ClpP agonists and POLRMT inhibitors have been shown to impair cellular proliferation in multiple cancer cell lines and models
[20,32,33,71,77,78][20][32][33][64][65][66]. Some evidence suggests that ClpP agonists and POLRMT inhibitors can induce cell death, though these effects are generally minimal compared to the cytostatic effects observed on cell proliferation
[20,32,74,75,77,79,80][20][32][63][65][67][68][69]. However, these results may depend on the specific ClpP agonist studied. For example, ONC201 treatment increased apoptosis in mantle cell lymphoma, AML, pancreatic ductal adenocarcinoma, multiple myeloma, and ovarian cancer cell lines, whereas there have been no reports of significant increases in apoptosis after TR compound treatment
[77,79,80,81,82][65][68][69][70][71]. ONC201 was previously reported to be both a ClpP agonist and a dopamine receptor D2 antagonist, while the TR compounds are selective ClpP agonists
[33,35,83][33][35][72], which may explain these differences.
5.2. Cytostatic to Cancer Cells, Harmless to Normal Cells
One of the unusual properties of ClpP agonists is that they appear to adversely affect cancer cells but have little or no effect on normal cells. Several groups have tested ClpP agonists on immortalized cell lines (i.e., HFF) or primary cells and observed no significant effects on cell proliferation, viability, or apoptosis as compared to the cancer cell models
[42,68,75,84][42][61][63][73]. Additionally, ClpP agonists were reported to be well tolerated clinically, with few side effects observed in patients with refractory solid tumors and neuroendocrine tumors
[64,65][57][58]. In contrast, other mitochondria-targeting drugs such as metformin and CPI-631 have shown negative side effects such as gastrointestinal tract disorders, nausea, and vomiting; however, these toxicities were not reported in ONC201 clinical trials
[64,65,85,86][57][58][74][75].
5.3. Dysregulation of Cancer Cell Metabolic Programs
Both ClpP agonists and POLRMT inhibitors induce major metabolic alterations in cancer cells, most prominently observed with the inhibition of OXPHOS
[20,34,75,78,83][20][34][63][66][72]. Both classes of compounds reduce the mitochondrially-encoded protein components required for OXPHOS
[20,34][20][34]. However, the mechanism by which OXPHOS is impaired in response to ClpP activation is less well understood than the proposed mechanism of POLRMT inhibitors. Loss of POLRMT activity results in the direct inhibition of mitochondrial transcription, which in turn prevents the expression of the 13 protein-coding genes essential for respiration
[20]. Significant decreases in mRNA levels of several of these genes have been shown following IMT1 treatment
[20[20][32],
32], confirming inhibition of mitochondrial transcription following POLRMT inhibition. This combined with the loss of their cognate proteins (i.e., COI) could explain the observed inhibition of OXPHOS in these cells
[20].
Another common metabolic alteration observed following ClpP activation or POLRMT inhibition is AMPK activation
[20,75][20][63]. Greer et al. (2018) observed a decline in ATP levels and an increase in AMPK phosphorylation (Thr172) following ONC201 treatment
[75][63]. Similar effects were observed with IMT1 treatment
[20]. AMPK activation is mediated by a high cellular AMP/ATP ratio
[87][76], further indicating that inhibition of OXPHOS and/or other mitochondrial processes required for ATP generation is occurring in response to these two different mitochondrial targeting approaches.
As an expected consequence of OXPHOS inhibition, ClpP agonists have been shown to cause a compensatory increase in glycolysis
[34,75,78][34][63][66]. A similar compensatory response has not been demonstrated following POLRMT inhibition, although it may be predicted.
5.4. Loss of mtDNA Content
An important similarity between ClpP agonists and POLRMT inhibitors is the corresponding reduction in mtDNA content, despite observations that the mtDNA copy number appears to change at different rates. IMT1 treatment reportedly took ~96 h to reduce the mtDNA copy number to ~25% of its original number
[20], whereas ONC201 treatment required less time (~48 h) to reduce the mtDNA copy number to the same level
[75][63]. This may in part be determined by the rate of TFAM protein loss. TFAM expression is closely correlated to the mtDNA copy number, likely because it binds to and packages mtDNA
[98][77]. ClpP activation results in a time- and dose-dependent decline in TFAM protein that correlates with observed decreases in mtDNA content
[33[33][34][63][66],
34,75,78], as do POLRMT inhibitors (Daglish, unpublished observations (IMT1)).
5.5. Inhibition of Mitochondrial Transcription
It has been shown that POLRMT and TFAM protein expression decreases after treatment with ClpP agonists, but it has not been established that mitochondrial transcription is inhibited. The scholars predicted that mitochondrial transcription would be impaired by ClpP agonists due to their ability to deplete POLRMT, TFAM, and TEFM proteins which are all essential for mitochondrial transcription (
Figure 1). Both TR-57 and IMT1 inhibited mitochondrial transcription in the tumorigenic immortalized HEK293T cells line (
Figure 3)
[101,102][78][79]. mRNA transcript levels of two mitochondrially-encoded protein genes required for OXPHOS (ND1 and ND6) were measured by quantitative PCR to monitor mitochondrial transcription from the heavy and light strands of mtDNA, respectively. Both treatments significantly decreased ND1 and ND6 transcript levels after 3 and 6 h, although there was a greater decrease observed following IMT1 treatment (
Figure 3).
