Top1 Inhibitors Research Articles

Crossing the border: Replication fork adducts move to lysosomes for autophagic repair

In a recent study in Cell, Lascaux etal.1 implicate TEX264 in the autophagy-driven resolution of nuclear topoisomerase 1 cleavage complexes (TOP1cc) in lysosomes, altering current concepts on the mechanism of action for clinically relevant doses of TOP1 inhibitors.

A phase I study of irinotecan combined with BAY1895344 (ATR inhibitor) in advanced solid tumors: Results of ETCTN 10402 dose escalation.

3077 Background: ATR protein kinase is activated by replication stress and recruited to stalled forks or single strand DNA defects in various cancers. The topoisomerase-I (Top1) inhibitor irinotecan induces DNA damage. The ATR inhibitor BAY1895344 (elimusertib) has demonstrated cytotoxic potential in SCLC and GI cancer xenografts when combined with Top1 inhibitors . Methods: This phase Ia trial assessed elimusertib in combination with irinotecan in adult patients with refractory advanced solid tumors for whom irinotecan can be considered standard care. Patients with previous irinotecan exposure were excluded. Two dosing schedules were used: 1) Biweekly - irinotecan IV (starting at 150 mg/m2) D1 + elimusertib (starting dose 20 mg PO BID) D1,D2 (cycle=14 days); 2) Weekly - irinotecan (starting at 50 mg/m2) IV on D1,8,15 with elimusertib (starting at 20 mg PO daily) on D2,D3,D9,D10 and D16,D17 (cycle=21 days). Dose escalation utilized a 3+3 design. Primary objectives were to assess safety and tolerability and estimate the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). Secondary objectives included estimating pharmacokinetic (PK) profiles and assessing anti-tumor activity. Results: A total of 21 patients were enrolled in dose escalation (N=9 [Biweekly] and N=12 [Weekly], median age 58 and 2 lines of prior therapy. For the Biweekly cohort: 3/3 patients enrolled at dose level (DL) 1 experienced hematologic dose-limiting toxicity (DLT); 6 patients received DL-1 without any DLTs. The weekly regimen escalation comprised of 12 patients: 6 enrolled in DL1 with 2/6 having hematologic DLT and 5/6 unable to complete ≥75% of cycle 1 dosing; 6 enrolled in DL-1 with no DLTs observed. Notable grade 3+ treatment-related adverse events and summary best response are summarized (Table 1). The majority of evaluable patients in both schedules had initial disease control (56% [biweekly] and 67% [weekly]). One confirmed partial response was seen in the biweekly RP2D. Median progression-free survival was 2.1 mo [biweekly] and 2.5 mo [weekly]. PK results will be presented at time of meeting. Conclusions: Both RP2D and MTD of elimusertib in combination with irinotecan were reached for both dosing schedules: irinotecan 150 mg/m2 IV D1 + elimusertib 10 mg BID PO D1,D2 (biweekly) and irinotecan 25 mg/m2 IV on D1 + elimusertib (20 mg PO daily) on D2,D3 (weekly). Dose escalation was notably limited by myelotoxicity. Due to sponsor decision, the study was halted prior to planned expansion but the concept of ATR + topo I combination remains scientifically relevant. Clinical trial information: NCT04514497 . [Table: see text]

A phase I study of ATR inhibitor BAY1895344 (elimusertib) plus topotecan (ETCTN 10402): Results of dose escalation.

3076 Background: Ataxia telangiectasia and Rad3-related (ATR) protein kinase is activated by replication stress and recruited to stalled forks or single strand DNA abnormalities in various cancers. The topoisomerase-I (Top1) inhibitor topotecan induces DNA damage. The ATR inhibitor BAY 1895344 (elimusertib) has demonstrated cytotoxic potential in small cell lung cancer and gastrointestinal cancer xenografts when combined with Top1 inhibitors . Methods: This is phase Ia study of elimusertib combined with topotecan in adult patients with refractory advanced solid tumors for whom topotecan can be considered as part of standard care. Patients with previous topotecan exposure were excluded. The study combination was assessed starting at Topotecan 1 mg/m2 IV (D1-D5) plus elimusertib 20 mg BID (D2, D5) (Cycle=21 days). Dose escalation utilized a 3+3 design. Primary objectives were to assess safety and tolerability and estimate the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of the combination. Secondary objectives included estimating pharmacokinetic (PK) profiles and assessing anti-tumor activity of the combinations. Results: Eight patients were treated in dose escalation. Participants were a median of 63 years old and had received 2 median lines of previous therapy. The initial two patients enrolled into dose level (DL) 1 had dose-limiting toxicities with one of patient experiencing respiratory failure and cardiac arrest in setting of pancytopenia related to study drug. Six patients were subsequently enrolled in and received DL-1 (elimusertib decreased from 20 mg BID to 20 mg Daily) and no DLTs were observed. Notable grade 3+ treatment-related adverse events and summary best response are summarized in Table. Disease control rate was 43% among evaluation patient including one unconfirmed partial response. Median progression-free survival in the RP2D cohort was 3.45 months. PK studies are in process and results will be presented at time of meeting. Conclusions: RP2D and MTD was established for elimusertib in combination with Topotecan: Topotecan 1 mg/m2 IV [D1-D5] plus elimusertib 20 mg Daily [D2, D5] every 3 weeks. Dose escalation was notably limited by myelotoxicity. Due to sponsor decision, the study was halted prior to planned expansions cohorts but the concept of ATR + topo I inhibition remains relevant. Clinical trial information: NCT04514497 . [Table: see text]

Phase II clinical trial of PLX038 in patients with platinum resistant ovarian, primary peritoneal and fallopian tube cancer.

