Abstract Microtubule-associated serine/threonine kinase-like is an essential regulator of mitosis and emerged as a novel oncogenic kinase. It is upregulated in several cancer types, correlating with chromosome instability and poor patient survival. Here, we are reporting a MASTL inhibitor, AD1208, as a new cancer therapeutics.AD1208 inhibits kinase activity of MASTL with IC50 value of 7nM in competitive manner against ATP and induces apoptosis of colorectal cancer cells with IC50 of 150nM. Selectivity index is higher than 100 folds when the cytotoxicity was measured in normal colon cells. Synthetic lethality with BRCA was observed in knock out studies. Cell panel analysis, consist of 300 cell lines, indicated that the AD1208 is effective in cancers originated from stomach, colon, leukemia, lymphoma, and soft tissue. In vivo efficacy was confirmed in mouse xenograft model in dose dependent manner and synergistic effect with PARP inhibitor was observed. Kinase Assay Panel screening revealed that AD1208 is highly selective against 468 kinases. Inhibition of MASTL resulted in decreasing phosphorylated ENSA, inactivating CDK1 and arresting cell cycle progression at G2/M phase. Clinical adverse drug reactions of AD1208 were predicted by in vitro Safety Functional Panel screening to exhibit no meaningful safety-related issues. Currently, AD1208 is being conducted in IND enabling GLP-Tox studies, including genotoxicity, mutagenicity, safety pharmacology as well as in vivo safety studies. Taken together, AD1208 is identified as a novel and selective MASTL inhibitor exhibiting significant therapeutic potential to be a development candidate. Citation Format: SOONGYU CHOI, JS Ahn, KS Baek, YL Choi, MY Im, JH Kim, JW Kim, JH Khoo, SH Lee, WL Yao, Young-Whan Park. Microtubule-associated serine/threonine kinase-likeinhibitor, AD1208, as a novel cancer therapeutics [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 5990.

Dihydroorotate dehydrogenase (DHODH) is the enzyme that catalyzes a rate-determining step during the de novo synthesis of uridine, an important source of cellular pyrimidine nucleotides. Ability to modulate the activity of this enzyme may be used to control diseases associated with rapid, out-of-control cell growth in oncology, immunology, and virology. Emvododstat (PTC299) is a tetrahydro-β-carboline DHODH inhibitor discovered through the GEMS technology (Gene Expression Modulation by Small-Molecules). Described in this paper is the lead optimization campaign that culminated in the discovery of this highly potent DHODH inhibitor.

Diabetic retinopathy is the leading cause of blindness which is associated with excessive angiogenesis. Using the structure of wondonin marine natural products, we previously created a scaffold to develop a novel type of antiangiogenesis agent that possesses minimized cytotoxicity. To overcome its poor pharmaceutical properties, we further modified the structure. A new scaffold was derived in which the stereogenic carbon was changed to nitrogen and the 1,2,3-triazole ring was replaced by an alkyl chain. By comparing the bioactivity versus cytotoxicity, compound 31 was selected, which has improved aqueous solubility and an enhanced selectivity index. Mechanistically, 31 suppressed angiopoietin-2 (ANGPT2) expression induced by high glucose in retinal cells and exhibited in vivo antiangiogenic activity in choroidal neovascularization and oxygen-induced retinopathy mouse models. These results suggest the potential of 31 as a lead to develop antiangiogenic small-molecule drugs to treat diabetic retinopathy and as a chemical tool to elucidate new mechanisms of angiogenesis.

