis or mitotic catastrophe and that p21 has a pivotal role in BTZ activity against RRCL.

Ofatumumab Exhibits Enhanced In Vitro and In Vivo Activity Compared to Rituximab in Preclinical Models of Mantle Cell Lymphoma.

Purpose: Mantle cell lymphoma (MCL) is a mature B-cell lymphoma considered to be incurable with current treatments, including first-line rituximab in combination with multiagent chemotherapy and for those eligible, high-dose chemotherapy and stem cell support or rituximab maintenance. On the other hand, achieving a complete remission by high-sensitive flow cytometry is associated with prolonged duration of remission, stressing the need to develop and/or incorporate novel agents into the management of MCL. To this end, we examined the activity of ofatumumab, an anti-CD20 monoclonal antibody with distinct binding and immunologic properties compared to rituximab, in MCL preclinical models. Experimental Design: MCL cells were labeled with 51Cr before incubation with rituximab or ofatumumab (10 μg/mL) plus human serum or effector cells. 51Cr-release was measured and the percentage of lysis was calculated. Surface CD20, CD55, and CD59 were measured by Imagestream analysis. SCID mice inoculated subcutaneously with Z138 cells were assigned to control versus four doses of ofatumumab or rituximab (10 mg/kg/dose). Results: Ofatumumab exhibited enhanced in vitro complement-dependent cytotoxicity activity compared with rituximab in MCL cell lines, despite a high degree of in vitro resistance to rituximab associated with low CD20 levels and/or high expression of complement inhibitory proteins. Ofatumumab also delayed tumor progression and prolonged survival in a murine model of MCL. Conclusions: Our results demonstrate that ofatumumab is more effective than rituximab in MCL preclinical models, including in the presence of rituximab resistance, and support the clinical investigation of ofatumumab in combination with standard systemic chemotherapy in MCL (NCT01527149). Clin Cancer Res; 21(19); 4391–7. ©2015 AACR.

MLN2238, a proteasome inhibitor, induces caspase-dependent cell death, cell cycle arrest, and potentiates the cytotoxic activity of chemotherapy agents in rituximab-chemotherapy-sensitive or rituximab-chemotherapy-resistant B-cell lymphoma preclinical models.

To further develop therapeutic strategies targeting the proteasome system, we studied the antitumor activity and mechanisms of action of MLN2238, a reversible proteasome inhibitor, in preclinical lymphoma models. Experiments were conducted in rituximab-chemotherapy-sensitive cell lines, rituximab-chemotherapy-resistant cell lines (RRCL), and primary B-cell lymphoma cells. Cells were exposed to MLN2238 or caspase-dependent inhibitors, and differences in cell viability, alterations in apoptotic protein levels, effects on cell cycle, and the possibility of synergy when combined with chemotherapeutic agents were evaluated. MLN2238 showed more potent dose-dependent and time-dependent cytotoxicity and inhibition of cell proliferation in lymphoma cells than bortezomib. Our data suggest that MLN2238 can induce caspase-independent cell death in RRCL. MLN2238 (and to a much lesser degree bortezomib) reduced RRCL S phase and induced cell cycle arrest in the G2/M phase. Exposure of rituximab-chemotherapy-sensitive cell lines and RRCL to MLN2238 potentiated the cytotoxic effects of gemcitabine, doxorubicin, and paclitaxel and overcame resistance to chemotherapy in RRCL. MLN2238 is a potent proteasome inhibitor active in rituximab-chemotherapy-sensitive and rituximab-chemotherapy-resistant cell models and potentiates the antitumor activity of chemotherapy agents and has the potential of becoming an effective therapeutic agent in the treatment of therapy-resistant B-cell lymphoma.

Pevonedistat, a NEDD8-activating enzyme inhibitor, is active in mantle cell lymphoma and enhances rituximab activity in vivo

Mantle cell lymphoma (MCL) is characterized by an aggressive clinical course and inevitable development of refractory disease, stressing the need to develop alternative therapeutic strategies. To this end, we evaluated pevonedistat (MLN4924), a novel potent and selective NEDD8-activating enzyme inhibitor in a panel of MCL cell lines, primary MCL tumor cells, and 2 distinct murine models of human MCL. Pevonedistat exposure resulted in a dose-, time-, and caspase-dependent cell death in the majority of the MCL cell lines and primary tumor cells tested. Of interest, in the MCL cell lines with lower half-maximal inhibitory concentration (0.1-0.5 μM), pevonedistat induced G1-phase cell cycle arrest, downregulation of Bcl-xL levels, decreased nuclear factor (NF)-κB activity, and apoptosis. In addition, pevonedistat exhibited additive/synergistic effects when combined with cytarabine, bendamustine, or rituximab. In vivo, as a single agent, pevonedistat prolonged the survival of 2 MCL-bearing mouse models when compared with controls. Pevonedistat in combination with rituximab led to improved survival compared with rituximab or pevonedistat monotherapy. Our data suggest that pevonedistat has significant activity in MCL preclinical models, possibly related to effects on NF-κB activity, Bcl-xL downregulation, and G1 cell cycle arrest. Our findings support further investigation of pevonedistat with or without rituximab in the treatment of MCL.