TPS5630 Background: Platinum-resistant ovarian cancer (PROC) portends a poor prognosis and, although treatments are available, response rates are low. Previous studies have demonstrated that DNA topoisomerase I (TOP1) inhibitors exhibit clinical activity in PROC. Irinotecan also has a 15-25% response rate in some studies of platinum-resistant epithelial ovarian cancer but does not have FDA approval for this indication. Additional studies in preclinical models suggest that TOP1 poisons are more active if administered at low doses on prolonged schedules. PLX038 is a long-acting prodrug of the potent TOP1 inhibitor SN-38. In PLX038, SN-38 is covalently conjugated to circulating polyethylene glycol and is slowly released at a constant rate to provide the active agent with a long half-life, a low Cmax and very high exposure – important facets for optimal safety and efficacy of this drug. Further, in preclinical studies the nanoparticle prodrug accumulates and is retained in tumors, where it slowly releases the SN-38. Methods: This single arm, phase 2 clinical trial is open at Mayo Clinic in Rochester, MN. The primary endpoint is overall response rate and secondary endpoints include progression-free survival and assessment of tolerability of PLX038. Correlative goals include i) measurement of TOP1-DNA covalent complexes in tumor biopsies and circulating tumor cells to assess successful target engagement; ii) assessment of homologous recombination status and SLFN11 expression for correlation with tumor response rate; and iii) pharmacokinetics of SN-38 and SN-38G as well as their association with GI toxicity. Assessment of gut microbiota and association with GI toxicity profile are also being investigated. Eligible patients include women with platinum resistant high grade serous ovarian, primary peritoneal, and fallopian tube cancer who have received fewer than two prior chemotherapy regimens in the platinum-resistant setting. All patients must have an ECOG performance status of 0, 1, or 2 and adequate bone marrow, renal, and hepatic function. As of January 31, 2024, 12 of planned 37 patients have enrolled. Clinical trial information: NCT05465941 .

Tazemetostat in combination with topotecan and pembrolizumab in patients with recurrent small cell lung cancer.

TPS8130 Background: Small-cell lung cancer (SCLC) is the most fatal type of lung cancer characterized by exquisite chemo-sensitivity at diagnosis and chemoresistance at relapse. Despite a highly mutated genome, patients with SCLC derive little benefit from immunotherapy. EZH2 (enhancer of zeste homolog 2) is a master epigenetic regulator of SCLC neuroendocrine cell fate and plasticity. EZH2 inhibition 1) promotes upregulation of Schlafen 11 (SLFN11) which irreversibly blocks replication in response to DNA damaging agents and 2) enhances intrinsic immune signaling, leading to constitutive MHC I recovery, sensitizing resistant SCLC models to DNA damaging chemotherapy and immunotherapy. Tazemetostat is a selective oral EZH2 inhibitor. Methods: This is an investigator-initiated, NCI Cancer Therapy Evaluation Program (CTEP) sponsored, phase I dose escalation and dose expansion study which will evaluate safety and tolerability of combination of tazemetostat with topotecan, a selective TOP1 inhibitor, and programmed cell death protein 1 (PD-1) inhibitor antibody pembrolizumab. Adult patients with relapsed/recurrent SCLC after at least platinum doublet (limited stage-SCLC) or chemo-immunotherapy (extensive stage-SCLC) and ECOG performance 0-1 are eligible. The regimen design involves a 7-day “run-in” of oral tazemetostat BID followed by 21-day cycles of tazemetostat (1-21 days), intravenous (IV) topotecan (day 1-5) and IV pembrolizumab (Day 1). The dose escalation cohort aims to determine safety and optimal doses of tazemetostat and topotecan (with standard dose of pembrolizumab) using a 3+3 design by assessing for dose limiting toxicities. The dose expansion cohort aims to assess safety, tolerability and preliminary efficacy of the combination in 15 additional patients with relapsed SCLC. The study involves collection of mandatory biopsies at pre-treatment and post-treatment (cycle 1) to gain insights into mechanism of action and resistance of the combination using single cell and spatial transcriptomic approaches. For more questions regarding enrollment and eligibility please contact Rasa.vilimas@nih.gov or anish.thomas@nih.gov . Clinical trial information: NCT05353439 .

Synergistic Nanomedicine Delivering Topoisomerase I Toxin (SN-38) and Inhibitors of Polynucleotide Kinase 3'-Phosphatase (PNKP) for Enhanced Treatment of Colorectal Cancer.

