Smarter Drug Discovery Yields Promising Results in Multiple Myeloma Therapy

An innovative screening assay developed by a Cleveland Clinic-led team of researchers has helped identify CCF642 — a small-molecule compound that has led to a patent for a new class of anticancer agents.

CCF642 has broad anti-myeloma activity in vivo, prolonging survival in a mouse model of multiple myeloma comparable to the survival extension achieved by the FDA-approved first-line therapeutic agent bortezomib. CCF642 appears to work at least in part by inhibiting protein disulfide-isomerase (PDI), the bottleneck enzyme that regulates protein folding prior to secretion, which is a highly myeloma-relevant process. Multiple myeloma cells secrete massive amounts of proteins, so disruption of the protein-folding process through PDI inhibition should increase cellular stress and trigger apoptosis, potentially augmenting existing therapies.

The researchers detailed their findings in the journal Cancer Research. A patent for an analog of CCF642 and its derivatives was issued in June.

“Our study aimed to identify novel mechanism to treat myeloma and avoid unnecessary animal experiments by simulating key aspects of an organism in the laboratory,” says Frederic Reu, MD, an oncologist in Cleveland Clinic Cancer Center’s Department of Translational Hematology and Oncology Research. “The drug we identified, CCF642, really does seem to work through a novel mechanism and it is effective across the board for myeloma cells, and in an animal model of myeloma. So, in general the screen seems to be effective and its first lead compound appears promising.”

Drug discovery tailored to myeloma

The assay screened candidate compounds by simulating variables such as renal clearance and hepatic metabolism, along with aspects of the myeloma microenvironment, normal bone marrow cell tolerability and activity against different multiple myeloma cell lines.

“We know that myeloma is genetically very heterogeneous. We therefore only pursued molecules that worked in a panel of genetically heterogeneous myeloma cell lines,” Dr. Reu says. “CCF642 fulfilled the mandate for activity across myeloma cell lines. It was tested in 10 different myeloma cell lines and killed them at similar drug concentrations.”

From many small molecules, three prospects

One of the strengths of this technique was that it was mechanistically unbiased — selection of candidate molecules was not based on the available literature with respect to biological processes, nor was it aimed at known, promising targets. (See Figure.)

• Out of 30,335 chemically diverse small molecules screened for anti-multiple myeloma activity, 225 compounds were identified.
• These 225 agents were filtered for clinical promise, based on relative tolerance of normal bone marrow mononuclear cells and ability to overcome pharmacokinetic and pharmacodynamic barriers to anti-myeloma activity, leaving three remaining drug candidates.
• These three compounds were tested for cytotoxicity in a panel of genetically heterogeneous myeloma cell lines, and CCF642 was the most potent of the candidates.

Dr. Reu explains part of the simulation as follows: “We introduced a liver model into the platform to resemble a mouse, and had myeloma grown on a cell line that models its natural supportive niche in an insert with a semi-permeable bottom that allowed diffusion of drugs and gave us the chance to move the entire unit into a drug-free well, so as to simulate clearance by the kidney.”

Only after confirming CCF642’s anti-multiple myeloma activity and tolerability did the researchers identify its interaction target as PDI.

“The downside of our approach is that although we’ve identified part of the mechanism of CCF642 as PDI inhibition, we still have an incomplete understanding of exactly how the drug works,” Dr. Reu says. “It might very well act through additional mechanisms.”

Figure. Schema of small-molecule screening algorithm. The in vivo model exposes normal bone marrow mononuclear (NLBM) cells constantly to small molecule (sm)–emitting liver, whereas luciferase-expressing MM1.S-luc cells, grown on resistance-conferring HS-5 bone marrow stroma cells in Transwell inserts, are exposed for 1 to 3 hours before they are placed with drug-equilibrated insert media (about 25% of well volume) into wells containing drug-free liver and NLBM with 75% small molecule–free media to simulate rapid single-dose clearance and the natural microenvironment. Three to 4 days later, drug-exposed NLBM cells are aspirated and analyzed by trypan blue flow cytometry, whereas MM1.S-luc survival is assessed by luminescence. The primary cytotoxicity screen and multiple myeloma cell line panel uses conventional constant small molecule exposure, with cellular ATP measurement and trypan blue flow cytometry after 3 to 4 days as readout of surviving cells, respectively. From Vatolin S, Phillips JG, Jha BK, et al. Novel Protein Disulfide Isomerase Inhibitor with Anticancer Activity in Multiple Myeloma. Cancer Res. 2016 Jun 1;76(11):3340-50.

CCF642’s characteristics

After the potency of CCF642 was established, the researchers evaluated the agent in an aggressive syngeneic mouse model of multiple myeloma. CCF642 at 10 mg/kg intraperitoneally 3 times/week yielded the most beneficial therapeutic effects, significantly prolonging the life of mice engrafted with myeloma and suppressing myeloma cell growth. In addition, it did not cause substantial bone marrow toxicity.

Survival prolongation in the CCF642-treated mice was statistically equivalent to extensions achieved in the same mice strain by the maximum tolerated dose of bortezomib, the most potent FDA-approved upfront myeloma drug. Looking to the future, Dr. Reu notes that an agent such as CCF642 might prove useful for patients with bortezomib resistance because its mechanism of action is different than that of the proteasome inhibitor.

Myeloma cells have the highest protein secretion rate in mammalian biology. This increased output of proteins rich in disulfide bonds results in the deposition of improperly or incompletely folded proteins in the endoplasmic reticulum (ER), exerting stress and eventually inducing apoptosis.

PDI corrects the misarrangement of disulfide bonds, so blocking that activity should further increase ER stress beyond repair and could thus improve multiple myeloma therapy. The researchers determined that CCF642 is 100 times more potent in multiple myeloma cell lines than the known PDI inhibitors PACMA 31 and LOC14. Computational modeling suggests a novel covalent binding mode, but more research is needed to clarify how CCF642 inhibits PDI.

Dr. Reu notes that it is not uncommon for drugs with clinical promise to have mechanisms of action that are detected years after clinical translation. Still, challenges in the clinical development of CCF642 exist, including formulation.

Future directions

“There are basically two pathways by which CCF642 can enter clinical trials,” Dr. Reu says. “Either we improve its pharmacologic properties by modifying some of the non-crucial side chains or packaging it in something that already exists, like nanoparticles, to make it more soluble, or we make it a stronger PDI-inhibitor.”

To advance these efforts, Dr. Reu’s team is using crystallography to identify the exact interaction of CCF642 with PDI, and validating PDI as a target using llama-derived intrabodies.

“If CCF642 continues to be an attractive candidate, it might be effectively combined with bortezomib in clinical translation because we’ve seen synergism between the two drugs in in vitro assays,” Dr. Reu says, “or it could potentially find a role for patients with refractory disease.”

Photo © Greg Mueller