Simultaneous targeting of mitochondrial metabolism and immune checkpoints as a new strategy for renal cancer therapy

Abstract

Cancer is a pathology still on the rise,1 with unmet need for efficient therapy, owing to factors such as considerable differences in mutational signature in the same patient in primary tumours and proximal/distal metastases, shown, for example, for renal cancer.2 What is needed then is an invariant target predominantly only affected by drugs in cancer cells. A thus far untested approach is targeting mitochondrial respiration using compounds from the group of mitocans,3 epitomised by mitochondrially targeted tamoxifen (MitoTam), that is, tamoxifen tagged with the mitochondrial vector triphenylphosphonium (TPP) (Figure S1A; see also Supporting Information for description of synthesis).4, 5 This strategy is based on the premise that cancer cells differ from their non-cancerous counterparts,3 making them selectively vulnerable to TPP-tagged anti-cancer agents,6 and on the premise that mitochondrial function is vital for tumour progression.7, 8 We have recently conducted Phase 1/1b MitoTam clinical trial for metastatic solid tumour patients, with all patients undergoing palliative therapy after exhaustion of established therapeutic regimens (MitoTam-01 trial; EudraCT 2017-004441-25). Although the Phase 1/1b clinical trial will be published in its entirety elsewhere, of the individual types of cancer, the greatest benefit was found for clear cell renal cancer patients represented here by two subjects (Tables S1 and S2). These patients underwent three and four rounds of MitoTam therapy, respectively, at 1 mg/kg three times per week followed by a week of rest, totalling four such cycles, with one patient showing tumour stabilisation and the other partial remission (Figure 1A). The trial revealed excellent safety profile of MitoTam, with only occasional grade 1 toxicity. The high efficiency for renal cancer was found to correlate with the highest level of MitoTam and its metabolites reached in kidneys (Figures 1B, S1B and S1C), being excreted via bile (Figure S1D). As renal cancer cells possess high activity of oxidative phosphorylation (OXPHOS) and its inhibition induces cell death in starvation-resistant tumours,7 we studied the effect of MitoTam on renal cancer in vitro and in vivo to better understand its benefit for patients. Using human and a mouse renal cancer cell lines, we found that MitoTam killed the cells with IC50 of ≈0.3–1.4 μM (Figure 1C, Figure S1E) even after its removal from cell culture media (Figure 1D). Analysing the mode of action using RenCa cells, we found necroptosis to play major role in elimination of cancer cells (Figures 1E and F, Figure S2A), while inhibition of caspases enhanced the MitoTam effect (Figure S2B). This indicates positive connotation for inhibiting renal cancer by killing cancer cells via multimodal manner. Importance of mitochondria for killing cancer cells by MitoTam was tested using cells with modified mitochondrial function8 (Figure S2C–S2E). Consistent with previous findings for breast cancer cells,5 MitoTam inhibited primarily CI-dependent respiration in RenCa cells (Figure 2A and B), which was accompanied by uncoupled respiration (Figure 2C), a drop in mitochondrial potential and a switch to glycolysis (Figure S2F–S2K). This was further supported by a drop in the respirasome level and CI- and ATPase-dependent activity upon MitoTam treatment (Figure 2D and E) as well as in levels of CI-CIV subunits (Figure 2F), in deregulation of the Krebs cycle and amino acid metabolism (Figure 2G and H, Table S3) and in alteration of mitochondrial morphology (Figure S2L). Using syngeneic model of RenCa cell-derived tumours, MitoTam showed anti-cancer activity in a concentration-dependent manner (Figure 3A–C), without any effect on healthy kidneys (Figure S3A and S3B) when suppressing orthotopic renal tumours (Figure 3D and E). A similar effect was observed for CAKI-1 cells in a xenograft model (Figure 3F and G). Although MitoTam did not inhibit proliferation of cells in syngeneic tumours, it promoted their death (Figure S3C and 3D). Selective toxicity for cancer cells was evident from suppression of CI-dependent respiration only in renal tumours (Figure 3H–J) compared to healthy tissue without any effect on mouse weight and blood count (Figure S3E–S3F). Interestingly, MitoTam suppressed metastases of RenCa cells into lungs (Figure 3K), depending on functional mitochondria (Figure 3L). The anti-metastatic