Inhibitors of a DNA repair enzyme known as polynucleotide kinase 3'-phosphatase (PNKP) are expected to show synergistic cytotoxicity in combination with topoisomerase I (TOP1) inhibitors in cancer. In this study, the synergistic cytotoxicity of a novel inhibitor of PNKP, i.e., A83B4C63, with a potent TOP1 inhibitor, i.e., SN-38, against colorectal cancer cells was investigated. Polymeric micelles (PMs) for preferred tumor delivery of A83B4C63, developed through physical encapsulation of this compound in methoxy poly(ethylene oxide)-poly(α-benzyl carboxylate-ε-caprolactone) (mPEO-b-PBCL) micelles, were combined with SN-38 in free or PM form. The PM form of SN-38 was prepared through chemical conjugation of SN-38 to the functional end group of mPEO-b-PBCL and further assembly of mPEO-b-PBCL-SN-38 in water. Moreover, mixed micelles composed of mPEO-b-PBCL and mPEO-b-PBCL-SN-38 were used to co-load A83B4C63 and SN-38 in the same nanoformulation. The loading content (% w/w) of the SN-38 and A83B4C63 to mPEO-b-PBCL in the co-loaded formulation was 7.91 ± 0.66 and 16.13 ± 0.11% (w/w), respectively, compared to 15.67 ± 0.34 (% w/w) and 23.06 ± 0.63 (% w/w) for mPEO-b-PBCL micelles loading individual drugs. Notably, the average diameter of PMs co-encapsulating both SN-38 and A83B4C63 was larger than that of PMs encapsulating either of these compounds alone but still lower than 60 nm. The release of A83B4C63 from PMs co-encapsulating both drugs was 76.36 ± 1.41% within 24 h, which was significantly higher than that of A83B4C63-encapsulated micelles (42.70 ± 0.72%). In contrast, the release of SN-38 from PMs co-encapsulating both drugs was 44.15 ± 2.61% at 24 h, which was significantly lower than that of SN-38-conjugated PMs (74.16 ± 3.65%). Cytotoxicity evaluations by the MTS assay as analyzed by the Combenefit software suggested a clear synergy between PM/A83B4C63 (at a concentration range of 10-40 μM) and free SN-38 (at a concentration range of 0.001-1 μM). The synergistic cytotoxic concentration range for SN-38 was narrowed down to 0.1-1 or 0.01-1 μM when combined with PM/A83B4C63 at 10 or 20-40 μM, respectively. In general, PMs co-encapsulating A83B4C63 and SN-38 at drug concentrations within the synergistic range (10 μM for A83B4C63 and 0.05-1 μM for SN-38) showed slightly less enhancement of SN-38 anticancer activity than a combination of individual micelles, i.e., A83B4C63 PMs + SN-38 PMs at the same molar concentrations. This was attributed to the slower release of SN-38 from the SN-38 and A83B4C63 co-encapsulated PMs compared to PMs only encapsulating SN-38. Cotreatment of cells with TOP1 inhibitors and A83B4C63 formulation enhanced the expression level of γ-HA2X, cleaved PARP, caspase-3, and caspase-7 in most cases. This trend was more consistent and notable for PMs co-encapsulating both A83B4C63 and SN-38. The overall result from the study shows a synergy between PMs of SN-38 and A83B4C63 as a mixture of two PMs for individual drugs or PMs co-encapsulating both drugs.

Crystal Structures, Molecular Docking and In Vitro Investigations of Two 4-Substituted 2-(5,5-dimethyl-3-styrylcyclohex-2-enylidene)malononitrile Derivatives as Potential Topoisomerase II Inhibitors

Type II topoisomerases (TOP2s) play a key role in altering the DNA topology by transiently cleaving both strands of a DNA duplex. Therefore, increased TOP2 activity is associated with many cancers. Herein, we present the synthesis, structural characterization, virtual screening, and structural exploration, as well as evaluation of the antiproliferative effects of two new 4-substituted 2-(5,5-dimethyl-3-styrylcyclohex-2-enylidene)malononitrile derivatives with potential application in the drug design of isoform-specific TOP2 inhibitors. Both compounds 1 and 2 were verified by ESI-TOF-MS, NMR, and single-crystal X-ray diffraction (SCXRD) analysis. Furthermore, we applied our recently proposed SCXRD/HYdrogen DEsolvation (HYDE) technology platform in order to perform molecular modeling, virtual screening, and structural exploration with 1 and 2. For this purpose, we used the crystal structure of human TOP2β complexed to DNA and the anticancer drug etoposide. Moreover, we further evaluated the antiproliferative activity of 1 and 2 on human hepatocarcinoma HepG2 cells and compared the observed effects with those of the reference hTOP2β inhibitor etoposide. Based on the obtained results, compounds 1 and 2 showed a virtually higher binding affinity (Ki HYDE values) over etoposide towards hTOP2β but lower antiproliferative activity compared to those of etoposide.

Abstract B027: The MAT2A inhibitor IDE397: a novel combination backbone for urothelial cancer subjects with MTAP deficiency

Abstract Methylthioadenosine phosphorylase (MTAP) loss, due to focal homozygous deletions in chromosome 9 (9p21.3), occurs in over 25% of urothelial carcinoma and is an adverse prognostic factor for overall survival. Furthermore, MTAP-deleted tumors are associated with a “cold” tumor immune microenvironment and are resistant to immune checkpoint inhibitor therapy. Loss of MTAP confers a dependency on methionine adenosyltransferase 2A (MAT2A), which catalyzes the production of the primary methyl donor for all cellular methyltransferase reactions, S-adenosyl methionine (SAM). IDE397, a potent small molecule inhibitor of MAT2A, was developed to selectively exploit this synthetic lethal vulnerability in MTAP −/− tumors. The accumulation of the metabolite MTA in MTAP −/− tumors combined with a reduction of SAM by MAT2A inhibition results in selective inhibition of PRMT5, a methyltransferase that governs spliceosome fidelity. This triggers pre-mRNA splicing defects that may induce RNA polymerase stalling and can cause R-loop accumulation. Furthermore, due to essential roles in 1-carbon metabolism, MAT2A inhibition together with MTAP loss leads to depletion of metabolites required for both de novo and salvage nucleotide biosynthesis, thus preventing effective management of DNA replication and DNA damage repair. Notably, R-loops must be resolved by topoisomerase activity (TOP1) to prevent replication conflict and mitotic catastrophe. These combinatorial mechanistic relationships underpin a hypothesis that dual inhibition of MAT2A and TOP1 will fully capitalize on the synthetic lethal vulnerabilities associated with MTAP loss in urothelial cancers (replication stress, genomic instability). Consistent with this, IDE397 in combination with TOP1 inhibitors demonstrated synergistic combination benefit in MTAP −/− bladder cancer cell lines in vitro and in cell-line derived xenograft (CDX) models in vivo. IDE397 exposure was associated with heavily disordered methionine metabolism and accumulation of DNA damage response intermediates. In the ongoing Phase 1 dose escalation/expansion study of IDE397 in MTAP −/− solid tumors (NCT04794699), measurable tumor shrinkage has been observed in urothelial cancers (including a CR per RECIST 1.1) along with molecular responses on serial ctDNA measurements. Supported by our preclinical data, and the clinical observation that MTAP −/− urothelial cancers may be more sensitive to sacituzumab govitecan (SG); a TROP-2-targeting TOP1 ADC (UNITE study; JCO.2023.41.16_suppl.4572), a combination of SG+IDE397 could represent a novel early-line therapy for these patients with high unmet need. We plan to evaluate the combination of SG+IDE397 in MTAP-/- urothelial cancers in the next stage of the ongoing IDE397 trial (NCT04794699). Citation Format: Claire L. Neilan, Marcus M. Fischer, Damien Garbett, Arjun A. Rao, Mili Mandal, Michael A. White, Jasgit C. Sachdev. The MAT2A inhibitor IDE397: a novel combination backbone for urothelial cancer subjects with MTAP deficiency [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B027.

