The overexpression of LSD1 is strongly linked to adverse clinical outcomes in cancer patients, thereby establishing LSD1 as a highly attractive target for therapeutic intervention. However, advancement of conventional small-molecule inhibitors of LSD1 in the clinical trials has been significantly impeded by challenges such as dose-limiting toxicities and off-target effects. In this context, PROTAC technology emerges as a powerful alternative strategy. By catalytically inducing the degradation of target proteins, PROTAC allows for lower dose, more complete and sustained target elimination, and a reduced potential for the development of drug resistance, thus holding the promise to overcome the limitations of existing small molecule inhibitors, and achieve superior therapeutic efficacy.
By conjugating the LSD1-binding ligand LI-1 with the CRBN-recruiting ligand thalidomide via linkers of various lengths, the authors designed and synthesized a series of PROTAC molecules. Through systematic SAR evaluation, LD-110 was identified as the most efficacious degrader of LSD1. Biochemically, LD-110 effectively reduced LSD1 protein levels and consequently increased the levels of LSD1 substrates, H3K4me1/me2 in a time- and dose-dependent manner across a panel of breast and lung cancer cell lines. The half-maximal degradation concentrations (DC₅₀) of LD-110 was 9.54, 7.08, and 446.0 nmol/L in MDA-MB-231, MDA-MB-453 breast cancer, and H520 lung cancer cells, respectively.
The authors confirmed that LD-110-mediated LSD1 degradation was in a PROTAC-UPS dependent process. Specifically, LI-1 warhead, thalidomide ligand as well as neddylation inhibitor, MLN4924 (to block CRBN) and proteasome inhibitor MG132, effectively abrogated LSD1 degradation. Furthermore, a methylated analog of LD-110 (LD-110Me), which is incapable of binding to CRBN, failed to trigger both LSD1 degradation and the subsequent accumulation of H3K4me2.
Biologically, LD-110 significantly inhibited the growth of cancer cells with half-maximal inhibitory concentrations (IC₅₀), ranging from 0.75 to 2506.33 nmol/L in a manner dependent of various cancer cell lines. More impressively, LD-110 displayed favorable pharmacokinetic profiles in vivo, and exerted potent suppressive effects on tumor growth in both breast and lung cancer xenograft models without inducing detectable cytotoxicity.
Mechanistically, LD-110 induced apoptosis by triggering endoplasmic reticulum (ER) stress through dual mechanisms that converged on the activation of the ATF4-CHOP signaling axis. On the transcriptional level, LD-110 facilitated ATF4 expression by degrading LSD1 to increase H3K4me2. On the translational level, LD-110 induced ROS generation to cause DNA damage, which in turn activated the GCN2-eIF2α pathway to enhance ATF4 protein synthesis. The activation of the ATF4-CHOP cascade subsequently altered the balance of BCL-2 family proteins by increasing pro-apoptotic factor NOXA and decreasing the anti-apoptotic protein MCL1, ultimately leading to apoptotic death of cancer cells.
CRBN-based PROTACs are intrinsically limited by its off-target activity, as CRBN E3 ligase degrades its nature-occurring substrates, such as GSPT1, IKZF1, and IKZF3. Indeed, LD-110 effectively degraded both LSD1 and GSPT1. Interestingly, the authors found that GSPT1 competed with LSD1 for LD-110 binding, thus attenuating its degradation activity on LSD1. Consistently, siRNA-based GSPT1 knockdown enhanced LD-110-induced LSD1 degradation and cell growth inhibition. Thus, it is likely that the combination of LD-110 with a GSPT1 degrader would yield a synergistic anti-tumor effect via dual engagement, potentially allowing for lower drug dose and reduced cytotoxicity.
In summary, this study reported the discovery of LD-110 as a highly potent PROTAC degrader of LSD1 with significant anti-tumor efficacy both in vitro and in vivo , positioning it as a promising candidate for future development as a novel anti-cancer therapeutic agent.
Science Bulletin
Experimental study