Proteasome inhibitors are the backbone of multiple myeloma therapy. However, disease progression or early relapse occur due to development of resistance to the therapy. One important cause of resistance to proteasome inhibition is the so-called bounce-back response, a recovery pathway driven by the TCF11/Nrf1 transcription factor, which activates proteasome gene re-synthesis upon impairment of the proteasome function. Thus, inhibiting this recovery pathway potentiates the cytotoxic effect of proteasome inhibitors and could benefit treatment outcomes. DDI2 protease, the 3D structure of which resembles the HIV protease, serves as the key player in TCF11/Nrf1 activation. Previous work found that some HIV protease inhibitors block DDI2 in cell-based experiments. Nelfinavir, an oral anti-HIV drug, inhibits the proteasome and/or pAKT pathway and has shown promise for treatment of relapsed/refractory multiple myeloma. Here, we describe how nelfinavir inhibits the TCF11/Nrf1-driven recovery pathway by a dual mode of action. Nelfinavir decreases the total protein level of TCF11/Nrf1 and inhibits TCF11/Nrf1 proteolytic processing, likely by interfering with the DDI2 protease, and therefore reduces the TCF11/Nrf1 protein level in the nucleus. We propose an overall mechanism that explains nelfinavir's effectiveness in the treatment of multiple myeloma.

1st Department Medicine Department of Hematology Charles University U Nemocnice 2 12808 Prague Czech Republic

1st Faculty of Medicine Charles University Kateřinská 32 12108 Prague Czech Republic

Department of Genetics and Microbiology Charles University Viničná 5 12843 Prague Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo n 2 16610 Prague Czech Republic

Zobrazit více v PubMed

Neznanov N., Komarov A.P., Neznanova L., Stanhope-Baker P., Gudkov A.V. Proteotoxic stress targeted therapy (PSTT): Induction of protein misfolding enhances the antitumor effect of the proteasome inhibitor bortezomib. Oncotarget. 2011;2:12. doi: 10.18632/oncotarget.246. PubMed DOI PMC

Obeng E.A., Carlson L.M., Gutman D.M., Harrington W.J., Jr., Lee K.P., Boise L.H. Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells. Blood. 2006;107:4907–4916. doi: 10.1182/blood-2005-08-3531. PubMed DOI PMC

Adams J. The proteasome: A suitable antineoplastic target. Nat. Rev. Cancer. 2004;4:349–360. doi: 10.1038/nrc1361. PubMed DOI

Rajkumar S.V., Kumar S. Multiple Myeloma: Diagnosis and Treatment. Mayo Clin. Proc. 2016;91:101–119. doi: 10.1016/j.mayocp.2015.11.007. PubMed DOI PMC

Radhakrishnan S.K., Lee C.S., Young P., Beskow A., Chan J.Y., Deshaies R.J. Transcription Factor Nrf1 Mediates the Proteasome Recovery Pathway after Proteasome Inhibition in Mammalian Cells. Mol. Cell. 2010;38:17–28. doi: 10.1016/j.molcel.2010.02.029. PubMed DOI PMC

Steffen J., Seeger M., Koch A., Kruger E. Proteasomal degradation is transcriptionally controlled by TCF11 via an ERAD-dependent feedback loop. Mol. Cell. 2010;40:147–158. doi: 10.1016/j.molcel.2010.09.012. PubMed DOI

Radhakrishnan S.K., den Besten W., Deshaies R.J. p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition. Elife. 2014;3:e01856. doi: 10.7554/eLife.01856. PubMed DOI PMC

Meiners S., Heyken D., Weller A., Ludwig A., Stangl K., Kloetzel P.M., Kruger E. Inhibition of proteasome activity induces concerted expression of proteasome genes and de novo formation of Mammalian proteasomes. J. Biol. Chem. 2003;278:21517–21525. doi: 10.1074/jbc.M301032200. PubMed DOI

