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Homozygous deletions of p16/CDKN2a (cyclin dependent kinase inhibitor 2A) locus are prevalent in cancer and often involve co-deletion of adjacent genes. Metabolic gene MTAP (methylthioadenosine phosphorylase) is localized at the 9p21 chromosome in the close proximity to p16/CDKN2A tumor-suppressor locus. Co-deletion of MTAP may be observed in 80-90% of all tumors harboring homozygous deletion of CDKN2A, which represents 10 - 15% of all human tumors. Many of these tumor types, such as non-small cell lung cancer, pancreatic adenocarcinoma, glioblastoma or mesothelioma are associated with poor prognosis, representing a significant unmet medical need. MTAP deletion results in a massive accumulation of methylthioadenosine (MTA) in cells. MTA in high concentrations is a very selective inhibitor of PRMT5 methyltransferase, competitive for the substrate: S-adenosylmethionine (SAM). Accumulation of MTA in cells with MTAP deletion causes partial inhibition of the methylation activity of PRMT5, which in turn reduces the level of symmetric arginine dimethylation of the whole proteome, and thus an increased sensitivity of cells to modulation of methylosome activity. Therapeutic targeting of PRMT5 in homozygous MTAP-deleted cancers constitute a promising strategy of selective killing of genetically defined cancer cells. Ryvu has identified a series of MTA-cooperative PRMT5 inhibitors which have good drug-like physicochemical properties and block methyltransferase activity with nanomolar IC50 values. Structurally enabled hit generation and optimization allowed quick expansion and delivery of several generations of compounds with novel IP, high target engagement in cells and selective potency in MTAP-deleted cell lines. Ryvu compounds selectively inhibit growth of MTAP-deleted cancer cells in prolonged 3D culture, which strongly correlates with inhibition of PRMT5-dependent protein symmetric dimethylation (SDMA) in those cells. Selectivity between effects observed in MTAP-deleted and WT cells exceeds 100-fold both for SDMA and growth inhibition. The DMPK profile of these compounds allows for oral administration, which enables testing antitumor activity in MTAP null tumor xenograft-bearing mice. Efficacy studies with our lead compound resulted in demonstration of tumor growth inhibition in MTAP -/- model, accompanied by significant inhibition of target proximal PD biomarker. Overall, these studies provide a rationale for further optimization of our chemical series of MTA-cooperative PRMT5 inhibitors towards a clinical candidate. Citation Format: Anna Bartosik, Adam Radzimierski, Aneta Bobowska, Oleksandr Levenets, Agata Stachowicz, Kamil Kuś, Kinga Michalik, Katarzyna Banaszak, Monika Madej, Marta Skoda, Kamila Kozłowska-Tomczyk, Igor Tomczyk, Karolina Pyziak, Dobrosława Krzemień, Mirosława Gładysz, Paulina Podkalicka, Aniela Gołas, Karolina Gluza, Grzegorz Satała, Andrzej Gondela, Marta Sowińska, Nicolas Boutard, Agata Chłopek, Aleksandra Więckowska, Daria Szukiel, Grzegorz Ćwiertnia, Iana Levenets, Karol Zuchowicz, Klara Korta-Piątek, Marcin Nowogródzki, Marek Wronowski, Marianna Girardi, Mateusz Świrski, Oleksandr Popika, Paulina Niedziejko-Ćwiertnia, Pierpaolo Cordone, Przemysław Wyrębek, Quỳnh Vũ, Sujit Sasmal, Svitlana Sukhomlinova, Magdalena Miodek, Jacek Faber, Anna Kowal-Chwast, Róża Starczak, Sanja Novak Ratajczak, Agnieszka Świrska, Dawid Gogola, Paweł Guzik, Martin Swarbrick, Mateusz Nowak. Discovery of novel MTA-cooperative PRMT5 inhibitors as targeted therapeutics for MTAP-deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 449.

Double-stranded DNA (dsDNA) fragments exhibit noncovalent attractive interactions between their tips. It is still unclear how DNA liquid crystal self-assembly is affected by such blunt-end attractions. It is demonstrated that stiff dsDNA fragments with moderate aspect ratio can specifically self-assemble in concentrated aqueous solutions into different types of smectic mesophases on the basis of selectively screening of blunt-end DNA stacking interactions. To this end, this type of attractions are engineered at the molecular level by constructing DNA duplexes where the attractions between one or both ends are screened by short hairpin caps. All-DNA bilayer and monolayer smectic-A type of phases, as well as a columnar phase, can be stabilized by controlling attractions strength. The results imply that the so far elusive smectic-A in DNA rod-like liquid crystals is a thermodynamically stable phase. The existence of the bilayer smectic phase is confirmed by Monte-Carlo simulations of hard cylinders decorated with one attractive terminal site. This work demonstrates that DNA blunt-ends behave as well-defined monovalent attractive patches whose strength and position can be potentially precisely tuned, highlighting unique opportunities concerning the stabilization of nonconventional DNA-based lyotropic liquid crystal phases assembled by all-DNA patchy particles with arbitrary geometry and composition.