Selective Small Molecule Inhibitor Research Articles

Abstract CT196: First-in-human phase 1/2a trial of a macrocyclic ATR inhibitor (ATRN-119) in patients with advanced solid tumors

Abstract Background: ATRN-119 is an orally bioavailable, potent, selective macrocyclic small molecule inhibitor of Ataxia Telangiectasia and Rad3- related (ATR) kinase. As the master regulator of DNA damage response (DDR), ATR orchestrates cellular response to DNA replication stress to preserve DNA replication fork integrity. Cancer cells with markedly increased oncogenic stress or dysfunctional DDR are more reliant on ATR for genome stabilization. Thus, inhibition of ATR can result in cancer cell-selective replication fork collapse and cancer cell death. Methods: This ongoing, first-in-human, multi-site phase 1/2a trial (NCT04905914) is investigating ATRN-119 safety and pharmacokinetic properties (primary objectives), preliminary efficacy in various solid tumors (secondary objective), and biomarker profile (exploratory objective). This study uses an open-label, standard 3+3 dose escalation and dose expansion study design. Key inclusion criteria include: advanced solid tumor, measurable disease (RECIST v1.1), tumor with at least 1 DDR mutation by next-generation-sequencing, at least 1 failed standard-of-care therapy, ECOG PS 0 or 1, and adequate organ function. Key exclusion criteria are cancer therapy within 4 weeks or at least 5 half-lives, unresolved therapy-related AEs, investigational agent within 5 half-lives or 30 days of study drug, CNS metastases or unstable involvement, and concomitant treatment with strong CYP3A4 or CYP2D6 inhibitors or inducers. Results: This trial is currently enrolling at Dose level 5 (550 mg). Among 12 patients in the first four dose levels, the median age is 62 years (range: 48 to 79), where 67% (n=8) are female, 75% (n=9) are White, and 25% (n=3) are Black. The median number of prior treatments is 3 (range: 1 to 6, and 92% have received platinum-based chemotherapy). The majority of tumors, 75% (n=9), had a TP53 mutation. There have been no reports of dose-limiting toxicities, treatment-related serious adverse events (AE), and treatment-related AEs Grade 3 or higher in these cohorts. The three most commonly reported all-cause AEs are fatigue (33%, n=4), nausea (25%, n=3), and diarrhea (25%, n=3). Increasing exposure and maximum serum concentrations (Cmax) were observed by dose level. The half-life of ATRN-119 is ~5 hours. Two of 12 patients achieved stable disease (SD): one patient from Dose level 1 (50 mg) who progressed on Day 112 and one patient from Dose level 3 (200 mg) who remains on treatment as of Day 118 (data cut off date: January 2, 2024). Conclusion: In these early cohorts, once-daily oral ATRN-119 appears to be well tolerated with a manageable safety profile, exhibits near dose proportionality, and demonstrates disease stabilization warranting further investigation. Further studies to explore a twice-daily schedule are being considered. Citation Format: Fiona Simpkins, Amit Mahipal, Patricia LoRusso, Reva Schneider, Crystal Miller, David Stenehjem, Eric J. Brown, Mike Carleton, Joachim Gullbo, Nadeem Q. Mirza. First-in-human phase 1/2a trial of a macrocyclic ATR inhibitor (ATRN-119) in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT196.

Abstract CT118: Phase I dose escalation of ASKC202, a novel cMET tyrosine kinase inhibitor, in patients with advanced solid tumors

Abstract Background ASKC202 is an oral and highly selective small molecule inhibitor which inhibits mesenchymal-epithelial transition. We report preliminary safety and efficacy data from an ongoing Phase I, FIH, dose-escalation study of ASKC202 in patients (pts) with advanced solid tumors (NCT05306132). Methods A two-part study was initiated to evaluate MTD, safety and tolerability, pharmacokinetics and preliminary antitumor activity of ASKC202, including dose escalation study (Part A) and dose expansion study (Part B). Responses were assessed per RECIST 1.1 every 2 cycles (6 weeks). AEs were graded using CTCAE v5.0.In Part A, dose escalation followed the accelerated titration combined with the "3+3" design. Patients with heavily pre-treated solid tumors received ASKC202 orally at doses of 50, 150, 300, 450 and 600 mg/day once daily. The treatment continued until disease progression, unacceptable toxicity or treatment termination. If the subject in the 50mg dose group developed DLT, the dose group was switched to a "3+3" design. Results As of November 30, 2023, 16 patients who are refractory or don’t respond to standard therapy were available received ASKC202 (50mg: N=1; 150mg: N=3; 300mg: N=6; 450mg: N=3; 600mg: N=3). 15/16 pts had pathological confirmation of non-small-cell lung cancer (except for 1 case of PSC). 8/16 pts had brain metastasis. 10/16 pts had cMET dysregulation including MET amplification (N=9; gene copy number 1.3-8.3) and MET missense mutation (N=1; p.S186L) with no patient having Exon 14 skipping mutation.ASKC202 was well-tolerated in all patients. No dose-limiting toxicity was observed in all dose levels (50 to 600 mg). The common TRAEs occurring in more than 20% of patients included: anemia (5/16), blood glucose elevation (5/16), ALT elevation (4/16), AST elevation (4/16) and peripheral edema (4/16). Grade 3 TRAEs were only observed in one patient (300mg QD), including anemia and hypokalemia. Among 14pts who had at least one post-baseline anti-tumor assessment, 5 pts achieved a confirmed partial response. The confirmed ORR is 35.7%. The median best percentage change in target lesion size from baseline (depth of response) was 51.8% and 3pts had duration of tumor response ≥ 6 months. ORR and DCR for patients with cMET alteration (N=8) was 62.5% (5/8) and 75.0% (6/8) respectively. One subject (150mg QD) with MET missense mutation (p.S186L) had a 67% tumor reduction in intracranial lesion from baseline. Exposure of ASKC202 increased dose-proportionally at dose range 50mg to 300mg, but less than dose-proportionally increased at 300mg to 600mg. Conclusions ASKC202 monotherapy was well-tolerated with manageable safety profile and showed anti-tumor signal and the central nervous system efficacy. Citation Format: Tingting Song, Caicun Zhou, Wei Li, Xingya Li, Lin Wu, Shan Zeng, Liyun Miao, Baogang Liu, Xuyu Wei, Jing Chen, Luwei Han. Phase I dose escalation of ASKC202, a novel cMET tyrosine kinase inhibitor, in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT118.

