Ubiquitination and regulation of AURKA identifies a hypoxia-independent E3 ligase activity of VHL.

The hypoxia-regulated tumor-suppressor von Hippel-Lindau (VHL) is an E3 ligase that recognizes its substrates as part of an oxygen-dependent prolyl hydroxylase (PHD) reaction, with hypoxia-inducible factor α (HIFα) being its most notable substrate. Here we report that VHL has an equally important function distinct from its hypoxia-regulated activity. We find that Aurora kinase A (AURKA) is a novel, hypoxia-independent target for VHL ubiquitination. In contrast to its hypoxia-regulated activity, VHL mono-, rather than poly-ubiquitinates AURKA, in a PHD-independent reaction targeting AURKA for degradation in quiescent cells, where degradation of AURKA is required to maintain the primary cilium. Tumor-associated variants of VHL differentiate between these two functions, as a pathogenic VHL mutant that retains intrinsic ability to ubiquitinate HIFα is unable to ubiquitinate AURKA. Together, these data identify VHL as an E3 ligase with important cellular functions under both normoxic and hypoxic conditions.

Integrated Two-Analyte Population Pharmacokinetic Model for Antibody-Drug Conjugates in Patients: Implications for Reducing Pharmacokinetic Sampling.

An integrated pharmacokinetics (PK) model that simultaneously describes concentrations of total antibody (Tab) and antibody-conjugated monomethyl auristatin E (acMMAE) following administration of monomethyl auristatin E (MMAE)-containing antibody-drug conjugates (ADCs) was developed based on phase I PK data with extensive sampling for two ADCs. Two linear two-compartment models that shared all parameters were used to describe the PK of Tab and acMMAE, except that the deconjugation rate was an additional clearance pathway included in the acMMAE PK model compared to Tab. Further, the model demonstrated its ability to predict Tab concentrations and PK parameters based on observed acMMAE PK and various reduced or eliminated Tab PK sampling schemes of phase II data. Thus, this integrated model allows for the reduction of Tab PK sampling in late-phase clinical development without compromising Tab PK characterization.

Loss of p27 Associated with Risk for Endometrial Carcinoma Arising in the Setting of Obesity.

Endometrial carcinoma (EC) exhibits the strongest association with obesity of all cancers. Growth of these tumors is driven by PI3K/AKT activation, and opposed by tumor suppressors, including the tuberous sclerosis complex 2 (TSC-2) and p27, with inactivation of TSC2 and loss or cytoplasmic mislocalization of p27 both being linked to PI3K/AKT activation. However, little is known about the involvement of p27 in the development of EC arising in the setting of obesity, especially its role early in disease progression. Using a panel of EC cell lines, in vitro studies using PI3K inhibitors provided evidence that p27 rescue contributes to the efficacy of interventions that inhibit endometrial cell growth. In "at risk" obese patients, and in an animal model of obesity-associated EC (Tsc2-deficient Eker rats), p27 was moderately-to-severely reduced in both "normal" endometrial glands as well as in endometrial complex atypical hyperplasia (obese women), and endometrial hyperplasia (obese rats). In obese Eker rats, an energy balance intervention; caloric restriction from 2-4 months of age, reduced weight, increased adiponectin and lowered leptin to produce a favorable leptin:adiponectin ratio, and reduced circulating insulin levels. Caloric restriction also increased p27 levels, relocalized this tumor suppressor to the nucleus, and significantly decreased hyperplasia incidence. Thus, dietary and pharmacologic interventions that inhibit growth and decrease risk for development of endometrial lesions are associated with increased expression and nuclear (re)localization of p27. These data suggest that p27 levels and localization may be useful as a biomarker, and possible determinant, of risk for EC arising in the setting of obesity.

Anti-CD22-MCC-DM1: an antibody-drug conjugate with a stable linker for the treatment of non-Hodgkin's lymphoma.

Antibody-drug conjugates (ADCs) are potent cytotoxic drugs linked to antibodies through chemical linkers, and allow specific targeting of drugs to neoplastic cells. The expression of CD22 is limited to B-cells, and we show that CD22 is expressed on the vast majority of non-Hodgkin's lymphomas (NHLs). An ideal target for an ADC for the treatment of NHL would have limited expression outside the B-cell compartment and be highly effective against NHL. We generated an ADC consisting of a humanized anti-CD22 antibody conjugated to the anti-mitotic agent maytansine with a stable linker (anti-CD22-MCC-DM1). Anti-CD22-MCC-DM1 was broadly effective in in vitro killing assays on NHL B-cell lines. We did not find a strong correlation between in vitro potency and CD22 surface expression, internalization of ADC or sensitivity to free drug. We show that anti-CD22-MCC-DM1 was capable of inducing complete tumor regression in NHL xenograft mouse models. Further, anti-CD22-MCC-DM1 was well tolerated in cynomolgus monkeys and substantially decreased circulating B-cells as well as follicle size and germinal center formation in lymphoid organs. These results suggest that anti-CD22-MCC-DM1 has an efficacy, safety and pharmacodynamic profile that support its use as a treatment for NHL.

Interaction of wild-type and catalytically inactive mutant forms of tissue-type plasminogen activator with human umbilical vein endothelial cell monolayers.

Several groups have demonstrated that radioiodinated tissue-type plasminogen activator (t-PA) binds to saturable sites on human umbilical vein endothelial cells (HUVECs) in culture (Hajjar, K. A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719; Beebe, D. P. (1987) Thromb. Res. 46, 241-254; Barnathan, E. S., Kuo, A., van der Keyl, H., McCrae, K. R., Larsen, G. L., and Cines, D. B. (1988) J. Biol. Chem. 263, 7792-7799). Here we report that most of the specific binding of 125I-t-PA to our HUVEC cultures is accounted for by binding to (i) plasminogen activator inhibitor type 1 (PAI-1), a t-PA inhibitor produced in abundance by HUVECs; and (ii) specific binding sites present on the plastic culture surface. The contribution of the sites on plastic can be eliminated by taking several precautions. Then, most or all of the specifically bound 125I-t-PA is present in a sodium dodecyl sulfate-stable 110-kDa 125I-t-PA.PAI-1 complex. Interestingly, a radioiodinated mutant form of t-PA, S478A, which is catalytically inactive and therefore unable to form the covalent complex with PAI-1, still binds to HUVECs. In fact, this ligand binds to HUVECs in 10-30-fold greater amounts than does wild-type 125I-t-PA (resulting in greater than 1 x 10(7) S478A 125I-t-PA molecules bound/cell at 12 nM ligand concentration). In contrast, diisopropyl fluorophosphate-treated t-PA binds to HUVECs in much smaller amounts than does wild-type t-PA. Several findings suggest that PAI-1 is a major binding site for S478A t-PA. The vast amount of binding observed with S478A t-PA, compared with wild-type t-PA, may be accounted for by an observed large scale release of wild-type 125I-t-PA.PAI-1 complexes from the solid phase (cells or extracellular matrix) into the culture medium. Immunoprecipitation experiments demonstrate that, in contrast to wild-type t-PA, S478A t-PA does not extract [35S]methionine-PAI antigen from metabolically labeled extracellular matrix. It is proposed that t-PA releases PAI-1 from the solid phase when it forms the irreversible covalent complex with the inhibitor, a process that does not occur with the catalytically inactive mutant form of t-PA.