Blocking the catalytic activity of proteasomes is a tried-and-true strategy in multiple myeloma, but use of marketed inhibitors is limited by drug resistance and lack of efficacy in solid tumors. Now, a team from The Johns Hopkins University has discovered a compound that suppresses tumor growth in vitro and in animal models of multiple myeloma and ovarian cancer by blocking the regulatory subunit of proteasomes.

The molecule, RA190, could represent a new class of proteasome inhibitors for use in solid tumors and drug-resistant MM.1

The team is now working on enhancing the compound's drug-like properties by optimizing its formulation and is testing the compound and other derivatives in clinical isolates from patients with MM.

Proteasome inhibition has emerged as a valuable strategy in cancer, in which rapid proliferation leads to the buildup of proteins synthesized during cell division. The proteasome controls the protein content of cells by eliminating proteins that are misfolded, damaged or present when not necessary.

As MM cells produce large amounts of immunoglobulin chains, many of which are misfolded and interrupt cell function, they are particularly sensitive to proteasome inhibition.2

The first generation of inhibitors, Velcade bortezomib and Kyprolis carfilzomib (ONO-7057), target the proteasome's catalytic activity. Velcade is a first-line treatment for MM, and Kyprolis is approved as a third-line treatment in patients who have received Velcade and immunomodulatory therapy.

Velcade is marketed by Takeda Pharmaceutical Co. Ltd., and Kyprolis is marketed by Amgen Inc.

Other proteasome compounds are in clinical development for MM or solid tumors by Amgen, Teva Pharmaceutical Industries Ltd. and Nereus Pharmaceuticals Inc.

Notably, Velcade and Kyprolis have shown little success in solid tumors despite suppressing growth of solid tumor-derived cells in vitro.

In addition, resistance to Velcade and Kyprolis is an emerging problem in MM. A subset of bortezomib-naïve patients fail to respond to therapy altogether, and a subset of treated patients develop resistance upon relapse. Velcade also causes side effects such as thrombocytopenia and peripheral neuropathy in a substantial portion of patients.

Richard Roden and his team at Johns Hopkins have now identified a compound, RA190, that potently inhibits the proliferation of MM cells and is effective in Velcade-resistant MM cell lines.

RA190 is a piperidone-based inhibitor derived from a series of compounds with amino acid substitutions on the amino group of 4-piperidone that Roden's team had previously shown inhibits protein degradation.3

Roden is a professor of pathology at The Sidney Kimmel Comprehensive Cancer Center at The Johns Hopkins University School of Medicine.

The activity of RA190 against Velcade-resistant cells was the first clue that it might be acting via a new mechanism to inhibit the proteasome.

The proteasome is composed of a large 20S catalytic subunit that resembles a barrel and is capped at each end by a 19S regulatory particle. The regulatory particles contain RPN10 and adhesion regulating molecule 1 (ADRM1; RPN13) subunits that accept ubiquitinated substrates and pass them into the barrel, where they are processed and destroyed by the proteolytic activity.4

Velcade acts by inhibiting the 20S catalytic activity of the proteasome. By contrast, the activity of RA190 appeared to be dependent upon binding to a cysteine residue (Cys88) in the RPN13 subunit of the regulatory particle, since RA190 did not bind a mutant of RPN13 containing an alanine substitution at Cys88.

In vitro, RA190 triggered apoptosis in HPV-driven cervical cancer cells. The compound suppressed the oncogenic effects of the proteasome-dependent E6 transforming protein (human papillomavirus-16; HpV16gp1) ubiquitin ligase, leading to stabilization of the p53 tumor suppressor and the proapoptotic proteins BCL2-antagonist/killer 1 (BAK1) and BCL2-associated X protein (BAX).

In addition, RA190 was effective in vitro at reducing the growth of a range of solid tumor-derived cells that included cervical, colon and ovarian cancer.

The team then went on to provide in vivo evidence for the efficacy of RA190.

Using bioluminescence assays with a ubiquitin-tagged reporter in mice, they showed that oral, i.p. or topical administration of RA190 inhibited proteasome function in muscle and skin tissue.

In tumor xenograft assays, RA190 caused regression of tumors derived from human myeloma cells and inhibited growth of solid tumors generated from human ovarian carcinoma cells.

Alfred Goldberg, a professor of cell biology at Harvard Medical School, said, "This is a very interesting study. All available proteasome inhibitors target active sites in the proteasome's larger 20S catalytic core particle. RA190 is targeting the regulatory subunit, opening up a new mechanistic approach to proteasome inhibition."

Goldberg was responsible for early insights that linked protein degradation with the proteasome and participated in the development of proteasome inhibitors including Velcade.

Other noncatalytic proteasome inhibitors have been isolated from Gram-positive bacteria and are in discovery at Cytomics Pharmaceuticals.

