George W. Small

Inhibition of Interleukin-6 Signaling with CNTO 328 Enhances the Activity of Bortezomib in Preclinical Models of Multiple Myeloma

Purpose: Inhibition of the proteasome leads to the activation of survival pathways in addition to those that promote cell death. We hypothesized that down-regulation of interleukin-6 (IL-6) signaling using the monoclonal antibody CNTO 328 would enhance the antitumor activity of the proteasome inhibitor bortezomib in multiple myeloma by attenuating inducible chemoresistance. Experimental Design: The cytotoxicity of bortezomib, CNTO 328, and the combination, along with the associated molecular changes, was assessed in IL-6–dependent and IL-6–independent multiple myeloma cell lines, both in suspension and in the presence of bone marrow stromal cells and in patient-derived myeloma samples. Results: Treatment of IL-6–dependent and IL-6–independent multiple myeloma cell lines with CNTO 328 enhanced the cytotoxicity of bortezomib in a sequence-dependent fashion. This effect was additive to synergistic and was preserved in the presence of bone marrow stromal cells and in CD138+ myeloma samples derived from patients with relative clinical resistance to bortezomib. CNTO 328 potentiated bortezomib-mediated activation of caspase-8 and caspase-9 and the common downstream effector caspase-3; attenuated bortezomib-mediated induction of antiapoptotic heat shock protein-70, which correlated with down-regulation of phosphorylated signal transducer and activator of transcription-1; and inhibited bortezomib-mediated accumulation of myeloid cell leukemia-1, an effect that was associated with down-regulation of phosphorylated signal transducer and activator of transcription-3. Conclusions: Taken together, our results provide a strong preclinical rationale for the clinical development of the bortezomib/CNTO 328 combination for patients with myeloma.

Evidence that inhibition of p44/42 mitogen-activated protein kinase signaling is a factor in proteasome inhibitor-mediated apoptosis

The proteasome is emerging as a target for cancer therapy because small molecule inhibitors of its catalytic activity induce apoptosis in both in vitro and in vivo models of human malignancies and are proving to have efficacy in early clinical trials. To further elucidate the mechanism of action of these inhibitors, their impact on signaling through the p44/42 mitogen-activated protein kinase (MAPK) pathway was studied. Proteasome inhibition with either carbobenzoxy-leucyl-leucyl-phenylalaninal or lactacystin led to a loss of dually phosphorylated, activated p44/42 MAPK in A1N4-mychuman mammary and MDA-MB-231 breast carcinoma cells in a dose- and time-dependent fashion. This correlated with an induction of the dual specificity MAPK phosphatases (MKP)-1 and -2, and blockade of MKP induction using either actinomycin D or Ro-31-8220 significantly decreased loss of activated p44/42 MAPK. Inhibition of p44/42 MAPK signaling by use of the MAPK kinase inhibitors PD 98059 or U0126, or by use of a dominant negative MAPK construct, enhanced proteasome inhibitor-mediated apoptosis. Conversely, activation of MAPK by epidermal growth factor, or use of a mutant MAPK resistant to MKP-mediated dephosphorylation, inhibited apoptosis. These studies support a role for inactivation of signaling through the p44/42 MAPK pathway in proteasome inhibitor-mediated apoptosis.

Mitogen-Activated Protein Kinase Phosphatase-1 Is a Mediator of Breast Cancer Chemoresistance

