Susan Demo

Antitumor Activity of PR-171, a Novel Irreversible Inhibitor of the Proteasome

Clinical studies with bortezomib have validated the proteasome as a therapeutic target for the treatment of multiple myeloma and non-Hodgkins lymphoma. However, significant toxicities have restricted the intensity of bortezomib dosing. Here we describe the antitumor activity of PR-171, a novel epoxyketone-based irreversible proteasome inhibitor that is currently in clinical development. In comparison to bortezomib, PR-171 exhibits equal potency but greater selectivity for the chymotrypsin-like activity of the proteasome. In cell culture, PR-171 is more cytotoxic than bortezomib following brief treatments that mimic the in vivo pharmacokinetics of both molecules. Hematologic tumor cells exhibit the greatest sensitivity to brief exposure, whereas solid tumor cells and nontransformed cell types are less sensitive to such treatments. Cellular consequences of PR-171 treatment include the accumulation of proteasome substrates and induction of cell cycle arrest and/or apoptosis. Administration of PR-171 to animals results in the dose-dependent inhibition of the chymotrypsin-like proteasome activity in all tissues examined with the exception of the brain. PR-171 is well tolerated when administered for either 2 or 5 consecutive days at doses resulting in >80% proteasome inhibition in blood and most tissues. In human tumor xenograft models, PR-171 mediates an antitumor response that is both dose and schedule dependent. The antitumor efficacy of PR-171 delivered on 2 consecutive days is stronger than that of bortezomib administered on its clinical dosing schedule. These studies show the tolerability, efficacy, and dosing flexibility of PR-171 and provide validation for the clinical testing of PR-171 in the treatment of hematologic malignancies using dose-intensive schedules.

A selective inhibitor of the immunoproteasome subunit LMP7 blocks cytokine production and attenuates progression of experimental arthritis

The immunoproteasome, a distinct class of proteasome found predominantly in monocytes and lymphocytes, is known to shape the antigenic repertoire presented on class I major histocompatibility complexes (MHC-I). However, a specific role for the immunoproteasome in regulating other facets of immune responses has not been established. We describe here the characterization of PR-957, a selective inhibitor of low–molecular mass polypeptide-7 (LMP7, encoded by Psmb8), the chymotrypsin-like subunit of the immunoproteasome. PR-957 blocked presentation of LMP7-specific, MHC-I–restricted antigens in vitro and in vivo. Selective inhibition of LMP7 by PR-957 blocked production of interleukin-23 (IL-23) by activated monocytes and interferon-γ and IL-2 by T cells. In mouse models of rheumatoid arthritis, PR-957 treatment reversed signs of disease and resulted in reductions in cellular infiltration, cytokine production and autoantibody levels. These studies reveal a unique role for LMP7 in controlling pathogenic immune responses and provide a therapeutic rationale for targeting LMP7 in autoimmune disorders.

Antitumor Activity of the Glutaminase Inhibitor CB-839 in Triple-Negative Breast Cancer

Glutamine serves as an important source of energy and building blocks for many tumor cells. The first step in glutamine utilization is its conversion to glutamate by the mitochondrial enzyme glutaminase. CB-839 is a potent, selective, and orally bioavailable inhibitor of both splice variants of glutaminase (KGA and GAC). CB-839 had antiproliferative activity in a triple-negative breast cancer (TNBC) cell line, HCC-1806, that was associated with a marked decrease in glutamine consumption, glutamate production, oxygen consumption, and the steady-state levels of glutathione and several tricarboxylic acid cycle intermediates. In contrast, no antiproliferative activity was observed in an estrogen receptor–positive cell line, T47D, and only modest effects on glutamine consumption and downstream metabolites were observed. Across a panel of breast cancer cell lines, GAC protein expression and glutaminase activity were elevated in the majority of TNBC cell lines relative to receptor positive cells. Furthermore, the TNBC subtype displayed the greatest sensitivity to CB-839 treatment and this sensitivity was correlated with (i) dependence on extracellular glutamine for growth, (ii) intracellular glutamate and glutamine levels, and (iii) GAC (but not KGA) expression, a potential biomarker for sensitivity. CB-839 displayed significant antitumor activity in two xenograft models: as a single agent in a patient-derived TNBC model and in a basal like HER2+ cell line model, JIMT-1, both as a single agent and in combination with paclitaxel. Together, these data provide a strong rationale for the clinical investigation of CB-839 as a targeted therapeutic in patients with TNBC and other glutamine-dependent tumors. Mol Cancer Ther; 13(4); 890–901. ©2014 AACR.

