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AKT is an important target for cancer therapeutics. Significant advancements have been made in developing ATP-competitive and allosteric AKT inhibitors. Recently, several AKT proteolysis targeting chimeras (PROTACs) derived from ATP-competitive AKT inhibitors have been reported, including MS21. While MS21 potently degraded AKT and inhibited the growth in tumor cells harboring PI3K/PTEN pathway mutation, it was largely ineffective in degrading AKT in KRAS/BRAF mutated cells as a single agent. To overcome the AKT degradation resistance in KRAS/BRAF mutated cells, we developed novel AKT PROTACs derived from an AKT allosteric inhibitor, including degrader

[Image: see text] Enhancer of zeste homolog 2 (EZH2), a catalytic subunit of polycomb repressive complex 2 (PRC2), is overexpressed in triple-negative breast cancer (TNBC), correlating with poor prognosis. However, EZH2 catalytic inhibitors are ineffective in suppressing the growth of TNBC cells that are dependent on EZH2. Knockdown of EZH2 inhibits the proliferation of these cells, suggesting that EZH2 protein overexpression but not its catalytic activity is critical for driving TNBC progression. Several proteolysis targeting chimera (PROTAC) degraders of EZH2, including the von Hippel–Lindau (VHL)-recruiting PROTAC YM281, have been reported. However, the effects of these EZH2 PROTACs in TNBC cells were not investigated. Here, we report the discovery and characterization of a novel, potent, and selective EZH2 PROTAC degrader, MS8815 (compound 16), which induced robust EZH2 degradation in a concentration-, time-, and proteasome-dependent manner in TNBC cells. Importantly, 16 effectively suppressed the cell growth in multiple TNBC cell lines and primary patient TNBC cells.

Using a panel of cancer cell lines, we characterized a novel degrader of AKT, MS21. In mutant PI3K–PTEN pathway cell lines, AKT degradation was superior to AKT kinase inhibition for reducing cell growth and sustaining lower signaling over many days. AKT degradation, but not kinase inhibition, profoundly lowered Aurora kinase B (AURKB) protein, which is known to be essential for cell division, and induced G2–M arrest and hyperploidy. PI3K activated AKT phosphorylation of AURKB on threonine 73, which protected it from proteasome degradation. A mutant of AURKB (T73E) that mimics phosphorylation and blocks degradation rescued cells from growth inhibition. Degrader-resistant lines were associated with low AKT phosphorylation, wild-type PI3K/PTEN status, and mutation of KRAS/BRAF. Pan-cancer analysis identified that 19% of cases have PI3K–PTEN pathway mutation without RAS pathway mutation, suggesting that these patients with cancer could benefit from AKT degrader therapy that leads to loss of AURKB.Significance:MS21 depletes cells of phosphorylated AKT (pAKT) and a newly identified AKT substrate, AURKB, to inhibit tumor growth in mice. MS21 is superior to prior agents that target PI3K and AKT due to its ability to selectively target active, pAKT and sustain repression of signaling to deplete AURKB.This article is highlighted in the In This Issue feature, p. 2945

Supplementary Figure from AKT Degradation Selectively Inhibits the Growth of PI3K/PTEN Pathway–Mutant Cancers with Wild-Type KRAS and BRAF by Destabilizing Aurora Kinase B

Supplementary Table from AKT Degradation Selectively Inhibits the Growth of PI3K/PTEN Pathway–Mutant Cancers with Wild-Type KRAS and BRAF by Destabilizing Aurora Kinase B

Supplementary Data from AKT Degradation Selectively Inhibits the Growth of PI3K/PTEN Pathway–Mutant Cancers with Wild-Type KRAS and BRAF by Destabilizing Aurora Kinase B

WD repeat domain 5 (WDR5), an integral component of the MLL/KMT2A lysine methyltransferase complex, is critically involved in oncogenesis and represents an attractive onco-target. Inhibitors targeting protein-protein interactions (PPIs) between WDR5 and its binding partners, however, do not inhibit all of WDR5-mediated oncogenic functions and exert rather limited antitumor effects. Here, we report a cereblon (CRBN)-recruiting proteolysis targeting chimera (PROTAC) of WDR5, MS40, which selectively degrades WDR5 and the well-established neo-substrates of immunomodulatory drugs (IMiDs):CRBN, the Ikaros zinc finger (IKZF) transcription factors IKZF1 and IKZF3. MS40-induced WDR5 degradation caused disassociation of the MLL/KMT2A complex off chromatin, resulting in decreased H3K4me2. Transcriptomic profiling revealed that targets of both WDR5 and IMiDs:CRBN were significantly repressed by treatment of MS40. In MLL-rearranged leukemias, which exhibit IKZF1 high expression and dependency, co-suppression of WDR5 and Ikaros by MS40 is superior in suppressing oncogenesis to the WDR5 PPI inhibitor, to MS40's non-PROTAC analog controls (MS40N1 and MS40N2, which do not bind CRBN and WDR5, respectively), and to a matched VHL-based WDR5 PROTAC (MS169, which degrades WDR5 but not Ikaros). MS40 suppressed the growth of primary leukemia patient cells in vitro and patient-derived xenografts in vivo. Thus, dual degradation of WDR5 and Ikaros is a promising anti-cancer strategy.

Canonically, EZH2 serves as the catalytic subunit of PRC2, which mediates H3K27me3 deposition and transcriptional repression. Here, we report that in acute leukaemias, EZH2 has additional noncanonical functions by binding cMyc at non-PRC2 targets and uses a hidden transactivation domain (TAD) for (co)activator recruitment and gene activation. Both canonical (EZH2-PRC2) and noncanonical (EZH2-TAD-cMyc-coactivators) activities of EZH2 promote oncogenesis, which explains the slow and ineffective antitumour effect of inhibitors of the catalytic function of EZH2. To suppress the multifaceted activities of EZH2, we used proteolysis-targeting chimera (PROTAC) to develop a degrader, MS177, which achieved effective, on-target depletion of EZH2 and interacting partners (that is, both canonical EZH2-PRC2 and noncanonical EZH2-cMyc complexes). Compared with inhibitors of the enzymatic function of EZH2, MS177 is fast-acting and more potent in suppressing cancer growth. This study reveals noncanonical oncogenic roles of EZH2, reports a PROTAC for targeting the multifaceted tumorigenic functions of EZH2 and presents an attractive strategy for treating EZH2-dependent cancers.

