DNMDP and related compounds, or velcrins, induce complex formation between the phosphodiesterase PDE3A and the SLFN12 protein, leading to a cytotoxic response in cancer cells that express elevated levels of both proteins. The mechanisms by which velcrins induce complex formation, and how the PDE3A-SLFN12 complex causes cancer cell death, are not fully understood. Here, we show that PDE3A and SLFN12 form a heterotetramer stabilized by binding of DNMDP. Interactions between the C-terminal alpha helix of SLFN12 and residues near the active site of PDE3A are required for complex formation, and are further stabilized by interactions between SLFN12 and DNMDP. Moreover, we demonstrate that SLFN12 is an RNase, that PDE3A binding increases SLFN12 RNase activity, and that SLFN12 RNase activity is required for DNMDP response. This new mechanistic understanding will facilitate development of velcrin compounds into new cancer therapies.

Over the past several decades, a trend toward delayed childbirth has led to increases in parental age at the time of conception. Sperm epigenome undergoes age-dependent changes increasing risks of adverse conditions in offspring conceived by fathers of advanced age. The mechanism(s) linking paternal age with epigenetic changes in sperm remain unknown. The sperm epigenome is shaped in a compartment protected by the blood-testes barrier (BTB) known to deteriorate with age. Permeability of the BTB is regulated by the balance of two mTOR complexes in Sertoli cells where mTOR complex 1 (mTORC1) promotes the opening of the BTB and mTOR complex 2 (mTORC2) promotes its integrity. We hypothesized that this balance is also responsible for age-dependent changes in the sperm epigenome. To test this hypothesis, we analyzed reproductive outcomes, including sperm DNA methylation in transgenic mice with Sertoli cell-specific suppression of mTORC1 (Raptor KO) or mTORC2 (Rictor KO). mTORC2 suppression accelerated aging of the sperm DNA methylome and resulted in a reproductive phenotype concordant with older age, including decreased testes weight and sperm counts, and increased percent of morphologically abnormal spermatozoa and mitochondrial DNA copy number. Suppression of mTORC1 resulted in the shift of DNA methylome in sperm opposite to the shift associated with physiological aging – sperm DNA methylome rejuvenation, and mild changes in sperm parameters. These results demonstrate for the first time that the balance of mTOR complexes in Sertoli cells regulates the rate of sperm epigenetic aging. Thus, mTOR pathway in Sertoli cells may be used as a novel target of therapeutic interventions to rejuvenate the sperm epigenome in advanced-age fathers. Ethics statement: All procedures followed the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the approval for this study was received from the Institutional Animal Care and Use Committee at University of Massachusetts, Amherst.

Background: Targeted methods that dominated toxicological research until recently did not allow for screening of all molecular changes involved in toxic response. Therefore, it is difficult to infer if all major mechanisms of toxicity have already been discovered, or if some of them are still overlooked. Objectives: To identify molecular mechanisms sensitive to chemical exposures in an unbiased manner. Methods: We used data on 641,516 unique chemical-gene interactions from the Comparative Toxicogenomic Database. Only data from high-throughput gene expression experiments with human, rat or mouse cells/tissues were extracted. The total number of chemical-gene interactions was calculated for every gene, and used as a measure of gene sensitivity to chemical exposures. These values were further used in enrichment analyses to identify molecular mechanisms sensitive to chemical exposures. Results: Remarkably, use of different input subsets with non-overlapping lists of chemical compounds identified largely the same genes and molecular pathways as most sensitive to chemical exposures, indicative of an unbiased nature of our analysis. One of the most important findings of this study is that almost every known molecular mechanism may be affected by chemical exposures. Predictably, xenobiotic metabolism pathways and mechanisms of cellular response to stress and damage were among the most sensitive. Additionally, our analysis identified a range of highly sensitive molecular pathways, which are not widely recognized by modern toxicology as major targets of toxicants, including lipid metabolism pathways, longevity regulation cascade and cytokine mediated signaling. Discussion: Molecular mechanisms identified as the most sensitive to chemical exposures are relevant for significant public health problems, such as aging, cancer, metabolic and autoimmune disease. Thus, public health system will likely benefit from future research focus on these sensitive molecular mechanisms. Additionally, approach used in this study may guide identification of priority adverse outcome pathways (AOP) for in-vitro and in-silico toxicity testing methods.

Velcrins are a novel class of compounds that kill cancer cells expressing elevated levels of PDE3A and SLFN12. These compounds induce PDE3A-SLFN12 heterotetramer complex formation, resulting in upregulation of the RNase activity of SLFN12. SLFN12 cleaves tRNA-Leu-TAA, which leads to the inhibition of global translation and, consequently, apoptosis of cells expressing sufficient levels of both proteins. Uveal melanoma, a type of melanoma that occurs in the eye, is genomically distinct from cutaneous melanoma. Whereas mutations of BRAF, NRAS, KRAS, and HRAS are common in cutaneous melanoma, uveal melanomas instead harbor mutually exclusive mutations of the G protein alpha subunits, GNAQ and GNA11. Around 50% of patients will develop metastatic disease with a very poor prognosis. Immunotherapy is not effective for these patients, nor are any targeted therapies available beyond tebentafusp, with an ORR of 9%. We found that a subset of uveal melanoma cell lines expresses elevated levels of PDE3A and SLFN12 and is sensitive to velcrin treatment. We are currently assessing whether velcrin treatment of these cell lines results in cleavage of tRNA-Leu-TAA, inhibition of protein synthesis, and tumor cell death, with the goal of developing a new treatment for uveal melanoma patients. To further understand the mechanism of velcrin-induced cancer cell death, we hypothesized that the primary function of PDE3A in the PDE3A-SLFN12 heterotetramer is the dimerization of SLFN12. Based on our cryo-EM structure of the PDE3A-SLFN12 complex, we identified SLFN12 I131 as a critical residue in the SLFN12 homodimer interface. We showed that a SLFN12 I131Q mutation disrupts SLFN12 dimerization and prevents tRNA-Leu-TAA cleavage in vitro. We have also shown that wild-type SLFN12 can dimerize independently of PDE3A or PDE3A-velcrin interactions. In PDE3A-depleted cells, the overexpression of wild-type SLFN12 results in cell death. We are currently testing SLFN12 I131Q for its ability to initiate SLFN12-dependent cytotoxicity in cells. Additionally, we are preparing different SLFN12 constructs that would allow us to induce chemical dimerization of SLFN12 and we plan to test if the forced dimerization of SLFN12 rescues its RNase activity and results in cell death. Citation Format: Kristyna Kotynkova, Sawyer Andersen, Colin Garvie, Xiaoyun Wu, Sooncheol Lee, Joseph McGaunn, Bethany Kaplan, Moony Tseng, Andrew D Cherniack, Matthew Meyerson, Heidi Greulich. Mechanisms of uveal melanoma sensitivity to velcrin treatment and SLFN12-mediated cancer cell death [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A159.