Drug Targets Research Articles

Structural insights into the orthosteric inhibition of P2X receptors by non-ATP analog antagonists

P2X receptors are extracellular ATP-gated ion channels that form homo- or heterotrimers and consist of seven subtypes. They are expressed in various tissues, including neuronal and nonneuronal cells, and play critical roles in physiological processes such as neurotransmission, inflammation, pain, and cancer. As a result, P2X receptors have attracted considerable interest as drug targets, and various competitive inhibitors have been developed. However, although several P2X receptor structures from different subtypes have been reported, the limited structural information of P2X receptors in complex with competitive antagonists hampers the understanding of orthosteric inhibition, hindering the further design and optimization of those antagonists for drug discovery. We determined the cryogenic electron microscopy (cryo-EM) structures of the mammalian P2X7 receptor in complex with two classical competitive antagonists of pyridoxal-5'-phosphate derivatives, pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS) and pyridoxal phosphate-6-azophenyl-2′,5′-disulfonic acid (PPADS), and performed structure-based mutational analysis by patch-clamp recording as well as molecular dynamics (MD) simulations. Our structures revealed the orthosteric site for PPADS/PPNDS, and structural comparison with the previously reported apo- and ATP-bound structures showed how PPADS/PPNDS binding inhibits the conformational changes associated with channel activation. In addition, structure-based mutational analysis identified key residues involved in the PPNDS sensitivity of P2X1 and P2X3, which are known to have higher affinity for PPADS/PPNDS than other P2X subtypes.

Abstract LB171: First-in-class inhibitors of the tRNA methyltransferase METTL1 for the treatment of cancer

Abstract METTL1 is an RNA methyltransferase that catalyzes N7-methylation on guanine at position 46 (m7G46) in a subset of transfer RNAs (tRNAs). These tRNAs are stabilized by m7G46, and this increases the translation of mRNAs containing the cognate codons. METTL1 is often over-expressed in cancer tissue, and its gene locus is frequently amplified in specific tumor types. Recent studies have shown that reducing METTL1 expression leads to tumor growth inhibition highlighting this enzyme as a potential novel target for anti-cancer drugs. We optimized small molecule inhibitors of METTL1 from a HTS hit using structure-guided medicinal chemistry. Representative compounds of two distinct chemical series exhibit potent in vitro METTL1 inhibition at low nanomolar concentrations while displaying exquisite selectivity over other RNA and protein methyltransferases. The effects of inhibiting METTL1 were measured by cellular mechanistic assays and modification-induced misincorporation tRNA sequencing (mim-tRNAseq). As expected, pharmacological inhibition led to decreased m7G46 methylation, lower levels of a subgroup of tRNAs and reduced cancer cell proliferation.The efficacy of METTL1 inhibition in a panel of cancer cell lines representing a wide range of solid and hematologic tumor types was determined. Cell proliferation was impaired in a subset of cell lines originating from various cancer types, including lymphoma, sarcoma, esophageal squamous cell carcinoma, glioblastoma, colorectal and lung adenocarcinoma. Importantly, viability was not affected in non-transformed cell lines, indicating a highly selective mechanism of action. The phenotypic consequences of METTL1 inhibition in selected cell lines were further investigated by flow cytometry and western blotting. Cell lines sensitive to METTL1 inhibition displayed reduced cell cycle progression and lowered expression of cell cycle regulators. RNA sequencing and proteome analyses identified molecular changes at both transcript and protein level. METTL1 inhibition led to induction of ATF4 expression and activation of the integrated stress response, as well as global changes to the translation machinery. Studies on cell-line derived xenograft or syngeneic in vivo mouse models were conducted to assess tumor growth inhibition after oral or intraperitoneal administration of the potent METTL1 small molecule inhibitor STM9005. Inhibition of METTL1 by this compound reduced tumor growth in both immune-deficient and immune-competent mouse models, effects accompanied by dose-dependent changes in pharmacodynamic biomarkers. Here, we describe the discovery of first-in-class inhibitors of METTL1 tRNA methyltransferase. To our knowledge, our data provide the first evidence that pharmacological inhibition of a tRNA methyltransferase reduces tumor growth in vivo. Citation Format: Alexandra Sapetschnig, Beth Thomas, Eliza Yankova, Harry Fischl, Aleksandra Azevedo, Sarah Bucknell, Richard Fosbeary, Sapphire Sawyer, Sian Evans, Carmen Livi, Byron Andrews, Jack Rogan, Natalie Webster, Matthew Fyfe, Konstantinos Tzelepis, Oliver Rausch. First-in-class inhibitors of the tRNA methyltransferase METTL1 for the treatment of cancer [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 LB171.