The mitogen-activated protein kinase (MAPK) phosphatase (MKP)-1 is overexpressed in a large proportion of breast cancers, and in some systems interferes with chemotherapy-mediated proapoptotic signaling through c-Jun-NH(2)-terminal kinase (JNK). We therefore sought to examine whether MKP-1 is a mediator of breast cancer chemoresistance using A1N4-myc human mammary epithelial cells, and BT-474 and MDA-MB-231 breast carcinoma cells. Transient or stable overexpression of MKP-1 reduced caspase activation and DNA fragmentation while enhancing viability in the face of treatment with alkylating agents (mechlorethamine), anthracylines (doxorubicin), and microtubule inhibitors (paclitaxel). This overexpression was associated with suppression of JNK activation, and JNK blockade alone induced similar effects. In contrast, reduction of MKP-1 levels using a small interfering RNA, or its targeted inactivation, enhanced sensitivity to these drugs, and this was associated with increased JNK activity. Pharmacologic reduction of MKP-1 by pretreatment with a novel p38 MAPK inhibitor, SD-282, suppressed MKP-1 activation by mechlorethamine, enhanced active JNK levels, and increased alkylating agent-mediated apoptosis. Combination treatment with doxorubicin and mechlorethamine had similar effects, and the enhanced efficacy of this regimen was abolished by forced overexpression of MKP-1. These results suggest that the clinical efficacy of combinations of alkylating agents and anthracyclines are due to the ability of the latter to target MKP-1. Moreover, they support the hypothesis that MKP-1 is a significant mediator of breast cancer chemoresistance, and provide a rationale for development and translation of other agents targeting MKP-1 into the clinical arena to overcome resistance and induce chemosensitization.

Targeted inhibition of interleukin-6 with CNTO 328 sensitizes pre-clinical models of multiple myeloma to dexamethasone-mediated cell death

Interleukin (IL)‐6‐mediated signalling attenuates the anti‐myeloma activity of glucocorticoids (GCs). We therefore sought to evaluate whether CNTO 328, an anti‐IL‐6 monoclonal antibody in clinical development, could enhance the apoptotic activity of dexamethasone (dex) in pre‐clinical models of myeloma. CNTO 328 potently increased the cytotoxicity of dex in IL‐6‐dependent and ‐independent human myeloma cell lines (HMCLs), including a bortezomib‐resistant HMCL. Isobologram analysis revealed that the CNTO 328/dex combination was highly synergistic. Addition of bortezomib to CNTO 328/dex further enhanced the cytotoxicity of the combination. Experiments with pharmacologic inhibitors revealed a role for the p44/42 mitogen‐activated protein kinase pathway in IL‐6‐mediated GC resistance. Although CNTO 328 alone induced minimal cell death, it potentiated dex‐mediated apoptosis, as evidenced by increased activation of caspases‐8, ‐9 and ‐3, Annexin‐V staining and DNA fragmentation. The ability of CNTO 328 to sensitize HMCLs to dex‐mediated apoptosis was preserved in the presence of human bone marrow stromal cells. Importantly, the increased activity of the combination was also seen in plasma cells from patients with GC‐resistant myeloma. Taken together, our data provide a strong rationale for the clinical development of the CNTO 328/dex regimen for patients with myeloma.

Gene Profiling of Canine B-Cell Lymphoma Reveals Germinal Center and Postgerminal Center Subtypes with Different Survival Times, Modeling Human DLBCL

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype, and fewer than half of patients are cured with standard first-line therapy. To improve therapeutic options, better animal models that accurately mimic human DLBCL (hDLBCL) are needed. Canine DLBCL, one of the most common cancers in veterinary oncology, is morphologically similar to hDLBCL and is treated using similar chemotherapeutic protocols. With genomic technologies, it is now possible to molecularly evaluate dogs as a potential large-animal model for hDLBCL. We evaluated canine B-cell lymphomas (cBCL) using immunohistochemistry (IHC) and gene expression profiling. cBCL expression profiles were similar in many ways to hDLBCLs. For instance, a subset had increased expression of NF-κB pathway genes, mirroring human activated B-cell (ABC)-type DLBCL. Furthermore, immunoglobulin heavy chain ongoing mutation status, which is correlated with ABC/germinal center B-cell cell of origin in hDLBCL, separated cBCL into two groups with statistically different progression-free and overall survival times. In contrast with hDLBCL, cBCL rarely expressed BCL6 and MUM1/IRF4 by IHC. Collectively, these studies identify molecular similarities to hDLBCL that introduce pet dogs as a representative model of hDLBCL for future studies, including therapeutic clinical trials.