Phosphatidylinositol 3-kinase (PI3K) plays critical roles in numerous intracellular processes. Of the four known subtypes, PI3Kδ subtype is primarily expressed in immune cells and has been reported to promote B cell differentiation and regulatory T (Treg) cell activation. Treg cell-mediated immunosuppression is thought to be a major resistance mechanism for immune checkpoint inhibitors. We therefore hypothesized that an effective PI3Kδ inhibitor could enhance anti-cancer immunity when combined with immune checkpoint inhibitors. Through recent efforts, we have discovered YH25248, an oral PI3Kδ-specific inhibitor. YH25248 effectively inhibits PI3Kδ (IC50 = 10nM) showing more than 100-fold selectivity over α, β, and γ subtypes, and exhibited favorable kinase selectivity in 463 kinase panels. In cellular experiments, phospho-AKT was effectively blocked (IC50 = 7 nM), with basophil activation, a known PI3Kδ specific signal, was also inhibited. YH25248 reduced the activity of Treg cells (CD4+/CD25+) isolated from the splenocytes of CT26 tumor-bearing mice, while sparing conventional T cells (CD4+/CD25-). When YH25248 was orally administered (10 mg/kg) to mice, drug exposure in plasma was reasonable, with a half-life of 4.3 hours, while once-daily oral administration of YH25248 (10 ~ 60 mg/kg) significantly inhibited tumor growth of CT26 colon carcinomas in mouse. When the immune cells of tumors were analyzed by flow cytometry, the number of Treg cells was reduced, CD8 + T cells increased. In this model, YH25248 (30 mg/kg, QD) showed synergistic efficacy when combined with an anti-PD-L1 antibody (200 μg/mice, 10F.9G2 clone). In other syngeneic models using 4T1 or MC38 cells, YH25248 (30 mg/kg, QD) also exhibited similar synergistic efficacy in combination with the anti-PD-L1 antibody. In conclusion, YH25248 is a potent, selective, oral inhibitor of PI3Kδ that reduces tumor growth in syngeneic mouse tumor models by increasing the ratio of CD8+ to Treg cells. The combination of YH25248 and anti-PD-L1 antibody resulted in substantially greater tumor growth inhibition in several syngeneic models. These data support the promising potential of YH25248 for combination therapy with immune checkpoint inhibitors to increase therapeutic response rates. Citation Format: Jinhwi Park, Ho-Woong Kang, Hyun-Mo Koo, Jongsuk Park, Tae-Wang Kim, Se-Woong Oh, Soongyu Choi. YH25248, a selective PI3K delta inhibitor, shows synergistic effect with an anti-PD-L1 antibody [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 3915.

In Gaucher disease, several macrophage-specific biomarkers have been validated for use in the clinic. However, Gaucher disease is more complex involving system-wide pathophysiology beyond the macrophage, and based on gene array analysis in our Gaucher disease mouse model and other emerging pathophysiologic insights, we evaluated serum levels of cathepsins D and S, YKL-40 and progranulin in Gaucher disease patients. We assessed their biomarker potential in Gaucher disease and compared them to established Gaucher disease biomarkers, chitotriosidase, chemokine ligand 18 (CCL18), and other indicators of disease severity and response to therapy. Mean YKL-40 and cathepsin D and S levels were significantly higher in Gaucher disease patients compared to healthy controls; in contrast, mean progranulin levels were lower in Gaucher disease patients compared to healthy controls. Enzyme replacement therapy resulted in a significant reversal of elevated cathepsin D and S but there was no change in progranulin and YKL-40 levels. Patients with persistent splenomegaly after long-term enzyme replacement therapy had significantly higher serum YKL-40 than patients with smaller spleens (63.0 ± 6.4 ng/ml vs. 46.4 ± 4.3 ng/ml, p = .03). Serum YKL-40 levels were higher in subjects with severe bone involvement (Hermann Score 3 to 5) compared to those with milder bone involvement (Hermann Score 1 to 2) (70.1 ± 4.3 ng/ml vs. 48.1 ± 3.7 ng/ml, p = .0002). YKL-40 was only weakly associated with chitotriosidase (r = 0.2, p = .008) and CCL18 (r = 0.3, p = .0004), and cathepsin S was moderately associated with chitotriosidase (r = 0.4, p = .01) and CCL18 (r = 0.6, p < .0001). Receiver operating curves for progranulin and YKL-40 demonstrated areas under the curves of 0.80 and 0.70, respectively. In conclusion, while these biomarkers do not meet robust properties of established macrophage-specific biomarkers, they may inform severity of skeletal disease, contribution of fibrosis to residual splenomegaly, and other disease manifestations. These findings, including markedly low progranulin levels that do not change upon enzyme replacement therapy, are intriguing to prompt further investigations to decipher their role in pathophysiology and relevance to diverse phenotypes of Gaucher disease.