Nuclear-penetrating scleroderma autoantibody inhibits topoisomerase 1 cleavage complex formation

The contributions of anti-Topoisomerase 1 (Top1) autoantibodies to the pathophysiology of diffuse cutaneous systemic sclerosis (dcSSc), the most aggressive scleroderma subtype, are unknown. Top1 catalyzes DNA relaxation and unwinding in cell nuclei, a site previously considered inaccessible to antibodies. The discovery of autoantibodies in systemic lupus erythematosus that penetrate nuclei and inhibit DNA repair raised the possibility that nuclear-penetrating autoantibodies contribute to mechanisms of autoimmunity. Here we show that an anti-Top1 autoantibody produced by a single B cell clone from a patient with dcSSc penetrates live cells and localizes into nuclei. Functionally, the autoantibody inhibits formation of the Top1 cleavage complex necessary for DNA nicking, which distinguishes it from cytotoxic camptothecin Top1 inhibitors used in cancer therapy that trap the cleavage complex rather than preventing its formation. Discovery of a patient-derived cell-penetrating scleroderma anti-Top1 autoantibody that inhibits Top1 cleavage complex formation supports the hypothesis that anti-Top1 autoantibodies contribute to cellular dysfunction in dcSSc and offers a valuable antibody reagent resource for future studies on anti-Top1 autoantibody contributions to scleroderma pathophysiology.

Identification of FTY720 and COH29 as novel topoisomerase I catalytic inhibitors by experimental and computational studies

The development of novel topoisomerase I (TOP1) inhibitors is crucial for overcoming the drawbacks and limitations of current TOP1 poisons. Here, we identified two potential TOP1 inhibitors, namely, FTY720 (a sphingosine 1-phosphate antagonist) and COH29 (a ribonucleotide reductase inhibitor), through experimental screening of known active compounds. Biological experiments verified that FTY720 and COH29 were nonintercalative TOP1 catalytic inhibitors that did not induce the formation of DNA-TOP1 covalent complexes. Molecular docking revealed that FTY720 and COH29 interacted favorably with TOP1. Molecular dynamics simulations revealed that FTY720 and COH29 could affect the catalytic domain of TOP1, thus resulting in altered DNA-binding cavity size. The alanine scanning and interaction entropy identified Arg536 as a hotspot residue. In addition, the bioinformatics analysis predicted that FTY720 and COH29 could be effective in treating malignant breast tumors. Biological experiments verified their antitumor activities using MCF-7 breast cancer cells. Their combinatory effects with TOP1 poisons were also investigated. Further, FTY720 and COH29 were found to cause less DNA damage compared with TOP1 poisons. The findings provide reliable lead compounds for the development of novel TOP1 catalytic inhibitors and offer new insights into the potential clinical applications of FTY720 and COH29 in targeting TOP1.

Minocycline and photodynamic priming significantly improve chemotherapy efficacy in heterotypic spheroids of pancreatic ductal adenocarcinoma

The prognosis for patients with advanced-stage pancreatic ductal adenocarcinoma (PDAC) remains dismal. It is generally accepted that combination cancer therapies offer the most promise, such as Folforinox, despite their associated high toxicity. This study addresses the issue of chemoresistance by introducing a complementary dual priming approach to attenuate the DNA repair mechanism and to improve the efficacy of a type 1 topoisomerase (Top1) inhibitor. The result is a regimen that integrates drug-repurposing and nanotechnology using 3 clinically relevant FDA-approved agents (1) Top1 inhibitor (irinotecan) at subcytotoxic doses (2) benzoporphyrin derivative (BPD) as a photoactive molecule for photodynamic priming (PDP) to improve the delivery of irinotecan within the cancer cell and (3) minocycline priming (MNP) to modulate DNA repair enzyme Tdp1 (tyrosyl-DNA phosphodiesterase) activity. We demonstrate in heterotypic 3D cancer models that incorporate cancer cells and pancreatic cancer-associated fibroblasts that simultaneous targeting of Tdp1 and Top1 were significantly more effective by employing MNP and photoactivatable multi-inhibitor liposomes encapsulating BPD and irinotecan compared to monotherapies or a cocktail of dual or triple-agents. These data are encouraging and warrant further work in appropriate animal models to evolve improved therapeutic regimens.

