Arginine Methyltransferase Inhibitor Research Articles

Abstract 7130: Dysregulated DNA repaired elicited by type 1 PRMT inhibitor in clear cell renal cell carcinoma enables development of novel combination therapies

Abstract Clear cell renal cell carcinoma (ccRCC) is the most prevalent form of kidney cancer, notorious for its resistance to treatment and dismal prognosis. Advanced or metastatic cases on average display an 8% 5-year survival rate. Given the considerable number of diagnoses in advanced stages, it is evident that we require innovative therapeutic approaches. Our lab has developed a methodology to establish cell lines from tumors and adjacent normal tissues sourced from patients. Consequently, we've amassed a collection of cell lines, including those from patient-derived ccRCC and normal kidney cells. Using these ccRCC models, we demonstrated that the type I protein arginine methyltransferase inhibitor, MS023, elicits a dysregulated DNA repair phenotype. RNA-Seq analysis of ccRCC cells treated with MS023 unveiled a marked downregulation of genes associated with the homologous recombination (HR) pathway for double-strand break repair and the Fanconi Anemia (FA) pathway for interstrand crosslink (ICL) repair. In a follow-up investigation using paired normal and cancerous cell lines from the same patient, we identified resistance to MS023 in normal kidney cells that is not mirrored in the corresponding ccRCC cell line. This hints at a potential therapeutic window and provides a strong rationale for clinical pursuit. Poly-ADP ribose polymerase (PARP) inhibitors are known to yield a synergistic effect when employed in HR-deficient diseases, such as BRCA-deficient breast cancers. Since MS023 induces an HR-deficient phenotype, we hypothesize that combining it with Talazoparib, a PARP inhibitor, will produce a similar synergistic effect. Additionally, mitomycin C (MMC) is a clinically approved compound that induces ICLs. As MS023 impedes ICL repair by downregulating crucial ICL-repairing genes, combining these two drugs should likewise generate a synergistic effect. Through a drug screen across two of our ccRCC cell lines, we discovered that the cells exhibit sensitivity to various PARP inhibitors. Employing synergy checkerboard assays and the highest single-agent model of synergy, we have found that the combination of MS023 and Talazoparib, as well as the combination of MS023 and MMC, demonstrate synergy, significantly reducing cellular viability at lower doses of the combination treatments compared to mono-treatment conditions at their respective doses. Preliminary in vivo studies using patient-derived xenograft models have revealed that both MS023 and Talazoparib are well-tolerated in mice, both when administered individually and in combination. Our current knowledge base indicates that MS023 holds promise for clinical investigation, both as a standalone treatment and in combination with Talazoparib or MMC. These results present an exciting opportunity to shed new light on the challenging therapeutic landscape faced by ccRCC patients today. Citation Format: Karen Arevalo, Joseph Walton, Jalna Meens, Angel Sau Ni Ng, Laurie Ailles. Dysregulated DNA repaired elicited by type 1 PRMT inhibitor in clear cell renal cell carcinoma enables development of novel combination therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7130.

PRMT1-mediated PGK1 arginine methylation promotes colorectal cancer glycolysis and tumorigenesis

Many types of cancer cells, including colorectal cancer cells (CRC), can simultaneously enhance glycolysis and repress the mitochondrial tricarboxylic acid (TCA) cycle, which is called the Warburg effect. However, the detailed mechanisms of abnormal activation of the glycolysis pathway in colorectal cancer are largely unknown. In this study, we reveal that the protein arginine methyltransferase 1 (PRMT1) promotes glycolysis, proliferation, and tumorigenesis in CRC cells. Mechanistically, PRMT1-mediated arginine asymmetric dimethylation modification of phosphoglycerate kinase 1 (PGK1, the first ATP-producing enzyme in glycolysis) at R206 (meR206-PGK1) enhances the phosphorylation level of PGK1 at S203 (pS203-PGK1), which inhibits mitochondrial function and promotes glycolysis. We found that PRMT1 and meR206-PGK1 expression were positively correlated with pS203-PGK1 expression in tissues from colorectal cancer patients. Furthermore, we also confirmed that meR206-PGK1 expression is positively correlated with the poor survival of patients with colorectal cancer. Our findings show that PRMT1 and meR206-PGK1 may become promising predictive biomarkers for the prognosis of patients with CRC and that arginine methyltransferase inhibitors have great potential in colorectal cancer treatment.

