Researchers in the UNC Lineberger Comprehensive Cancer Center led by Lee Graves, PhD, have uncovered the mechanism that explains how an investigational cancer therapeutic might be working.
The research to on the method of action of cancer drug, ONC201, and its analogs, led by Lee Graves, Professor in the Department of Pharmacology, was highlighted in “The Scientist” May 21issue.
ONC201, which blocks cancer cells from proliferating, is currently in clinical trials, but its mechanism has not been well understood.
“ONC201 was originally identified as a potential cancer drug in a screen for molecules that induce the transcription of TRAIL, a gene that leads to apoptosis in tumors. Yet ONC201 doesn’t activate TRAIL in all of the cancer cells against which it is effective: In a 2018 paper, Yoshimi Greer and colleagues in the Lipkowitz lab reported that ONC201 worked against several lines of breast cancer cells—without upregulating TRAIL. Instead, they found, ONC201 was hindering the cancers’ mitochondrial function. But just how ONC201 was doing its mitochondrial damage was unclear.
“To find out, Graves, a University of North Carolina School of Medicine pharmacologist, and colleagues studied ONC201 as well as very similar molecules, called ONC201 analogs, generated by the Chapel Hill–based company Madera Therapeutics, of which one of Graves’s coauthors is president. They attached the ONC201 analog TR-80 to agarose beads to construct a column and ran the innards of HeLa cells, the immortal cervical cancer–derived cell line, through it to see what would stick.
“We came at it from an old-fashioned affinity-chromatography approach—you know, ‘let’s make some bait and go fishing and see what we catch,’” Graves tells The Scientist. Mass spectrometry identified the protein they caught as ClpP. They repeated the experiments using cell lysates from other cancers, including breast, pancreatic, and lung, and in every case, they found that the ONC201 analogs bound ClpP, they report in ACS Chemical Biology. ” (~the above is an excerpt from an article by Ashley Taylor, in The Scientist, May 21, 2019 issue, “Found: A Cancer Drug’s Mechanism of Action”)
ACS Chemical Biology article abstract
ONC201 is a first-in-class imipridone molecule currently in clinical trials for the treatment of multiple cancers. Despite enormous clinical potential, the mechanism of action is controversial. To investigate the mechanism of ONC201 and identify compounds with improved potency, we tested a series of novel ONC201 analogues (TR compounds) for effects on cell viability and stress responses in breast and other cancer models. The TR compounds were found to be ∼50-100 times more potent at inhibiting cell proliferation and inducing the integrated stress response protein ATF4 than ONC201. Using immobilized TR compounds, we identified the human mitochondrial caseinolytic protease P (ClpP) as a specific binding protein by mass spectrometry. Affinity chromatography/drug competition assays showed that the TR compounds bound ClpP with ∼10-fold higher affinity compared to ONC201. Importantly, we found that the peptidase activity of recombinant ClpP was strongly activated by ONC201 and the TR compounds in a dose- and time-dependent manner with the TR compounds displaying a ∼10-100 fold increase in potency over ONC201. Finally, siRNA knockdown of ClpP in SUM159 cells reduced the response to ONC201 and the TR compounds, including induction of CHOP, loss of the mitochondrial proteins (TFAM, TUFM), and the cytostatic effects of these compounds. Thus, we report that ClpP directly binds ONC201 and the related TR compounds and is an important biological target for this class of molecules. Moreover, these studies provide, for the first time, a biochemical basis for the difference in efficacy between ONC201 and the TR compounds.
In addition to Graves, other researchers are Paul R. Graves, Lucas J. Aponte-Collazo, Emily M.J. Fennell, Adam C. Graves, Andrew E. Hale, Nedyalka Dicheva, Laura E. Herring, Thomas S.K. Gilbert, Michael P. East, Ian M. McDonald, Matthew R. Lockett, Hani AShamalla, Nathaniel J. Moorman, Donald S. Karanewsky, Edwin J. Iwanowicz, and Ekhson Holmuhamedov. UNC Proteomics, of which Graves is faculty director, also played an important role in this study.