We recently reported a potent, selective, and in vivo efficacious AKT degrader, MS21, which is a von Hippel–Lindau (VHL)-recruiting proteolysis targeting chimera (PROTAC) based on the AKT inhibitor AZD5363. However, no structure–activity relationship (SAR) studies that resulted in this discovery have been reported. Herein, we present our SAR studies that led to the discovery of MS21, another VHL-recruiting AKT degrader, MS143 (compound 20) with similar potency as MS21, and a novel cereblon (CRBN)-recruiting PROTAC, MS5033 (compound 35). Compounds 20 and 35 induced rapid and robust AKT degradation in a concentration- and time-dependent manner via hijacking the ubiquitin-proteasome system. Compound 20 suppressed cell growth more effectively than AZD5363 in multiple cancer cell lines. Furthermore, 20 and 35 displayed good plasma exposure levels in mice and are suitable for in vivo efficacy studies. Lastly, compound 20 effectively suppressed tumor growth in vivo in a xenograft model without apparent toxicity.

The serine/threonine kinase AKT functions as a critical node of the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (m-TOR) signaling pathway. Aberrant activation and overexpression of AKT are strongly correlated with numerous human cancers. To date, only two AKT degraders with no structure–activity relationship (SAR) results have been reported. Through extensive SAR studies on various linkers, E3 ligase ligands, and AKT binding moieties, we identified two novel and potent AKT proteolysis targeting chimera (PROTAC) degraders: von Hippel–Lindau (VHL)-recruiting degrader 13 (MS98) and cereblon (CRBN)-recruiting degrader 25 (MS170). These two compounds selectively induced robust AKT protein degradation, inhibited downstream signaling, and suppressed cancer cell proliferation. Moreover, these two degraders exhibited good plasma exposure levels in mice through intraperitoneal injection. Overall, our comprehensive SAR studies led to the discovery of degraders 13 and 25, which are potentially useful chemical tools to investigate biological and pathogenic functions of AKT in vitro and in vivo.

Proteolysis targeting chimeras (PROTACs) represent a new class of promising therapeutic modalities. PROTACs hijack E3 ligases and the ubiquitin-proteasome system (UPS), leading to selective degradation of the target proteins. However, only a very limited number of E3 ligases have been leveraged to generate effective PROTACs. Herein, we report that the KEAP1 E3 ligase can be harnessed for targeted protein degradation utilizing a highly selective, noncovalent small-molecule KEAP1 binder. We generated a proof-of-concept PROTAC, MS83, by linking the KEAP1 ligand to a BRD4/3/2 binder. MS83 effectively reduces protein levels of BRD4 and BRD3, but not BRD2, in cells in a concentration-, time-, KEAP1- and UPS-dependent manner. Interestingly, MS83 degrades BRD4/3 more durably than the CRBN-recruiting PROTAC dBET1 in MDA-MB-468 cells and selectively degrades BRD4 short isoform over long isoform in MDA-MB-231 cells. It also displays improved antiproliferative activity than dBET1. Overall, our study expands the limited toolbox for targeted protein degradation.

Using a panel of cancer cell lines, we characterized a novel degrader of AKT, MS21. In mutant PI3K–PTEN pathway cell lines, AKT degradation was superior to AKT kinase inhibition for reducing cell growth and sustaining lower signaling over many days. AKT degradation, but not kinase inhibition, profoundly lowered Aurora kinase B (AURKB) protein, which is known to be essential for cell division, and induced G2–M arrest and hyperploidy. PI3K activated AKT phosphorylation of AURKB on threonine 73, which protected it from proteasome degradation. A mutant of AURKB (T73E) that mimics phosphorylation and blocks degradation rescued cells from growth inhibition. Degrader-resistant lines were associated with low AKT phosphorylation, wild-type PI3K/PTEN status, and mutation of KRAS/BRAF. Pan-cancer analysis identified that 19% of cases have PI3K–PTEN pathway mutation without RAS pathway mutation, suggesting that these patients with cancer could benefit from AKT degrader therapy that leads to loss of AURKB. Significance: MS21 depletes cells of phosphorylated AKT (pAKT) and a newly identified AKT substrate, AURKB, to inhibit tumor growth in mice. MS21 is superior to prior agents that target PI3K and AKT due to its ability to selectively target active, pAKT and sustain repression of signaling to deplete AURKB. This article is highlighted in the In This Issue feature, p. 2945

Several epidermal growth factor receptor (EGFR) proteolysis-targeting chimeras (PROTACs), including MS39 and MS154 developed by us, have been reported to effectively degrade the mutant but not the wild-type (WT) EGFR. However, the mechanism underlying the selectivity in degrading the mutant over the WT EGFR has not been elucidated. Here, we report comprehensive structure–activity relationship studies that led to the discovery of two novel EGFR degraders, 31 (MS9449) and 72 (MS9427), and mechanistic studies of these EGFR degraders. Compounds 31 and 72 selectively degraded the mutant but not the WT EGFR through both ubiquitination/proteasome and autophagy/lysosome pathways. Interestingly, we found that the mutant but not the WT EGFR can effectively form EGFR–PROTAC–E3 ligase ternary complexes. Furthermore, we found that PI3K inhibition sensitized WT EGFR to PROTAC-induced degradation and combination treatment with a PI3K inhibitor enhanced antiproliferation activities of EGFR degraders in cancer cells harboring WT EGFR, providing a potential therapeutic strategy for patients with WT EGFR overexpression.