Abstract LB172: A cytosolic complex between exportin-7(XPO7) and ste20-like kinase (SLK) regulates PI3K-AKT-mTOR signaling in cholangiocarcinoma

Abstract Cholangiocarcinoma (CCA) is a deadly malignancy with an evolving therapeutic landscape. Although nucleic acid-based evaluations have unveiled some druggable targets, limited options are available for the majority of patients with this disease. We therefore evaluated biliary exosomes by mass spectrometry from 16 patients with CCA, which revealed a 17-fold enhancement of the nuclear export protein XPO7 in CCA-derived biliary exosomes. IHC analysis of XPO7 expression in CCA tumors from 170 patients unexpectedly demonstrated intense cytoplasmic staining in 30% of the samples, which correlated with abbreviated survival. Within the cytosol, we demonstrate that XPO7 exists in a molecular complex with the serine/threonine kinase SLK. shRNA-mediated knockdown of either XPO7 or SLK abrogated tumor organoid formation in vitro, and reduced orthotopic tumor formation and substantially altered tumor morphology using athymic mice. A kinome screen of FDA-approved drugs revealed tivozanib inhibits SLK (IC50= 81.8 nM), which we confirmed using crystallography. Tivozanib treatment reduced tumor organoid formation in vitro and induced tumor regression in vivo using murine xenografts with resulting morphology similar to XPO7 and SLK knockdown tumors. Importantly, site-directed mutagenesis of SLK was used to confirm the observed phenotypic alterations secondary to SLK inhibition. Signal transduction studies with SLK knockdown in CCA cell lines demonstrated downregulation of PI3K-AKT-mTOR signaling with further effects on DNA damage resulting in G2 cell cycle arrest. Moreover, tivozanib demonstrated anti-tumor activity and increased apoptosis in established CCA patient-derived xenografts with cytoplasmic XPO7 expression. Using ex vivo tumor slice cultures, tivozanib significantly increased tumor cell apoptosis in XPO7/SLK-expressing tumors (N=3) but not in tumors without this expression pattern (N=6). Further evaluation of these tivozanib-treated tumor slice cultures demonstrated a reduction in the expression of phosphorylated mTOR (S2448), AKT (T308), S6 (S235/236) in the high XPO7/SLK-expressing tumors but not in tumors without this expression pattern. Lastly, tivozanib monotherapy demonstrated in vivo efficacy with arrest of tumor progression (RECIST stable disease) in an ongoing clinical trial (NCT04645160). Citation Format: Priyanka P. Desai, Tahsin M. Khan, Reed I. Ayabe, Emily Smith, Surajit Sinha, Ashley Rainey, Yuri Lin, Tracey Pu, Kenneth Lubrice, Leila Sarvestani, Sarfaraz Akmal, Shahyan Rehman, Kirsten Remmert, Michael Yaffe, Craig Thomas, John Brognard, Jonathan M. Hernandez. A cytosolic complex between exportin-7(XPO7) and ste20-like kinase (SLK) regulates PI3K-AKT-mTOR signaling in cholangiocarcinoma [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 LB172.

Abstract LB216: Static and microfluidic platforms for novel therapeutic testing in high-grade serous ovarian cancer