The study was published in Cancer Cell.

Nailing the lid on the mechanism

Raymond Deshaies, a professor of biology at the California Institute of Technology, an investigator at the Howard Hughes Medical Institute and cofounder of the protein degradation-focused biotech Cleave Biosciences Inc., told SciBX that he "would like to see an unambiguous demonstration that in vitro toxicity and in vivo efficacy for RA190 are indeed due to targeting RPN13."

Deshaies' lab investigates the mechanism and regulation of the ubiquitin proteasome system.

Goldberg and Deshaies both pointed out that it will be important to understand how the toxic effect of RA190 on different cancer cells can be reconciled with the fact that Rpn13 is not an essential gene in mice.

Goldberg said, "It is surprising that RPN13 knockout is viable but the drug is toxic. This discrepancy eventually needs to be resolved. The reason could be that there are additional targets of RA190 or that there is cellular compensation to the loss of RPN13 that happens in the embryo but does not occur in the cancer cells studied."

Deshaies also cited the need for genetic studies to support a differential requirement for RPN13 in normal versus cancer cells. "This paper suggests this may be the case, which would be very exciting and raise the level of enthusiasm for RA190 considerably."

According to Wolfgang Baumeister and Pawel Sledz, RPN13 is an attractive target for developing cancer therapeutics because RPN13 is overexpressed in many cancers, which may increase their sensitivity to the compound compared with normal cells.

"Normal cells would be affected by RPN13 inhibition to a lesser extent, as RPN13 is typically substoichiometric and another ubiquitin receptor, RPN10, is sufficient to execute ubiquitin recognition at the proteasome," Sledz told SciBX.

Baumeister is a professor at the Technical University Munich and director at the Max Planck Institute of Biochemistry. Sledz is a research associate in Baumeister's group. Baumeister has contributed key insights into structural aspects of proteasome function.

Advancing the compound

Mark Rolfe, president and CSO of Cleave Biosciences, told SciBX that development of the compound should include further study of its toxicology profile because the molecule contains a reactive moiety that could interact with other cysteine residues. He added that it would need to be well differentiated from Velcade.

Rolfe also suggested investigating whether there are synergistic effects between RA190 and bortezomib. "For example, the Vk*Myc transgenic mouse model of MM developed at the Mayo Clinic would be very predictive of activity in humans."

The Vk*Myc model faithfully recapitulates MM by constitutive overexpression of the c-Myc (MYC) oncogene in B cells.

Roden said that the team's strategy is to begin by differentiating the unique properties of RA190 as an RPN13-specific inhibitor from the marketed drugs targeting the 20S proteasome and to examine its effectiveness in clinical isolates of MM refractory to bortezomib and/or carfilzomib.

In addition, Roden and his coworkers are planning to examine RA190's efficacy in HPV-associated cancer models, which could pave the way for proteasome inhibition in solid cancers.

Johns Hopkins has filed a provisional patent covering RA190, several related compounds and chemical features of these inhibitors and RPN13 blockade. The team is looking to license the IP.

Boettner, B. SciBX 7(2); doi:10.1038/scibx.2014.42
Published online Jan. 16, 2014

REFERENCES

1.   Anchoori, R.K. et al. Cancer Cell; published online Dec. 9, 2013; doi:10.1016/j.ccr.2013.11.001
Contact: Richard B.S. Roden, The Johns Hopkins University, Baltimore, Md.
e-mail: roden@jhmi.edu

2.   Goldberg, A.L. J. Cell Biol. 199, 583-588 (2012)

3.   Bazzaro, M. et al. J. Med. Chem. 54, 449-456 (2011)

4.   Förster, F. et al. Structure 21, 1551-1562 (2013)

COMPANIES AND INSTITUTIONS MENTIONED

Amgen Inc. (NASDAQ:AMGN), Thousand Oaks, Calif.

California Institute of Technology, Pasadena, Calif.

Cytomics Pharmaceuticals, Orsay, France

Cleave Biosciences Inc., Burlingame, Calif.

Harvard Medical School, Boston, Mass.

Howard Hughes Medical Institute, Pasadena, Calif.

The Johns Hopkins University, Baltimore, Md.

Max Planck Institute of Biochemistry, Martinsried, Germany

Mayo Clinic, Scottsdale, Ariz.

Nereus Pharmaceuticals Inc., San Diego, Calif.

The Sidney Kimmel Comprehensive Cancer Center at The Johns Hopkins University School of Medicine, Baltimore, Md.

Takeda Pharmaceutical Co. Ltd. (Tokyo:4502), Osaka, Japan

Technical University Munich, Munich, Germany

Teva Pharmaceutical Industries Ltd. (NYSE:TEVA), Petah Tikva, Israel