Abstract 3148: ABBV-706 is a novel SEZ6-targeted topoisomerase 1 inhibitor ADC for SCLC and other neuroendocrine cancers

Abstract Lung cancer has the highest rates of incidence (2.1 million cases/year) and mortality (1.8 million deaths/year) among all cancers worldwide. Approximately 15% of lung cancers are small cell lung cancer (SCLC). SCLC is typically diagnosed at late stages, with approximately 70% of patients having extensive stage disease at time of diagnosis. Unfortunately, these patients have a dismal 5-year survival rate of approximately 3%. In contrast to non-small cell lung cancer (NSCLC), there has been little advancement in the treatment of SCLC, with no biomarker directed therapies available for patients. SEZ6 is overexpressed in most SCLC’s and other neuroendocrine tumor types. This overexpression and limited normal tissue expression suggest SEZ6 would be a good target for the development of an antibody drug conjugate (ADC). Validation of SEZ6 as an ADC target was supported by signs of clinical activity for ABBV-011, a SEZ6-targeting calicheamicin ADC. Preclinical and clinical data suggest an increased therapeutic index for Top1i ADCs relative to calicheamicin ADCs. Furthermore, Top1i inhibition is an effective mechanism of action in SCLC with topotecan approved in 2L, although significant toxicity limits broad utility. As such, we hypothesized that a SEZ6-Top1i ADC would enable effective Top1 inhibition in tumor cells and reduce toxicity relative to ABBV-011 or systemic chemotherapy, driving increased therapeutic benefit. ABBV-706 is an ADC comprised of the same SEZ6 targeting antibody as ABBV-011 conjugated to AbbVie’s topoisomerase-1 (Top1) inhibitor. ABBV-706 is a potent anti-proliferative agent in SEZ6 positive SCLC cell lines, with sub- to low-nanomolar activity. SEZ6 dependence was demonstrated by the correlation of SEZ6 mRNA expression to ABBV-706 growth inhibition. On-target Top1i activity was confirmed in cells treated with ABBV-706 as measured by dose-dependent induction of γ-H2AX, a marker of DNA damage, and G2/M arrest. ABBV-706 possessed superior anti-tumor activity compared to ABBV-011 in a panel of SCLC cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models with a range of SEZ6 expression levels. In addition, ABBV-706 had activity in the setting of resistance to cisplatin/etoposide (1L SoC in SCLC), in combination with cisplatin, and in SEZ6-expressing neuroendocrine liver and cervical PDX models. ABBV-706 has favorable pharmacokinetics and is well-tolerated in cynomolgus monkeys with bone marrow and gastrointestinal tract toxicities common to other Top1 inhibitors. ABBV-706 shows promise as an agent for the treatment of SCLC and other difficult to treat SEZ6 expressing neuroendocrine tumors. ABBV-706 has completed dose escalation, and dose expansion is currently ongoing in a Phase 1 FIH clinical study (NCT05599984). Citation Format: Emily J. Faivre, Marybeth A. Pysz, Eoghainín Ó hAinmhire, Laura Kreckler, Kelly Doyle, Aloma D'Souza, Zhiyong Ding, Trusha Sondkar, Dolonchampa Maji, Fan Zhao, Andrew Phillips. ABBV-706 is a novel SEZ6-targeted topoisomerase 1 inhibitor ADC for SCLC and other neuroendocrine cancers [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 3148.

Abstract 3130: In vitro characterization of a novel TROP2-targeting antibody-drug conjugate OBI-992

Abstract BACKGROUND TROP2, a transmembrane glycoprotein widely expressed on epithelial cancers, has emerged as an attractive target for antibody-drug conjugate (ADC) development. Sacituzumab govitecan, a TROP2 ADC conjugated to topoisomerase inhibitor (TOP1) SN-38, has been approved for patients with triple-negative breast cancer. This study aims to investigate the characteristics of a novel TROP2 antibody (R4702) and its ADC OBI-992, which is derived from the conjugation of R4702 with a TOP1 inhibitor exatecan. METHODS Clinical study: Prognostic roles of TROP2 and TOP1 in patients were accessed through the “Kaplan-Meier plotter” database from more than 1055 colon cancer patients. In vitro study: An ELISA study was conducted to investigate the binding epitope of R4702. Cell binding assay and Surface Plasmon Resonance were carried out to measure non-specific binding activity and binding affinity, respectively. DNA damage activity, cytotoxicity, and immunogenic cell death (ICD) effects of three TOP1 inhibitors exatecan, deruxtecan (Dxd), and SN-38, were evaluated. Cytotoxicity of OBI-992 along with other ADC was measured in both 2D and 3D spheroids cancer cells (gastric cancer NCI-N87, pancreatic cancer Capan1, and prostate cancer DU145). TROP2 and BCRP expression as well as potential mutations in TOP1 were investigated in TROP2 ADCs-induced resistant colon cancer cells. The synergistic effects of the combination of OBI-992 with PARP inhibitors were evaluated. RESULTS Clinical study: Positive correlations between high mRNA expressions of both TROP2 (p=0.0075) and TOP1 (p=0.043) with poor overall survival for colon cancer patients were found. In vitro: The R4702 antibody displayed different binding epitopes from sacituzumab and datopotamab. In addition, R4702 showed lower non-specific binding and better binding affinity to TROP2 antigen than datopotamab. The payload exatecan demonstrated excellent ICD effects. Relatively higher cytotoxicity was observed by exatecan when compared to that of Dxd and SN-38. OBI-992 exhibited excellent activities on cell growth inhibition in 2D cell cultures and 3D cell spheroids. TROP2 down-regulation and BCRP upregulation were not found in OBI-992-induced resistant cells. A mutation R364H was identified in TOP1 in the resistance study with OBI-992, which is critical for the binding to TOP1 inhibitors camptothecin analogs. Finally, a synergistic effect of OBI-992 and PARP inhibitors (talazoparib, rucaparib, niraparib, and olaparib) on the enhancement of cancer cell killing and DNA damage was observed. CONCLUSION High expression of both TROP2 and TOP1 is associated with poor survival in patients with colon cancer. R4702 is a novel TROP2 antibody with a unique binding epitope and low non-specific binding. Exatecan and exatecan-conjugated ADC OBI-992 showed high cytotoxic activities. Combination of OBI-992 with PARP inhibitors have potential in TROP2-expressing malignancies. Citation Format: Tzer-Min Kuo, Ting-Yu Chang, Jye-Yu Huang, Wei-Chien Tang, Chun-Jung Lin, Yi-Chen Wu, Chi-Huan Lu, Hao-Cheng Weng, Yu-Jung Chen, Yu-Hsuan Tsao, Cheng-Yen Wei, Lifen Shen, Wan-Fen Li, Ming-Tain Lai. In vitro characterization of a novel TROP2-targeting antibody-drug conjugate OBI-992 [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 3130.