Abstract Background: High-Grade Serous Ovarian Cancer (HGSOC) is the most lethal OC type because of its silent progression and lack of specific markers for early detection. In this context, the timely initiation of effective treatment is imperative to prevent disease progression extending life expectancy. However, responses to treatment can vary significantly among individuals, presenting clinical challenges in selecting effective treatments, necessitating the interpretation of molecular and genomic data of the patient on a case-by-case basis. The aim of this study is to establish a highly sensitive platform, implemented with microfluidic technology, to predict patient drug responses with the goal of assisting oncologists in shaping effective treatments. Moreover, validating this platform through a retrospective study, enabling the development of innovative drugs. Methods: In this work, patient-derived ovarian cancer organoids (PDOs) were engineered for drug screening and twenty FDA approved drugs were assessed on seven PDOs derived from both primary tumors and ascites. The fidelity of PDOs in reproducing the histopathology of the primary tumour was confirmed by immunohistochemistry. Proteomics and repeated screening on high- and low-passage PDOs confirmed the reliability of the platform over time. Furthermore, to bridge the gap between static systems and the dynamic of human tumours, a microfluidic platform was employed and using fluorescence microscopy the patients' drug sensitivity was predicted (PMID: 37143603). Results: PDOs were able to replicate patients' response to the drug in a retrospective study and surprisingly, in some cases, to predict resistance to certain drugs administered on the basis of strong, clinically validated predictive biomarkers, which proved ineffective in the clinic. The platform could predict innovative therapeutic strategies as demonstrated by inhibiting the activity of Peptidylprolyl Cis/Trans Isomerase, NIMA-Interacting 1 (Pin1), a novel druggable target for OC (PMID: 22322860, 29746956, 30697729). The Pin1 inhibitor (VS10) is effective in a subset of patients resistant to platinum-based chemotherapy and synergize with doxorubicin. Finally, a microfluidic platform fluorescence based, which improved drug penetration in PDOs was developed for efficient and faster drug screening. Conclusions: This study underscores the significance of utilizing engineered PDOs to develop predictive and easy to use platforms that can truly aid oncologists in devising patient-specific treatments while circumventing the use of drugs that, despite strong recommendations, may prove ineffective. Furthermore, these platforms have demonstrated their effectiveness in the development of innovative therapeutics, such as Pin1 inhibitors, which have been proven both effective and safe in animal models, underscoring the need for clinical studies. Acknowledgments: AIRC IG23566 Citation Format: Enrico Cavarzerani, Gloria Saorin, Matteo Mauceri, Isabella Caligiuri, Michele Bartoletti, Vincenzo Canzonieri, Flavio Rizzolio. Static and microfluidic platforms for novel therapeutic testing in high-grade serous ovarian cancer [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 LB216.

Abstract CT153: Phase I trial of intratumoral administration of autologous CCL21 gene-modified dendritic cells in combination with pembrolizumab for advanced NSCLC: Feasibility of repeated IT injections

Abstract Background: Effective immunotherapy options are lacking for patients with advanced non-small cell lung cancer (NSCLC) who progress on a programmed cell death-(ligand)1 [PD-(L)1] inhibitor and for those who are epidermal growth factor receptor (EGFR) mutation or anaplastic lymphoma kinase (ALK) rearrangement positive after progression on tyrosine kinase inhibitor (TKI) therapy. One potential approach to improve immune checkpoint efficacy in these patient populations is to promote cytolytic T cell infiltration into tumors. This can be accomplished via in situ vaccination with chemokine gene-engineered functional antigen presenting cells (APCs) which can take advantage of the full repertoire of tumor antigens and convert the tumor into a lymph node-like environment to promote both local and systemic antitumor responses. The chemokine CCL21 promotes co-localization of naive T cells and antigen-experienced dendritic cells (DCs) to facilitate T cell activation. Our preclinical studies and phase I trial of intratumoral (IT) administration of DC genetically modified to overexpress CCL21 (CCL21-DC) revealed augmentation of tumor antigen presentation in situ, resulting in systemic antitumor immunity. However, increased PD-L1 expression was observed in some patient tumors, suggesting that tumor-mediated impairment of T cell function may be forestalling a more robust CCL21-DC mediated antitumor response. Similarly, improved PD-(L)1 inhibitor efficacy may be possible with enhanced T cell infiltration and augmented APC function following IT CCL21-DC. Therefore, we are conducting a phase I trial, combining IT CCL21-DC with pembrolizumab in patients with advanced NSCLC. Methods: Phase I, dose-escalating, multi-cohort trial followed by dose expansion. Maximum of 24 patients (9-12 escalation + 12 expansion) with stage IV NSCLC will be evaluated who have tumors accessible for IT injection and are either (1) EGFR/ALK wild-type after progression on a PD-(L)1 inhibitor or (2) EGFR/ALK mutant after progression on TKI therapy. Three IT injections of autologous CCL21-DC (days 0, 21, 42) will be concurrently administered with pembrolizumab, followed by pembrolizumab once every three weeks for up to 1 year. Primary objective of dose escalation is safety and determination of maximum tolerated dose (MTD) of IT CCL21-DC when combined with pembrolizumab. Primary objective of dose expansion is objective response rate at MTD. Secondary objectives include adverse event profiling and determination of drug target activity by immune monitoring studies. This trial, NCT03546361, is currently open for enrollment, and this updated abstract will highlight the feasibility of repeated IT injections in treated patients. Citation Format: Bin Liu, Aaron Lisberg, Ramin Salehi-Rad, Michael Oh, Jay M. Lee, Linh M. Tran, Kostyantyn Krysan, Raymond J. Lim, Camelia Dumitras, Zhe Jing, Fereidoun Abtin, Robert D. Suh, Scott J. Genshaft, Scott S. Oh, Gregory A. Fishbein, Anita Kaul, Kanwarpal S. Kahlon, Shahryar A. Ashouri, Jonathan W. Goldman, David A. Elashoff, Edward Garon, Steven M. Dubinett. Phase I trial of intratumoral administration of autologous CCL21 gene-modified dendritic cells in combination with pembrolizumab for advanced NSCLC: Feasibility of repeated IT injections [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 CT153.

