CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing’s sarcoma

Abstract

The post-genomic era has seen many advances in our understanding of cancer pathways, yet resistance and tumor heterogeneity necessitate multiple approaches to target even monogenic tumors. Here, we combine phenotypic screening with chemical genetics to identify pre-messenger RNA endonuclease cleavage and polyadenylation specificity factor 3 (CPSF3) as the target of JTE-607, a small molecule with previously unknown target. We show that CPSF3 represents a synthetic lethal node in a subset of acute myeloid leukemia (AML) and Ewing’s sarcoma cancer cell lines. Inhibition of CPSF3 by JTE-607 alters expression of known downstream effectors in AML and Ewing’s sarcoma lines, upregulates apoptosis and causes tumor-selective stasis in mouse xenografts. Mechanistically, it prevents the release of newly synthesized pre-mRNAs, resulting in read-through transcription and the formation of DNA-RNA hybrid R-loop structures. This study implicates pre-mRNA processing, and specifically CPSF3, as a druggable target providing an avenue to therapeutic intervention in cancer.

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Fig. 1: In vitro growth of AML and Ewing’s sarcoma cancer cell lines is sensitive to compound 1.
Fig. 2: Chemical genetics and proteomics studies combine to identify mRNA processing and CPSF3 as the target of compound 2.
Fig. 3: Compound 2 binds directly to CPSF3, inhibiting its endonuclease activity and conferring growth inhibitory effects.
Fig. 4: Compound 1 induces transcript accumulation and RNA Pol II read-through.
Fig. 5: Compound 1 results in accumulation of nuclear R-loops.
Fig. 6: Proposed model for compound 2 MoA.

Data availability

The datasets generated during and/or analyzed during the current study are included in this published article (and its Supplementary Information files). The raw RNA-sequencing reads are available in the NCBI Sequence Read Archive under accession number SRP158650. X-ray structure data have been deposited to the PDB with the code 6M8Q. All other relevant data are available from the corresponding author on reasonable request.

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Acknowledgements

We thank D. Porter, R. Tiedt, J. Baryza and G. Rice for helpful discussions through the course of this research study. This work was supported by the Novartis Research Foundation (J.A.C.), the SNF-NCCR RNA & Disease network (J.A.C.) and the Medical Research Council (MRC) grant no. MC_U105192715 (L.A.P.).

Author information

R.E.J.B., F.L. and N.T.R conceptualized research, interpreted data and wrote the manuscript. S.C. conducted and interpreted biomarker, variomics and MoA studies. A.F. synthesized all compounds. W.A.W. conducted crystallography experiments and solved structures. M.H. conducted the siRNA synergy experiment. M.S. and G.M. conducted and interpreted CPSF3 biochemical binding studies. J.T., S.C. and F.A.A. prepared RNA samples. J.K. and W.C. conducted RNA-sequencing experiments. S.H.C., S.G., S.S. and G.R. interpreted RNA-seq results. S.B., J.M., J.T. and M.S. conducted and interpreted chemical proteomics experiments. G.M. and S.C. conducted and interpreted FACS experiments. Y.W. and J.J. contributed to in silico target predictions. F.S. analyzed siRNA experiments. H.M. conducted cell line cytotoxicity studies. G.B. and E.G. conducted and interpreted in vivo xenograft experiments. A.C. and K.X. conducted variomics cloning experiments. J.S.R.-H. designed variomics cloning strategy. M.Z. and M.S. conducted mass spectrometry experiments. C.S. and E.T.W. conducted in vivo compound pharmacokinetics experiments. J.H.W., C.A.-R., M.J. and J.A.C. conducted and interpreted FISH and R-loop experiments. J.B.R.M. and L.A.P. conducted and interpreted in vitro cleavage experiments. J.A.C. and J.T. helped write the manuscript.

Correspondence to Rohan E. J. Beckwith.

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Competing interests

All authors (except otherwise noted) were employees of Novartis Institutes for BioMedical Research at the time of their involvement in this study and may hold stock in Novartis.

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Supplementary information

Supplementary information

Supplementary Figures 1–21 and Supplementary Tables 1–4

Reporting Summary

Supplementary Note

Synthetic Procedures

Supplementary Dataset 1

siRNA library.

Supplementary Dataset 2

siRNA plus compound 1 synergy screen gene level activity per compound 1, DMSO or differential.

Supplementary Dataset 3

Chemoproteomics data.

Supplementary Dataset 4

PAL data.

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Ross, N.T., Lohmann, F., Carbonneau, S. et al. CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing’s sarcoma. Nat Chem Biol 16, 50–59 (2020). https://doi.org/10.1038/s41589-019-0424-1

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