Proteasome Inhibitors Induce a p38 Mitogen-activated Protein Kinase (MAPK)-dependent Anti-apoptotic Program Involving MAPK Phosphatase-1 and Akt in Models of Breast Cancer

Proteasome inhibitors represent a novel class of anti-tumor agents that have clinical efficacy against hematologic malignancies, but single-agent activity against solid tumors such as breast cancer has been disappointing, perhaps due to activation of anti-apoptotic survival signals. To evaluate a possible role for the p38 mitogen-activated protein kinase (MAPK), A1N4-myc human mammary epithelial, and BT-474 and MDA-MB-231 breast carcinoma cells, were studied. Exposure of these lines to pharmacologic p38 blockade enhanced proteasome inhibitor-mediated apoptosis, as did overexpression of dominant negative (DN)-p38-α and -β-MAPK isoforms. Inhibition of p38 resulted in suppression of induction of anti-apoptotic MAPK phosphatase (MKP)-1, in association with enhanced activation of the pro-apoptotic c-Jun-N-terminal kinase (JNK). Moreover, infection of cells treated with a proteasome inhibitor/p38 inhibitor combination with Adenovirus (Ad) inducing over-expression of MKP-1 suppressed apoptosis compared with controls. Further targets of p38 MAPK were also studied, and proteasome inhibition activated phosphorylation of MAPK-activated protein kinase-2, heat shock protein (HSP)-27, and the AKT8 virus oncogene cellular homolog (Akt). Inhibition of p38 MAPK resulted in decreased phospho-HSP-27 and phospho-Akt, while down-regulation of HSP-27 with a small interfering RNA decreased phosphorylation of Akt, directly linking activation of p38 to Akt. Finally, inhibition of Akt with phosphatidylinositol-3-kinase inhibitors increased apoptosis, as did over-expression of DN-Akt. These studies support the hypothesis that proteasome inhibitors activate an anti-apoptotic survival program through p38 MAPK that involves MKP-1 and Akt. Further, they suggest that strategies targeting MKP-1 and Akt could enhance the anti-tumor efficacy of proteasome inhibitors against breast cancer.

Evidence for involvement of calpain in c-Myc proteolysis in vivo

Precise control of the level of c-Myc protein is important to normal cellular homeostasis, and this is accomplished in part by degradation through the ubiquitin-proteasome pathway. The calpains are a family of calcium-dependent proteases that play important roles in proteolysis of some proteins, and their possible participation in degradation of intracellular c-Myc was therefore investigated. Activation of calpain with the cell-permeable calcium ionophore A23187 in Rat1a-myc or ts85 cells in culture induced rapid cleavage of c-Myc. This degradation was both calpain- and calcium-dependent since it was inhibited by preincubation with either the calpain-inhibitory peptide calpeptin or the calcium-chelating agent EGTA. A23187-induced c-Myc cleavage occurred in a time-dependent manner comparable to that of FAK, a known calpain substrate, and while calpeptin was able to significantly protect c-Myc from degradation, inhibitors of the proteasome or caspase proteases could not. Exposure of Rat1a-myc or ts85 cells in culture to calpeptin, or to the thiol-protease inhibitor E64d, resulted in the accumulation of c-Myc protein without an impact on ubiquitin-protein conjugates. Using an in vitro assay, calpain-mediated degradation occurred rapidly with wild-type c-Myc as the substrate, but was significantly prolonged in some c-Myc mutants with increased transforming activity derived from lymphoma patients. Those mutants with a prolonged half-life in vitro were also more resistant to A23187-induced cleavage in intact cells. These studies support a role for calpain in the control of c-Myc levels in vivo, and suggest that mutations impacting on sensitivity to calpain may contribute to c-Myc-mediated tumorigenesis.

FLT3 mutations in canine acute lymphocytic leukemia.