Lethal metabolism of Candida albicans respiratory mutants.

The destructive impact of fungi in agriculture and animal and human health, coincident with increases in antifungal resistance, underscores the need for new and alternative drug targets to counteract these trends. Cellular metabolism relies on many intermediates with intrinsic toxicity and promiscuous enzymatic activity generates others. Fuller knowledge of these toxic entities and their generation may offer opportunities of antifungal development. From this perspective our observation of media-conditional lethal metabolism in respiratory mutants of the opportunistic fungal pathogen Candida albicans was of interest. C. albicans mutants defective in NADH:ubiquinone oxidoreductase (Complex I of the electron transport chain) exhibit normal growth in synthetic complete medium. In YPD medium, however, the mutants grow normally until early stationary phase whereupon a dramatic loss of viability occurs. Upwards of 90% of cells die over the subsequent four to six hours with a loss of membrane integrity. The extent of cell death was proportional to the amount of BactoPeptone, and to a lesser extent, the amount of yeast extract. YPD medium conditioned by growth of the mutant was toxic to wild-type cells indicating mutant metabolism established a toxic milieu in the media. Conditioned media contained a volatile component that contributed to toxicity, but only in the presence of a component of BactoPeptone. Fractionation experiments revealed purine nucleosides or bases as the synergistic component. GC-mass spectrometry analysis revealed acetal (1,1-diethoxyethane) as the active volatile. This previously unreported and lethal synergistic interaction of acetal and purines suggests a hitherto unrecognized toxic metabolism potentially exploitable in the search for antifungal targets.

Abstract LB411: Investigating the dependency of androgen receptor-positive prostate cancer on class I myosins

Abstract Prostate adenocarcinoma is characterized by high expression and activity of the androgen receptor (AR). AR is a nuclear hormone receptor that stimulates anabolic cell metabolism. We hypothesized that the high activity of AR in prostate cancer creates unique dependencies on cellular machinery to support this high rate of anabolism. We observed that androgen treatment of patients with metastatic castration-resistant prostate cancer resulted in increased abundance of MYO1E, a class I myosin that facilitates intracellular vesicular trafficking, in tumor biopsies. Similarly, MYO1E protein increased in AR+ prostate cancer cell lines upon treatment with androgens. Notably, a specific small molecule inhibitor of MYO1E, pentachloropseudilin (PClP), induced cell death of AR-positive prostate cancer cell lines with an IC50 of 2-4uM, but was non-toxic to AR-negative prostate cancer cell lines. Expression of AR in these AR-negative lines conferred sensitivity to PClP, while deletion of AR in an AR-positive line conferred resistance to PClP, indicating that AR expression was required and sufficient for sensitivity of prostate cancer cell lines to PClP. Previous studies have suggested that AR globally increases protein synthesis and proteotoxic stress, and we asked whether this may create a dependency on MYO1E for secretion of protein-containing vesicles to manage proteotoxic stress. Initial experiments suggest that AR increases global rates of protein synthesis, as assessed by puromycin-incorporation assays, and inhibition of MYO1E increases phosphorylation of EIF2A, suggestive of activation of the integrated stress response. Further experiments will determine the essential function of MYO1E in AR-positive prostate cancer. Altogether our studies suggest that the class I myosin MYO1E is an essential protein in AR-positive cell lines and may represent a novel drug target for prostate adenocarcinoma. Citation Format: Anoushka Kadam, Shivani Kumar, Varsha Vakkala, Rajendra Kumar, Angelo M. De Marzo, Edward J. Pearce, Laura A. Sena. Investigating the dependency of androgen receptor-positive prostate cancer on class I myosins [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 LB411.