Abstract CT115: A first-in-human phase I study of CTX-712 in patients with advanced, relapsed or refractory malignant tumors (CTX-712-CL-01 study): Efficacy and safety in a hematologic malignancies cohort

Abstract Background: Mutations in genes encoding RNA splicing factors such as SF3B1, SRSF2, U2AF1, and ZRSR2, are common genetic aberrations in many forms of myeloid neoplasms. CTX-712 is a first-in-class, orally available, highly potent, and selective small molecule inhibitor of CDC2-like kinase (CLK), a key regulator of RNA splicing. Preclinical data show that CTX-712 demonstrated anti-proliferative activity in in-vitro and in-vivo models derived from hematologic malignancies. A phase I study of CTX-712 was conducted to evaluate the safety and the preliminary efficacy of CTX-712 in patients with relapsed or refractory acute myeloid leukemia (AML) and higher-risk myelodysplastic syndrome (MDS). Methods: The objectives of this cohort were to evaluate the dose-limiting toxicity (DLT), safety, pharmacokinetic (PK)/pharmacodynamic (PD) profiles, and preliminary efficacy of CTX-712 in patients with hematologic malignancies. A 3+3 design was used in two dose levels (70 mg and 105 mg twice a week (TW)) based on the safety data from the previous dose escalation cohort in patients with solid tumors. Results: As of data cutoff on November 20, 2023, a total of 14 patients (12 AML and 2 MDS) were enrolled in 70 mg TW (n=8) and 105 mg TW dose levels (n=6). DLT of Grade 4 pneumonia was observed in 1 of 3 DLT-evaluable patients in the 105 mg TW group. In the 70 mg TW group, no DLT was observed in 6 DLT-evaluable patients. Among the safety analysis population (n=14), median treatment duration was 76 days (Range 15-974 days). At data cutoff, treatment was ongoing in 1 patient and discontinued in 13 patients. The common any-grade adverse events (AEs) (≥30%) were diarrhea (57.1%) and nausea (42.9%). The most common Grade 3 or higher AE were white blood cell count decreased and tumor lysis syndrome (14.3%). In AML patients (n=12), complete remission (CR) was observed in 3 patients (25.0%), and CR with incomplete hematologic recovery (CRi) was observed in 1 patient (8.3%). The median time to remission was 67 days (Range 29-113 days), and the median duration of response was 281 days (Range 56-924 days). In MDS patients (n=2), CR was observed in 1 patient (50.0%). PK/PD profiles of the hematologic malignancies dose escalation cohort were comparable with those of the solid tumor cohort at the same dosage. Conclusions: CTX-712 demonstrated a manageable and tolerable safety profile and showed anti-tumor efficacy in patients with hematologic malignancies. Currently, a Phase I/II Study of CTX-712 in relapsed/refractory AML and higher risk MDS is ongoing using a new tablet formulation in the United States. Clinical trial information: jRCT2080224127 Citation Format: Hisayuki Yokoyama, Noriko Fukuhara, Koji Ando, Hiroatsu Iida, Takahiro Yamauchi, Suguru Fukuhara, Koji Izutsu, Yasushi Tanoue, Maki Yamamoto, Hirokazu Tozaki, Eiji Takahara, Shingo Shoji, Akio Mizutani, Daisuke Morishita, Robert W. Oda, Hiroshi Miyake, Noboru Yamamoto. A first-in-human phase I study of CTX-712 in patients with advanced, relapsed or refractory malignant tumors (CTX-712-CL-01 study): Efficacy and safety in a hematologic malignancies cohort [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT115.

Role of TBK1 Inhibition in Targeted Therapy of Cancer.

TANK-binding kinase 1 (TBK1) is a serine/threonine protein that plays a crucial role in various biological processes like immunity, autophagy, cell survival, and proliferation. The level and kinase activity of the TBK1 protein is regulated through post-translational modifications (PTMs). TBK1 mainly mediates the activation of IRF3/7 and NF-κB signaling pathways while also participating in the regulation of cellular activities such as autophagy, mitochondrial metabolism, and cell proliferation. TBK1 regulates immune, metabolic, inflammatory, and tumor occurrence and development within the body through these cellular activities. TBK1 kinase has emerged as a promising therapeutic target for tumor immunity. However, its molecular mechanism of action remains largely unknown. The identification of selective TBK1 small molecule inhibitors can serve as valuable tools for investigating the biological function of TBK1 protein and also as potential drug candidates for tumor immunotherapy. The current research progress indicates that some TBK1 inhibitors (compounds 15,16 and 21) exhibit certain antitumor effects in vitro culture systems. Here, we summarize the mechanism of action of TBK1 in tumors in recent years and the progress of small molecule inhibitors of TBK1.

NIBR-LTSi is a selective LATS kinase inhibitor activating YAP signaling and expanding tissue stem cells in vitro and in vivo

The YAP/Hippo pathway is an organ growth and size regulation rheostat safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. Here, we report the identification and characterization of the selective small-molecule LATS kinase inhibitor NIBR-LTSi. NIBR-LTSi activates YAP signaling, shows good oral bioavailability, and expands organoids derived from several mouse and human tissues. In tissue stem cells, NIBR-LTSi promotes proliferation, maintains stemness, and blocks differentiation invitro and invivo. NIBR-LTSi accelerates liver regeneration following extended hepatectomy in mice. However, increased proliferation and cell dedifferentiation in multiple organs prevent prolonged systemic LATS inhibition, thus limiting potential therapeutic benefit. Together, we report a selective LATS kinase inhibitor agonizing YAP signaling and promoting tissue regeneration invitro and invivo, enabling future research on the regenerative potential of the YAP/Hippo pathway.

