Neutrophils are closely involved in the regulation of tumor progression and formation of pre-metastatic niches. However, the mechanisms of their involvement and therapeutic regulation of these processes remain elusive. Here, we report a critical role of neutrophil peptidylarginine deiminase 4 (PAD4) in neutrophil migration in cancer. In several transplantable and genetically engineered mouse models, tumor growth was accompanied by significantly elevated enzymatic activity of neutrophil PAD4. Targeted deletion of PAD4 in neutrophils markedly decreased the intratumoral abundance of neutrophils and led to delayed growth of primary tumors and dramatically reduced lung metastases. PAD4 mediated neutrophil accumulation by regulating the expression of the major chemokine receptor CXCR2. PAD4 expression and activity as well as CXCR2 expression were significantly upregulated in neutrophils from patients with lung and colon cancers compared to healthy donors, and PAD4 and CXCR2 expression were positively correlated in neutrophils from cancer patients. In tumor-bearing mice, pharmacological inhibition of PAD4 with the novel PAD4 isoform-selective small molecule inhibitor JBI-589 resulted in reduced CXCR2 expression and blocked neutrophil chemotaxis. In mouse tumor models, targeted deletion of PAD4 in neutrophils or pharmacological inhibition of PAD4 with JBI-589 reduced both primary tumor growth and lung metastases and substantially enhanced the effect of immune checkpoint inhibitors. Taken together, these results suggest a therapeutic potential of targeting PAD4 in cancer.

Protein arginine deiminases (PAD) 4 is an enzyme that catalyzes citrullination of protein and its role in autoimmune diseases has been established through clinical genetics and gene knock out studies in mice. Further, studies with PAD4 – deficient mice have shown that PAD4 deficiency does not lead to increased infection or immune suppression, which makes PAD4 an attractive therapeutic target for auto-immune and inflammatory diseases. PAD4 has critical enzymatic role of promoting chromatin decondensation and neutrophil extracellular traps (NETs) formation that is associated with a number of immune-mediated pathological conditions. Here, we present a non-covalent PAD4 inhibitor JBI-589 with high PAD4 isoform selectivity and delineated its binding mode at 2.88 Å resolution by X-ray crystallography. We confirmed its effectiveness in inhibiting NET formation in vitro. Additionally, by using two mouse arthritis models for human rheumatoid arthritis (RA), the well-known disease associated with PAD4 clinically, we established its efficacy in vivo. These results suggest that JBI-589 would be beneficial for both PAD4 and NET-associated pathological conditions.

Background The PD-1/PD-L1 molecular pathway is one of the primary mechanisms of immune evasion deployed by cancer cells. Activation of PD-1/PD-L1 pathway induces anergy and exhaustion of cytotoxic T-cells and enhances the function of regulatory T-cells causing an immune suppressive environment. Therefore, blocking this pathway restores T-cell proliferation and enhanced tumor cell killing. Approved monoclonal antibodies targeting PD-1/PD-L1 pathway require intravenous injections and have a long half-life that could contribute to the well-documented drug-related adverse effects. Also, efficacy of these antibodies appears to be marginal in malignancies associated with CNS due to poor brain penetrance. Therefore, small molecule inhibitors of the PD-1/PD-L1 pathway with oral bioavailability, better tumor and brain penetrance and shorter half-life will be highly valuable in cancer therapy. In this regard, JBI-2174 shows excellent ADME properties, brain exposure, pharmacokinetics and comparable efficacy as approved mAbs in preclinical studies. Methods Structure based drug design was used to design PD-L1 inhibitors; potency of these inhibitors was assessed in an in-vitro TR-FRET assay. Reporter assays and ex-vivo co-culture assays were used to assess T-cell proliferation and function. Pharmacokinetics were performed in multiple pre-clinical species to derive at bioavailability and brain penetration. In vivo efficacy was assessed in partially humanized mice efficacy models.Results JBI-2174 showed strong in vitro IC 50 of 1.5 nM in TR-FRET assay that measures interaction between PD-1 and PD-L1 and let to stabilization of PD-L1 as measured by thermal shift assay. This molecule also augmented T-cell co-inhibitory signalling as observed by Jurkat cell/SHP-1 assay. Competition study between anti-PD-L1 blocking antibody and x-ray crystal structure studies clearly demonstrated that JBI-2174 leads to dimerization of PD-L1. More importantly, JBI-2174 showed excellent oral bioavailability across pre-clinical species and strong and sustained (up to 24 h) brain exposure (0.66 to 2.1 fold plasma vs. brain ratio). JBI-2174 showed comparable or better efficacy to the anti-PD-L1 antibody Atezolizumab in hPD-L1/MC38 syngeneic and brain orthotopic models by oral administration with a concomitant increase in tumor infiltrating lymphocytes. Toxicological studies conducted in non-human primates clearly show that the molecule is well tolerated at exposures much higher than efficacious exposure. Conclusions Oral administration and brain exposure of these small molecule PD-L1 inhibitors provides an attractive option to be used in the treatment of glioblastoma and other solid tumors with brain metastasis. IND enabling studies are being initiated for this molecule to initiate clinical trials in humans.