PRMT5 inhibition shows in vitro efficacy against H3K27M-altered diffuse midline glioma, but does not extend survival in vivo

H3K27-altered Diffuse Midline Glioma (DMG) is a universally fatal paediatric brainstem tumour. The prevalent driver mutation H3K27M creates a unique epigenetic landscape that may also establish therapeutic vulnerabilities to epigenetic inhibitors. However, while HDAC, EZH2 and BET inhibitors have proven somewhat effective in pre-clinical models, none have translated into clinical benefit due to either poor blood–brain barrier penetration, lack of efficacy or toxicity. Thus, there remains an urgent need for new DMG treatments. Here, we performed wider screening of an epigenetic inhibitor library and identified inhibitors of protein arginine methyltransferases (PRMTs) among the top hits reducing DMG cell viability. Two of the most effective inhibitors, LLY-283 and GSK591, were targeted against PRMT5 using distinct binding mechanisms and reduced the viability of a subset of DMG cells expressing wild-type TP53 and mutant ACVR1. RNA-sequencing and phenotypic analyses revealed that LLY-283 could reduce the viability, clonogenicity and invasion of DMG cells in vitro, representing three clinically important phenotypes, but failed to prolong survival in an orthotopic xenograft model. Together, these data show the challenges of DMG treatment and highlight PRMT5 inhibitors for consideration in future studies of combination treatments.

Development of selective class I protein arginine methyltransferase inhibitors through fragment-based drug design approach

Protein arginine methyltransferases (PRMTs) catalyze the methylation of the terminal nitrogen atoms of the guanidino group of arginine of protein substrates. The aberrant expression of these methyltransferases is linked to various diseases, making them promising therapeutic targets. Currently, PRMT inhibitors are at different stages of clinical development, which validated their significance as drug targets. Structural Genomics Consortium (SGC) has reported several small fragment inhibitors as Class I PRMT inhibitors, which can be the starting point for rational drug development. Herein, we report the successful application of a fragment-based approach toward the discovery of selective Class I PRMT inhibitors. Structure-based ligand optimization was performed by strategic incorporation of fragment hits on the drug-like quinazoline core and subsequent fragment growth in the desired orientation towards identified hydrophobic shelf. A clear SAR was established, and the lead compounds 55 and 56 displayed potent inhibition of Class I PRMTs with IC50 values of 92 nM and 37 nM against PRMT4. We report the systematic development of potent Class I PRMT inhibitors with good potency and about 100-fold selectivity when tested against a panel of 31 human DNA, RNA, and protein lysine and arginine methyltransferases. These improved small molecules will provide new options for the development of novel potent and selective PRMT4 inhibitors.

Research progress on small-molecule inhibitors of protein arginine methyltransferase 5 (PRMT5) for treating cancer.

The protein arginine methyltransferase family includes nine members, with PRMT5 being the major type II arginine methyltransferase. PRMT5 is upregulated in a variety of tumors and promotes tumorigenesis and tumor cell proliferation and metastasis, making it a potential tumor therapy target. Recently, PRMT5 inhibitor research and development have become hotspots in the tumor therapy field. We classified and summarized PRMT5 inhibitors according to different binding mechanisms. We mainly analyzed the structure, biological activity, and binding interactions of PRMT5 inhibitors with the PRMT5 enzyme. At present, many PRMT5 inhibitors with various mechanisms of action have been reported, including substrate-competitive inhibitors, SAM-competitive inhibitors, dual substrate-/SAM-competitive inhibitors, allosteric inhibitors, PRMT5 degraders, MTA-cooperative PRMT5 inhibitors and PPI inhibitors. These inhibitors are beneficial to the treatment of tumors. Some drugs are being used in clinical trials. PRMT5 inhibitors have broad application prospects in tumor therapy.

Reduced C9orf72 expression exacerbates polyGR toxicity in patient iPSC-derived motor neurons and a Type I protein arginine methyltransferase inhibitor reduces that toxicity.

