A Small Molecule Stabilizer of the MYC G4-Quadruplex Induces Endoplasmic Reticulum Stress, Senescence and Pyroptosis in Multiple Myeloma

SIMPLE SUMMARY: The DNA G-quadruplex (G4) present in the promoter of the MYC oncogene, commonly amplified in cancers, including multiple myeloma, represents a potential anti-cancer target. A previously identified MYC G4-stablizer, which demonstrated cytotoxicity and senescence in myeloma cells, was discovered to induce endoplasmic reticulum stress and non-apoptotic cell death, pyroptosis. Cancers including myeloma escape apoptosis through upregulation of anti-apoptotic proteins and drug resistance; therefore, induction of pyroptosis provides an alternate therapeutic option. Thus, our study provides a disease-specific experimental strategy for identifying new investigational drugs in cancer treatment. ABSTRACT: New approaches to target MYC include the stabilization of a guanine-rich, G-quadruplex (G4) tertiary DNA structure in the NHE III region of its promoter. Recent screening of a small molecule microarray platform identified a benzofuran, D089, that can stabilize the MYC G4 and inhibit its transcription. D089 induced both dose- and time-dependent multiple myeloma cell death mediated by endoplasmic reticulum induced stress. Unexpectedly, we uncovered two mechanisms of cell death: cellular senescence, as evidenced by increased levels of p16, p21 and γ-H2AX proteins and a caspase 3-independent mechanism consistent with pyroptosis. Cells treated with D089 exhibited high levels of the cleaved form of initiator caspase 8; but failed to show cleavage of executioner caspase 3, a classical apoptotic marker. Cotreatment with the a pan-caspase inhibitor Q-VD-OPh did not affect the cytotoxic effect of D089. In contrast, cleaved caspase 1, an inflammatory caspase downstream of caspases 8/9, was increased by D089 treatment. Cells treated with D089 in addition to either a caspase 1 inhibitor or siRNA-caspase 1 showed increased IC(50) values, indicating a contribution of cleaved caspase 1 to cell death. Downstream effects of caspase 1 activation after drug treatment included increases in IL1B, gasdermin D cleavage, and HMGB1 translocation from the nucleus to the cytoplasm. Drug treated cells underwent a ‘ballooning’ morphology characteristic of pyroptosis, rather than ‘blebbing’ typically associated with apoptosis. ASC specks colocalized with NLRP3 in proximity ligation assays after drug treatment, indicating inflammasome activation and further confirming pyroptosis as a contributor to cell death. Thus, the small molecule MYC G4 stabilizer, D089, provides a new tool compound for studying pyroptosis. These studies suggest that inducing both tumor senescence and pyroptosis may have therapeutic potential for cancer treatment.