Immune checkpoint blockade therapy has revolutionized cancer care, including the treatment of advanced metastatic disease. However, most patients derive little or no clinical benefit from these therapies and many cancer types are notoriously non-responsive. Motivated by (1) the correlation between tumor neoantigen abundance and anti-tumor immunity and (2) that most cancers are characterized by widespread dysregulation of RNA processing, we reasoned that pharmacologic modulation of RNA splicing might increase cancer cell immunogenicity via the generation of splicing-derived neoantigens. We demonstrated that two compounds which modulate RNA splicing via distinct mechanisms, inhibited tumor growth and enhanced response to immune checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Critical for their clinical translatability, therapeutic doses of splicing inhibitors were non-toxic, tolerated by the host immune system, and did not affect T cell activation, proliferation, and anti-cancer killing activities. Mechanistically, splicing modulation induced stereotyped, dose-dependent “splicing failure” — dramatic intron retention, alternative exon skipping, etc. — that was consistent across multiple mouse and human tumor types. By combining RNA-seq-based peptide predictions and mass spectrometry of the MHC I-bound immunopeptidome, we identified drug-induced, splicing-derived peptides that promote the expansion of antigen-specific CD8+ T cells and trigger anti-tumor T cell responses in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic. Citation Format: James D. Thomas, Sydney X. Lu, Emma De Neef, Erich Sabio, Benoit Rousseau, Mathieu Gigoux, David A. Knorr, Benjamin Greenbaum, Yuval Elhanati, Simon J. Hogg, Andrew Chow, Arnab Ghosh, Abigail Xie, Dmitriy Zamarin, Daniel Cui, Caroline Erickson, Michael Singer, Hana Cho, Eric Wang, Bin Lu, Benjamin H. Durham, Harshal Shah, Diego Chowell, Austin M. Gabel, Yudao Shen, Jing Liu, Jian Jin, Matthew C. Rhodes, Richard E. Taylor, Henrik Molina, Jedd D. Wolchok, Taha Merghoub, Luis A. Diaz Jr, Omar Abdel-Wahab, Robert K. Bradley. Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. [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 5742.

Protein arginine methyltransferase 6 (PRMT6) catalyzes monomethylation and asymmetric dimethylation of arginine residues in various proteins, plays important roles in biological processes, and is associated with multiple cancers. To date, a highly selective PRMT6 inhibitor has not been reported. Here we report the discovery and characterization of a first-in-class, highly selective allosteric inhibitor of PRMT6, (R)-2 (SGC6870). (R)-2 is a potent PRMT6 inhibitor (IC(50) = 77 ± 6 nM) with outstanding selectivity for PRMT6 over a broad panel of other methyltransferases and nonepigenetic targets. Notably, the crystal structure of the PRMT6−(R)-2 complex and kinetic studies revealed (R)-2 binds a unique, induced allosteric pocket. Additionally, (R)-2 engages PRMT6 and potently inhibits its methyltransferase activity in cells. Moreover, (R)-2’s enantiomer, (S)-2 (SGC6870N), is inactive against PRMT6 and can be utilized as a negative control. Collectively, (R)-2 is a well-characterized PRMT6 chemical probe and a valuable tool for further investigating PRMT6 functions in health and disease.

The aberrant expression of protein arginine methyltransferase 5 (PRMT5) has been associated with multiple cancers. Using the proteolysis targeting chimera technology, we discovered a first-in-class PRMT5 degrader 15 (MS4322). Here, we report the design, synthesis, and characterization of compound 15 and two structurally similar controls 17 (MS4370) and 21 (MS4369), with impaired binding to the von Hippel-Lindau E3 ligase and PRMT5, respectively. Compound 15, but not 17 and 21, effectively reduced the PRMT5 protein level in MCF-7 cells. Our mechanism studies indicate that compound 15 degraded PRMT5 in an E3 ligase- and proteasome-dependent manner. Compound 15 also effectively reduced the PRMT5 protein level and inhibited growth in multiple cancer cell lines. Moreover, compound 15 was highly selective for PRMT5 in a global proteomic study and exhibited good plasma exposure in mice. Collectively, compound 15 and its two controls 17 and 21 are valuable chemical tools for exploring the PRMT5 functions in health and disease.

Protein arginine methyltransferase 6 (PRMT6) plays important roles in several biological processes associated with multiple cancers. Well-characterized potent, selective, and cell-active PRMT6 inhibitors are invaluable tools for testing biological and therapeutic hypotheses. Although there are several known reversible PRMT6 inhibitors, covalent PRMT6 inhibitors have not been reported. Based on a cocrystal structure of PRMT6-MS023 (a type I PRMT inhibitor), we discovered the first potent and cell-active irreversible PRMT6 inhibitor, 4 (MS117). The covalent binding mode of compound 4 to PRMT6 was confirmed by mass spectrometry and kinetic studies and by a cocrystal structure. Compound 4 did not covalently modify other closely related PRMTs, potently inhibited PRMT6 in cells, and was selective for PRMT6 over other methyltransferases. We also developed two structurally similar control compounds, 5 (MS167) and 7 (MS168). We provide these valuable chemical tools to the scientific community for further studying PRMT6 physiological and pathophysiological functions.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and few effective therapies. Here, we undertook a screen of epigenetic chemical probes to systematically uncover the epigenetic regulators critical for TNBC growth. We identified MS023, an inhibitor of type I protein arginine methyltransferases (PRMTs), as having anti-tumor growth activity in TNBC in vitro and in vivo. Pathway analysis of TNBC cell lines indicates that the activation of interferon responses pre- and post-MS023 treatment is a functional biomarker and determinant of response; and these observations extend to a panel of patient-derived organoids. Inhibition of type I PRMT triggers an interferon response through the antiviral defense pathway with the induction of double-stranded RNA (dsRNA). The observed dsRNA accumulation is derived, at least in part, from inverted-repeat Alus (IR-Alus), many of which are expressed from retained introns induced by MS023, which inhibits arginine methylation of RNA-binding proteins and alters mRNA splicing machinery. Together, our results represent a shift in understanding the anti-tumor mechanism of type I PRMT inhibitors and provide a novel rationale and biomarker approach for the clinical development of type I PRMT inhibitors.