Abstract 2828: Combining neratinib with trastuzumab deruxtecan to treat HER2;TP53 co-mutated breast cancers

Abstract In metastatic breast cancer, HER2 activating mutations frequently co-occur with mutations in the TP53 gene and patients with these two co-occuring mutations have a lower response rate to neratinib. In order to address this clinical problem, we bred HER2 activating mutant transgenic mice with two different alleles of TP53. We used mice with the TP53 R172H mutation, which is the murine homolog of the human hotspot TP53 R175H mutation, and mice with TP53flox/flox allele, which mimics TP53 truncating mutations seen in human breast cancers. All three H53 genotypes (H53null, H53172/WT, and H53172/null) developed breast cancers faster than H mice, with H53172/null mice developing breast tumors the fastest, at just 6 weeks after adenoviral-Cre injection into the mammary fat pad. We developed organoids from these murine breast cancers and found that all H53 genotype organoids were less sensitive to neratinib compared to H, however, neratinib was able to synergize with trastuzumab deruxtecan (T-DXd), likely through neratinib mediated increased internalization of T-DXd. Prior literature studies suggest TP53 mutant cancer cells may have therapeutic vulnerabilities to the topoisomerase 1 (Top1) inhibitors. Organoids from all three H53 genotypes were sensitive to the Top1 inhibitor, exatecan. H53172/WT behaved like H organoids with G1 phase cell cycle arrest and induction of p21 expression after exatecan treatment. In contrast, H53null and H53172/null failed to induce p21 and arrested at late-S/G2 phase. Additional animal experiments with these H53 genotypes are in progress and results will be presented at the AACR Annual Meeting. Abbreviation Genotype Median tumor formation (weeks) Neratinib IC50 (nM) Neratinib +T-DXd H HER2V777L 28 10 Not determined H53null HER2V777L;TP53flox/flox 10 240 Synergy H53172/WT HER2V777L;TP53R172H/WT 11 100 Synergy H53172/null HER2V777L;TP53R172H/flox 6 150 Synergy Citation Format: Xiaoqing (Cathy) Cheng, Maureen Highkin, Jacob Hsia, Julie L. Prior, Ian S. Hagemann, Ron Bose. Combining neratinib with trastuzumab deruxtecan to treat HER2;TP53 co-mutated breast cancers [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 2828.

Abstract 5733: Development of a topoisomerase 1 inhibitor platform technology for efficacious and well tolerated ADCs

Abstract Topoisomerase 1 (TOP1) inhibitors are a class of cytotoxic small molecules that have demonstrated effectiveness as therapeutic agents in cancer. However, the broad clinical application has been hindered by poor pharmacokinetic profiles and off-target toxicities. Antibody-drug conjugates (ADCs) enable the targeted delivery of cytotoxic payloads by specifically targeting cell-surface antigens expressed on cancers reducing systemic exposure and toxicity. TOP1-ADCs have received FDA approval for HER2- (trastuzumab-deruxtecan) and Trop2- (sacituzumab-govitecan) expressing solid tumors, demonstrating the promise for this drug class to improve the lives of patients. We have developed a novel drug-linker using a potent TOP1 inhibitor with desirable ADME properties, and a hydrophilic glycoside linker as a platform technology enabling ADCs with favorable physicochemical and biological properties. The TOP1 inhibitor payload was optimized for potency, with reduced P-glycoprotein efflux and enhanced permeability. The hydrophilicity of the linker facilitates the development of highly loaded ADCs (8 drugs/mAb) with high plasma stability, low aggregation, and pharmacokinetics similar to parental antibody. The resultant ADCs are potent and immunologically specific with activity in multidrug-resistant tumor models and bystander activity in heterogenous antigen models. Human cancer cell derived xenograft models demonstrated potent and specific ADC anti-tumor activity with several targets and indications. Improved activity was observed compared to corresponding govitecan- and deruxtecan-based ADCs. ADCs prepared from this drug-linker technology were well tolerated in rats after repeat administration of 60 mg/kg every 4 days for 4 doses. The excellent anti-tumor activity in several models and favorable safety profile demonstrates this platform TOP1 drug-linker technology has the potential for development as ADCs against many cancer targets. Citation Format: Ryan Lyski, Lauren Bou, Johann Sigurjonsson, David Meyer, Calvin Neace, David Ortiz, Katie Snead, Kaleb Smith, Steven Jin, Nicole Stevens, Julia Cochran, Kaveh Alizadeh, Narayana Yeddula, Erica McKinney, Adam Peterson, Lindsay Dotloe, Jessica Simmons, Christopher Carosino, Kim Emmerton, Nancy Everds, Scott Jeffrey, Peter Senter. Development of a topoisomerase 1 inhibitor platform technology for efficacious and well tolerated ADCs [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 5733.

