Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia

The MUSASHI family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. We found that the small molecule Ro 08–2750 (Ro) directly binds to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in mouse and human myeloid leukemia cells resulted in an increase in differentiation and apoptosis, inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA depletion of MSI2. Ro demonstrated in vivo inhibition of c-MYC and reduced disease burden in a murine AML leukemia model. Thus, we have identified a small molecule that targets MSI’s oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.

INTRODUCTION post-transcriptionally. Ribonucleoprotein complexes are essential for all steps of mRNA 4 characterization of Ro 08-2750. Using biochemical and structural approaches, we find that Ro 83 binds to the MSI2 RRM1 RNA-binding site and inhibits MSI RNA-binding activity and regulation of 84 downstream oncogenic targets. Furthermore, we demonstrate that Ro 08-2750 has efficacy in 85 inhibiting leukemogenesis by in vitro and in vivo models of myeloid leukemia. 7 interact via the RRM/RNA binding site and that the drug can displace RNA from its binding site, 161 thus likely inhibiting MSI-related translational regulation.

Ro 08-2750 demonstrates therapeutic efficacy in murine MLL-AF9 leukemic cells 164
To test the MSI-inhibitory effect of Ro in a murine AML of leukemia, we used MLL-AF9 165 expressing leukemic BM cells from secondary transplants previously established in the lab 8 . We 166 first assessed the cytotoxicity effects of Ro and the two analogs against these leukemia cells. 8 NGF) revealed a >4.5-fold weaker potency. Ro induced myeloid differentiation and apoptosis in both K562 and MOLM13 cells based on flow cytometry and by morphology (Fig. 4b-d and  9 12). Thus, Ro treatment after a short administration recapitulated a large portion of the MSI2-234 associated gene expression program.

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To determine how Ro affects previously determined MSI targets, we treated both K562 and 237 MOLM13 cells with increasing concentrations of Ro (up to 20 μ M at 4 hours). In previous studies,

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MSI was demonstrated to maintain the protein levels of TGFβR1, c-MYC, SMAD3 and HOXA9 8,31 239 while suppressing P21 abundance 41,42 . Consistent with this, we observed a significant and dose 240 dependent reduction of TGFβR1, c-MYC, SMAD3, HOXA9 and an increase in the protein 241 abundance of P21, while the non-target control β-ACTIN remained unchanged ( Fig. 5d and 5e).

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Additionally, Ro could inhibit MSI2 targets in a time-dependent manner with c-MYC, a short half-243 life protein, being reduced in 1 hour of treatment ( Fig. 5f and 5g). In support of Ro altering 244 translation of specific MSI2 targets but not generally inhibiting global translation, we found

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suggesting that Ro mainly influences its direct targets through a post-transcriptional mechanism.

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Thus, these results support our hypothesis that Ro acts in the MSI-related translational program.

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Finally, we sought to determine if Ro has activity in vivo using an aggressive murine MLL-AF9 253 murine leukemia model. Acute treatment of Ro (4h and 12hr) reduced c-KIT protein abundance 254 and intracellular c-MYC (Fig. 6a-c). To determine if Ro treament could affect disease burden we 255 next treated a second cohort of animals and monitored them for disease progression for 19 days 256 after transplantation (Fig. 6d). Ro administration every 3 days was well tolerated (Extended Data 257 Fig. 7a, b, c) demonstrating little to no weight loss and equivalent red blood cells and platelets 258 counts compared to control group. When control mice succumbed to disease (day 19 post-259 transplantation), we assessed the disease in both groups and found a significant reduction in 260 spleen weights (Fig. 6e), white blood cell counts (Fig. 6f)

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For binding affinity studies of RNA and small-molecules to proteins of interest, purified