Interactions between WD40 repeat domain protein 5 (WDR5) and its various partners such as mixed lineage leukemia (MLL) and c-MYC are essential for sustaining oncogenesis in human cancers. However, inhibitors that block protein-protein interactions (PPIs) between WDR5 and its binding partners exhibit modest cancer cell killing effects and lack in vivo efficacy. Here, we present pharmacological degradation of WDR5 as a promising therapeutic strategy for treating WDR5-dependent tumors and report two high-resolution crystal structures of WDR5-degrader-E3 ligase ternary complexes. We identified an effective WDR5 degrader via structure-based design and demonstrated its in vitro and in vivo antitumor activities. On the basis of the crystal structure of an initial WDR5 degrader in complex with WDR5 and the E3 ligase von Hippel–Lindau (VHL), we designed a WDR5 degrader, MS67, and demonstrated the high cooperativity of MS67 binding to WDR5 and VHL by another ternary complex structure and biophysical characterization. MS67 potently and selectively depleted WDR5 and was more effective than WDR5 PPI inhibitors in suppressing transcription of WDR5-regulated genes, decreasing the chromatin-bound fraction of MLL complex components and c-MYC, and inhibiting the proliferation of cancer cells. In addition, MS67 suppressed malignant growth of MLL-rearranged acute myeloid leukemia patient cells in vitro and in vivo and was well tolerated in vivo. Collectively, our results demonstrate that structure-based design can be an effective strategy to identify highly active degraders and suggest that pharmacological degradation of WDR5 might be a promising treatment for WDR5-dependent cancers.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype with the worst prognosis and few effective therapies. Here we identified MS023, an inhibitor of type I protein arginine methyltransferases (PRMTs), which has antitumor growth activity in TNBC. Pathway analysis of TNBC cell lines indicates that the activation of interferon responses before and after MS023 treatment is a functional biomarker and determinant of response, and these observations extend to a panel of human-derived organoids. Inhibition of type I PRMT triggers an interferon response through the antiviral defense pathway with the induction of double-stranded RNA, which is derived, at least in part, from inverted repeat Alu elements. Together, our results represent a shift in understanding the antitumor mechanism of type I PRMT inhibitors and provide a rationale and biomarker approach for the clinical development of type I PRMT inhibitors.

Relapse remains the main cause of MLL-rearranged (MLL-r) acute lymphoblastic leukemia (ALL) treatment failure resulting from persistence of drug-resistant clones after conventional chemotherapy treatment or targeted therapy. Thus, defining mechanisms underlying MLL-r ALL maintenance is critical for developing effective therapy. PRMT1, which deposits an asymmetric dimethylarginine mark on histone/non-histone proteins, is reportedly overexpressed in various cancers. Here, we demonstrate elevated PRMT1 levels in MLL-r ALL cells and show that inhibition of PRMT1 significantly suppresses leukemic cell growth and survival. Mechanistically, we reveal that PRMT1 methylates Fms-like receptor tyrosine kinase 3 (FLT3) at arginine (R) residues 972 and 973 (R972/973), and its oncogenic function in MLL-r ALL cells is FLT3 methylation dependent. Both biochemistry and computational analysis demonstrate that R972/973 methylation could facilitate recruitment of adaptor proteins to FLT3 in a phospho-tyrosine (Y) residue 969 (Y969) dependent or independent manner. Cells expressing R972/973 methylation-deficient FLT3 exhibited more robust apoptosis and growth inhibition than did Y969 phosphorylation-deficient FLT3-transduced cells. We also show that the capacity of the type I PRMT inhibitor MS023 to inhibit leukemia cell viability parallels baseline FLT3 R972/973 methylation levels. Finally, combining FLT3 tyrosine kinase inhibitor PKC412 with MS023 treatment enhanced elimination of MLL-r ALL cells relative to PKC412 treatment alone in patient-derived mouse xenografts. These results indicate that abolishing FLT3 arginine methylation through PRMT1 inhibition represents a promising strategy to target MLL-r ALL cells.

Functionally selective G protein-coupled receptor ligands are valuable tools for deciphering the roles of downstream signaling pathways that potentially contribute to therapeutic effects versus side effects. Recently, we discovered both Gi/o-biased and β-arrestin2-biased D2 receptor agonists based on the Food and Drug Administration (FDA)-approved drug aripiprazole. In this work, based on another FDA-approved drug, cariprazine, we conducted a structure-functional selectivity relationship study and discovered compound 38 (MS1768) as a potent partial agonist that selectively activates the Gi/o pathway over β-arrestin2. Unlike the dual D2R/D3R partial agonist cariprazine, compound 38 showed selective agonist activity for D2R over D3R. In fact, compound 38 exhibited potent antagonism of dopamine-stimulated β-arrestin2 recruitment. In our docking studies, compound 38 directly interacts with S1935.42 on TM5 but has no interactions with extracellular loop 2, which appears to be in contrast to the binding poses of D2R β-arrestin2-biased ligands. In in vivo studies, compound 38 showed high D2R receptor occupancy in mice and effectively inhibited phencyclidine-induced hyperlocomotion.

