A Novel Ribonuclease Targeting Small Molecule RNA-Degrader (MYC-RiboTAC) Overcomes MYC Dependency in Multiple Myeloma

c-MYC (MYC) is an important oncogene in multiple myeloma (MM), driving MM cell proliferation, metabolism, and survival. However, MYC has been challenging to therapeutically target because of its protein structure lacking well-defined binding sites. In this study, we investigated a novel Ribonuclease Targeting Chimera (MYC-RiboTAC) to degrade MYC mRNA in MM cells. This dimeric small molecule binds both the Internal Ribosomal Entry Site (IRES) of MYC mRNA and RNase L. RNase L then dimerizes into its active conformation and degrades the MYC mRNA. First, by immunohistochemistry (IHC) analysis of MM tumor microarrays from 24 MM patients. we confirmed significant expression of RNase L in all patients compared to healthy donors (n=11). Notably, RNase L was selectively expressed by plasma cells in the bone marrow. Based on RNA-seq and Western blot analyses of MYC and RNase L expression, to test MYC-RiboTAC, we selected AMO1, H929, R8226, MOLP8, MM1S, OPM2 (MYC+/RNase L+), KMS12BM (MYC+/RNase L-), and U266 (MYC-/RNase L-) MM cell lines. MYC-RiboTAC significantly reduced MYC mRNA levels in cell lines expressing both MYC and RNase L, with the most substantial reduction observed in OPM2 (~75%) and similar reductions in R8226, MOLP8, MM1S, AMO1, and H929 cells (~35%). The compound exerted more pronounced effects at the protein level, downregulating MYC protein in all cell lines expressing MYC and RNase L, with no effect on RNase L negative KMS12BM cell line. Additionally, single-molecule RNA FISH to detect MYC mRNA and immunofluorescence to detect MYC protein confirmed that MYC-RiboTAC significantly reduced both MYC mRNA (located in the cytosol) and MYC protein (located in the nucleus). Transcriptomic analysis in AMO1 cells further supported the selective action of MYC-RiboTAC on the MYC pathway. We further demonstrated the RNase L-dependent effect of MYC-RiboTAC by i) RNase L knockout in AMO1 cells, which abrogated the downregulation of MYC by MYC-RiboTAC, and ii) ectopic expression of RNase L in KMS12BM cells, which restored the MYC downregulating activity of MYC-RiboTAC. MYC-RiboTAC inhibited growth in all six MM cell lines that expressed both MYC and RNase L but had no impact on the growth of KMS12BM and U266 cell lines. It suppressed colony formation in AMO1 and H929 cells in a dose-dependent manner. Its anti-MM activity was not abrogated when MM cells were co-cultured with HS5 stromal cells. Additionally, MYC-RiboTAC showed synergistic effects with clinically active drugs such as Lenalidomide, Pomalidomide, and Melphalan, which were found to upregulate RNase L and potentially enhance MYC-RiboTAC efficacy. The essential role of RNase L was further confirmed by treating MM tumor cells from three patients with MYC-RiboTAC, which reduced MYC protein levels only in the RNase L-expressing patient. Moreover, the compound reduced the viability of CD138+ cells from an MM patient without affecting the viability of CD138- cells and normal cells such as PBMCs or human fibroblasts. We performed a DMPK study in mice and found that daily intraperitoneal administration of MYC-RiboTAC at 30 mg/kg, achieved an active concentration of 6 µM in the blood and a half-life of 5.2 hours. In H929 xenograft mouse model a significant reduction (~64% p<0.05) in tumor growth was observed in MYC-RiboTAC treated animals compared to the vehicle-treated group, with no overt toxicity. Western blot analysis of retrieved tumors from mice revealed significantly lower MYC protein levels. In conclusion, we report functional effects of a first RNA-targeting small molecules in MM and establish MYC-RiboTAC as a potential therapeutic molecule targeting MYC. This study opens new frontiers in drug discovery for targets long considered undruggable.