Design and synthesis of an orally bioavailable and selective peptide epoxyketone proteasome inhibitor (PR-047).

Proteasome inhibition has been validated as a therapeutic modality in the treatment of multiple myeloma and non-Hodgkins lymphoma. Carfilzomib, an epoxyketone currently undergoing clinical trials in malignant diseases, is a highly selective inhibitor of the chymotrypsin-like (CT-L) activity of the proteasome. A chemistry effort was initiated to discover orally bioavailable analogues of carfilzomib, which would have potential for improved dosing flexibility and patient convenience over intravenously administered agents. The lead compound, 2-Me-5-thiazole-Ser(OMe)-Ser(OMe)-Phe-ketoepoxide (58) (PR-047), selectively inhibited CT-L activity of both the constitutive proteasome (beta5) and immunoproteasome (LMP7) and demonstrated an absolute bioavailability of up to 39% in rodents and dogs. It was well tolerated with repeated oral administration at doses resulting in >80% proteasome inhibition in most tissues and elicited an antitumor response equivalent to intravenously administered carfilzomib in multiple human tumor xenograft and mouse syngeneic models. The favorable pharmacologic profile supports its further development for the treatment of malignant diseases.

Targeting glutaminolysis has antileukemic activity in acute myeloid leukemia and synergizes with BCL-2 inhibition.

Cancer cells require glutamine to adapt to increased biosynthetic activity. The limiting step in intracellular glutamine catabolism involves its conversion to glutamate by glutaminase (GA). Different GA isoforms are encoded by the genes GLS1 and GLS2 in humans. Herein, we show that glutamine levels control mitochondrial oxidative phosphorylation (OXPHOS) in acute myeloid leukemia (AML) cells. Glutaminase C (GAC) is the GA isoform that is most abundantly expressed in AML. Both knockdown of GLS1 expression and pharmacologic GLS1 inhibition by the drug CB-839 can reduce OXPHOS, leading to leukemic cell proliferation arrest and apoptosis without causing cytotoxic activity against normal human CD34(+) progenitors. Strikingly, GLS1 knockdown dramatically inhibited AML development in NSG mice. The antileukemic activity of CB-839 was abrogated by both the expression of a hyperactive GAC(K320A) allele and the addition of the tricarboxyclic acid cycle product α-ketoglutarate, indicating the critical function of GLS1 in AML cell survival. Finally, glutaminolysis inhibition activated mitochondrial apoptosis and synergistically sensitized leukemic cells to priming with the BCL-2 inhibitor ABT-199. These findings show that targeting glutamine addiction via GLS1 inhibition offers a potential novel therapeutic strategy for AML.

Selective inhibition of chymotrypsin-like activity of the immunoproteasome and constitutive proteasome in Waldenström macroglobulinemia

Proteasome inhibition represents a valid antitumor approach and its use has been validated in Waldenström macroglobulinemia (WM), where bortezomib has been successfully tested in clinical trials. Nevertheless, a significant fraction of patients relapses, and many present toxicity due to its off-target effects. Selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive proteasome 20S (c20S) and immunoproteasome 20S (i20S) represents a sufficient and successful strategy to induce antineoplastic effect in hematologic tumors. We therefore studied ONX0912, a novel selective, irreversible inhibitor of the CT-L activity of i20S and c20S. Primary WM cells express higher level of i20S compared with c20S, and that ONX0912 inhibited the CT-L activity of both i20S and c20S, leading to induction of toxicity in primary WM cells, as well as of apoptosis through c-Jun N-terminal kinase activation, nuclear factor kappaB (NF-kappaB) inhibition, caspase cleavage, and initiation of the unfolded protein response. Importantly, ONX0912 exerted toxicity in WM cells, by reducing bone marrow (BM)-derived interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1) secretion, thus inhibiting BM-induced p-Akt and phosphorylated extracellular signal-related kinase (p-ERK) activation in WM cells. These findings suggest that targeting i20S and c20S CT-L activity by ONX0912 represents a valid antitumor therapy in WM.

Drug discovery in the ubiquitin regulatory pathway.