Protein methyltransferases (PMTs) comprise a major class of epigenetic regulatory enzymes with therapeutic relevance. Here we present a collection of chemical probes and associated reagents and data to elucidate the function of human and murine PMTs in cellular studies. Our collection provides inhibitors and antagonists that together modulate most of the key regulatory methylation marks on histones H3 and H4, providing an important resource for modulating cellular epigenomes. We describe a comprehensive and comparative characterization of the probe collection with respect to their potency, selectivity, and mode of inhibition. We demonstrate the utility of this collection in CD4+ T cell differentiation assays revealing the potential of individual probes to alter multiple T cell subpopulations which may have implications for T cell-mediated processes such as inflammation and immuno-oncology. In particular, we demonstrate a role for DOT1L in limiting Th1 cell differentiation and maintaining lineage integrity. This chemical probe collection and associated data form a resource for the study of methylation-mediated signaling in epigenetics, inflammation and beyond., Protein methyltransferases (PMTs) are epigenetic regulatory enzymes with significant therapeutic relevance. Here the authors describe a collection of chemical inhibitors and antagonists to modulate most of the key methylation marks on histones H3 and H4, and use the collection to study of the role of PMTs in mouse and human T cell differentiation.

PI3K/AKT/mTOR signaling pathway is one of the most frequently dysregulated pathway in cancer development and the serine/threonine kinase AKT functions as the key node in this pathway to regulate multiple cellular and physiological processes. AKT inhibitors that competitively bind the ATP pocket have been evaluated in clinical trials and have a tolerable toxicity profile with greater efficacy for tumors with pathway mutations; however, many PI3K/AKT pathway mutant tumors remain resistant. Proteolysis targeting chimeras (PROTACs) are a powerful targeted protein degradation technology that hijacks the cellular ubiquitin-proteasome system to induce selective polyubiquitination and degradation of the target proteins. Using this technology, we designed and developed a library of novel small-molecules putative degraders to degrade AKT by recruiting either the cereblon (CRBN) or von Hippel-Lindau (VHL) E3 ligase, that have the ability to degrade AKT in cells and lower downstream signaling to various extents. Though extensive structure-activity relationship studies on various linkers, E3 ligase ligands, and AKT binding moieties, we identified a novel VHL-recruiting AKT degrader, MS21, and characterized it using a panel of 38 cancer cell lines with diverse genotypes and tissue origins. Our results suggest that efficient pharmacologic degradation of AKT phosphorylated on threonine 308 and serine 473 leads to selective inhibition of the growth of tumor cells with alterations of HER2, PIK3CA, PTEN, or AKT1. In these PI3K/PTEN pathway mutant lines, AKT degradation by MS21 was superior to AKT kinase inhibition for reducing cell growth and sustaining lower signaling over many days, and inhibited tumor cell growths through lowering the level of Aurora Kinase B, which we found to be an AKT substrate protein. AKT degradation but not kinase inhibition profoundly lowered Aurora kinase B (AURKB) protein, which is known to be essential for cell division, and induced G2/M arrest and hyperploidy. PI3K activated AKT phosphorylation of AURKB on threonine 73, which protected it from proteasome degradation. A mutant of AURKB (T73E) that mimics phosphorylation and blocks its degradation rescued cells from growth inhibition by MS21. In addition, resistance to MS21 was found to be associated with low levels of baseline AKT phosphorylation in cells as well as mutation of either KRAS or BRAF, and resistance to MS21 could be overcome by the combination treatment of a MEK inhibitor trametinib with MS21, which not only inhibited MEK but also increased AKT phosphorylation and enhanced AKT degradation. Pan-cancer analysis identified that 19% of cases have PI3K/PTEN pathway mutation without RAS pathway mutation, suggesting that these cancer patients could benefit from AKT degrader therapy that leads to loss of AURKB. Citation Format: Jia Xu, Xufen Yu, Tiphaine C. Martin, Ankita Bansal, Kakit Cheung, Abigail Lubin, Elias Stratikopoulos, Kaitlyn M. Cahuzac, Li Wang, Ling Xie, Royce Zhou, Yudao Shen, Xuewei Wu, Shen Yao, Ruifang Qiao, Poulikos I. Poulikakos, Xian Chen, Jing Liu, Jian Jin, Ramon Parsons. AKT degradation selectively inhibits the growth of PI3K/PTEN pathway mutant cancers with wild type KRAS and BRAF by destabilizing Aurora kinase B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB195.

PROTACs (proteolysis targeting chimeras) are an emerging class of promising therapeutic modalities that degrade intracellular protein targets by hijacking the cellular ubiquitin-proteasome system. However, potential toxicity of PROTACs in normal cells due to the off-tissue on-target degradation effect limits their clinical applications. Precise control of a PROTAC's on-target degradation activity in a tissue-selective manner could minimize potential toxicity/side-effects. To this end, we developed a cancer cell selective delivery strategy for PROTACs by conjugating a folate group to a ligand of the VHL E3 ubiquitin ligase, to achieve targeted degradation of proteins of interest (POIs) in cancer cells versus noncancerous normal cells. We show that our folate-PROTACs, including BRD PROTAC (folate-ARV-771), MEK PROTAC (folate-MS432), and ALK PROTAC (folate-MS99), are capable of degrading BRDs, MEKs, and ALK, respectively, in a folate receptor-dependent manner in cancer cells. This design provides a generalizable platform for PROTACs to achieve selective degradation of POIs in cancer cells.

Summary Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic.

