半胱氨酸积累作为硼替佐米耐药的驱动因素

Covalent drugs targeting key oncogenic drivers are promising cancer therapies, but resistance limits their clinical benefit. Many covalent inhibitors rely on reacting with nucleophilic residues on proteins, and thus we hypothesized that these inhibitors could interact directly with intracellular pools of nucleophiles, like cysteine, diminishing the drug's efficacy. To investigate the possibility that intracellular cysteine drives resistance to chemotherapies, we performed a high throughput screen of clinically available chemotherapeutics in high and low cysteine conditions. We identified cells to be more resistant to boronic-acid proteasome inhibitors in high cysteine conditions (Bortezomib (Btz) and Ixazomib (Ixa)), but not to the epoxyketone-containing proteasome inhibitor Carfilzomib (Cfz). We confirmed the potential of proteasome inhibitors to react with free cysteine and found that Btz and Ixa both formed a covalent conjugate with cysteine as detected via LC-MS. Upon treating cells with the cysteine-drug conjugate, we find nearly all of the toxicity of the drug has been abolished, supporting the hypothesis that cysteine could be a detoxification mechanism in cells. Using cancer cell lines derived from diverse cancer types, we modulated the cysteine availability and treated cells with two classes of proteasome inhibitors: those with either a boronic-acid or an epoxyketone moiety. In conditions known to increase intracellular cysteine such as high media cystine or co-treatment with the cysteine pro-drug NAC, we found that cells are more resistant to boronic acid-containing inhibitors. Conversely, when we decrease intracellular cysteine levels by co-treating with erastin, an inhibitor of SLC7A11, we measured that cells become more sensitive to Btz and Ixa. In either low or high cysteine, sensitivity to Cfz was unchanged. Collectively, these results indicate that a direct interaction between cysteine with the boronic acid group of Btz and Ixa is responsible for cysteine mediated resistance to these compounds, upstream of proteasome inhibition. We next explored cysteine's role in rescuing proteasome function by measuring proteasome activity and ubiquitylation in cells. In both assays, we observed that high cysteine prevented the effects of proteosome in cells treated with boronic acid-containing inhibitors. We did not measure a difference in proteasome function in carfilzomib-treated cells in high cysteine, once again implying a unique direct interaction between boronic acid-containing inhibitors and cysteine. In summary, we uncovered a novel mechanism of resistance to boronic acid-containing proteasome inhibitors with straightforward possibilities to reverse resistance. This work has clinical implications for cancer treatment, especially those with ATF4 or NRF2 stabilization which both drive cysteine accumulation in a SLC7A11-dependent manner.