Abstract 5921: PH027-1, a potent and selective small-molecule WRN inhibitor that targets MSI-H tumors

Abstract Background: High microsatellite instability (MSI-H) is highly prevalent across many cancer types including endometrium, gastric, ovary and colorectal cancer. It is well known that there is a clear synthetic lethality (SL) interaction between MSI-H and Werner syndrome protein (WRN) inhibition. WRN is a member of RecQ helicase family and involved in the maintenance of genome integrity. Different from other RecQ family members, WRN has two activity centers responsible for helicase and exonuclease activity, respectively. Further studies confirmed that an inhibition on helicase activity, but not exonuclease activity, is responsible for SL interaction with MSI-H. Also, Recent clinical observations supported that though loss-of-function of WRN could cause an early-aged disease Werner syndrome, selective loss of helicase but not exonuclease leads to only mild to moderate symptoms, suggesting a selective inhibitor of helicase activity has the potential to induce the SL interaction with MSI-H and keep patients safe. Methods: Biochemical assays and cell-based assays were performed to evaluate in vitro activity and selectivity of PH027-1. The in vivo anti-tumor efficacy was tested in two cell-derived xenograft (CDX) mouse models with tumors harboring MSI-H. The in vivo pharmacokinetics (PK) properties were evaluated in mice, rats and dogs, and other in vitro and in vivo PK and safety properties were assessed with corresponding assay methods. Results: Biochemical assays showed that PH027-1 potently inhibited helicase activity of WRN (IC50, 0.8 nM), but had no effect on other RecQ family members even at the highest tested concentration of 10 μM (IC50, all≥10 μM for BLM, RecQ4 and RecQ5). At the same time, PH027-1 did not affect the exonuclease activity of WRN at all tested concentrations. In cell-based assays, PH027-1 potently inhibited cell viability of MSI-H cancer cells SW48 and HCT116 at tens of nM, but had no effect on MSS cells (HT29 cancer cells and CD34+ hematopoietic stem cells) even at 10 μM. The tests were further expanded to 18 cancer cell lines covering uterus, gastric, ovary and colorectal cancer. Nine of twelve MSI-H cell lines were very sensitive to WRN inhibition, and all six MSS cell lines were totally resistant to the treatment. PH027-1 possessed excellent PK profiles as shown by in vivo PK studies, with absolute bioavailability of 58%~102% in mice, rats and dogs. In two CDX models (SW48 and HCT116), oral administration of PH027-1 dose-dependently inhibited tumor growth, and a nearly complete inhibition was observed at a dose of 20 mg/kg QD. Tumor regression was clearly shown at higher doses. All other in vitro and in vivo PK and safety tests showed favorable results and supported for preclinical development of PH027-1. Conclusion: As a highly selective and potent inhibitor on helicase activity of WRN, PH027-1 has the potential to become a novel and safe approach for treatment of MSI-H tumors. Citation Format: Feng Gao, Bin Liu, Liandong Jing, Jing Wang, Yongyong Wu, Jun Wan, Pengzhi Zhang, Yu Gao, Zhizhong Li, Yongqi Guo. PH027-1, a potent and selective small-molecule WRN inhibitor that targets MSI-H tumors [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 5921.

Abstract 1970: Discovery and characterization of potent and selective HER2 exon20 insertion mutant inhibitors

Abstract Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for over 85% of all cases. Among NSCLC, approximately 2-4% of patients have mutations in the HER2, a member of the ErbB family of receptor tyrosine kinases and key proliferation driver, with exon 20 insertions making up about 90% of these mutations. HER2 exon mutations can have various alterations, and among them, the most common HER2 mutation is in-frame insertions in exon 20, especially the A775_G776insYVMA insertion/duplication. These mutations can affect the structure and function of the HER2 protein and may contribute to tumor development and growth. HER2 mutations have often shown resistance to EGFR tyrosine kinase inhibitors (TKIs), further highlighting the need for HER2-specific treatments. Also, inhibitors targeting HER2 mutations are selective, meaning they specifically target HER2 alterations without causing toxicities or affecting EGFRWT. Therefore, there is a need for the development of selective HER2 exon20 insertion mutant inhibitors. Herein, Hanmi has identified selective HER2 exon20 insertion mutant small molecule inhibitors, designed based on affinity and binding mode starting from a known crystal structure using GLIDE (Schrödinger), and the screening for the viability of cell lines including HER2A775_G776insYVMA, HER2G776delinsVC , HER2WT and EGFRWT was investigated. Hanmi's HER2 exon20 insertion mutant inhibitors selectively target mutant HER2 receptor tyrosine domains through shared binding, sparing EGFRWT and minimizing EGFR-related toxicities. With highly selectivity against EGFRWT and desirable pharmacokinetic properties, the Hanmi compounds inhibit HER2 mutant cells and HER2 mutant enzymes. Additionally, the compounds have an oral administration-compatible DMPK profile and demonstrated anti-tumor activity in HER2 mutant and HER2WT tumor xenograft mice. In conclusion, based on our study, Hanmi compounds, a novel HER2 exon20 insertion mutant inhibitors could be suggested as an appropriate therapeutic agent for inhibitory activity against HER2 exon20 insertion mutant NSCLC. Citation Format: Ho Yeon Nam, Sun Young Jang, Jiyoung Jeon, HyungSeok Yoo, Jooyun Byun, Gunwoo Lee, Youngjoo Lee, Yu-Yon Kim, Soye Jeon, Young Gil Ahn. Discovery and characterization of potent and selective HER2 exon20 insertion mutant inhibitors [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 1970.