Supplementary Data from A Novel Selective Inhibitor JBI-589 Targets PAD4-Mediated Neutrophil Migration to Suppress Tumor Progression

Introduction: Protein arginine methyltransferases (PRMTs) transfer a methyl group from the cofactor S-adenosyl-L-methionine (SAM) to the arginine of a substrate protein (1,2). PRMT5 is a type II protein arginine methyltransferase that catalyzes the symmetrical dimethylation. PRMT5 drives transcriptional suppression of cell cycle control and tumor suppressor genes by hypermethylating promoter histones H3R8 and H4R3. Recently, dysregulation of the splicing machinery has been identified to be one of the therapeutic vulnerabilities for PRMT5 inhibition in multiple cancers. Therefore, inhibitors selectively targeting PRMT5 could be of high clinical value in cancers with defects in spliceosome machinery. In this regard, we are developing a series of novel substrate competitive PRMT5 inhibitors to target brain cancer and solid tumors with brain metastasis. Methods: Structure based drug design was used to identify novel PRMT5 inhibitors. To assess in vitro potency, flash plate assay was used. Cell based activity was assessed by measuring the symmetrical dimethylation of SmD3, intron retention and long-term cell proliferation assays. Tumor growth inhibition was measured in orthotopic glioblastoma models in mice. Results: Our lead PRMT5 inhibitor JBI-778 had a strong in vitro potency against PRMT5 where the EC50 was ~5 nM and a similar cellular potency (< 10 nM) in inhibiting symmetric dimethylation of arginine. JBI-778 showed a strong anti-proliferative activity in select Neuroblastoma, Medulloblastoma, NSCLC, and glioma cell lines where the IC50s ranged from 27 to 700 nM. Of note, NSCLC cell lines with mutation in the spliceosome protein RBM10 appeared to be more sensitive to JBI-778 treatment, which is consistent with enhanced retention of introns that are reported to be PRMT5 substrates including ATM, POLD and PNISR. JBI-778 showed an excellent oral bioavailability with a very high and sustained brain exposure in rodents. In U87-MG human GBM orthotopic model, oral administration of the JBI-778 resulted in strong and dose dependent efficacy with complete (~95 %) tumor growth inhibition and regression in few mice and translated into significant survival advantage. In 005 mouse GBM orthotopic model that mimics human GBM, JBI-778 showed a strong tumor growth inhibition and significantly extended survival (40 in control vs 58 days in 778 treated, p<0.05). IND-enabling GLP toxicology studies conducted in rodent and non-rodent species did not detect anemia or thrombocytopenia even at higher than efficacious concentrations. Conclusion: PRMT5 inhibitor JBI-778 is able to penetrate the brain in all animal species tested and it is highly efficacious in orthotopic glioma models. Its differentiated mechanism makes it a potential option for the treatment of gliomas and brain metastasis with a focus on patients with spliceosome mutations. IND application for JBI-778 has been approved by FDA and a FIH trial is being planned. Citation Format: Dhanalakshmi Sivanandhan, Chandru Gajendran, Naveen Sadhu M, Zainuddin Mohammed, Ramachandraiah Gosu, Divsha Sher, Shahar Mansur, Dinorah Friedmann-Morvinski, Sridharan Rajagopal, Luca Rastelli. JBI-778, a novel brain-penetrant small molecule PRMT5 inhibitor for treatment of cancer [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 6269.