PRMT6 catalyzes monomethylation and asymmetric dimethylation of arginine residues in various proteins, plays important roles in biological processes and is associated with multiple cancers. While there are several reported PRMT6 inhibitors, a highly selective PRMT6 inhibitor has not been reported to date. Furthermore, allosteric inhibitors of protein methyltransferases are rare. Here we report the discovery and characterization of a first-in-class, highly selective allosteric inhibitor of PRMT6, SGC6870. SGC6870 is a potent PRMT6 inhibitor (IC50 = 77 ± 6 nM) with outstanding selectivity for PRMT6 over a broad panel of other methyltransferases and non-epigenetic targets. Notably, the crystal structure of the PRMT6–SGC6870 complex and kinetic studies revealed SGC6870 binds a unique, induced allosteric pocket. Additionally, SGC6870 engages PRMT6 and potently inhibits its methyltransferase activity in cells. Moreover, SGC6870’s enantiomer, SGC6870N, is inactive against PRMT6 and can be utilized as a negative control. Collectively, SGC6870 is a well-characterized PRMT6 chemical probe and valuable tool for further investigating PRMT6 functions in health and disease.

The balance between pro- and anti-inflammatory signals maintains tissue homeostasis and defines the outcome of chronic inflammatory diseases such as periodontitis, a condition that afflicts the tooth-supporting tissues and exerts an impact on systemic health. The induction of tissue inflammation relies heavily on Toll-like receptor (TLR) signaling, which drives a proinflammatory pathway through recruiting myeloid differentiation primary response gene 88 (MyD88) and activating nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). TLR-induced production of proinflammatory cytokines and chemokines is reined in by anti-inflammatory cytokines, including the transforming growth factor β (TGFβ) family of cytokines. Although Smad6 is a key mediator of TGFβ-induced anti-inflammatory signaling, the exact mechanism by which TGFβ regulates TLR proinflammatory signaling in the periodontal tissue has not been addressed to date. In this study, we demonstrate for the first time that the ability of TGFβ to inhibit TLR-NFκB signaling is mediated by protein arginine methyltransferase 1 (PRMT1)–induced Smad6 methylation. Upon methylation, Smad6 recruited MyD88 and promoted MyD88 degradation, thereby inhibiting NFκB activation. Most important, Smad6 is expressed and methylated in the gingival epithelium, and PRMT1-Smad6 signaling promotes tissue homeostasis by limiting inflammation. Consistent with this, disturbance of Smad6 methylation exacerbates inflammation and bone loss in experimental periodontitis. The dissected mechanism is therapeutically important, as it highlights the manipulation of PRMT1-Smad6 signaling as a novel promising strategy to modulate the host immune response in periodontitis.

Summary: Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.

The presence of FMS-like receptor tyrosine kinase-3 internal tandem duplication (FLT3-ITD) mutations in patients with acute myeloid leukemia (AML) is associated with poor clinical outcome. FLT3 tyrosine kinase inhibitors (TKIs), although effective in kinase ablation, do not eliminate primitive FLT3-ITD+ leukemia cells, which are potential sources of relapse. Thus, understanding the mechanisms underlying FLT3-ITD+ AML cell persistence is essential to devise future AML therapies. Here, we show that expression of protein arginine methyltransferase 1 (PRMT1), the primary type I arginine methyltransferase, is increased significantly in AML cells relative to normal hematopoietic cells. Genome-wide analysis, coimmunoprecipitation assay, and PRMT1-knockout mouse studies indicate that PRMT1 preferentially cooperates with FLT3-ITD, contributing to AML maintenance. Genetic or pharmacological inhibition of PRMT1 markedly blocked FLT3-ITD+ AML cell maintenance. Mechanistically, PRMT1 catalyzed FLT3-ITD protein methylation at arginine 972/973, and PRMT1 promoted leukemia cell growth in an FLT3 methylation–dependent manner. Moreover, the effects of FLT3-ITD methylation in AML cells were partially due to cross talk with FLT3-ITD phosphorylation at tyrosine 969. Importantly, FLT3 methylation persisted in FLT3-ITD+ AML cells following kinase inhibition, indicating that methylation occurs independently of kinase activity. Finally, in patient-derived xenograft and murine AML models, combined administration of AC220 with a type I PRMT inhibitor (MS023) enhanced elimination of FLT3-ITD+ AML cells relative to AC220 treatment alone. Our study demonstrates that PRMT1-mediated FLT3 methylation promotes AML maintenance and suggests that combining PRMT1 inhibition with FLT3 TKI treatment could be a promising approach to eliminate FLT3-ITD+ AML cells.

Functionally selective G protein-coupled receptor ligands are valuable tools for deciphering the roles of downstream signaling pathways that potentially contribute to therapeutic effects versus side effects. Recently, we discovered both G(i/o)-biased and β-arrestin2-biased D(2) receptor agonists based on the Food and Drug Administration (FDA)-approved drug aripiprazole. In this work, based on another FDA-approved drug, cariprazine, we conducted a structure–functional selectivity relationship study and discovered compound 38 (MS1768) as a potent partial agonist that selectively activates the G(i/o) pathway over β-arrestin2. Unlike the dual D(2)R/D(3)R partial agonist cariprazine, compound 38 showed selective agonist activity for D(2)R over D(3)R. In fact, compound 38 exhibited potent antagonism of dopamine-stimulated β-arrestin2 recruitment. In our docking studies, compound 38 directly interacts with S193(5.42) on TM5 but has no interactions with extracellular loop 2, which appears to be in contrast to the binding poses of D(2)R β-arrestin2-biased ligands. In in vivo studies, compound 38 showed high D(2)R receptor occupancy in mice and effectively inhibited phencyclidine-induced hyperlocomotion.

Cancer-associated mutations in genes encoding RNA splicing factors (SFs) commonly occur in leukemias, as well as in a variety of solid tumors, and confer dependence on wild-type splicing. These observations have led to clinical efforts to directly inhibit the spliceosome in patients with refractory leukemias. Here, we identify that inhibiting symmetric or asymmetric dimethylation of arginine, mediated by PRMT5 and type I protein arginine methyltransferases (PRMTs), respectively, reduces splicing fidelity and results in preferential killing of SF-mutant leukemias over wild-type counterparts. These data identify genetic subsets of cancer most likely to respond to PRMT inhibition, synergistic effects of combined PRMT5 and type I PRMT inhibition, and a mechanistic basis for the therapeutic efficacy of PRMT inhibition in cancer.