Abstract 7265: ETS-005, a highly selective TEAD4 palmitoylation inhibitor with potent anti-tumor activity and brain penetrating capability

Abstract The Hippo pathway is a highly conserved network that regulates cell proliferation, tissue regeneration, and organ size by orchestrating YAP/TAZ-TEADs, the core effectors of signaling. Aberrant activation of YAP/TAZ has been implicated in tumorigenesis, tumor progression, metastasis and the resistance to targeted therapeutics, including MAPK and RTK inhibitors. Among the four TEAD paralogs, TEAD4 is found widely overexpressed in various solid tumors and potentially less associated with YAP/TEAD-related renal toxicity. Here we reported the identification of ETS-005, a highly selective TEAD4 small molecule inhibitor that shows preference for binding to the palmitoylation pocket of TEAD4 than other TEAD paralogs over 15 folds. ETS-005 displayed low nanomolar inhibition of cell proliferation in NF2 or LATS1/2 mutant mesothelioma in vitro, and robust anti-tumor efficacy in vivo. ETS-005 showed favorable oral availability, high brain penetration capability and broad safety window. Moreover, ETS-005 had relative long projected T1/2 in human, supporting a more convenient clinical QD dosing schedule. Our study demonstrated that selective TEAD4 palmitoylation inhibitor ETS-005 exhibited potent anti-tumor activity as much as pan-TEAD palmitoylation inhibitors while may gain broader therapeutic index. Additionally, ETS-005 also exhibited profound synergistic effects in drug combination studies, offering broad opportunities combined with MAPK and RTK therapies. ETS-005 is currently in IND-enabling study and expected to enter Phase 1 in 2024. Citation Format: Jiajun Lu, Wenpei Du, Ming Gao, Xiaofei Fan, Jinping Li, Lijian Feng, Mengya Wang, Yingjie Li, Xiaofeng Zhong, Wenqi Cui, Qiangang Zheng, Jidong Zhu. ETS-005, a highly selective TEAD4 palmitoylation inhibitor with potent anti-tumor activity and brain penetrating capability [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 7265.

Abstract 1965: 3HP-2827-high selective inhibitor of FGFR2, a best-in-class therapy for FGFR2-driven solid tumors

Abstract FDA-approved pan-FGFR inhibitors bring encouraging results in solid tumors. However, resistance mechanisms and side effect profiles may limit their clinical utility. We designed 3HP-2827, a highly selective and potent small molecule inhibitor of FGFR2, to treat solid tumors harboring FGFR2 alterations, including amplifications, mutations, and fusions to overcome these limitations. 3HP-2827 inhibits FGFR2 enzyme activity with more than 1623-fold selectivity over FGFR1 and FGFR3. 3HP-2827 inhibits cell proliferation with IC50 <10 nM and 30 nM in FGFR2 amplified and FGFR2-mutant cancer cell lines, respectively. 3HP-2827 demonstrates high kinome selectivity for FGFR2 against a panel of 485 human kinases, resulting in 90.1% inhibition of FGFR2, and no other kinases showed greater than 75% inhibition. 3HP-2827 leads only modest activity at 10 µM against PDE4D2 and LCK in 44 distinct common adverse drug reaction targets, supporting its high degree of safety. 3HP-2827 also demonstrates a dose-dependent inhibition of phosphorylation of FGFR2 and FGFR2 signaling pathway nodes, including ERK and AKT, in the FGFR2-dependent cancer cell line. In vivo, 3HP-2827 demonstrates potent antitumor activity in cell- and patient-derived xenograft mouse models, including FGFR2-amplified gastric cancer, FGFR2-mutant (N549K) endometrial cancer, FGFR2 fusion ICC and gastric cancers. Strikingly, 3HP-2827 demonstrates superior activity in patient-derived ICC and gastric cancer, cell-derived endometrial cancer (CDX) in tumor and pFGFR2 inhibitions. In the ICC PDX model, 3HP-2827 also induces the regression of pemigatinib-resistant tumors. 3HP-2827 is well tolerated in the toxicology studies, and no hyperphosphatemia or tissue mineralization is observed at 150 mg/kg/kg/day in rats and 120 mg/kg/day in dogs. The data support that 3HP-2827, a potential to be a best-in-class selective FGFR2 inhibitor, will be studying clinically at the beginning of 2024. Citation Format: Susan S. Liu-Chen, Jufang Lin, Jingyu Hu, Yanchao Liu, Weibo Zhang, Zhixiong Zhang, Yan Ding, Jinqiu Liu, Kevin Chen, Shaojing Hu. 3HP-2827-high selective inhibitor of FGFR2, a best-in-class therapy for FGFR2-driven solid tumors [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 1965.

Abstract 4484: Discovery of a potential first-in-class Za targeted small molecule inhibitor of ADAR1 p150 which demonstrates strong anti-tumor efficacy

Abstract Background: Adenosine deaminase, RNA specific (ADAR1), catalyzes the hydrolytic deamination of adenosine (A) to inosine (I) in double-stranded (ds) RNAs. ADAR1 has two isoforms: p110 in the nucleus and p150 in the cytoplasm. Both isoforms alter the coding and non-coding sections of self-dsRNA. The ADAR1 p150 isoform is expressed from an interferon (IFN)-response promoter and has a Z-DNA/Z-RNA binding domain at the N-terminus. ADAR1 has been implicated in promoting cell survival and resistance to immune response in several cancer types like multiple myeloma, breast, lung, liver, and skin cancer. Inhibition of ADAR1 p150 has the potential to enhance anti-tumor efficacy as monotherapy and in combination with other therapeutic modalities. The Zα domain of ADAR1 p150 provides exclusivity to p150 compared to the other isoforms. Herein, we outline the discovery of a potential first-in-class Zα targeted ADAR1 p150 inhibitor and degrader for cancer immunotherapy. Methods: AVA-ADR-001 was identified through a high throughput p110 knockout cell-based assay. The ability of AVA-ADR-001 to induce interferons was confirmed in various cell lines. The anti-tumor efficacy of AVA-ADR-001 was evaluated in B16F10 syngeneic melanoma mice model as monotherapy and in combination with anti-PD-1. PROTAC AVA-ADR-703 was used to demonstrate selective degradation of the ADAR1 p150 protein. Results: We have discovered AVA-ADR-001, a novel small molecule inhibitor of ADAR1 which significantly increases the IFN response in vitro in an MDA5-dependent manner. In vitro binding studies confirm that AVA-ADR-001 binds to the Zα domain of ADAR1 with low nM potency. AVA-ADR-001 demonstrates anti-tumor efficacy 1.5X superior to anti-PD1 ICB non-responsive tumor models. In addition, the AVA-ADR-001 monotherapy and combination groups showed a significant upregulation of numerous interferon-stimulated genes, including IFIH1, IFN-β, and CXCL-10. Finally, targeted protein degradation of ADAR1 p150 while sparing ADAR1 p110 was confirmed when cells were treated with AVA-ADR-703 with nM DC50, the PROTAC variant of AVA-ADR-001. Conclusions: To our knowledge, AVA-ADR-001 and AVA-ADR-703 are the first selective Zα-targeted small molecule inhibitor and degrader of ADAR1 p150 respectively to be disclosed. As a powerful and targeted first-in-class ADAR1 inhibitor, AVA-ADR-001 has demonstrated a strong induction of interferon (IFN) in a number of cancer cell lines and in vivo. AVA-ADR-001 has also demonstrated strong in vivo anti-tumor efficacy as monotherapy and in combination with anti-PD1. Furthermore, AVA-ADR-703 demonstrates selective and highly efficient degradation of ADAR1 p150 while sparing ADAR1 p110. Given that ADAR1 has a pro-metastatic and immune-suppressive role, AVA-ADR-001 and AVA-ADR-703 have significant potential to emerge as strong cancer immunotherapy candidates. Citation Format: Avijit Goswami, Sandeep Goyal, Kawaljit Singh, Princy Khurana, Barnali Deb, Aditya Kulkarni. Discovery of a potential first-in-class Za targeted small molecule inhibitor of ADAR1 p150 which demonstrates strong anti-tumor efficacy [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 4484.