Abstract 7168: PBI-410 (GQ1010), A novel Trop-2-targeted ADC demonstrates a favorable safety and toxicokinetic profile in multiple preclinical assessments

Abstract Introduction: Trop-2-targeting ADCs (Trop-2 ADC) have demonstrated notable clinical efficacy; however, safety concerns necessitate frequent dose adjustments or discontinuations. PBI-410 (GQ1010) is a next-generation TROP-2 ADC developed using GeneQuantum's Ligase-Dependent Conjugation (iLDC) technology, that integrates a highly stable linker and a novel camptothecin analogue payload, TopoIx. This study included comprehensive preclinical evaluations that demonstrated a favorable safety and TK profile and potential for broader therapeutic margin versus other Trop-2 ADCs approved or in development. Methods: TopoIx underwent extensive Good Laboratory Practice toxicity assessments in Sprague-Dawley (SD) rats (payload alone) and as part of the intact ADC in Non-Human Primates. Rats were administered repeated intravenous infusions of 0.5, 1.25, and 2.5 mg/kg of H0011 once a week over 5 doses. The potential toxicity profile of PBI-410 was evaluated in cynomolgus monkeys via repeated intravenous infusions of 10, 30, and 60 mg/kg doses every 3 weeks for 3 doses, followed by a six-week recovery phase. Results: TopoIx administration in SD rats exhibited good tolerance, with slight reductions in body weight gain and food consumption. Microscopic tissue changes resolved post-drug withdrawal, except for persistent findings in the sciatic nerve. Repeated doses of PBI-410 in cynomolgus monkeys were well-tolerated, primarily manifesting as skin pigmentation and associated microscopic alterations in specific tissues, such as eyes, esophagus, thymus, and lymph nodes typical for topoisomerase inhibitors. Importantly, these changes were reversible upon drug cessation. The HNSTD/NOAEL for PBI-410 was determined to be 60 mg/kg. This corresponds to mean Cmax of 1760 µg/mL and AUCinf of 235000 µg*hr/mL for ADC and for TopoIx mean Cmax of 16.4 ng/mL and AUCinf of 2610 ng*hr/mL. The lower exposure of TopoIx suggests minimum payload shedding in the systemic circulation. Notably, PBI-410 demonstrated a significantly enhanced safety profile compared to other Topo1-based Trop-2 ADCs, including an absence of hematopoietic and intestinal toxicities, and no pulmonary toxicity or interstitial lung disease (ILD) observed. Conclusion: PBI-410 demonstrated a favorable and differentiated nonclinical safety profile and favorable TK relative to those reported to date for other Trop-2 ADCs in development. This profile, in addition to the potent anti-tumor response and synergistic activity in combination with anti-PD1 reported previously, position PBI-410 as a best-in-class potential Trop-2 ADC and partner of choice for anti-PD1 combination therapy. Additionally, TopoIx has displayed excellent properties among the TOP1 inhibitors, making it suitable for a broader therapeutic application based on exceptional potency while maintaining a favorable safety profile. Citation Format: Yajun Sun, Vijayapal Reddy, Palaniappan Kulanthaivel, Yanwen Feng, Jie Zhao, Zhiyan Chi, Junhao Wang, Lili Shi, Gang Qin, Paul H. Song. PBI-410 (GQ1010), A novel Trop-2-targeted ADC demonstrates a favorable safety and toxicokinetic profile in multiple preclinical assessments [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 7168.

Abstract 5067: Exploring the genomic landscape of de novo neuroendocrine prostate cancer: Insights for innovative therapies from spatial gene expression analysis

Abstract Introduction & Objectives Neuroendocrine prostate cancer (NEPC) accounts for less than 1% of all prostate cancers but has a worse prognosis than androgen receptor-positive prostate adenocarcinoma (ARPC). Rare cases exist where both de novo NEPC and ARPC are found in the same tissue sample. Our study examines one such unique case: a 78-year-old male treated at Ehime University Hospital who was diagnosed with both ARPC and adjacent de novo metastatic NEPC. This case provides an excellent opportunity for studying gene expression in these two types of prostate cancer. Materials & Methods We used Visium CytAssist Spatial Gene Expression technology from 10x Genomics to analyze formalin-fixed, paraffin-embedded (FFPE) tissue samples from this unique patient. Results We observed elevated expression levels of specific neuroendocrine markers like PEG10, DLL3, NCAM1, SYP, and CHGA in de novo NEPC regions. In contrast, androgen receptor markers such as KLK3, ACPP, TMPRSS2, and NKX3.1 were more prevalent in ARPC areas. Furthermore, genes like Chek1, BRCA1/2, TOP2A, and FANCA were also elevated in NEPC, suggesting that PARP and TOP2 inhibitors could be effective. Expression levels of Rbfox3 and SFRTM2 were lower in NEPC areas, implying possible roles in epithelial-mesenchymal transition (EMT) and Wnt signaling pathways. Additionally, fibrotic markers like HGF, HMOX1, ELN, and GREM1 were upregulated, suggesting that de novo NEPC tumors might be 'immune cold,' thus requiring treatments to boost the effectiveness of immune checkpoint inhibitors. Conclusion The data from additional cases could open the door for new treatment options for NEPC and improve survival rates for castration-resistant prostate cancer patients. Our research serves as a pioneering application of Visium CytAssist on FFPE samples and is the first to examine gene expression in both ARPC and de novo NEPC. A subset of these findings was published in the International Journal of Molecular Science (IJMS) in May 2023. Citation Format: Ryuta Watanabe, Noriyoshi Miura, Mie Kurata, Riko Kitazawa, Tadahiko Kikugawa, Takashi Saika. Exploring the genomic landscape of de novo neuroendocrine prostate cancer: Insights for innovative therapies from spatial gene expression analysis [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 5067.