BackgroundFMS-like tyrosine kinase 3 (FLT3) is a commonly mutated protein in a variety of human acute leukemias. Mutations leading to constitutively active FLT3, including internal tandem duplications of the juxtamembrane domain (ITD), result in continuous cellular proliferation, resistance to apoptotic cell death, and a poorer prognosis. A better understanding of the molecular consequences of FLT3 activation would allow improved therapeutic strategies in these patients. Canine lymphoproliferative diseases, including lymphoma and acute leukemias, share evolutionarily conserved chromosomal aberrations and exhibit conserved mutations within key oncogenes when compared to their human counterparts. A small percentage of canine acute lymphocytic leukemias (ALL) also exhibit FLT3 ITD mutations.MethodsWe molecularly characterized FLT3 mutations in two dogs and one cell line, by DNA sequencing, gene expression analysis via quantitative real-time PCR, and sensitivity to the FLT3 inhibitor lestaurtinib via in vitro proliferation assays. FLT 3 and downstream mediators of FLT3 activation were assessed by Western blotting.ResultsThe canine B-cell leukemia cell line, GL-1, and neoplastic cells from 2/7 dogs diagnosed cytologically with ALL were found to have FLT3 ITD mutations and FLT3 mRNA up-regulation. Lestaurtinib, a small molecule FLT3 inhibitor, significantly inhibited the growth of GL-1 cells, while not affecting the growth of two other canine lymphoid cell lines without the FLT3 mutation. Finally, western blots were used to confirm the conserved downstream mediators of FLT3 activating mutations.ConclusionsThese results show that ALL and FLT3 biology is conserved between canine and human patients, supporting the notion that canine ALL, in conjunction with the GL-1 cell line, will be useful in the development of a relevant large animal model to aid in the study of human FLT3 mutant leukemias.

Analysis of innate and acquired resistance to anti-CD20 antibodies in malignant and nonmalignant B cells.

The anti-CD20 monoclonal antibody, rituximab, provides a significant therapeutic benefit for patients with B-cell disorders. However, response to therapy varies and relapses are common, so an understanding of both inherited and acquired rituximab resistance is needed. In order to identify mechanisms of inherited resistance, sensitive versus resistant individuals were selected from a survey of 92 immortalized lymphoblastoid B-cell lines from normal individuals. Levels of CD20 protein and surface expression were lower in the resistant group. In contrast, CD20 mRNA levels were not correlated with susceptibility, suggesting regulation at a post-transcriptional level. To examine acquired resistance, resistant sublines were selected from both lymphoblastoid as well as lymphoma cell lines. Confirming previous findings, there was significant down-regulation of CD20 protein expression in all the resistant sublines. CD20 mRNA splice variants are reported to be associated with development of resistance. Three splice variants were observed in our cell lines, each lacking the binding epitope for rituximab, but none were associated with rituximab resistance. The second generation anti-CD20 mAb, ofatumumab, was more active compared with rituximab in vitro in the survey of all B-cell lines, mirroring results that have been reported previously with malignant B-cells. These studies show that normal B-lymphoblastoid cell lines can be used to model both innate and acquired mechanisms of resistance. They validate the important role of CD20 expression and enable future genetic studies to identify additional mediators of anti-CD20 mAb resistance.

VH1-44 gene usage defines a subset of canine B-cell lymphomas associated with better patient survival

The use of specific immunoglobulin heavy chain variable region (VH) genes has been associated with increased patient survival in human B-cell lymphomas (hBCL). Given the similarity of human and canine BCL (cBCL) in morphology and clinical treatment, we examined the choice of VH in cBCL and determined whether VH gene selection was a distinct feature associated with survival time in dogs. VH gene selection and mutational status in 52 cBCL, including 29 diffuse large B-cell lymphomas (cDLBCL, the most common subtype of cBCL), were analyzed by comparison with the 80 published canine germline VH gene sequences. We further examined the prognostic impact of the subgroups defined by these features on canine survival. We found that VH1-44 was preferentially expressed in the majority of the 52 cBCLs (60%) as well as in the majority of the cDLBCL subset (59%). VH1-44 gene expression was associated with a statistically better overall survival (p=0.039) in cBCL patients, as well as in the cDLBCL subset of patients (p=0.038). These findings suggest that VH gene selection in cBCL is not random and may therefore have functional implications for cBCL lymphomagenesis, in addition to being a useful prognostic biomarker.