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

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 IC50 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.

Inflammation-related pyroptosis, a novel programmed cell death pathway, and its crosstalk with immune therapy in cancer treatment

“…Moreover, its activation inhibits the recruitment of CD4+ T lymphocytes, contributing to angiogenesis, a loss of apoptosis, and an uncontrolled cell cycle 149 . The G-quadruplex (G4), a stable secondary structure formed by guanine-rich regions of DNA and RNA 150 , inhibits the transcription of Myc by forming a stable structure within the Myc promoter 151 . Gaikwad et al suggested that the benzofuran scaffold D089 (MYC G-quadruplex ligand) induces cell cycle arrest at G1 phase and cell senescence and triggers pyroptosis in the multiple myeloma cell line L363 through cleavage mediated by caspase-1 instead of caspase-3 151 , 152 .…”

On the Road to Fight Cancer: The Potential of G-Quadruplex Ligands as Novel Therapeutic Agents

“…Notably, the compound exhibited an antitumor activity on a xenograft model of squamous cell carcinoma that could be related to its binding to MYC G4, [ 60 ]. Similarly, the benzofuran derivative D089 has been reported to inhibit MYC transcription and to interfere with the survival of multiple myeloma cells through the direct binding to MYC G4 [ 61 ]. In particular, the exposure of myeloma cells to D089 induced endoplasmic reticulum stress, thus resulting in pyroptotic cell death, as evidenced by the appearance of a “ballooning” morphology associated with increased levels of the cleaved form of the inflammatory caspase 1 and of IL1-β as well as of gasdermin D cleavage and of HMGB1 cytoplasm translocation [ 61 ].…”

“…Similarly, the benzofuran derivative D089 has been reported to inhibit MYC transcription and to interfere with the survival of multiple myeloma cells through the direct binding to MYC G4 [ 61 ]. In particular, the exposure of myeloma cells to D089 induced endoplasmic reticulum stress, thus resulting in pyroptotic cell death, as evidenced by the appearance of a “ballooning” morphology associated with increased levels of the cleaved form of the inflammatory caspase 1 and of IL1-β as well as of gasdermin D cleavage and of HMGB1 cytoplasm translocation [ 61 ]. Recently, a Curcumin derivative, the synthetic 3,4-dimethoxy benzaldehyde Cur-4, has been recognized as a promising candidate for direct G4-mediated inhibition of MYC expression [ 62 ].…”

Transcription/Replication Conflicts in Tumorigenesis and Their Potential Role as Novel Therapeutic Targets in Multiple Myeloma

“…Overall, these structures are rare in tumor-suppressor genes and frequent in proto-oncogenes [238]. A recent study showed that MYC-G4 stabilization using a small molecule G4 stabilizer in MM cells reduces MYC transcription and increases endoplasmic reticulum stress [239].…”