Intronic repeat expansions in the C9orf72 gene are the most frequent known single genetic causes of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These repeat expansions are believed to result in both loss-of-function and toxic gain-of-function. Gain-of-function results in the production of toxic arginine-rich dipeptide repeat proteins (DPRs), namely polyGR and polyPR. Small-molecule inhibition of Type I protein arginine methyltransferases (PRMTs) has been shown to protect against toxicity resulting from polyGR and polyPR challenge in NSC-34 cells and primary mouse-derived spinal neurons, but the effect in human motor neurons (MNs) has not yet been explored. To study this, we generated a panel of C9orf72 homozygous and hemizygous knockout iPSCs to examine the contribution of C9orf72 loss-of-function toward disease pathogenesis. We differentiated these iPSCs into spinal motor neurons (sMNs). We found that reduced levels of C9orf72 exacerbate polyGR15 toxicity in a dose-dependent manner. Type I PRMT inhibition was able to partially rescue polyGR15 toxicity in both wild-type and C9orf72-expanded sMNs. This study explores the interplay of loss-of-function and gain-of-function toxicity in C9orf72 ALS. It also implicates type I PRMT inhibitors as a possible modulator of polyGR toxicity.

Comparative Study of Adenosine Analogs as Inhibitors of Protein Arginine Methyltransferases and a Clostridioides difficile-Specific DNA Adenine Methyltransferase.

S-Adenosyl-l-methionine (SAM) analogs are adaptable tools for studying and therapeutically inhibiting SAM-dependent methyltransferases (MTases). Some MTases play significant roles in host-pathogen interactions, one of which is Clostridioides difficile-specific DNA adenine MTase (CamA). CamA is needed for efficient sporulation and alters persistence in the colon. To discover potent and selective CamA inhibitors, we explored modifications of the solvent-exposed edge of the SAM adenosine moiety. Starting from the two parental compounds (6e and 7), we designed an adenosine analog (11a) carrying a 3-phenylpropyl moiety at the adenine N6-amino group, and a 3-(cyclohexylmethyl guanidine)-ethyl moiety at the sulfur atom off the ribose ring. Compound 11a (IC50 = 0.15 μM) is 10× and 5× more potent against CamA than 6e and 7, respectively. The structure of the CamA-DNA-inhibitor complex revealed that 11a adopts a U-shaped conformation, with the two branches folded toward each other, and the aliphatic and aromatic rings at the two ends interacting with one another. 11a occupies the entire hydrophobic surface (apparently unique to CamA) next to the adenosine binding site. Our work presents a hybrid knowledge-based and fragment-based approach to generating CamA inhibitors that would be chemical agents to examine the mechanism(s) of action and therapeutic potentials of CamA in C. difficile infection.

PRMT5 Inhibition Enhances Elimination of FLT3-ITD AML Stem Cells in Combination with TKI Treatment

