Inhibition of the p53 E3 ligase HDM-2 induces apoptosis and DNA damage--independent p53 phosphorylation in mantle cell lymphoma.

PURPOSE: The ubiquitin-proteasome pathway has been validated as a target in non-Hodgkin's lymphoma through demonstration of the activity of the proteasome inhibitor bortezomib. EXPERIMENTAL DESIGN: Another potentially attractive target is the human homologue of the murine double minute-2 protein, HDM-2, which serves as the major p53 E3 ubiquitin ligase; we therefore evaluated the activity of a novel agent, MI-63, which disrupts the HDM-2/p53 interaction. RESULTS: Treatment of wild-type p53 mantle cell lymphoma (MCL) cell lines with MI-63 resulted in a dose- and time-dependent inhibition of proliferation, with an IC(50) in the 0.5 to 5.0 micromol/L range. MI-63 induced p53 and HDM-2 accumulation, as well as other downstream p53 targets such as p53 up-regulated modulator of apoptosis and p21(Cip1). This was associated with cell cycle arrest at G(1)-S; activation of caspase-3, caspase-8, and caspase-9; cleavage of poly-(ADP-ribose) polymerase; and loss of E2F1. HDM-2 inhibition caused phosphorylation of p53 at multiple serine residues, including 15, 37, and 392, which coincided with low levels of DNA strand breaks. DNA damage occurred in a small percentage of cells and did not induce phosphorylation of the DNA damage marker H2A.X(Ser139). Combinations of MI-63 with the molecularly targeted agents bortezomib and rapamycin showed synergistic, sequence-dependent antiproliferative effects. Treatment of primary MCL patient samples resulted in apoptosis and induction of p53 and p21, which was not seen in normal controls. CONCLUSIONS: These findings support the hypothesis that inhibition of the HDM-2/p53 interaction may be a promising approach both by itself and in combination with currently used chemotherapeutics against lymphoid malignancies.

Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitin-proteasome pathway, against preclinical models of multiple myeloma.

The proteasome has emerged as an important target for cancer therapy with the approval of bortezomib, a first-in-class, reversible proteasome inhibitor, for relapsed/refractory multiple myeloma (MM). However, many patients have disease that does not respond to bortezomib, whereas others develop resistance, suggesting the need for other inhibitors with enhanced activity. We therefore evaluated a novel, irreversible, epoxomicin-related proteasome inhibitor, carfilzomib. In models of MM, this agent potently bound and specifically inhibited the chymotrypsin-like proteasome and immunoproteasome activities, resulting in accumulation of ubiquitinated substrates. Carfilzomib induced a dose- and time-dependent inhibition of proliferation, ultimately leading to apoptosis. Programmed cell death was associated with activation of c-Jun-N-terminal kinase, mitochondrial membrane depolarization, release of cytochrome c, and activation of both intrinsic and extrinsic caspase pathways. This agent also inhibited proliferation and activated apoptosis in patient-derived MM cells and neoplastic cells from patients with other hematologic malignancies. Importantly, carfilzomib showed increased efficacy compared with bortezomib and was active against bortezomib-resistant MM cell lines and samples from patients with clinical bortezomib resistance. Carfilzomib also overcame resistance to other conventional agents and acted synergistically with dexamethasone to enhance cell death. Taken together, these data provide a rationale for the clinical evaluation of carfilzomib in MM.

Emerging data on the use of anthracyclines in combination with bortezomib in multiple myeloma.

Since the inception of infusional vincristine/doxorubicin/pulsed dexamethasone (VAD) for the treatment of multiple myeloma, anthracyclines have remained an important class of antimyeloma agents. More recently, the introduction of pegylated liposomal doxorubicin with improved pharmacokinetic characteristics has led to the development of newer anthracycline-containing regimens with improved toxicity profiles. Bortezomib, a first in class reversible inhibitor of the proteasome, has also emerged as an important novel agent for the treatment of multiple myeloma and is currently approved for patients with relapsed/refractory disease progressing after 1 previous therapy. Although both classes of agents have potent proapoptotic activity, they also induce activation of an antiapoptotic prosurvival program that limits their own efficacy, a process known as inducible chemotherapy resistance. Importantly, in preclinical studies, each of these drugs has been shown to attenuate chemotherapy resistance induced by the other, and combinations of the 2 have demonstrated striking synergistic activity. Furthermore, early phase I/II clinical trials have shown impressive activity of pegylated liposomal doxorubicin and conventional doxorubicin in combination with bortezomib in patients with newly diagnosed and relapsed/refractory myeloma. Phase II/III clinical trials evaluating these regimens in patients with newly diagnosed and relapsed/refractory disease have recently completed accrual and will better define the role of these combinations in myeloma therapy. Herein, we review the preclinical data supporting the use of bortezomib with anthracyclines and the promising clinical data with these combinations.