Abstract 1059: Biomarkers analysis on samples from patients in EVT801 clinical trial: Patient characterization and immunomonitoring

Abstract Introduction: EVT801 is a highly selective small molecule inhibitor of VEGFR3 that acts by inducing tumor (lymph)-angiogenesis normalization in and around the tumor. EVT801 has shown compelling activity in a wide range of cancer models showing a decrease of tumor hypoxia and an increase of CD8pos T-cells infiltration into the tumor micro-environment. EVT801 was well-tolerated in preclinical toxicology studies. A phase I study is underway focusing primarily on understanding the safety, tolerability, and pharmacokinetics of EVT801 across a range of doses (NCT05114668). Methods: The first stage of the phase I trial aims to determine the maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) for EVT801. Clinical samples from enrolled patients have been collected to explore preliminary signals of the biological activity of the drug and characterize tumor phenotypes. Patient characterization includes histology and mRNA signature in archival biopsies. Target engagement and pharmacodynamics effects are investigated by immunomonitoring as well as assessment of a defined set of proteins as markers of inflammation and angiogenesis as identified in preclinical in vivo models. Conclusion: Our first biomarkers analyses on a subpopulation of patients with High Grade Serous Ovarian Cancer (HGS-OC) enrolled in the first stage of the study showed that levels of VEGFR3 expression had a negative correlation with CD8 positive T-cells infiltration in the tumor microenvironment and a tendency to positive correlation with a PD1 mAb resistance signature. VEGFR3 expression also tends to be correlated with higher levels of hypoxia (CAIX labelling). Our working hypothesis is that patients with hypoxic HGS-OC tumors with high VEGFR3 expression may benefit from EVT801 treatment; this will need to be reinforced by inclusion of additional patients in dedicated PD biomarkers cohorts during stage 2 of the clinical trial. Citation Format: Lise Davenne, Michael Fitzgerald, Pierre-Benoit Ancey, Oona Delpuech, Céline Poussereau-Pomié, Michael Esquerre, Michael R. Paillasse, Marie Mandron, Philippe Rochaix, Maha Ayyoub, Clara Maria Scarlata, Christine Caux, Christophe Caux, Philippe Cassier, Carlos Gomez-Roca, Jean-Pierre Delord, John Friend, Pierre Fons. Biomarkers analysis on samples from patients in EVT801 clinical trial: Patient characterization and immunomonitoring [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 1059.

Abstract 446: Integrated spatial transcriptomics and lipidomics analyses of intraductal papillary mucinous neoplasm identifies enrichment of long chain sulfatide as an early metabolic alteration

Abstract Purpose: To define the metabolic alterations associated with malignant progression of intraductal papillary mucinous neoplasm (IPMN). Experimental Procedures: Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) was conducted for spatial characterization of lipid profiles on frozen tissue sections from resected human IPMN patients. The specimen set consisted of either low-grade dysplasia (LG) (n=15), high-grade (HG) dysplasia (n=4), or with an associated pancreatic ductal adenocarcinoma (PDAC) (n=4) as well as 15 PDAC tissues. Utilizing serial sections to the same human IPMN cases analyzed with MALDI-MS imaging, we used the Visium Spatial Gene Expression technology platform to characterize the spatiomolecular underpinnings of LG and HG IPMN. H&E images of the same tissue section used for the Visium workflow, were used to annotate the spots in the dataset covering the epithelial lining of the IPMN lesions (“epilesional” areas) and the spots covering the adjacent microenvironment surrounding the lesion (“perilesiopecific” area). Findings were compared with lipidomic analyses of cystic fluid from 89 IPMN patients. The anti-cancer efficacy of UGT8-IN-1, a selective small molecule inhibitor of ceramide galactosyltransferase (CGT, also known as UGT8), was assessed in vitro and in mutant Kras;Gnas allografts. Results: MALDI/MS-based spatial imaging of human resected IPMN tissues and pancreata from a mutant Kras;Gnas mouse model of IPMN revealed long-chain hydroxylated sulfatides, particularly the C24:0(OH) and C24:1(OH) species, to be selectively enriched in the IPMN and PDAC neoplastic epithelium. Visium spatial transcriptomics data confirmed that the cognate transcripts engaged in sulfatide biosynthesis, including UGT8, Gal3St1, and FA2H, were co-localized with areas of sulfatide enrichment. Lipidomic analyses of cystic fluid from 89 patients with IPMN identified several sulfatide species to be significantly elevated in patients with IPMN/PDAC compared to those with low-grade IPMN. Inhibition of sulfatide metabolism via UGT8-IN-1 reduced viability of Kras;Gnas murine cell lines in vitro and attenuated tumor growth in an allograft IPMN model. UGT8-IN-1 treatment resulted in reduced levels of several sulfatide species, accompanied by a concomitant increase in a subset of quantified sulfatides precursor ceramides, accumulation of mitochondrial mass and reactive oxygen species and time-dependent increases in autophagy/mitophagy markers p62/SQSTM1 and LC-3B, suggesting impaired mitophagy. Conclusion: Enhanced sulfatide metabolism is an early alteration in cystic pre-cancerous lesions of the pancreas that persists through invasive neoplasia. Targeting sulfatide biosynthesis might represent an actionable vulnerability for cancer interception. Citation Format: Yihui Chen, Marta Sans, Fredrik Thege, Rongzhang Dou, Jimin Min, Michele Yip-Schneider, Jianjun Zhang, Ranran Wu, Ehsan Irajizad, Yuki Makino, Kimal Rajapakshe, Mark Hurd, Ricardo León-Letelier, Jody Vykoukal, Jennifer Dennison, Kim-Anh Do, Robert Wolff, Paola Guerrero, Michael Kim, Max Schmidt, Anirban Maitra, Samir Hanash, Johannes Fahrmann. Integrated spatial transcriptomics and lipidomics analyses of intraductal papillary mucinous neoplasm identifies enrichment of long chain sulfatide as an early metabolic alteration [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 446.