Biased ligands (also known as functionally selective ligands) of G protein-coupled receptors are valuable tools for dissecting the roles of G protein-dependent and independent signaling pathways in health and disease. Biased ligands have also been increasingly pursued by the biomedical community as promising therapeutics with improved efficacy and reduced side effects compared with unbiased ligands. We previously discovered first-in-class β-arrestin-biased agonists of dopamine D2 receptor (D2R) by extensively exploring multiple regions of aripiprazole, a balanced D2R agonist. In our continuing efforts to identify biased agonists of D2R, we unexpectedly discovered a G protein-biased agonist of D2R, compound 1, which is the first G protein-biased D2R agonist from the aripiprazole scaffold. We designed and synthesized novel analogues to explore two regions of 1 and conducted structure-functional selectivity relationship (SFSR) studies. Here we report the discovery of 1, findings from our SFSR studies, and characterization of novel G protein-biased D2R agonists.

Well-characterized selective inhibitors of protein arginine methyltransferases (PRMTs) are invaluable chemical tools for testing biological and therapeutic hypotheses. Based on 4, a fragment-like inhibitor of type I PRMTs, we conducted structure–activity relationship (SAR) studies and explored three regions of this scaffold. The studies led to the discovery of a potent, selective and cell-active dual inhibitor of PRMT4 and PRMT6, 17 (MS049). As compared to 4, 17 displayed much improved potency for PRMT4 and PRMT6 in both biochemical and cellular assays. It was selective for PRMT4 and PRMT6 over other PRMTs and a broad range of other epigenetic modifiers and non-epigenetic targets. We also developed 46 (MS049N), which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. Considering possible overlapping substrate specificity of PRMTs, 17 and 46 are valuable chemical tools for dissecting specific biological functions and dysregulation of PRMT4 and PRMT6 in health and disease.

Protein arginine methyltransferases (PRMTs) play a crucial role in a variety of biological processes. Overexpression of PRMTs has been implicated in various human diseases including cancer. Consequently, selective small-molecule inhibitors of PRMTs have been pursued by both academia and the pharmaceutical industry as chemical tools for testing biological and therapeutic hypotheses. PRMTs are divided into three categories: type I PRMTs which catalyze mono- and asymmetric dimethylation of arginine residues, type II PRMTs which catalyze mono- and symmetric dimethylation of arginine residues, and type III PRMT which catalyzes only monomethylation of arginine residues. Here, we report the discovery of a potent, selective, and cell-active inhibitor of human type I PRMTs, MS023, and characterization of this inhibitor in a battery of biochemical, biophysical, and cellular assays. MS023 displayed high potency for type I PRMTs including PRMT1, -3, -4, -6, and -8 but was completely inactive against type II and type III PRMTs, protein lysine methyltransferases and DNA methyltransferases. A crystal structure of PRMT6 in complex with MS023 revealed that MS023 binds the substrate binding site. MS023 potently decreased cellular levels of histone arginine asymmetric dimethylation. It also reduced global levels of arginine asymmetric dimethylation and concurrently increased levels of arginine monomethylation and symmetric dimethylation in cells. We also developed MS094, a close analog of MS023, which was inactive in biochemical and cellular assays, as a negative control for chemical biology studies. MS023 and MS094 are useful chemical tools for investigating the role of type I PRMTs in health and disease.

SUMMARYProtein methyltransferases (PMTs) comprise a major class of epigenetic regulatory enzymes with therapeutic relevance. Here we present a collection of chemical probes and associated reagents and data to elucidate the function of human and murine PMTs in cellular studies. Our collection provides inhibitors and antagonists that together modulate most of the key regulatory methylation marks on histones H3 and H4, providing an important resource for modulating cellular epigenomes. We describe a comprehensive and comparative characterization of the probe collection with respect to their potency, selectivity, and mode of inhibition. We demonstrate the utility of this collection in CD4+ T cell differentiation assays revealing the remarkable potential of individual probes to alter multiple T cell subpopulations with important implications for T cell-mediated processes such as inflammation and immuno-oncology. In particular, we demonstrate a role for DOT1L in limiting Th1 cell differentiation and maintaining lineage integrity.

Mutations in RNA splicing factors commonly occur in myeloid leukemia and solid tumors. These mutations occur in a heterozygous manner and confer dependency on the wild-type allele. Studies have shown that splicing factor mutant cancers are vulnerable to further perturbation of splicing, by pharmacological intervention that directly targets core splicing factors. Our project identifies the use of PRMT inhibitors, as plausible alternative therapeutic strategy to treat spliceosomal mutant leukemia. The data show that splicing factor mutant leukemia exhibit greater sensitivity to the use of inhibitors against Type I PRMTs and PRMT5, in comparison to their wild-type counterparts. As the need for new therapeutic strategies in cancer treatment increases, this study identifies PRMT inhibitors as a potential therapeutic intervention against cancers with splicing factor mutations. Citation Format: Jia Yi Fong, Diana Low, Luca Pignata, Kimihito Cojin Kawabata, Stanley CW Lee, Cheryl Koh, Daniele Musiani, Enrico Massignani, Cheng Mun Wun, Pierre-Alexis V. Goy, Yudao Shen, Heike Wollmann, Florence PH Gay, Genna Luciani, Dalia Barsyte, Jian Jin, Ari M. Melnick, Tiziana Bonaldi, Omar Abdel-Wahab, Ernesto Guccione. Therapeutic targeting of RNA splicing through inhibition of protein arginine methylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4731.