Introduction: The FMS-like tyrosine kinase-3 (FLT3) internal tandem duplication (ITD) is a commonly observed molecular abnormality in AML and is associated with increased risk of relapse and poor survival. FLT3 tyrosine kinase inhibitors (TKIs) have shown benefit in the treatment of FLT3-ITD AML but do not eliminate primitive leukemia stem cells and are limited by frequent relapse and resistance. There is considerable interest in understanding mechanisms of FLT3-ITD AML stem cell persistence to develop strategies to enhance their elimination. Here we evaluated the role of epigenetic regulators in maintenance of human FLT3-ITD AML cells. Methods: We performed an epigenetic screen using a well-curated collection of epigenetic probes (Epigenetics Probes Collection, Structural Genomics Consortium, n=41). FLT3-ITD (MOLM-13 and MV4-11), and FLT3-WT AML cell lines (OCI-AML3) were exposed to inhibitors for 7 days and CellTiter-Glo and Annexin V labeling were used to evaluate response. We further studied the effects of the PRMT5 inhibitors, GSK-591and LLY-283, with and without FLT3 TKIs, Quizartinib and Giltertinib, on FLT3-ITD+ and FLT3-WT AML cell lines. Synergy between PRMT5 inhibitors and TKIs was analyzed with Calcusyn software using the median effects method of Chou and Talalay. We performed in vivo analysis of the effects of PRMT5 and FLT3 kinase inhibition in a FLT3-ITD Tet2 deleted genetic mouse model of FLT3-ITD AML. Finally, we studied in vitro and in vivo effects of PRMT5 and FLT3 inhibition on primary CD34+ cells from FLT3-ITD AML patients. Results: Screening with epigenetic probes identified PRMT5 arginine methyl transferase inhibitors, GSK591 and LLY-283, amongst the probes with the greatest inhibitory effects on growth and survival of FLT3-ITD MOLM-13 and MV4-11 AML cells. In contrast, inhibitors of PRMT1, PRMT3. PRMT4 and PRMT7 were associated with significantly less growth inhibition of FLT3-ITD AML cell lines. FLT3-WT OCI-AML3 AML cells were also sensitive to PRMT5 inhibition. However, ectopic FLT3-ITD expression in FLT3-WT THP1 cells led to enhanced sensitivity to PRMT5 inhibition. The combination of GSK-591 or LLY-283 with either of the FLT3 TKIs Quizartinib or Giltertinib resulted in synergistically enhanced inhibition of FLT3-ITD+ MOLM-13 and MV4-11 AML cells, compared to TKI or PRMT5 inhibitor alone. On the other hand, FLT3-WT cells were insensitive to TKI alone, and the combination of PRMT5 inhibitor and TKI did not lead to synergistic enhancement of inhibition. We next evaluated the effect of PRMT5 inhibition in a genetic mouse model of FLT3-ITD AML. AML mice were treated with vehicle (control), Giltertinib, LLY283 or the combination of Giltertinib and LLY283 for 2 weeks. AML cells from LLY-283 treated mice showed significantly reduced symmetric dimethylarginine in SmB protein. Significant inhibition of mature AML cells and of AML stem and progenitor cells was seen in the bone marrow and spleen of mice treated with LLY283, and significantly enhanced inhibition was seen with combination of LLY283 and Giltertinib (Figure 1a). Finally, we evaluated the effect of TKI (Giltertinib or Quizartinib), LLY283 or the combination on primary FLT3-ITD AML CD34+ cells with diverse co-mutations (n=8). LLY283 significantly inhibited FLT3-ITD AML CD34+ cell growth and survival in vitro by itself, and the combination of TKI and LLY283 resulted in significantly increased inhibition compared to TKI alone, including both CD34+CD38- and CD34+CD38+ cell populations (Figure 1b). In addition, in vivo treatment of immunodeficient NRGS mice xenografted with human FLT3-ITD AML cells with LLY283 or the combination of Giltertinib and LLY283 led to significant depletion of AML cells in BM and spleen. Conclusion: An epigenetic probe screen identified PRMT5 as a key regulator of FLT3-ITD AML cell viability. We show an important role for PRMT5 in maintenance of murine and human FLT3-ITD AML stem cells and in their persistence following FLT3 TKI treatment. Treatment with a PRMT5 inhibitor in combination with a FLT3 TKI could be a promising approach to enhance elimination of FLT3-ITD AML stem cells. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A PRMT5 inhibitor protects against noise-induced hearing loss by alleviating ROS accumulation

The aim of this study was to investigate the effect of LLY-283, a selective inhibitor of protein arginine methyltransferase 5 (PRMT5), on a noise-induced hearing loss (NIHL) mouse model and to identify a potential target for a therapeutic intervention against NIHL. Eight-week-old male C57BL/6 mice were used. The auditory brainstem response was measured 2 days after noise exposure. The apoptosis of hair cells (HCs) was detected by caspase-3/7 staining, whereas the accumulation of reactive oxygen species (ROS) was measured by 4-HNE staining. We demonstrated that the death of HCs and loss of cochlear synaptic ribbons induced by noise exposure could be significantly reduced by the presence of LLY-283. LLY-283 pretreatment before noise exposure notably decreased 4-HNE and caspase-3/7 levels in the cochlear HCs. We also noticed that the number of spiral ganglion neurons (SGNs) was notably increased after LLY-283 pretreatment. Furthermore, we showed that LLY-283 could increase the expression level of p-AKT in the SGNs. The underlying mechanism involves alleviation of ROS accumulation and activation of the PI3K/AKT pathway, indicating that LLY-283 might be a potential candidate for therapeutic intervention against NIHL.

A peptoid-based inhibitor of protein arginine methyltransferase 1 (PRMT1) induces apoptosis and autophagy in cancer cells

Protein arginine methyltransferases (PRMTs) are S-adenosylmethionine-dependent enzymes that transfer a methyl group to arginine residues within proteins, most notably histones. The nine characterized PRMT family members are divided into three types depending on the resulting methylated product: asymmetric dimethylarginine (Type I PRMT), symmetric dimethylarginine (Type II PRMT), or monomethylated arginine (Type III PRMT). In some cancers, the resulting product can lead to either increased or decreased transcription of cancer-related genes, suggesting PRMT family members may be valid therapeutic targets. Traditionally, peptide-based compounds have been employed to target this family of enzymes, which has resulted in multiple tool and lead compounds being developed. However, peptide-based therapeutics suffer from poor stability and short half-lives, as proteases can render them useless by hydrolytic degradation. Conversely, peptoids, which are peptide-mimetics composed of N-substituted glycine monomers, are less susceptible to hydrolysis, resulting in improved stability and longer half-lives. Herein, we report the development of a bioavailable, peptoid-based PRMT1 inhibitor that induces cell death in MDA468 and HCT116 cancer cell lines while not exhibiting any significant impact on nontumorigenic HepaRG or normal human mammary epithelial cells. Furthermore, the inhibitor described herein appears to induce both apoptosis and autophagy, suggesting it may be a less toxic cytostatic agent. In conclusion, we propose this peptoid-based inhibitor has significant anticancer and therapeutic potential by reducing cell viability, growth, and size in breast and colon cancer. Further experimentation will help determine the mechanism of action and downstream effects of this compound.

Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction-Reconstruction and Fragment-Growing Approach.

Protein arginine methyltransferases (PRMTs) are important therapeutic targets, playing a crucial role in the regulation of many cellular processes and being linked to many diseases. Yet, there is still much to be understood regarding their functions and the biological pathways in which they are involved, as well as on the structural requirements that could drive the development of selective modulators of PRMT activity. Here we report a deconstruction–reconstruction approach that, starting from a series of type I PRMT inhibitors previously identified by us, allowed for the identification of potent and selective inhibitors of PRMT4, which regardless of the low cell permeability show an evident reduction of arginine methylation levels in MCF7 cells and a marked reduction of proliferation. We also report crystal structures with various PRMTs supporting the observed specificity and selectivity.

Purification and Identification of Natural Inhibitors of Protein Arginine Methyltransferases from Plants

Protein arginine methyltransferase (PRMT) enzymes catalyze posttranslational modifications of target proteins and are often upregulated in human cancers. In this study, we purified two chemical compounds from seeds of Foeniculum vulgare based on their ability to inhibit the enzymatic activity of PRMT5. These two compounds were identified as Pheophorbide a (PPBa) and Pheophorbide b (PPBb), two breakdown products of chlorophyll. PPBa and PPBb inhibited the enzymatic activity of both Type I and Type II PRMTs with IC50 values at sub micromole concentrations, inhibited the arginine methylation of histones in cells, and suppressed proliferation of prostate cancer cells. Molecular docking results predicted that PPBa binds to an allosteric site in the PRMT5 structure with a high affinity (ΔG = -9.0 kcal/mol) via hydrogen bond, ionic, and π-π stacking interactions with amino acid residues in PRMT5. Another group of natural compounds referred to as protoporphyrins and sharing structural similarity with pheophorbide also inhibited the PRMT enzymatic activity. This study is the first report on the PRMT-inhibitory activity of the tetrapyrrole macrocycles and provides useful information regarding the application of these compounds as natural therapeutic reagents for cancer prevention and treatment.

Acquired resistance to PRMT5 inhibition induces concomitant collateral sensitivity to paclitaxel

Epigenetic regulators play key roles in cancer and are increasingly being targeted for treatment. However, for many, little is known about mechanisms of resistance to the inhibition of these regulators. We have generated a model of resistance to inhibitors of protein arginine methyltransferase 5 (PRMT5). This study was conducted in KrasG12D;Tp53-null lung adenocarcinoma (LUAD) cell lines. Resistance to PRMT5 inhibitors (PRMT5i) arose rapidly, and barcoding experiments showed that this resulted from a drug-induced transcriptional state switch, not selection of a preexisting population. This resistant state is both stable and conserved across variants arising from distinct LUAD lines. Moreover, it brought with it vulnerabilities to other chemotherapeutics, especially the taxane paclitaxel. This paclitaxel sensitivity depended on the presence of stathmin 2 (STMN2), a microtubule regulator that is specifically expressed in the resistant state. Remarkably, STMN2 was also essential for resistance to PRMT5 inhibition. Thus, a single gene is required for both acquisition of resistance to PRMT5i and collateral sensitivity to paclitaxel in our LUAD cells. Accordingly, the combination of PRMT5i and paclitaxel yielded potent and synergistic killing of the murine LUAD cells. Importantly, the synergy between PRMT5i and paclitaxel also extended to human cancer cell lines. Finally, analysis of The Cancer Genome Atlas patient data showed that high STMN2 levels correlate with complete regression of tumors in response to taxane treatment. Collectively, this study reveals a recurring mechanism of PRMT5i resistance in LUAD and identifies collateral sensitivities that have potential clinical relevance.

Protein Arginine Methyltransferase (PRMT) Inhibitors-AMI-1 and SAH Are Effective in Attenuating Rhabdomyosarcoma Growth and Proliferation in Cell Cultures.