Figure 3. TR-57 and IMT1 both inhibit mitochondrial transcription from the heavy and light strand. HEK293T cells were plated at 4 × 10
5 cells/well in 6 well plates, allowed to adhere overnight, and treated with TR-57 (150 nM) and IMT1 (10 µM) for indicated times. Cells were washed 3 times with cold DPBS, scraped, and pelleted. RNA was isolated using the RNeasy Plus Mini Kit (Qiagen) and residual DNA was removed using the RNase-Free DNase Set (Qiagen). cDNA was synthesized from 2 μg of extracted RNA in 20 μL total volume using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and the Bio-Rad T100 thermal cycler. Quantitative PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad). The nuclear reference gene 18S, mitochondrial heavy strand gene, ND1, and mitochondrial light strand gene, ND6, were quantified using the CFX96 Touch Real-Time System. A one-way ANOVA analysis was performed to determine significance;
p-value < 0.05 (*), 0.01 (**), 0.001 (***). N = 2 biological replicates.
6. Mechanistic Differences between ClpP Agonists and POLRMT Inhibitors
6.1. Differences in Treatment Response Times
Despite the many mechanistic similarities between ClpP agonists and POLRMT inhibitors, there are several notable differences. Primarily, ClpP agonists inhibit cell proliferation much more robustly than POLRMT inhibitors. TR-57 strongly inhibits HEK293T cell proliferation following 72 h treatment, whereas IMT1 treatment requires a minimum of 120 h to show a similar inhibition of cell proliferation (
Figure 4). In terms of potency, the TR compounds are more potent inhibitors of HEK293T growth; the IC
50 of TR-57 in HEK293T cells is ~15 nM after 72 h, whereas the POLRMT inhibitor IMT1 has an IC
50 of ~190 nM after 120 h of treatment (
Figure 4).
Figure 4. The ClpP agonist TR-57 inhibits cell proliferation more potently and rapidly than the POLRMT inhibitor IMT1. HEK293T cells were cultured in DMEM media supplemented with 10% fetal bovine serum and 1% antibiotic/antimycotic (Gibco). Cells were either plated in 96 well plates at 5 × 10
3 cells/well, allowed to adhere overnight, and treated for 72 h with indicated concentrations of TR-57 and IMT1, or cells were plated at 1.5 × 10
3 cells/well, allowed to adhere overnight, and treated with indicated concentrations of IMT1 for 120 h. After drug treatment for 72 (
A) or 120 h (
B), cells were stained with 2.5 µg/mL of Hoechst 33,342 and incubated for 20 min at 37 °C and 5% CO
2. Total cell number was counted using the Celígo S imager (Nexcelom Biosciences). N = 2 biological replicates. The reported IC
50 values are averaged from the 2 replicates.
6.2. Known and Unknown Mechanisms of Resistance
There are a few known cancer cell mechanisms of resistance to ClpP agonists, including p0 cells, fumarate hydratase knockout cell lines, and ClpP knockout or ClpP mutant cell lines [61][80]. The specific ClpP mutation, D190A, is known to confer resistance to ClpP agonists [68][61]. Comparatively, there are specific POLRMT mutations that confer resistance to IMT1. Bonekamp et al. (2020) reported six mutations in POLRMT that lead to IMT1-resistance [20]. A CRISPR screen aimed at identifying mechanisms of resistance against IMT1 found that loss of von Hippel-Landau protein (VHL) and mammalian target of rapamycin complex 1 (mTORC1) expression produced IMT1-resistance in RKO cells [104][81].
6.3. Inhibiting One Protein versus Degrading Many Proteins
Major differences distinguishing the effects of POLRMT inhibitors from ClpP agonists may result from IMT1 selectively inhibiting mitochondrial transcription
[20], whereas ClpP agonists dysregulate multiple mitochondrial processes
[35,39,68,76,79][35][39][61][62][68]. As such, elucidating the specific mechanisms of action of ClpP agonists may prove to be more difficult. While efforts continue to identify specific ClpP substrates and related metabolic processes, the connection between loss of a specific ClpP substrate and its effects on cell proliferation remains to be established. Comparatively, a potential drawback to POLRMT inhibition is that these inhibitors only primarily impact one process or cancer cell vulnerability (mitochondrial transcription)
[20]. Since pharmacological activation of ClpP proteolysis affects multiple growth-related cellular processes, this intuits an overall more disruptive mechanism than POLRMT inhibition. In the context of cancer cell growth, there may be an advantage to eliminating multiple events required for cancer cell growth to prevent the development of resistance more effectively.