Summary Tumor hypoxia is associated with therapy resistance and poor patient prognosis. Hypoxia-activated prodrugs, designed to selectively target hypoxic cells while sparing normal tissue, represent a promising treatment strategy. We report the pre-clinical efficacy of 1-methyl-2-nitroimidazole panobinostat (NI-Pano, CH-03), a novel bioreductive version of the clinically used lysine deacetylase inhibitor, panobinostat. NI-Pano was stable in normoxic (21% O 2 ) conditions and underwent NADPH-CYP-mediated enzymatic bioreduction to release panobinostat in hypoxia (<0.1% O 2 ). Treatment of cells grown in both 2D and 3D with NI-Pano increased acetylation of histone H3 at lysine 9, induced apoptosis, and decreased clonogenic survival. Importantly, NI-Pano exhibited growth delay effects as a single agent in tumor xenografts. Pharmacokinetic analysis confirmed the presence of sub-micromolar concentrations of panobinostat in hypoxic mouse xenografts, but not in circulating plasma or kidneys. Together, our pre-clinical results provide a strong mechanistic rationale for the clinical development of NI-Pano for selective targeting of hypoxic tumors.

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Tumor hypoxia contributes resistance to chemo- and radiotherapy, while oxygenated tumors are sensitive to these treatments. The indirect detection of hypoxic tumors is possible by targeting carbonic anhydrase IX (CA IX), an enzyme overexpressed in hypoxic tumors, with sulfonamide-based imaging agents. In this study, we present the design and synthesis of novel gallium-radiolabeled small-molecule sulfonamides targeting CA IX. The compounds display favorable in vivo pharmacokinetics and stability. We demonstrate that our lead compound, [(68)Ga]-2, discriminates CA IX-expressing tumors in vivo in a mouse xenograft model using positron emission tomography (PET). This compound shows specific tumor accumulation and low uptake in blood and clears intact to the urine. These findings were reproduced in a second study using PET/computed tomography. Small molecules investigated to date utilizing (68)Ga for preclinical CA IX imaging are scarce, and this is one of the first effective (68)Ga compounds reported for PET imaging of CA IX.

Due to compromised homologous recombination ( HR ) repair, BRCA 1‐ and BRCA 2‐ mutated tumours accumulate DNA damage and genomic rearrangements conducive of tumour progression. To identify drugs that target specifically BRCA 2‐deficient cells, we screened a chemical library containing compounds in clinical use. The top hit was chlorambucil, a bifunctional alkylating agent used for the treatment of chronic lymphocytic leukaemia ( CLL ). We establish that chlorambucil is specifically toxic to BRCA 1/2‐deficient cells, including olaparib‐resistant and cisplatin‐resistant ones, suggesting the potential clinical use of chlorambucil against disease which has become resistant to these drugs. Additionally, chlorambucil eradicates BRCA 2‐deficient xenografts and inhibits growth of olaparib‐resistant patient‐derived tumour xenografts ( PDTX s). We demonstrate that chlorambucil inflicts replication‐associated DNA double‐strand breaks ( DSB s), similarly to cisplatin, and we identify ATR , FANCD 2 and the SNM 1A nuclease as determinants of sensitivity to both drugs. Importantly, chlorambucil is substantially less toxic to normal cells and tissues in vitro and in vivo relative to cisplatin. Because chlorambucil and cisplatin are equally effective inhibitors of BRCA 2‐compromised tumours, our results indicate that chlorambucil has a higher therapeutic index than cisplatin in targeting BRCA ‐deficient tumours.