Rhabdomyosarcoma (RMS) is a malignant soft tissue cancer that develops mostly in children and young adults. With regard to histopathology, four rhabdomyosarcoma types are distinguishable: embryonal, alveolar, pleomorphic and spindle/sclerosing. Currently, increased amounts of evidence indicate that not only gene mutations, but also epigenetic modifications may be involved in the development of RMS. Epigenomic changes regulate the chromatin architecture and affect the interaction between DNA strands, histones and chromatin binding proteins, thus, are able to control gene expression. The main aim of the study was to assess the role of protein arginine methyltransferases (PRMT) in the cellular biology of rhabdomyosarcoma. In the study we used two pan-inhibitors of PRMT, called AMI-1 and SAH, and evaluated their effects on proliferation and apoptosis of RMS cells. We observed that AMI-1 and SAH reduce the invasive phenotype of rhabdomyosarcoma cells by decreasing their proliferation rate, cell viability and ability to form cell colonies. In addition, microarray analysis revealed that these inhibitors attenuate the activity of the PI3K-Akt signaling pathway and affect expression of genes related to it.

Abstract 1145: AGX323 - A SAM-competitive, orally available inhibitor of protein arginine methyltransferase 5 (PRMT5) with potent cellular antiproliferative and in vivo antitumor activity against selected solid cancer types

Abstract Background: Protein arginine methyltransferase 5 (PRMT5) is an attractive therapeutic target because of its involvement in key oncogenic mechanisms including epigenetics, DNA response and RNA processing. Early development of PRMT5 inhibitors has focused on hematological malignancies although more recently several compounds have entered the clinic for both liquid and solid malignancies. Early Phase I clinical data indicate dose-limiting hematological toxicity, particularly anemia and thrombocytopenia. We focused our effort in discovering compounds with potent inhibitory activity against cells from solid cancer types known to overexpress PRMT5 and which correlated with poor prognosis (NSCLC, breast, bladder and pancreatic cancers). Methods: Enzyme inhibition was conducted using a PRMT5:MEP50 flash plate assay, with S-adenosylmethionine (SAM) as the methyl donor and histone H4 as substrate. Inhibition of cell proliferation was assessed in various cancer lines using a 7-day cellTitre-Glo (CTG) assay. Binding mode of the inhibitors to PRMT5 was investigated using surface plasmon resonance (SPR). In vivo antitumor activity was evaluated in various human cancer models grown in immunocompromised mice. Results: Structure activity relationship of a series of over 50 close analogs demonstrated that in vitro anticancer activities correlate poorly with enzyme inhibition potency. To understand this discrepancy we used SPR to study the PRMT5 binding mode of cell active and inactive compounds in the presence of various endogenous substrates (SAM, SAH or MTA). We obtained binding affinity (KD), association constant (Ka) and dissociation constant (Kd) of exemplars of active and inactive inhibitors and correlated their relationship to cell activity. The lead compound AGX323 has a KD of 1.8 x 10-10 M to SAM-bound PRMT5 and exhibited potent antiproliferative activity against a panel of over 20 human solid epithelial cancer cell lines from lung, breast, bladder and pancreas, with nM potency. For example, in the H1048 human lung cancer model it has an enzyme inhibition IC50 of 0.8 nM, a cell proliferation IC50 of 1.7 nM and when given to tumor bearing mice it demonstrated robust antitumor activity at well-tolerated dose levels. In nonclinical ADME studies, AGX323 demonstrated favorable drug-like properties: including acceptable oral bioavailability (in mouse, dog and monkey) and systemic exposure following oral administration, excellent metabolic stability, lack of significant drug-drug interaction liabilities (transporters, CYP inhibition, CYP induction). Conclusion: AGX323 demonstrated potentially best-in-class activity in PRMT5 dependent human solid cancer models and exhibited excellent drug-like properties. IND enabling studies are currently planned. Citation Format: Francis Y. F. Lee, Wen-Lian Wu, Zhiqiang Yang, John Tan. AGX323 - A SAM-competitive, orally available inhibitor of protein arginine methyltransferase 5 (PRMT5) with potent cellular antiproliferative and in vivo antitumor activity against selected solid cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1145.

PRMT5 Selective Inhibitor Enhances Therapeutic Efficacy of Cisplatin in Lung Cancer Cells.