The current view is that treatment failures of AML patients are due to persistence of leukemia stem cells (LSCs). The presence of FMS-like tyrosine kinase-3 (FLT3) Internal tandem duplication (ITD) is associated with poor prognosis. But, FLT3 tyrosine kinase inhibitors (TKI) demonstrate transient clinical activity in FLT3-ITD+ AML patients. Persistent FLT3-ITD+ AML LSC represent a source of relapse. There is a pressing need to target LSC and improve outcomes for FLT3-ITD+ AML patients. PRMT1, the predominant arginine methyltransferase, has been implicated in pathogenesis of AML rare subtype (i.e., acute megakaryoblastic leukemia). Herein, we show that PRMT1 protein expression is significantly increased in LSC-enriched AML CD34+CD38- cells relative to the counterparts of normal peripheral blood stem cells (PBSC) (AML n=9, normal n=8, p=0.0004,). Following PRMT1-knockdown (KD), AML CD34+ cells (n=15) demonstrated varying degrees of apoptosis while the survival of normal cells were not affected. Interestingly, we observed significant apoptosis-induction in a subset of samples bearing FLT3-ITD mutation (6 out of total 15) upon PRMT1-KD (ShCtrl 13.9±3.6%, ShPRMT1 32.5±4.6%, p Given that PRMT1 directly interacts with FLT3-ITD, we next asked whether PRMT1 catalyzes FLT3-ITD protein methylation. Through mass-spectrometry analysis of a FLT3-ITD+ AML specimen and in vitro methylation assay, we identified that PRMT1 catalyzes FLT3-ITD arginine (R) methylation (Me) at two conserved residues, 972 and 973. Using in-house R972/973 Me antibody, we validated the expression of FLT3 R-Me in FLT3-ITD AML speciemens (7 out of 7). To test R-Me function, we transduced MLL-AF9 (MA9) overexpressing murine c-Kit+ cells with methylation-deficient FLT3-ITD (R972/973K, arginine [R] to lysine [K]) construct, and found that MA9 cells expressing R972/973K underwent more apoptosis than that of WT FLT3-ITD (WT FLT3-ITD 9.7±1.1%, R972/973K 23.7±2.1%, p=0.003). The double transformed cells were further transplanted into recipients for leukemia development. Mice receiving MA9 cells expressing R972/973K exhibited longer survival (median survival: WT FLT3-ITD 36 days, R972/973K 50 days, p=0.002, n=6). Mechanistically, expression of R972/973K did not affect the total tyrosine phosphorylation level of FLT3-ITD. Additionally, FLT3-ITD R-Me expression persisted after a TKI (AC220) treatment. These facts indicated that FLT3-ITD R-Me function is independent of FLT3-ITD kinase activity. We then used a FLT3-ITD+ patient derived xenograft (PDX) model to assess the effects of TKI and PRMT1 inhibition. Following engraftment >1% in peripheral blood, we divided mice (n=24) into 4 groups and treated each with vehicle, MS023 (a type I PRMT inhibitor, ACS Chem Biol. 2016;11:772-781) (160 mg/kg/i.p), AC220 (10 mg/kg/i.g) or combination for 4 weeks. MS023 treatment downregulating FLT3 R-Me levels enhanced elimination of FLT3-ITD AML cells by AC220 treatment (AC220 24.6±13.4% vs combination 7.6±6.5%, p=0.02, n=6). At 16 weeks post-secondary BMT, significant AML burden in single drug treated transplants was observed, but less AML cells were detected in combination-treated transplants (AC220 52.3%, vs combination 25.4%, p In summary, our study demonstrated PRMT1 overexpression contributes to AML stem/progenitor cell survival possibly through FLT3-ITD methylation, supporting further exploration into how PRMT1-mediated FLT3 methylation governs LSC survival. Disclosures Khaled: Alexion: Consultancy, Speakers Bureau; Daiichi: Consultancy; Juno: Other: Travel Funding.

Mixed-lineage leukemia-rearranged (MLL-r) ALL, seen in 70% of infant ALL, has a dismal prognosis compared to those with wild type MLL1 gene. Transcriptional profiling has identified Fms-like receptor tyrosine kinase 3 (FLT3) as one of the most significantly upregulated genes in MLL-r ALL. The highly expressed FLT3 protein is activated by the autocrine ligand, making the kinase a therapeutic target. FLT3 tyrosine kinase inhibitors (TKIs) such as PKC412, although effective in kinase inhibition, partially impair survival of MLL-r ALL cells and clinical trial results are not promising, promoting us to ask whether FLT3 regulates the ALL cells survival also through a kinase-independent mechanism. Herein, we report the finding of dimethylated arginines on FLT3, detected through mass spectrometry analysis of a MLL-r ALL specimen and a MLL-r ALL line SEM. The most conserved and enriched of dimethylated arginines are residues R972/R973. Using home-made arginine methylation (R-Me) antibody, we found that PRMT1, which is responsible for most type I arginine methyltransferases activity, catalyzes FLT3 methylation. Immunoblot (IB) analysis validated the expression of FLT3 R-Me in MLL-r ALL samples (6 out of 6) and MLL-r ALL lines (4 out of 4). Analysis of the GEO dataset (GSE13204) revealed that PRMT1 mRNA levels are increased in MLL-r ALL relative to normal cells (MLL-r, n=70 vs. normal, n=73, p1% in peripheral blood, mice were subdivided into four groups and treated with vehicle, PKC412 (100 mg/kg, i.g.), MS023 (80 mg/kg, i.p, bid), or the combination (n=7/group) for 4 weeks. The BM tumor burden of CD45+ CD19+ cells was reduced in single drug-treated mice cohorts, with further reduction after combination treatment (vehicle, 94.4±0.5%, PKC412, 50.2±6.3%, MS023, 55.6±4.5%, combination, 30.7±4.9%, PKC412 vs. combination, p Our results support further exploring the molecular function of FLT3 R-Me. We will determine whether PRMT1 and FLT3 methylation are potential druggable targets in MLL-r ALL. Disclosures Konopleva: Stemline Therapeutics: Research Funding.