The Novel ATR Inhibitor M1774 Induces Replication Protein Overexpression and Broad Synergy with DNA-targeted Anticancer Drugs.

Ataxia telangiectasia and Rad3-related (ATR) checkpoint kinase inhibitors are in clinical trials. Here we explored the molecular pharmacology and therapeutic combination strategies of the oral ATR inhibitor M1774 (Tuvusertib) with DNA-damaging agents (DDA). As single agent, M1774 suppressed cancer cell viability at nanomolar concentrations, showing greater activity than ceralasertib and berzosertib, but less potency than gartisertib and elimusertib in the small cell lung cancer H146, H82, and DMS114 cell lines. M1774 also efficiently blocked the activation of the ATR-CHK1 checkpoint pathway caused by replication stress induced by TOP1 inhibitors. Combination with non-toxic dose of M1774 enhanced TOP1 inhibitor-induced cancer cell death by enabling unscheduled replication upon replicative damage, thereby increasing genome instability. Tandem mass tag-based quantitative proteomics uncovered that M1774, in the presence of DDA, forces the expression of proteins activating replication (CDC45) and G2-M progression (PLK1 and CCNB1). In particular, the fork protection complex proteins (TIMELESS and TIPIN) were enriched. Low dose of M1774 was found highly synergistic with a broad spectrum of clinical DDAs including TOP1 inhibitors (SN-38/irinotecan, topotecan, exatecan, and exatecan), the TOP2 inhibitor etoposide, cisplatin, the RNA polymerase II inhibitor lurbinectedin, and the PARP inhibitor talazoparib in various models including cancer cell lines, patient-derived organoids, and mouse xenograft models. Furthermore, we demonstrate that M1774 reverses chemoresistance to anticancer DDAs in cancer cells lacking SLFN11 expression, suggesting that SLFN11 can be utilized for patient selection in upcoming clinical trials.

Unleashing the Potential of Camptothecin: Exploring Innovative Strategies for Structural Modification and Therapeutic Advancements.

Camptothecin (CPT) is a potent anti-cancer agent targeting topoisomerase I (TOP1). However, CPT has poor pharmacokinetic properties, causes toxicities, and leads to drug resistance, which limit its clinical use. In this paper, to review the current state of CPT research. We first briefly explain CPT's TOP1 inhibition mechanism and the key hurdles in CPT drug development. Then we examine strategies to overcome CPT's limitations through structural modifications and advanced delivery systems. Though modifications alone seem insufficient to fully enhance CPT's therapeutic potential, structure-activity relationship analysis provides insights to guide optimization of CPT analogs. In comparison, advanced delivery systems integrating controlled release, imaging capabilities, and combination therapies via stimulus-responsive linkers and targeting moieties show great promise for improving CPT's pharmacological profile. Looking forward, multifaceted approaches combining selective CPT derivatives with advanced delivery systems, informed by emerging biological insights, hold promise for fully unleashing CPT's anti-cancer potential.

Anthracyclines induce cardiotoxicity through a shared gene expression response signature.

TOP2 inhibitors (TOP2i) are effective drugs for breast cancer treatment. However, they can cause cardiotoxicity in some women. The most widely used TOP2i include anthracyclines (AC) Doxorubicin (DOX), Daunorubicin (DNR), Epirubicin (EPI), and the anthraquinone Mitoxantrone (MTX). It is unclear whether women would experience the same adverse effects from all drugs in this class, or if specific drugs would be preferable for certain individuals based on their cardiotoxicity risk profile. To investigate this, we studied the effects of treatment of DOX, DNR, EPI, MTX, and an unrelated monoclonal antibody Trastuzumab (TRZ) on iPSC-derived cardiomyocytes (iPSC-CMs) from six healthy females. All TOP2i induce cell death at concentrations observed in cancer patient serum, while TRZ does not. A sub-lethal dose of all TOP2i induces limited cellular stress but affects calcium handling, a function critical for cardiomyocyte contraction. TOP2i induce thousands of gene expression changes over time, giving rise to four distinct gene expression response signatures, denoted as TOP2i early-acute, early-sustained, and late response genes, and non-response genes. There is no drug- or AC-specific signature. TOP2i early response genes are enriched in chromatin regulators, which mediate AC sensitivity across breast cancer patients. However, there is increased transcriptional variability between individuals following AC treatments. To investigate potential genetic effects on response variability, we first identified a reported set of expression quantitative trait loci (eQTLs) uncovered following DOX treatment in iPSC-CMs. Indeed, DOX response eQTLs are enriched in genes that respond to all TOP2i. Next, we identified 38 genes in loci associated with AC toxicity by GWAS or TWAS. Two thirds of the genes that respond to at least one TOP2i, respond to all ACs with the same direction of effect. Our data demonstrate that TOP2i induce thousands of shared gene expression changes in cardiomyocytes, including genes near SNPs associated with inter-individual variation in response to DOX treatment and AC-induced cardiotoxicity.