Introduction: We have discovered novel non-toxic rifamycins that are extremely potent at sensitizing many drug-resistant cancers, including DLBCL, to standard-of-care chemotherapeutics.
Methods: CHOP-resistant DLBCL cell lines were derived from CHOP-sensitive cells by on-off cycles of CHOP treatment, analogous to clinical therapy. The CHOP-resistant cells were used in high-throughput screening of a highly diverse collection of approximately 50,000 drug-like molecules to identify small molecules that reverse CHOP-resistance. The FDA-approved drug, Rifabutin, was identified as a non-cytotoxic compound that potently reversed resistance to CHOP. Structure-Activity-Relationships (SAR) on Rifabutin were conducted, which led to the generation of a new more potent CHOP-chemosensitizing agent, designated RTI.
Results: RTI was highly synergistic with variety of chemotherapeutics, including doxorubicin (DOX), epirubicin, vinblastine, etoposide in drug-resistant NHL cells. Combination therapy of DOX+RTI in mouse xenograft models of DLBCL was much more effective at repressing tumor growth than with DOX alone. RTI lowered the IC90 in a dose-dependent manner in CD20-positive B cells in bone marrow aspirates from both CHOP-nave and CHOP-relapsed patients, and in a metastatic lymph node. RTIs PK characteristics are similar to rifabutin, and it exhibited no overt toxicity in mice or pigs at high doses. RTI rapidly induced mitochondrial superoxide, membrane potential, and fission. The superoxide dismutase, FeTCP, antagonized RTI-induced ROS and potentiation of DOX cytotoxicity. RTI reduced the activity of the Nrf-2 antioxidant protein, upregulated proteins involved in the unfolded protein response (UPR), and induced metabolic reprogramming as indicated by Seahorse assays that showed increased glycolysis and decreased oxygen consumption from 3 to 24 hrs.
Conclusions: RTI has a broad spectrum of action in both double- and triple-hit DLBCL and synergizes with many different chemotherapeutics to restore drug sensitivity. RTI-79 works by dramatically increasing intracellular superoxide through redox cycling, triggers UPR, and downregulates Nrf-2 activity. Thus, RTI-79 increases oxidative stress through the squelching of Nrf-2s ability to respond to chemotherapeutic stress. Since the parent compound, rifabutin, binds to the aryl hydrocarbon receptor (AhR) and modulates its activity, we hypothesize that RTI mediates its unique pleiotropic chemosensitizing mechanism through targeting of AhR, and provides for a broad, safe, and novel approach to treating drug resistant cancers. Ongoing experiments are investigating the effect of RTI on the AhR-signaling pathway and the role it plays in RTI-mediated chemosensitization and potentiation of chemotherapeutics. A clinical trial of companion dogs with CHOP-relapsed DLBCL treated with combination CHOP+RTI is currently in progress.
Citation Format: Steve A. Maxwell, Deeann Wallis, Nian Zhou, Dwight Baker, Seyed H. Mousavi-Fard, Kimberly Loesch, Stacy Galaviz, Liam Guthrie, Thomas Snavely, Qingan Sun, Carolina M. Rojas, David W. Threadgill, Thomas Ioerger, Wen Dong, Gwen Seemann, Theresa W. Fossum, James C. Sacchettini. Development of novel, non-toxic rifamycins that reverse drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 410.