Blocking Protein Secretion with Novel Small Molecule Inhibitors of Sec61 Represents a Potential Treatment Strategy Against Hematologic Malignancies

Secreted and membrane proteins play key roles in cancer development, including as autocrine growth factors, receptors in oncogenic signaling cascades, and checkpoints in immune system evasion. The biogenesis of most secreted and transmembrane proteins involves cotranslational translocation of nascent polypeptides ("clients") into the endoplasmic reticulum (ER) through the Sec61 translocon. Multiple inhibitors of protein secretion have been described that target Sec61 and have antitumor activity but lack adequate pharmaceutical properties or tolerability to be studied clinically. Here we describe novel small molecule inhibitors of Sec61 which exhibit activity against hematologic tumor cells in vitro and in vivo.

KZR-8834 (8834) was identified through a screening campaign for novel inhibitors of Sec61-dependent protein secretion, where it exhibited nanomolar potency against multiple Sec61 client proteins of therapeutic value, including several immune checkpoint proteins. The broad Sec61 client inhibition profile of 8834 led to its in vitro assessment of anti-cancer activity against a panel of 346 human cancer cell lines. 8834 displayed broad cytotoxic activity against both solid and hematologic tumor types with high potency for hematologic malignancies including acute lymphoid leukemia (mean IC₅₀=317nM, n=8 cell lines), acute myeloid leukemia (IC₅₀=359nM, n=14), lymphoma (IC₅₀=250nM, n=15) and multiple myeloma (IC₅₀=352nM, n=11). In mouse xenograft models of multiple myeloma (H929) and mantle cell lymphoma (Mino), once weekly administration of 8834 or KZR-9261 (8834 analog) resulted in >90% tumor growth inhibition without significant clinical toxicity.

Two multiple myeloma cell lines, H929 and U266, were chosen to assess cellular response due to their sensitivity and resistance to 8834, respectively. In H929 cells, 250nM 8834 induced activation of caspase 3/7 (>15 fold) within 8 hours of exposure which corresponded with a cell viability IC₅₀ of 98nM at 24 hours. In contrast, no caspase 3/7 activation was noted in U266 cells through 24 hours of exposure, which corresponded with minimal effects on cell viability. Gene expression profiling by RNAseq and quantitative proteomic profiling by mass spectrometry was performed on these cell lines to elucidate mechanisms of sensitivity and resistance to Sec61 inhibition. Both methods revealed rapid upregulation of ER stress response genes/proteins and activation of the unfolded protein response, which was greater in H929 cells and confirmed by immunoblot and QPCR. Gene set enrichment analysis revealed significantly higher basal levels of ER stress-related genes in H929 vs U266 cells, suggesting ER stress response capacity as a possible predictive biomarker.

In summary, blockade of Sec61-dependent translocation of secreted and membrane proteins with novel small molecule inhibitors exhibits a broad antitumor profile in vitro, potentially in part through activation of proteotoxic stress. These effects translate into therapeutic activity in multiple mouse xenograft models, demonstrating a potential novel treatment for hematologic malignancies.