Fluorescently labelled vedolizumab to visualise drug distribution and mucosal target cells in inflammatory bowel disease

ObjectiveImproving patient selection and development of biological therapies such as vedolizumab in IBD requires a thorough understanding of the mechanism of action and target binding, thereby providing individualised treatment strategies....

DPI_CDF: druggable protein identifier using cascade deep forest

BackgroundDrug targets in living beings perform pivotal roles in the discovery of potential drugs. Conventional wet-lab characterization of drug targets is although accurate but generally expensive, slow, and resource intensive. Therefore, computational methods are highly desirable as an alternative to expedite the large-scale identification of druggable proteins (DPs); however, the existing in silico predictor’s performance is still not satisfactory.MethodsIn this study, we developed a novel deep learning-based model DPI_CDF for predicting DPs based on protein sequence only. DPI_CDF utilizes evolutionary-based (i.e., histograms of oriented gradients for position-specific scoring matrix), physiochemical-based (i.e., component protein sequence representation), and compositional-based (i.e., normalized qualitative characteristic) properties of protein sequence to generate features. Then a hierarchical deep forest model fuses these three encoding schemes to build the proposed model DPI_CDF.ResultsThe empirical outcomes on 10-fold cross-validation demonstrate that the proposed model achieved 99.13 % accuracy and 0.982 of Matthew’s-correlation-coefficient (MCC) on the training dataset. The generalization power of the trained model is further examined on an independent dataset and achieved 95.01% of maximum accuracy and 0.900 MCC. When compared to current state-of-the-art methods, DPI_CDF improves in terms of accuracy by 4.27% and 4.31% on training and testing datasets, respectively. We believe, DPI_CDF will support the research community to identify druggable proteins and escalate the drug discovery process.AvailabilityThe benchmark datasets and source codes are available in GitHub: http://github.com/Muhammad-Arif-NUST/DPI_CDF.

Abstract LB436: Using FCCS to monitor target occupancy in cells and patient biopsies

Abstract Lack of target engagement is one of the major reasons for failures in clinical studies. We developed an assay platform to detect and quatify target occupancy values in patient and animal biopsies to guide the development and dosing during the drug development process. FCCS (fluorescence cross correlation spectroscopy) is a powerful tool to characterize drug target interaction in physiological environments. Over the last decade this technology was used to understand the binding and kinetics of small molecules and antibodies to challenging target proteins in great details and accuracy. Over the last decade Intana Bioscience expanded the application of FCCS from affinity measurements to comprehensive interaction studies including kinetics, multi component complex formation, assessing stoichiometry and quantification of aggregation, PK studies and target engagement. This presentation will highlight examples of results obtained with FCCS in projects ranging from early stages in the drug discovery up to the support of clinical trials to quantify target occupancies in patient samples. Citation Format: Frank Becker. Using FCCS to monitor target occupancy in cells and patient biopsies [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 LB436.

Abstract LB317: TNBC cell co-culture with primary human lung fibroblasts identifies a paracrine-juxtacrine signaling loop promoting fibroblast activation and autophagy-dependent breast cancer cell therapy resistance