The ubiquitin system has been implicated in the pathogenesis of numerous disease states, including oncogenesis, inflammation, viral infection, CNS disorders and metabolic dysfunction. Ubiquitin conjugation and deconjugation to substrate proteins is carried out by multiple families of proteins, each with a defined role in the enzymatic cascade. This conjugation-deconjugation system parallels the kinase-phosphatase system in that both alter protein function by the addition and removal of post-translational modifiers. Our understanding of ubiquitin biology and strategies to interfere pharmacologically with the ubiquitin regulatory machinery is progressing rapidly. In light of increased interest in ubiquitin pathways as drug targets, we review the ubiquitin enzymatic cascades, highlighting therapeutic opportunities and enzymatic mechanisms. We also discuss the challenges of targeting this class of enzymes with small molecules, as well as current approaches and progress in drug discovery.

Cell cycle regulatory E3 ubiquitin ligases as anticancer targets

Disregulation of the cell cycle and proliferation play key roles in cellular transformation and tumorigenesis. Such processes are intimately tied to the concentration, localization and activity of enzymes, adapters, receptors, and structural proteins in cells. Ubiquitination of these cellular regulatory proteins, governed by specific enzymes in the ubiquitin (Ub) conjugation cascade, has profound effects on their various functions, most commonly through proteasome targeting and degradation. This review will focus on a variety of E3 Ub ligases as potential oncology drug targets, with particular emphasis on the role of these molecules in the regulation of stability, localization, and activity of key proteins such as tumor suppressors and oncoproteins. E3 ubiquitin ligases that have established roles in cell cycle and apoptosis, such as the anaphase-promoting complex (APC), the Skp-1-Cul1-F-box class, and the murine double minute 2 (MDM2) protein, in addition to more recently discovered E3 ubiquitin ligases which may be similarly important in tumorigenesis, (e.g. Smurf family, CHFR, and Efp), will be discussed. We will present evidence to support E3 ligases as good biological targets in the development of anticancer therapeutics and address challenges in drug discovery for these targets.

Molecular Mechanisms of Bortezomib Resistant Adenocarcinoma Cells

Bortezomib (Velcade™) is a reversible proteasome inhibitor that is approved for the treatment of multiple myeloma (MM). Despite its demonstrated clinical success, some patients are deprived of treatment due to primary refractoriness or development of resistance during therapy. To investigate the role of the duration of proteasome inhibition in the anti-tumor response of bortezomib, we established clonal isolates of HT-29 adenocarcinoma cells adapted to continuous exposure of bortezomib. These cells were ∼30-fold resistant to bortezomib. Two novel and distinct mutations in the β5 subunit, Cys63Phe, located distal to the binding site in a helix critical for drug binding, and Arg24Cys, found in the propeptide region were found in all resistant clones. The latter mutation is a natural variant found to be elevated in frequency in patients with MM. Proteasome activity and levels of both the constitutive and immunoproteasome were increased in resistant cells, which correlated to an increase in subunit gene expression. These changes correlated with a more rapid recovery of proteasome activity following brief exposure to bortezomib. Increased recovery rate was not due to increased proteasome turnover as similar findings were seen in cells co-treated with cycloheximide. When we exposed resistant cells to the irreversible proteasome inhibitor carfilzomib we noted a slower rate of recovery of proteasome activity as compared to bortezomib in both parental and resistant cells. Importantly, carfilzomib maintained its cytotoxic potential in the bortezomib resistant cell lines. Therefore, resistance to bortezomib, can be overcome with irreversible inhibitors, suggesting prolonged proteasome inhibition induces a more potent anti-tumor response.

Validation of the proteasome as a therapeutic target in Plasmodium using an epoxyketone inhibitor with parasite-specific toxicity

The Plasmodium proteasome has been suggested to be a potential antimalarial drug target; however, toxicity of inhibitors has prevented validation of this enzyme in vivo. We report a screen of a library of 670 analogs of the recent US Food and Drug Administration-approved inhibitor, carfilzomib, to identify compounds that selectively kill parasites. We identified one compound, PR3, that has significant parasite killing activity in vitro but dramatically reduced toxicity in host cells. We found that this parasite-specific toxicity is not due to selective targeting of the Plasmodium proteasome over the host proteasome, but instead is due to a lack of activity against one of the human proteasome subunits. Subsequently, we used PR3 to significantly reduce parasite load in Plasmodium berghei infected mice without host toxicity, thus validating the proteasome as a viable antimalarial drug target.