Abstract 720: Combining zelasudil, a small molecule ROCK2 inhibitor, with chemotherapy or immunotherapy improves response in preclinical models of pancreatic cancer

Abstract Zelasudil (RXC007) is an orally available, highly selective small molecule inhibitor of Rho-associated coiled-coil containing protein kinase 2 (ROCK2), which has demonstrated pre-clinical anti-fibrotic efficacy and is currently being tested in a Phase 2a clinical trial for the treatment of idiopathic pulmonary fibrosis (IPF) (NCT05570058). Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and therapeutically challenging disease with a dense and highly fibrotic tumor microenvironment which may limit efficacy of conventional therapies. Here, we investigate the benefit of combining an anti-fibrotic, RXC007, with either standard of care chemotherapy or immunotherapy in mouse models of PDAC.In a fibrotic patient-derived metastatic PDAC orthotopic model, RXC007 (10mg/kg, 50mg/kg and 100mg/kg BID 5d on/2d off PO) in combination with SoC chemotherapy Gemcitabine (100mg/kg IP QW)/Abraxane (30 mg/kg IP QW) improved survival in a dose-dependent manner (all P<0.0001) compared to Gemcitabine/Abraxane alone.In the immunocompetent orthotopic LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) model of metastatic PDAC, survival was significantly increased (P<0.0001) upon treatment with a combination of RXC007 (50 mg/kg BID 5d on/2d off PO) and anti-PD1 (200 ug BIW) compared to anti-PD1 alone. ROCK2 inhibition elicited positive immunomodulatory effects in KPC pancreatic tumors, with increase of CD8+ (P=0.0031) and CD4+ (P=0.008) T cell infiltrate into the tumor cortex and reduction in immunosuppressive FOXP3+ regulatory T cells at the tumor border. Furthermore, RXC007 monotherapy exhibited anti-fibrotic effects, via a decrease in collagen content and organization, effects associated with CAF reprogramming and reduced intra-tumoral aSMA+ PDPN+ myofibroblast-like CAFs (myCAFs; P=0.0406).This data highlights the potential for treating highly fibrotic tumours with RXC007 in combination with SoC chemotherapy or immunotherapy in order to improve patient outcomes in this aggressive and treatment-refractory cancer. Citation Format: Marina Pajic, Benjamin McLean, Diego Chacon Fajardo, Sean Porazinski, Dannielle H. Upton, Diana Schuhmacher, Aji Istadi, Thomas Cox, Paul Timpson, Daniel J. Wilcock, Katie J. Anderson, Helen Mason, Nicolas E. Guisot, Clifford D. Jones, Caroline Phillips, Richard Armer. Combining zelasudil, a small molecule ROCK2 inhibitor, with chemotherapy or immunotherapy improves response in preclinical models of pancreatic cancer [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 720.

Abstract 613: Oral CHK1 inhibitor BBI-355 allows flexibility of dose and schedule with demonstration of monotherapy and combinational antitumor activity in extrachromosomal DNA (ecDNA) driven preclinical models

Abstract Focal, high copy number amplifications of prevalent oncogenes (e.g., MYC, EGFR) frequently occur on extrachromosomal DNA (ecDNA), highly transcribed units of circular, non-chromosomal DNA. While use of targeted therapies is associated with improved survival in patients with cancers that have activating oncogene mutations or fusions, they have demonstrated limited activity for patients whose tumors harbor oncogene amplifications, including those mediated by ecDNA. We have previously identified Checkpoint Kinase 1 (CHK1) as a target essential for ecDNA function in cancer, as tumor cells with oncogene amplification on ecDNA exhibit increased DNA replication stress (RS) and are hypersensitive to inactivation of CHK1, cells’ master regulator of RS. Thus, we have developed BBI-355, a novel, orally bioavailable, and selective small molecule inhibitor of CHK1, which is currently being studied in the first-in-human Phase 1/2 POTENTIATE clinical trial for patients with cancer harboring oncogene amplifications, including on ecDNA (NCT05827614). In vitro antiproliferation activity of BBI-355 was observed in a panel of ecDNA+ oncogene amplified tumor lines and non-amplified lines, with heightened sensitivity and induction of RS observed in ecDNA+ cells. Oral administration of BBI-355 resulted in dose dependent on-target activity, with induced p-CHK1-S345 protein expression, in agreement with measured plasma and tumor concentrations. Subsequent pharmacokinetic/pharmacodynamic (PK/PD) modeling guided continuous and intermittent dose/schedules of single agent BBI-355 that demonstrated durable antitumor activity, including tumor regressions, in multiple ecDNA+ tumor models. Single agent targeted therapy treatment of cancers with oncogene amplification on ecDNA can result in acquired resistance via ecDNA-based mechanisms, further increasing reliance on CHK1. Enhanced antitumor activity of BBI-355 in combination with agents targeting the protein products of amplified oncogenes was demonstrated in multiple in vivo tumor models; these included combination with a pan-FGFR inhibitor in FGFR2 ecDNA+ amplified gastric cancer and combination with CDK4/6 inhibitors in CDK4 ecDNA+ amplified sarcoma models. The first ecDNA-directed therapy (ecDTx) in development, BBI-355 demonstrated significant antitumor activity as a single agent and in combination with targeted therapies in multiple ecDNA+ oncogene amplified tumor models. Oral dosing of BBI-355 provides dose/schedule flexibility to attempt to optimize antitumor activity with a tolerable safety profile in patients with highly aggressive cancers and significant unmet need. The POTENTIATE clinical trial is open and currently enrolling. Citation Format: Ryan J. Hansen, Auzon Steffy, Joshua Plum, Ardalan Ardeshiri, Edison Tse, Salvador Garcia, Ben Norman, Jafar Moininazeri, AnneMarie Pferdekamper, Evan Holmes, Debbie Liao, Rachelle Elsdon, Joshua Lange, Anthony Pinkerton, Julius Apuy, Sudhir Chowdhry, Christian Hassig, Shailaja Kasibhatla. Oral CHK1 inhibitor BBI-355 allows flexibility of dose and schedule with demonstration of monotherapy and combinational antitumor activity in extrachromosomal DNA (ecDNA) driven preclinical models [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 613.

