Joseph B. Bolen

The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck

The CD4 and CD8 T cell antigens are thought to transduce an independent signal during the process of T cell activation. We report our evaluation of the possible involvement of the lymphocyte-specific tyrosine kinase p56lck in these transduction pathways. Our data demonstrate that p56lck is specifically modulated with either CD4 or CD8 following antibody-mediated cross-linking of these molecules and that a large fraction of the total cellular lck protein can be coimmunoprecipitated with these surface glycoproteins. These results suggest that p56lck is functionally and physically associated with CD4/CD8 in normal murine T lymphocytes and support the concept that an independent signal is transduced by the interaction of these surface molecules with major histocompatibility complex determinants.

An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer

The clinical development of an inhibitor of cellular proteasome function suggests that compounds targeting other components of the ubiquitin–proteasome system might prove useful for the treatment of human malignancies. NEDD8-activating enzyme (NAE) is an essential component of the NEDD8 conjugation pathway that controls the activity of the cullin-RING subtype of ubiquitin ligases, thereby regulating the turnover of a subset of proteins upstream of the proteasome. Substrates of cullin-RING ligases have important roles in cellular processes associated with cancer cell growth and survival pathways. Here we describe MLN4924, a potent and selective inhibitor of NAE. MLN4924 disrupts cullin-RING ligase-mediated protein turnover leading to apoptotic death in human tumour cells by a new mechanism of action, the deregulation of S-phase DNA synthesis. MLN4924 suppressed the growth of human tumour xenografts in mice at compound exposures that were well tolerated. Our data suggest that NAE inhibitors may hold promise for the treatment of cancer.

Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8-AMP mimetic in situ.

The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.

Antitumor activity of the investigational proteasome inhibitor MLN9708 in mouse models of B-cell and plasma cell malignancies.

Purpose: The clinical success of the first-in-class proteasome inhibitor bortezomib (VELCADE) has validated the proteasome as a therapeutic target for treating human cancers. MLN9708 is an investigational proteasome inhibitor that, compared with bortezomib, has improved pharmacokinetics, pharmacodynamics, and antitumor activity in preclinical studies. Here, we focused on evaluating the in vivo activity of MLN2238 (the biologically active form of MLN9708) in a variety of mouse models of hematologic malignancies, including tumor xenograft models derived from a human lymphoma cell line and primary human lymphoma tissue, and genetically engineered mouse (GEM) models of plasma cell malignancies (PCM). Experimental Design: Both cell line–derived OCI-Ly10 and primary human lymphoma–derived PHTX22L xenograft models of diffuse large B-cell lymphoma were used to evaluate the pharmacodynamics and antitumor effects of MLN2238 and bortezomib. The iMycCα/Bcl-XL GEM model was used to assess their effects on de novo PCM and overall survival. The newly developed DP54-Luc–disseminated model of iMycCα/Bcl-XL was used to determine antitumor activity and effects on osteolytic bone disease. Results: MLN2238 has an improved pharmacodynamic profile and antitumor activity compared with bortezomib in both OCI-Ly10 and PHTX22L models. Although both MLN2238 and bortezomib prolonged overall survival, reduced splenomegaly, and attenuated IgG2a levels in the iMycCα/Bcl-XL GEM model, only MLN2238 alleviated osteolytic bone disease in the DP54-Luc model. Conclusions: Our results clearly showed the antitumor activity of MLN2238 in a variety of mouse models of B-cell lymphoma and PCM, supporting its clinical development. MLN9708 is being evaluated in multiple phase I and I/II trials. Clin Cancer Res; 17(23); 7313–23. ©2011 AACR.

Effects of the tyrosine‐kinase inhibitor geldanamycin on ligand‐induced HER‐2/NEU activation, receptor expression and proliferation of HER‐2‐positive malignant cell lines

Geldanamycin belongs to the family of benzoquinoid ansamycin tyrosine‐kinase inhibitors. We have examined its effects on Her‐2/neu kinase activity, protein expression level, and proliferation of Her‐2+ malignant cells. In SK‐BR‐3 breast‐cancer cells, short‐time treatment with geldanamycin completely abrogated gp30‐ligand‐induced activation of Her‐2 without a change of receptor‐expression level. Longer treatment of intact cells with geldanamycin induced decreased steady‐state Her‐2 autophosphorylation activity, which correlated with reduction of Her‐2 protein expression and phosphotyrosine content of several proteins. The decrease was time‐ and dose‐dependent, starting after 1 hr at 100 nM concentration and reaching completion by 24 hr. The reduction of the Her‐2 protein level probably resulted from increased degradation, since the Her‐2 mRNA level remained constant. Geldanamycin effects were not specific for Her‐2, since the non‐receptor tyrosine‐kinase fyn was inhibited equally. In contrast to these results, protein‐kinase‐C activity was not affected. In 3 other malignant cell lines expressing different amounts of Her‐2 (SK‐BR‐3 > SK‐OV‐3 > OVCAR3 > MCF7), geldanamycin also effectively reduced Her‐2‐kinase activity proportionally to the decrease of protein expression. In contrast, in a [3H]‐thymidine‐uptake assay, cell growth was meaningfully inhibited by geldanamycin at nanomolar concentrations only in SK‐BR‐3 (IC50 2nM) and MCF7 (IC50 20nM), while OVCAR3 was only moderately sensitive (IC50 2μM) and SK‐OV‐3 was clearly resistant to geldanamycin. In direct comparison with herbimycin A, another benzoquinoid ansamycin that has been more thoroughly characterized, the biologic effects of geldanamycin were more pronounced. Int. J. Cancer, 70:221–229, 1997.

Treatment-Emergent Mutations in NAEβ Confer Resistance to the NEDD8-Activating Enzyme Inhibitor MLN4924

MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE) in clinical trials for the treatment of cancer. MLN4924 is a mechanism-based inhibitor, with enzyme inhibition occurring through the formation of a tight-binding NEDD8-MLN4924 adduct. In cell and xenograft models of cancer, we identified treatment-emergent heterozygous mutations in the adenosine triphosphate binding pocket and NEDD8-binding cleft of NAEβ as the primary mechanism of resistance to MLN4924. Biochemical analyses of NAEβ mutants revealed slower rates of adduct formation and reduced adduct affinity for the mutant enzymes. A compound with tighter binding properties was able to potently inhibit mutant enzymes in cells. These data provide rationales for patient selection and the development of next-generation NAE inhibitors designed to overcome treatment-emergent NAEβ mutations.

Takeda's Oncology Discovery Strategy

Takedas Oncology Discovery Strategy is tightly integrated and focused on first and fast-best-in-class products and product combinations. Core areas of expertise include hormones, protein homeostasis, biotherapeutics and signal transduction. Strategic imperatives for research success are understanding of unmet needs, focus on biological expertise in foundational areas of leadership and flexibility to adapt to new information.