Vangala J.R., Sotzny F., Kruger E., Deshaies R.J., Radhakrishnan S.K. Nrf1 can be processed and activated in a proteasome-independent manner. Curr. Biol. 2016;26:R834–R835. doi: 10.1016/j.cub.2016.08.008. PubMed DOI PMC

Koizumi S., Irie T., Hirayama S., Sakurai Y., Yashiroda H., Naguro I., Ichijo H., Hamazaki J., Murata S. The aspartyl protease DDI2 activates Nrf1 to compensate for proteasome dysfunction. Elife. 2016;5 doi: 10.7554/eLife.18357. PubMed DOI PMC

Northrop A., Vangala J.R., Feygin A., Radhakrishnan S.K. Disabling the Protease DDI2 Attenuates the Transcriptional Activity of NRF1 and Potentiates Proteasome Inhibitor Cytotoxicity. Int. J. Mol. Sci. 2020;21 doi: 10.3390/ijms21010327. PubMed DOI PMC

Siva M., Svoboda M., Veverka V., Trempe J.F., Hofmann K., Kozisek M., Hexnerova R., Sedlak F., Belza J., Brynda J., et al. Human DNA-Damage-Inducible 2 Protein Is Structurally and Functionally Distinct from Its Yeast Ortholog. Sci. Rep. 2016;6:30443. doi: 10.1038/srep30443. PubMed DOI PMC

White R.E., Powell D.J., Berry C. HIV proteinase inhibitors target the Ddi1-like protein of Leishmania parasites. FASEB J. 2011;25:1729–1736. doi: 10.1096/fj.10-178947. PubMed DOI PMC

Gills J.J., Lopiccolo J., Tsurutani J., Shoemaker R.H., Best C.J., Abu-Asab M.S., Borojerdi J., Warfel N.A., Gardner E.R., Danish M., et al. Nelfinavir, A lead HIV protease inhibitor, is a broad-spectrum, anticancer agent that induces endoplasmic reticulum stress, autophagy, and apoptosis in vitro and in vivo. Clin. Cancer Res. 2007;13:5183–5194. doi: 10.1158/1078-0432.CCR-07-0161. PubMed DOI

Bono C., Karlin L., Harel S., Mouly E., Labaume S., Galicier L., Apcher S., Sauvageon H., Fermand J.P., Bories J.C., et al. The human immunodeficiency virus-1 protease inhibitor nelfinavir impairs proteasome activity and inhibits the proliferation of multiple myeloma cells in vitro and in vivo. Haematologica. 2012;97:1101–1109. doi: 10.3324/haematol.2011.049981. PubMed DOI PMC

Brüning A., Rahmeh M., Gingelmaier A., Friese K. The mitochondria-independent cytotoxic effect of nelfinavir on leukemia cells can be enhanced by sorafenib-mediated mcl-1 downregulation and mitochondrial membrane destabilization. Mol. Cancer. 2010;9 doi: 10.1186/1476-4598-9-19. PubMed DOI PMC

Driessen C., Kraus M., Joerger M., Rosing H., Bader J., Hitz F., Berset C., Xyrafas A., Hawle H., Berthod G., et al. Treatment with the HIV protease inhibitor nelfinavir triggers the unfolded protein response and may overcome proteasome inhibitor resistance of multiple myeloma in combination with bortezomib: A phase I trial (SAKK 65/08) Haematologica. 2016;101:346–355. doi: 10.3324/haematol.2015.135780. PubMed DOI PMC

Driessen C., Muller R., Novak U., Cantoni N., Betticher D., Mach N., Rufer A., Mey U., Samaras P., Ribi K., et al. Promising activity of nelfinavir-bortezomib-dexamethasone in proteasome inhibitor-refractory multiple myeloma. Blood. 2018;132:2097–2100. doi: 10.1182/blood-2018-05-851170. PubMed DOI PMC

