A chemical probe of CARM1 alters epigenetic plasticity against breast cancer cell invasion

Acceptance summary:

This manuscript describes the development of the small molecular weight compound SKI-73 as a prodrug that releases two potent and selective inhibitors of protein arginine methyltransferase 4 (CARM1) in cells. Using a previously reported inhibitor from natural sources as a scaffold, the authors developed the two inhibitors that display sub-nanomolar potency against the target enzyme and >10-fold selectivity over 7 other human protein arginine methyltransferase and 26 methyltransferases of other classes. Two other chemical probes for the target enzyme have been identified previously. Importantly, all three of these chemical probes have distinct molecular scaffolds and mechanisms. The current compounds compete for the cofactor binding site, unlike the previous two inhibitors that target the substrate arginine binding pocket. The authors next added chemical groups to a primary amine of the initial non-cell permeable inhibitor to develop the cell-permeable pro-drug SKI-73. This strategy potentially delivers a new class of compounds for inhibiting methyltransferases. Detailed single-cell transcription analysis revealed that SKI-73 alters epigenetic plasticity and that the subpopulation of cells that is reduced upon SKI-73 treatment is similar to that of freshly isolated invasive cells. The experimental approach used to arrive at this conclusion will be applicable to studies involving other epigenetic tool compounds. Overall this is an excellent study on the development of CARM1-selective chemical probe with potential for the treatment of metastatic breast cancer.

Decision letter after peer review:

Thank you for submitting your article "A chemical probe of CARM1 alters epigenetic plasticity against breast cancer cell invasion" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by Wilfred van der Donk as Reviewing Editor and Philip Cole as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Paul R Thompson; Mark Bedford.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

This manuscript describes the development of SKI-73 as a prodrug that releases two potent and selective inhibitors of protein arginine methyltransferase 4 (PRMT4 or CARM1) in cells. Using 6'-homosinefungin (HSF) as a core scaffold, the authors developed two inhibitors (2a and 5a) with sub-nanomolar potency for CARM1 and >10-fold selectivity over 7 human PRMTs and 26 methyltransferases of other classes. A related compound that lacks a 6'-methylene group (5b) exhibited less activity. Two other CARM1 chemical probes have been identified previously – EZM2302 and TP-064. Importantly, all three of these chemical probes have distinct molecular scaffolds. The current study shows that compounds 2a and 5a compete for the SAM binding site, unlike the other two inhibitors that target the substrate arginine binding pocket. The authors masked the primary amine of 5a to develop SKI-73, a cell permeable pro-drug. Surprisingly, SKI-73 generated the parent inhibitor 5a in MDA-MB-231 cells along with 2a and these compounds accumulated to relatively high concentrations. They potently inhibited methylation of BAF155 in MCF-7 and MDA-MB-231 cells and PABP1 in MDA-MB-231 cells, two well-known CARM1 substrates. Furthermore, SKI-73 efficiently blocked invasion of MDA-MB-231 without affecting proliferation. This strategy potentially delivers a new class of compounds for inhibiting methyltransferases. Detailed single-cell RNA-seq analysis revealed that SKI-73 alters epigenetic plasticity and that the subpopulation of cells that is lost/reduced upon SKI-73 treatment is similar to that of freshly isolated invasive cells. This approach will be applicable to studies involving other epigenetic tool compounds. Overall this is an excellent study on the development of CARM1-selective chemical probe with potential for the treatment of metastatic breast cancer. The work is potentially suitable for publication in eLife after the authors address a number of points raised during review.

Essential revisions:

1) The authors should determine the stability of 6a in microsomes to better account for the formation of 2a from 6a in cells.

2) In subsection “Characterization of 6a (SKI-73) as a chemical probe of CARM1” the authors mention that 6b, the negative control, also is processed to 5b and 2b. Therefore, the authors should provide information on the selectivity of 2b in Figure 1. Only data is shown for 5b, which appears quite selective and has a relatively low IC₅₀. The assumption in the discussion is that 2b and 5b will not bind (the authors state that they interact poorly with CARM1), but this is not actually shown.

3) Compound 2a seems to be a fairly good inhibitor of SMYD2 (Figure 1C). When SKI-73 is taken up by cells and processed, it primarily results in the accumulation of 2a (Figure 5—figure supplement 6). Thus, part of the phenotype seen in the epigenetic reprogramming studies may be due to SMYD2 inhibition. This issue needs to be addressed in the following way:

a) Are SMYD2 methylation sites impacted by the treatment of SKI-73? For this experiment, methyl-specific antibodies to known non-histone substrates (like p53, Rb or HSP90) should be tested;

b) Tagged SMYD2 (or endogenous) should be tested in the CETSA assay as performed in Figure 4F;

c) The issue of potential SKI-73 off-target effects should be addressed in the discussion.

4) Figure 6C shows a major shift in the subpopulation of cells that display SKI-73 specific depletion. This finding is nicely linked to the subpopulation of freshly isolated invasive cells. The other dramatic change is in the "SKI-73 specific emerging" cell population (3,4,5,6,16). Is there anything unique about these subpopulations of cells?

5) In general, the medicinal chemistry and compound design are not clearly described and this part of the paper is not easy to follow. The authors should be able to be explain the reasoning and design in a clearer manner. Similarly, the details of the biochemical and cellular assays should be better described and discussed.