As a therapeutic approach, epigenetic modifiers have the potential to enhance the efficacy of chemotherapeutic agents. Protein arginine methyltransferase 5 (PRMT5), highly expressed in lung adenocarcinoma, was identified to be involved in tumorigenesis. In the current study, we examined the potential antineoplastic activity of PRMT5 inhibitor, arginine methyltransferase inhibitor 1 (AMI-1), and cisplatin on lung adenocarcinoma. Bioinformatic analyses identified apoptosis, DNA damage, and cell cycle progression as the main PRMT5-associated functional pathways, and survival analysis linked the increased PRMT5 gene expression to worse overall survival in lung adenocarcinoma. Combined AMI-1 and cisplatin treatment significantly reduced cell viability and induced apoptosis. Cell cycle arrest in A549 and DMS 53 cells was evident after AMI-1, and was reinforced after combination treatment. Western blot analysis showed a reduction in demethylation histone 4, a PRMT5- downstream target, after treatment with AMI-1 alone or in combination with cisplatin. While the combination approach tackled lung cancer cell survival, it exhibited cytoprotective abilities on HBEpC (normal epithelial cells). The survival of normal bronchial epithelial cells was not affected by using AMI-1. This study highlights evidence of novel selective antitumor activity of AMI-1 in combination with cisplatin in lung adenocarcinoma cells.

A First-in-Class, Highly Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 6.

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 (IC50 = 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.

Licochalcone A is a Natural SelectiveInhibitor of Arginine Methyltransferase 6.

Arginine methylation is a post-translational modification that is implicated in multiple biological functions including transcriptional regulation. The expression of protein arginine methyltransferases (PRMT) has been shown to be up-regulated in various cancers. PRMTs have emerged as attractive targets for the development of new cancer therapies. Here, we describe the identification of a natural compound, licochalcone A, as a novel, reversible and selective inhibitor of PRMT6. Since expression of PRMT6 is up-regulated in human breast cancers and is associated with oncogenesis, we used the human breast cancer cell line system to study the effect of licochalcone A treatment on PRMT6 activity, cell viability, cell cycle, and apoptosis. We demonstrated that licochalcone A is a non-S-adenosyl L-methionine (SAM) binding site competitive inhibitor of PRMT6. In MCF-7 cells, it inhibited PRMT6-dependent methylation of histone H3 at arginine 2 (H3R2), which resulted in a significant repression of estrogen receptor activity. Licochalcone A exhibited cytotoxicity towards human MCF-7 breast cancer cells, but not MCF-10A human breast epithelial cells, by up-regulating p53 expression and blocking cell cycle progression at G2/M, followed by apoptosis. Thus, licochalcone A has potential for further development as a therapeutic agent against breast cancer.

Arginine methylation: the promise of a \u2018silver bullet\u2019 for brain tumours?

Despite intense research efforts, our pharmaceutical repertoire against high-grade brain tumours has not been able to increase patient survival for a decade and life expectancy remains at less than 16 months after diagnosis, on average. Inhibitors of protein arginine methyltransferases (PRMTs) have been developed and investigated over the past 15 years and have now entered oncology clinical trials, including for brain tumours. This review collates recent advances in the understanding of the role of PRMTs and arginine methylation in brain tumours. We provide an up-to-date literature review on the mechanisms for PRMT regulation. These include endogenous modulators such as alternative splicing, miRNA, post-translational modifications and PRMT–protein interactions, and synthetic inhibitors. We discuss the relevance of PRMTs in brain tumours with a particular focus on PRMT1, -2, -5 and -8. Finally, we include a future perspective where we discuss possible routes for further research on arginine methylation and on the use of PRMT inhibitors in the context of brain tumours.

Targeting the Synthetic Lethality Interaction of MTAP and PRMT5 to Overcome Drug Resistance and Enhance Anti-Cancer Immunity in Mantle Cell Lymphoma