Abstract Purpose: Given that osimertinib is the only approved third-generation EGFR-TKI against EGFR activating and resistant T790M mutated non–small cell lung cancer (NSCLC), additional mutant-selective inhibitors with a higher efficacy, especially for brain metastases, with favorable toxicity profile are still needed. In this study, we investigated the antitumor efficacy of YH25448, an oral, mutant-selective, irreversible third-generation EGFR-TKI in preclinical models. Experimental Design: Antitumor activity of YH25448 was investigated in vitro using mutant EGFR-expressing Ba/F3 cells and various lung cancer cell lines. In vivo antitumor efficacy, ability to penetrate the blood–brain barrier (BBB), and skin toxicity of YH25448 were examined and compared with those of osimertinib using cell lines and PDX model. Results: Compared with osimertinib, YH25448 showed a higher selectivity and potency in kinase assay and mutant EGFR-expressing Ba/F3 cells. In various cell line models harboring EGFR activating and T790M mutation, YH25448 effectively inhibited EGFR downstream signaling pathways, leading to cellular apoptosis. When compared in vivo at equimolar concentrations, YH25448 produced significantly better tumor regression than osimertinib. Importantly, YH25448 induced profound tumor regression in brain metastasis model with excellent brain/plasma and tumor/brain area under the concentration–time curve value. YH25448 rarely suppressed the levels of p-EGFR in hair follicles, leading to less keratosis than osimertinib in animal model. The potent systemic and intracranial activity of YH25448 has been shown in an ongoing phase I/II clinical trial for advanced EGFR T790M mutated NSCLC (NCT03046992). Conclusions: Our findings suggest that YH25448 is a promising third-generation EGFR inhibitor, which may be more effective and better tolerated than the currently approved osimertinib.

Abstract EGFR mutated lung cancer shows approximately 10-15% of non-small cell lung cancer (NSCLC). Although the best therapeutic EGFR tyrosine kinase inhibitors (TKIs) targeting mutant EGFR, such as gefitinib and erlotinib, are used in the first line treatment of patients with advanced EGFR mutated NSCLC, the acquired resistance to the drugs usually appears in 10-12 months of therapy by the occurrence of a second EGFR mutation T790M. YH25448, a highly mutant-selective and irreversible 3rd generation EGFR TKI potently penetrating blood-brain barrier (BBB) penetration, targets both activating EGFR mutations Del19, L858R and T790M mutation while sparing wild type. In NSCLC cell lines and primary cancer cells from patients harboring EGFR mutations, YH25448 showed more potent inhibition of cancer cell growth and significantly increased tumor cell apoptosis compared to osimertinibs, which is one of 3rd generation EGFR TKIs. In vivo mouse model implanted with H1975 cells, YH25448 treatment at the once-daily showed a dramatic dose-dependent tumor regression in both subcutaneous and intracranial lesions with no abnormal signs such as skin keratosis shown in osimertinib-treated mice. Plasma half life of YH25448 was 5.9-6.8 hr and tumor to plasma AUC0-last ratio was 3.0-5.1 in tumor bearing mice. YH25448 also showed excellent penetration of the BBB, achieving CSF concentrations exceeding the IC50 value for pEGFR inhibition in the tumor-bearing mice. Taken together, these findings suggest important role for the further development of YH25448 as a novel therapeutic for the treatment of EGFR mutant-positive NSCLC patients with brain metastases. Citation Format: Jiyeon Yun, Min Hee Hong, Seok-Young Kim, Chae Won Park, So-Young Kim, Mi Ran Yun, Han Na Kang, Kyoung-Ho Pyo, Jong Sung Koh, Ho-Juhn Song, Young- Sung Lee, Se-Woong Oh, Soongyu Choi, Byoung-Chul Cho. YH25448, an irreversible 3rd generation EGFR TKI, exhibits superior anticancer effects with potent brain BBB penetration in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4790.