Abstract 594: ORIC-613, a potential first- and best-in-class, orally bioavailable, potent and selective PLK4 inhibitor with synthetic lethality in TRIM37 high cancer models

Abstract A synthetic lethal interaction between polo-like kinase 4 (PLK4) and elevated TRIM37 was discovered in breast cancer and neuroblastoma (Meitinger et al. 2020; Yeow et al. 2020). High TRIM37 renders cells dependent on PLK4, a kinase that controls centriole duplication. Amplification and copy number gains of the 17q23 amplicon, where TRIM37 resides, are common in breast cancer and neuroblastoma and associated with early relapse and poor prognosis, making PLK4 a promising new synthetic lethal target for these patients. The development candidate ORIC-613 is an orally bioavailable, potent and exquisitely selective small molecule inhibitor of PLK4, which is synthetic lethal in TRIM37 high cells. ORIC-613 is highly selective against the kinome, including against the closely related aurora and PLK families. ORIC-613 ADME/PK profile includes high cell permeability and low potential for drug-drug interactions based on CYP profiling. ORIC-613 blocked PLK4 trans-autophosphorylation of phosphodegrons, leading to stabilization of PLK4, thus directly demonstrating target engagement in cells. PLK4 protein stabilization correlated with cell viability for ORIC-613, providing a quantifiable pharmacodynamic (PD) association with antitumor activity. Cell viability assessment in cancer cell lines revealed that ORIC-613 showed greater potency in TRIM37 high versus TRIM37 low cell lines. Importantly, ORIC-613 induced significantly greater apoptosis in TRIM37 high versus low cancer cell lines as measured by caspase 3/7 assay. We confirmed that ORIC-613 activity is on-target using an engineered cell line system of PLK4 G95L, in which the leucine mutation maintains enzymatic function but blocks compound binding. In cell viability assays, ORIC-613 was potent in the parental cells and lost activity in G95L cells. Analysis of genomic data from tumors indicates that TRIM37 copy number amplification and gain is prevalent across numerous cancers. Oral dosing of ORIC-613 resulted in tumor growth inhibition and regressions in TRIM37 high xenograft breast tumors with no body weight loss. In summary, ORIC-613 is a potential first- and best-in-class development candidate, with exquisite selectivity for PLK4, which demonstrates synthetic lethality in TRIM37 high tumors and has the potential to benefit these patients. Citation Format: Kyle A. Edgar, Siobhan McRee, Gina Andretta, Chelsea Chen, Christophe Colas, Wei Fang, Wayne Kong, Jason Long, Fang Liu, Jared Moore, Aleskandr Pankov, Dan Shore, Joanne Tan, Robert Warne, Livia Ulicna, Rakesh Vekariya, Anneleen Daemen, F Romero, Melissa R. Junttila, Lori S. Friedman. ORIC-613, a potential first- and best-in-class, orally bioavailable, potent and selective PLK4 inhibitor with synthetic lethality in TRIM37 high cancer models [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 594.

Abstract 3337: Inhibition of KIF18A leads to mitotic arrest and robust anti-tumor activity in chromosomally instable tumors

Abstract KIF18A is a plus-end directed kinesin known to play a role in mitosis by facilitating chromosome alignment and spindle microtubule dynamics. Knockdown or knockout of KIF18A has been shown to inhibit proliferation in cells with whole genome doubling or aneuploidy, cellular states characterized by chromosomal rearrangements. These alterations render cells particularly vulnerable to disrupted mitosis upon KIF18A loss. As such, KIF18A is a compelling target in a subset of tumors with chromosomal instability (CIN), which have ongoing chromosomal segregation defects. We have identified potent and selective small molecule inhibitors of KIF18A. A proprietary tool compound, ATX-21020, inhibits KIF18A ATPase activity with an IC50 of 14.5 nM; KIF18A is selectively inhibited over other kinesins including CENPE (IC50 > 10 µM) and EG5 (IC50 5.87 µM). ATX-21020 exhibits robust cellular activity, with anti-proliferative activity observed in chromosomally instable ovarian cancer cell lines such as OVCAR-3 (IC50 53.3 nM) and OVCAR-8 (IC50 0.54 µM). Upon treatment with ATX-21020, Annexin V is robustly induced in sensitive cell lines, demonstrating that KIF18A-dependent cell lines undergo apoptosis upon KIF18A inhibition. In KIF18Ai sensitive cells, a dose-dependent upregulation of phospho-Histone H3 and gamma-H2AX is observed upon ATX-21020 treatment in OVCAR-3 and OVCAR-8 cells, consistent with the induction of mitotic arrest and DNA damage. In contrast, CIN negative Ovarian cancer cells that are insensitive to KIF18A loss, such as A2780 and OVK18, proliferate normally upon ATX-21020 treatment, and Annexin V, phospho-Histone H3, and gamma-H2AX are not induced. In sensitive cell lines, ATX-21020 induces a dose-dependent cell cycle arrest in G2/M, consistent with the role of KIF18A in facilitating mitosis. OVCAR-3 cells treated with ATX-21020 exhibit fragmented nuclei and malformed mitotic spindles, in contrast to A2780 cells where cell division is unaffected, suggesting that KIF18A is dispensable in CIN negative cells. This selective dependency is also observed in vivo, where administration of ATX-21020 leads to dose-dependent tumor growth inhibition in an OVCAR-3 cancer cell line-derived xenograft model, with significant tumor regression observed at 100 mpk/day. In contrast, OVK18 tumors are unaffected by ATX-21020 administration, as expected based on the lack of cell cycle and proliferation effects in that cell line. Together, these data demonstrate a critical role for KIF18A in facilitating mitosis in a subset of chromosomally instable cells. These effects are selective, with KIF18A-independent cell lines proceeding through the cell cycle normally in the presence of inhibitors. Small molecule inhibition of KIF18A is therefore expected to have robust efficacy in solid tumors with high levels of chromosomal instability such as Ovarian cancer. Citation Format: Maureen S. Lynes, Sanjoy Khan, Taylor Hotz, Brian A. Sparling, Mary-Margaret Zablocki, Deepali Gotur, P Ann Boriack-Sjodin, Kenneth W. Duncan, Stephen J. Blakemore, Serena J. Silver, Robert A. Copeland. Inhibition of KIF18A leads to mitotic arrest and robust anti-tumor activity in chromosomally instable tumors [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 3337.