Background: Complex genetic, epigenetic alterations and metabolic reprogramming are among the hallmarks of mantle cell lymphoma, an aggressive B-cell type of non-Hodgkin's lymphoma. CDKN2A and MTAP are frequently co-deleted in human cancers including MCL. Deletion of MTAP gene results in the accumulation of methylthioadenosine (MTA), a selective inhibitor of protein arginine methyltransferase 5 (PRMT5), signifying a dependence of MTAP deleted tumors on PRMT5. PRMT5 plays an important role in normal cellular processes via epigenetic and post-translational modification of RNA splicing, DNA repair and cell cycle regulators. These pathways may be activated in response to targeted therapies to maintain tumor growth and survive chemotherapy. Sustained PRMT5 expression and activity could be therapeutically targeted in MTAP-deficient MCL. Mutations in RNA splicing factors are common in lymphoma and increase the susceptibility to perturbations in splicing events. Deregulated PRMT5 might induce alternative splicing, leading to loss of expression of proteins or epitopes, and subsequent immune evasion and failure of tumor specific immune therapy. Multiple mechanisms may co-exist. Results: CDKN2A and MTAP were commonly co-deleted in ibrutinib-resistant MCL tumors (71%, p = 0.01). To corroborate with this finding, we found that PRMT5 was overexpressed in both primary ibrutinib-resistant MCL and cell lines, with its expression levels inversely correlated with the patient responses to ibrutinib treatment (p = 0.004). Metabolomics analysis revealed elevated S-Adenosyl-methionine (SAM) and S-Adenosyl-homocysteine (SAH) (t-test p <0.001 and p <0.05), PRMT5 cofactor and product, respectively, in ibrutinib-resistant MCL cell lines. PRMT5 plays an important role in the regulation of cell cycle progression. Consistently, treatment with PRMT5 inhibitors induced cell cycle arrest in MCL cell lines (20% and 47% reduction in S phase for JVM13 and JVM2 respectively). Interestingly, treatment with the catalytic inhibitors (GSK3226595 and EPZ015666) led to S-phase stasis in which MCL cells seem incapable of replication. Meanwhile, PRMT5 inhibition with these inhibitors prevented the growth of MCL cell lines as indicated by remarkable decreases in the cell viability (IC50 = 1-1.4 µM and 0.58-0.95µM, respectively). PRMT5 enzymatic activity is required for its function in promoting cell cycle progression and proliferation, as treatment with its inhibitors leads to dramatic reduction in the level of SDMA-containing proteins, especially that of H4R3me2s. More importantly, pharmacological blockade of PRMT5 with single agent GSK3226595 significantly inhibited tumor growth in ibrutinib-resistant MCL PDX models (n = 5, p <0.05), implicating PRMT5 as an actionable target in MCL. PRMT5-mediated alternative splicing may contribute to immune escape and immune suppression. Indeed, our preliminary results revealed that T cells from MCL specimen with high expression of PRMT5 displayed decreased glucose uptake (fold MFI <1-1.23 vs. 2.2), mitochondria mass (p <0.01) and ΔΨm (fold MFI <1-1.26 vs. 2.3), by flow cytometry. Strikingly, the same patient-derived cytotoxic T-lymphocytes (CTL) exhibit reduced cytotoxicity towards autologous lymphoma cells, compared to those derived from healthy donors (p <0.05), strongly implicating impaired T cell metabolic fitness plus immune escape and suppression diminished T cell effector function. Conclusion: We presented evidence that MCL with MTAP deletion confers synthetic lethal dependence on PRMT5, making it an attractive therapeutic target. As an important metabolic and epigenetic regulator, loss of PRMT5 leads to malfunction of the constitutive splicing machinery that induces alternative splicing events, genetic instability and cell cycle arrest. Sustained PRMT5 expression and activity confers tumor aggressiveness and therapeutic resistance in MTAP-deficient MCL. Pharmacological blockade of PRMT5 may dramatically enhance T-cell immunity, while suppressing MCL cell proliferation and tumor growth in vivo. Further successful mechanistic studies would facilitate the identification of novel therapeutics and strategies to overcome resistance to targeted therapies and significantly improve the efficacy of immunotherapy for much better clinical outcomes of MCL. Disclosures Wang: Oncternal: Consultancy, Research Funding; Lu Daopei Medical Group: Honoraria; Juno: Consultancy, Research Funding; Guidepoint Global: Consultancy; Dava Oncology: Honoraria; MoreHealth: Consultancy; Targeted Oncology: Honoraria; Beijing Medical Award Foundation: Honoraria; Verastem: Research Funding; OncLive: Honoraria; Acerta Pharma: Research Funding; VelosBio: Research Funding; Molecular Templates: Research Funding; Nobel Insights: Consultancy; AstraZeneca: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Pulse Biosciences: Consultancy; Loxo Oncology: Consultancy, Research Funding; Kite Pharma: Consultancy, Other: Travel, accommodation, expenses, Research Funding; BioInvent: Research Funding; Celgene: Consultancy, Other: Travel, accommodation, expenses, Research Funding; OMI: Honoraria, Other: Travel, accommodation, expenses; InnoCare: Consultancy.