Guan M., Fousek K., Jiang C., Guo S., Synold T., Xi B., Shih C.C., Chow W.A. Nelfinavir induces liposarcoma apoptosis through inhibition of regulated intramembrane proteolysis of SREBP-1 and ATF6. Clin. Cancer Res. 2011;17:1796–1806. doi: 10.1158/1078-0432.CCR-10-3216. PubMed DOI

Guan M., Su L., Yuan Y.C., Li H., Chow W.A. Nelfinavir and nelfinavir analogs block site-2 protease cleavage to inhibit castration-resistant prostate cancer. Sci. Rep. 2015;5:9698. doi: 10.1038/srep09698. PubMed DOI PMC

Gupta A.K., Cerniglia G.J., Mick R., McKenna W.G., Muschel R.J. HIV protease inhibitors block Akt signaling and radiosensitize tumor cells both in vitro and in vivo. Cancer Res. 2005;65:8256–8265. doi: 10.1158/0008-5472.CAN-05-1220. PubMed DOI

Plastaras J.P., Vapiwala N., Ahmed M.S., Gudonis D., Cerniglia G.J., Feldman M.D., Frank I., Gupta A.K. Validation and toxicity of PI3K/Akt pathway inhibition by HIV protease inhibitors in humans. Cancer Biol. Ther. 2008;7:628–635. doi: 10.4161/cbt.7.5.5728. PubMed DOI

Yang Y., Ikezoe T., Nishioka C., Bandobashi K., Takeuchi T., Adachi Y., Kobayashi M., Takeuchi S., Koeffler H.P., Taguchi H. NFV, an HIV-1 protease inhibitor, induces growth arrest, reduced Akt signalling, apoptosis and docetaxel sensitisation in NSCLC cell lines. Br. J. Cancer. 2006;95:1653–1662. doi: 10.1038/sj.bjc.6603435. PubMed DOI PMC

Kraus M., Bader J., Overkleeft H., Driessen C. Nelfinavir augments proteasome inhibition by bortezomib in myeloma cells and overcomes bortezomib and carfilzomib resistance. Blood Cancer J. 2013;3:e103. doi: 10.1038/bcj.2013.2. PubMed DOI PMC

Gupta A.K., Li B., Cerniglia G.J., Ahmed M.S., Hahn S.M., Maity A. The HIV protease inhibitor nelfinavir downregulates Akt phosphorylation by inhibiting proteasomal activity and inducing the unfolded protein response. Neoplasia. 2007;9:271–278. doi: 10.1593/neo.07124. PubMed DOI PMC

Vangala J.R., Radhakrishnan S.K. Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex. J. Biol. Chem. 2019;294:2036–2045. doi: 10.1074/jbc.RA118.006290. PubMed DOI PMC

Dantuma N.P., Lindsten K., Glas R., Jellne M., Masucci M.G. Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells. Nat. Biotechnol. 2000;18:538–543. doi: 10.1038/75406. PubMed DOI

Heaslet H., Rosenfeld R., Giffin M., Lin Y.C., Tam K., Torbett B.E., Elder J.H., McRee D.E., Stout C.D. Conformational flexibility in the flap domains of ligand-free HIV protease. Acta Crystallogr. D Biol. Crystallogr. 2007;63:866–875. doi: 10.1107/S0907444907029125. PubMed DOI

McQuin C., Goodman A., Chernyshev V., Kamentsky L., Cimini B.A., Karhohs K.W., Doan M., Ding L., Rafelski S.M., Thirstrup D., et al. CellProfiler 3.0: Next-generation image processing for biology. PLoS Biol. 2018;16:e2005970. doi: 10.1371/journal.pbio.2005970. PubMed DOI PMC

Kawabata S., Gills J.J., Mercado-Matos J.R., Lopiccolo J., Wilson W., 3rd, Hollander M.C., Dennis P.A. Synergistic effects of nelfinavir and bortezomib on proteotoxic death of NSCLC and multiple myeloma cells. Cell Death Dis. 2012;3:e353. doi: 10.1038/cddis.2012.87. PubMed DOI PMC