9033 Background: The epidermal growth factor receptor (EGFR) T790M mutation is one of the most common acquired resistance mechanism to EGFR tyrosine kinase inhibitors (TKIs). YH25448 is an oral, potent, irreversible EGFR TKI that is highly selective for activating (EGFRm) and T790M resistance mutations. Methods: Patients with EGFRm advanced NSCLC with acquired resistance to EGFR-TKIs with or without asymptomatic brain metastases were enrolled in an open-label, multicenter, phase I/II study with dose escalation and expansion cohorts. YH25448 was administered once daily at doses of 20 to 240 mg in a 21 day cycle. Patients were assessed for safety, tolerability, pharmacokinetics and efficacy. T790M status was confirmed in the expansion cohorts. Results: A total of 105 patients (median age 62 years, female 61%) were enrolled. The dose escalation cohort included 33 patients administered with 20 to 240 mg once daily across 6 dose levels, and 72 patients in the dose expansion cohort were administered with 40 to 240 mg. No dose-limiting toxicities were observed. The most common treatment-emergent adverse events (AEs) were pruritus (12%), decreased appetite (11%), rash (11%), and constipation (10%). AEs of grade 3 or higher were observed in 5% of the patients. Systemic exposure increased dose-dependently. Of the evaluable patients (n = 91) at data cut-off, the objective response rate (ORR) was 64% (95% confidence interval [CI], 53.0 to 73.6). The ORR for 76 of the T790M-positive patients was 67% (95% CI, 55.4 to 77.5), and for 15 of the T790M-negative patients was 47% (95% CI, 21.3 to 73.4). In patients with brain metastases (n = 9), the overall intracranial ORR was 56% (95% CI, 21.2 to 86.3). Conclusions: YH25448 was well tolerated and exhibits promising systemic and intracranial antitumor activity at multiple dose levels in EGFR T790M+ NSCLC patients. Clinical trial identifier: NCT03046992. ORR in T790M+ patients. Dose, QD 20 mg 40 mg 80 mg 120 mg 160 mg 240 mg Evaluable patients*, n 2 25 18 21 8 2 ORR, n (%) 2 (100) 17 (68) 11 (61) 15 (71) 4 (50) 2 (100) *Patients are evaluable for response if they have post-baseline radiological assessment (RECIST 1.1) or patients who discontinued prior to the post-baseline assessment.

The underlying cause of spinal muscular atrophy (SMA) is a deficiency of the survival motor neuron (SMN) protein. Starting from hits identified in a high-throughput screening campaign and through structure-activity relationship investigations, we have developed small molecules that potently shift the alternative splicing of the SMN2 exon 7, resulting in increased production of the full-length SMN mRNA and protein. Three novel chemical series, represented by compounds 9, 14, and 20, have been optimized to increase the level of SMN protein by >50% in SMA patient-derived fibroblasts at concentrations of <160 nM. Daily administration of these compounds to severe SMA Δ7 mice results in an increased production of SMN protein in disease-relevant tissues and a significant increase in median survival time in a dose-dependent manner. Our work supports the development of an orally administered small molecule for the treatment of patients with SMA.

Abstract BMI1 (B-cell specific Moloney Murine Leukemia Virus Insertion 1) is a gene predominantly expressed in stem cells. A deficiency of BMI1 protein leads to a reduction in the adult stem cell population. Decreasing of BMI1 protein levels by siRNA causes apoptosis and/or senescence in tumor cells in vitro and increases susceptibility to cytotoxic agents. In tumors, the level of BMI1 protein is often elevated. Consequently, the expression level of BMI1 is highly prognostic in many types of cancers. Since BMI1 is non-enzymatic, targeting this protein by traditional drug discovery approaches has not met with success. PTC Therapeutics has developed a novel screening technology referred to as GEMSTM (Gene Expression Modulation by Small molecules) to identify small molecules that modulate the levels of a protein of interest by targeting post-transcriptional regulatory processes. Using this screening technology, we have identified a number of compounds that can selectively reduce levels of BMI1 protein. Schematically, the lead scaffold could be represented by four key components: a bicyclic right core, a heterocyclic middle ring (thiazole), a linking group and a left aryl region. Structure-activity relationships associated with this series have been investigated through medicinal chemistry efforts. Incorporation of a small electron-donating para- substituent at the left part of the molecule is highly effective in decreasing levels of BMI1, indicating the strong influence of both steric and electronic effects on activity. An amino (linking group) moiety was found to be essential for activity. Variation at the right-hand moiety appears to be more tolerated, allowing improvements in both pharmacokinetic and activity profiles. These SAR findings have revealed important pharmacophoric and structural features required for the desired biological activity with this series, and provide a foundation for further lead optimization efforts. Citation Format: Nadiya Sydorenko, Ramil Baiazitov, Soongyu Choi, Chang-Sun Lee, Liangxian Cao, Thomas W. Davis, Neil G. Almstead, Young-Choon Moon. Optimization of small molecules targeting BMI1 protein expression. Part 1. Amino-thiazoles: the first-in-class highly potent inhibitors of BMI1 protein. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2529. doi:10.1158/1538-7445.AM2014-2529