effect of MitoTam is supported by suppression of migration and invasion of renal cancer cells in vitro using three- and two-dimensional settings (Figure S3G–S3I). Renal cell carcinoma is an immunogenic tumour featuring abundant infiltration of lymphocytes. Despite promising results of immunotherapy, resistance to this treatment occurs in several tumour types providing an opportunity for improved immunotherapeutic approaches. Since targeting of immune checkpoints (ICIs) PD-L1 and PD-1, currently representing first-line therapy in renal cancer,9 induces metabolic reprogramming of cancer cells by decreased ability to use glycolysis and increased OXPHOS importance,10 we combined their effect with MitoTam. Based on testing different PD-L1 concentrations on tumour growth and CD8+ T-cell activity and recruitment (Figure S4A–S4D), we document additive effect of MitoTam and PD-L1 used at 2 mg/kg and 400 μg/mouse, respectively, compared to single agent therapy (Figure 4A), supported by significantly greater survival (Figure 4B) and increased level of tumour cell death (Figure 4C). PD-L1 treatment enhanced infiltration and activity of CD8+ T-cells, which was not affected by MitoTam (Figure 4D–F). Moreover, MitoTam affected the level of PD-L1 neither in tumours of mice with syngeneic carcinomas (Figure 4G) nor in the two cancer patients presented in Figure 1A (Figure S4E). PD-1 showed an additive affect with MitoTam in RenCa cell tumours (Figure 4H and I) similar to that for the combination of MitoTam with PD-L1 presented above. To conclude, we document that MitoTam efficiently kills a range of renal cancer cells and suppresses renal carcinomas in a mouse model by a complex effect on mitochondria, resulting in decreased tumorigenesis. The fact that monotherapy of MitoTam proved to be as efficient in our animal model as immunotherapy with ICIs is highly encouraging, especially given the fact that renal cancer patients respond poorly to standard chemotherapy. Our study therefore suggests a novel strategy for treatment of renal cancer by targeting mitochondria in combination with immunotherapy presented by ICIs. As MitoTam as well as clinically approved ICIs show excellent safety profiles, these results have direct clinical implications in designing rational combination treatments and, together with Phase1/1b results, encourage further development of MitoTam in future Phase 2 trial. We thank Karel Harant, Pavel Talacko and Petr Zacek from the BIOCEV OMICS/Proteomics Core Facility, Faculty of Science, Charles University, for performing proteomic and metabolomics analysis; and Jana Juhasova, Jiri Klima, Patricia Jandurova, Lenka Travnickova and Irena Deylova from Institute of Animal Physiology and Genetics, Czech Academy of Sciences, for minipigs ADME study. This research was supported in part by grants from Czech Science Foundation 20-05942S and 21-04607X to JN, 18-02550S to SSH and 19-08772S to LA, and from Czech Heath Foundation NU21-03-00195 to JN, and by the BIOCEV European Regional Development Fund CZ.1.05/1.100/02.0109 and the Institute of Biotechnology fund RVO: 86652036. MD, EV and KK were supported by the Grant Agency of Charles University (GA UK 1242120 to MD, 1560218 to EV, 1306120 to KK). The Core Facility for Electron Microscopy (Institute of Molecular Genetics, Prague, Czech Republic) is supported by the Czech-BioImaging large RI project (LM2018129 funded by MEYS CR) and by OP RDE (CZ.02.1.01/0.0/0.0/16_013/0001775 ‘Modernization and support of research activities of the national infrastructure for biological and medical imaging Czech-BioImaging’). VF and PH were supported by the MEYS CR COST Inter-excellence internship LTC19048 and by the Institute of Molecular Genetics (RVO: 68378050). JSt and LW were supported by Strategy AV21 (Research Programme No.18. Preclinical Testing of Potential Pharmaceuticals). The whole project was supported in part by the National Sustainability Program I, project number LO1609 (Czech Ministry of Education, Youth and Sports) and by the Institutional Research Concept of IAPG CAS (RVO:67985904). JN, JSt and LW are co-owners of MitoTax s.r.o. and the MitoTam intellectual property. JN, JSt and LW are also co-inventors of MitoTam as an oncolytic agent. Other authors declare no conflict of interest. Private investors KKCG a.s. and SmartBrain s.r.o. (both Prague, Czech Republic) financed the MitoTam-01 clinical trial. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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