Tomlin F.M., Gerling-Driessen U.I.M., Liu Y.C., Flynn R.A., Vangala J.R., Lentz C.S., Clauder-Muenster S., Jakob P., Mueller W.F., Ordonez-Rueda D., et al. Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity. ACS Cent. Sci. 2017;3:1143–1155. doi: 10.1021/acscentsci.7b00224. PubMed DOI PMC

Onchieku N.M., Mogire R., Ndung′u L., Mwitari P., Kimani F., Matoke-Muhia D., Kiboi D., Magoma G. Deciphering the targets of retroviral protease inhibitors in Plasmodium berghei. PLoS ONE. 2018;13:e0201556. doi: 10.1371/journal.pone.0201556. PubMed DOI PMC

Yip M.C.J., Bodnar N.O., Rapoport T.A. Ddi1 is a ubiquitin-dependent protease. Proc. Natl. Acad. Sci. USA. 2020 doi: 10.1073/pnas.1902298117. PubMed DOI PMC

Hitz F., Kraus M., Pabst T., Hess D., Besse L., Silzle T., Novak U., Seipel K., Rondeau S., Studeli S., et al. Nelfinavir and lenalidomide/dexamethasone in patients with lenalidomide-refractory multiple myeloma. A phase I/II Trial (SAKK 39/10) Blood Cancer J. 2019;9:70. doi: 10.1038/s41408-019-0228-2. PubMed DOI PMC

Soprano M., Sorriento D., Rusciano M.R., Maione A.S., Limite G., Forestieri P., D′Angelo D., D′Alessio M., Campiglia P., Formisano P., et al. Oxidative Stress Mediates the Antiproliferative Effects of Nelfinavir in Breast Cancer Cells. PLoS ONE. 2016;11:e0155970. doi: 10.1371/journal.pone.0155970. PubMed DOI PMC

Bruning A., Burger P., Vogel M., Rahmeh M., Gingelmaiers A., Friese K., Lenhard M., Burges A. Nelfinavir induces the unfolded protein response in ovarian cancer cells, resulting in ER vacuolization, cell cycle retardation and apoptosis. Cancer Biol. Ther. 2009;8:226–232. doi: 10.4161/cbt.8.3.7339. PubMed DOI

Ben-Romano R., Rudich A., Etzion S., Potashnik R., Kagan E., Greenbaum U., Bashan N. Nelfinavir induces adipocyte insulin resistance through the induction of oxidative stress: Differential protective effect of antioxidant agents. Antivir. Ther. 2006;11:1051–1060. PubMed

Zhang Y., Manning B.D. mTORC1 signaling activates NRF1 to increase cellular proteasome levels. Cell Cycle. 2015;14:2011–2017. doi: 10.1080/15384101.2015.1044188. PubMed DOI PMC

Besse A., Stolze S.C., Rasche L., Weinhold N., Morgan G.J., Kraus M., Bader J., Overkleeft H.S., Besse L., Driessen C. Carfilzomib resistance due to ABCB1/MDR1 overexpression is overcome by nelfinavir and lopinavir in multiple myeloma. Leukemia. 2018;32:391–401. doi: 10.1038/leu.2017.212. PubMed DOI PMC

Dyer B.W., Ferrer F.A., Klinedinst D.K., Rodriguez R. A noncommercial dual luciferase enzyme assay system for reporter gene analysis. Anal. Biochem. 2000;282:158–161. doi: 10.1006/abio.2000.4605. PubMed DOI

Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., Preibisch S., Rueden C., Saalfeld S., Schmid B., et al. Fiji: An open-source platform for biological-image analysis. Nat. Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Impact of proteostasis workload on sensitivity to proteasome inhibitors in multiple myeloma

DDI2 protease controls embryonic development and inflammation via TCF11/NRF1

HIV-protease inhibitors potentiate the activity of carfilzomib in triple-negative breast cancer

Viral proteases as therapeutic targets