Integrated Proteomics Characterization of NLRP3 Inflammasome Inhibitor MCC950 in Monocytic Cell Line Confirms Direct MCC950 Engagement with Endogenous NLRP3.

Inhibition of the NLRP3 inflammasome is a promising strategy for the development of new treatments for inflammatory diseases. MCC950 is a potent and selective small-molecule inhibitor of the NLRP3 pathway and has been validated in numerous species and disease models. Although the capacity of MCC950 to block NLRP3 signaling is well-established, it is still critical to identify the mechanism of action and molecular targets of MCC950 to inform and derisk drug development. Quantitative proteomics performed in disease-relevant systems provides a powerful method to study both direct and indirect pharmacological responses to small molecules to elucidate the mechanism of action and confirm target engagement. A comprehensive target deconvolution campaign requires the use of complementary chemical biology techniques. Here we applied two orthogonal chemical biology techniques: compressed Cellular Thermal Shift Assay (CETSA) and photoaffinity labeling chemoproteomics, performed under biologically relevant conditions with LPS-primed THP-1 cells, thereby deconvoluting, for the first time, the molecular targets of MCC950 using chemical biology techniques. In-cell chemoproteomics with inlysate CETSA confirmed the suspected mechanism as the disruption of inflammasome formation via NLRP3. Further cCETSA (c indicates compressed) in live cells mapped the stabilization of NLRP3 inflammasome pathway proteins, highlighting modulation of the targeted pathway. This is the first evidence of direct MCC950 engagement with endogenous NLRP3 in a human macrophage cellular system using discovery proteomics chemical biology techniques, providing critical information for inflammasome studies.

Asymmetric Synthesis of CIDD-0072424 via an Enantioselective Nitro-Mannich Reaction: A Central Nervous System Penetrant, Selective Small Molecule Inhibitor of Protein Kinase C Epsilon.

CIDD-0072424 is a novel small molecule developed in silico with remarkable activity for the inhibition of protein kinase C (PKC)-epsilon to treat alcohol use disorder. We developed a concise synthesis of (S)-2 that is highly enantioselective, scalable, and amenable for 3-point structure-activity relationship (SAR) studies for compound optimization. The highly enantioselective nitro-Mannich reaction was achieved through a dual-reagent catalysis system. The overall utility and the efficiency of the enantioselective route provided a scalable synthesis of both PKCε inhibitors 1 and 2.

Discovery of a Potent and Cell-Active Inhibitor of DNA 6mA Demethylase ALKBH1.

N6-Methyladenine (6mA) of DNA has emerged as a novel epigenetic mark in eukaryotes, and several 6mA effector proteins have been identified. However, efforts to selectively inhibit the biological functions of these effector proteins with small molecules are unsuccessful to date. Here we report the first potent and selective small molecule inhibitor (13h) of AlkB homologue 1 (ALKBH1), the only validated 6mA demethylase. 13h showed an IC50 of 0.026 ± 0.013 μM and 1.39 ± 0.13 μM in the fluorescence polarization (FP) and enzyme activity assay, respectively, and a KD of 0.112 ± 0.017 μM in the isothermal titration calorimetry (ITC) assay. The potency of 13h was well explained by the cocrystal structure of the 13h-ALKBH1 complex. Furthermore, 13h displayed excellent selectivity for ALKBH1. In cells, compound 13h and its derivative 16 were able to engage ALKBH1 and modulate the 6mA levels. Collectively, our study identified the first potent, isoform selective, and cell-active ALKBH1 inhibitor, providing a tool compound for exploring the biological functions of ALKBH1 and DNA 6mA.

Discovery of a selective TRF2 inhibitor FKB04 induced telomere shortening and senescence in liver cancer cells.

Telomere repeat binding factor 2 (TRF2), a critical element of the shelterin complex, plays a vital role in the maintenance of genome integrity. TRF2 overexpression is found in a wide range of malignant cancers, whereas its down-regulation could cause cell death. Despite its potential role, the selectively small-molecule inhibitors of TRF2 and its therapeutic effects on liver cancer remain largely unknown. Our clinical data combined with bioinformatic analysis demonstrated that TRF2 is overexpressed in liver cancer and that high expression is associated with poor prognosis. Flavokavain B derivative FKB04 potently inhibited TRF2 expression in liver cancer cells while having limited effects on the other five shelterin subunits. Moreover, FKB04 treatment induced telomere shortening and increased the amounts of telomere-free ends, leading to the destruction of T-loop structure. Consequently, FKB04 promoted liver cancer cell senescence without modulating apoptosis levels. In corroboration with these findings, FKB04 inhibited tumor cell growth by promoting telomeric TRF2 deficiency-induced telomere shortening in a mouse xenograft tumor model, with no obvious side effects. These results demonstrate that TRF2 is a potential therapeutic target for liver cancer and suggest that FKB04 may be a selective small-molecule inhibitor of TRF2, showing promise in the treatment of liver cancer.