Abstract Triple negative breast cancer (TNBC) carries a poorer prognosis than other breast cancer subtypes due to high metastatic incidence, lack of molecular drug targets, and rapid development of chemoresistance. Cancer-associated fibroblasts (CAFs) are key extrinsic drivers of chemoresistance in primary TNBC tumors, but it is unknown whether resident fibroblasts in distant metastatic tissues also influence TNBC drug sensitivity. Since the lung is a top metastatic site in TNBC, we developed a breast cancer cell (BCC) - primary human donor-derived lung fibroblast (LF) co-culture model to identify therapeutic vulnerabilities and mechanisms of fibroblast-mediated extrinsic chemoresistance in lung-metastatic TNBC. We characterized the growth and chemoresistance phenotypes of BCC co-cultured with primary LFs via bioluminescence and fluorescence imaging and evaluated their transcriptional and secretory profiles using bulk and scRNAseq and multiplex cytokine analysis. Co-culture of human BCC and primary LFs resulted in BCC molecular subtype-dependent changes in cell growth and drug resistance compared with conventional monoculture. Moreover, we also observed increased LF secretion of the immunomodulatory cytokines IL-6 and CCL2 in response to both BCC co-culture and BCC-conditioned media. Transcriptomic analysis of LFs sorted from co-culture identified upregulation of pro-inflammatory and pro-fibrotic iCAF and myCAF-like gene signatures, while co-cultured TNBC cells upregulated interferon response-mediated multi-therapy resistance genes and cellular dormancy signatures. We applied this model in a high throughput screen of 846 kinase inhibitor compounds, which identified reproducible differential BCC responses to kinase inhibitors between monoculture and co-culture conditions. When co-cultured with LFs, BCC were broadly more resistant to kinase inhibitor compounds but showed increased sensitivity to inhibitors of the lipid kinase VPS34, an important initiator of autophagy. We subsequently established that autophagy is increased in co-cultured TNBC cells and contributes to LF-mediated resistance to kinase inhibitors. Moreover, we determined that while paracrine signals from BCC can promote LF activation indicated by enhanced cytokine secretion, cell-cell contact is required for LF modulation of TNBC therapy responses in co-culture. Thus, we conclude that breast cancer cell co-culture with primary lung fibroblasts is a scalable in vitro model that can facilitate therapeutic discovery and mechanistic evaluation of extrinsic modulation of therapy responses in metastatic breast cancer, and further demonstrate that BCC can promote paracrine activation of co-cultured LFs in association with juxtacrine induction of BCC autophagy and extrinsic therapy resistance. Citation Format: Marika L. Klosowski, Kathryn Cronise, Claire Stratton, Qiong Zhou, Hector Esquer, Daniel LaBarbera, Daniel Regan. TNBC cell co-culture with primary human lung fibroblasts identifies a paracrine-juxtacrine signaling loop promoting fibroblast activation and autophagy-dependent breast cancer cell therapy resistance [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 LB317.

Abstract LB289: Single-cell CRISPR screens at scale to understand T cell biology

Abstract CRISPR screens have become the primary discovery engine in modern biology and are widely used to uncover novel targets in immuno-oncology. However, these genetic screens are usually coupled to rather simplistic read-outs such as cell fitness. In contrast, CROP-Seq (or “CRISPR Droplet” Sequencing) screens combine CRISPR perturbation with single-cell transcriptomics, enabling high-content phenotyping at single-cell resolution. Briefly, cells are perturbed with a pooled sgRNA library and transcriptomic profiles of each cell are recorded using conventional single-cell RNA sequencing platforms. Currently, the scale of these screens is limited to the perturbation of a couple of hundred genes, possibly up to 1.000 genes, because of increased costs for single-cell library preparation and next generation sequencing (NGS). At Myllia, we have built a platform allowing high-content CROP-Seq screens in both CRISPR-ready cancer cell lines as well as primary immune cells, e.g., human T cells. Using CRISPR KO, CRISPR interference or CRISPR activation workflows, we can help accelerate the identification of target genes and regulatory networks involved in immuno-oncology. Primary T cells are of great interest in the immunotherapy community as they are key players in autoimmune and inflammatory disease. Engineered T-lymphocytes such as CAR-T cells are currently developed as novel cellular medicines, yet many T cell-intrinsic features involved in CAR-T potency remain elusive. To enable the discovery of novel targets, we have performed multiple customized CROP-Seq CRISPR KO screens in primary human T cells investigating T cell stemness and effector phenotypes as well as CD4+ T helper cell differentiation towards Th1, Th2, or Th17 subsets. Here, we present screens for regulators of functional T cell plasticity and Th2 commitment in which we aimed to identify genes determining critical T cell fate decisions, potentially linking gene (dys)function to either autoimmune/inflammatory disease or functional persistence of cytotoxic T cells for cancer immunotherapy. Overall, the advanced CROP-Seq platform combining large-scale perturbations and targeted sequencing (TA) read-outs will catalyse a paradigm shift for CROP-Seq enabling high-throughput functional genomic screens that support the validation of drug targets in autoimmunity, inflammation and immuno-oncology. Citation Format: Nicole Untermoser, Johanna Irnstorfer, Anatoly Vasilyev, Nikola Vinko, Sumit Pawar, Anke Loregger, Adam Krejci, Henrik Schmidt, Tilmann Bürckstümmer. Single-cell CRISPR screens at scale to understand T cell biology [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 LB289.

Abstract LB422: Specific activation of the Epstein-Barr Virus BGLF4 locus via lowered DNA methylation in EBV-associated CNS lymphoproliferative disease

Abstract Introduction: Epstein-Barr virus positive (EBV+) central nervous system lymphoproliferative diseases (CNS-LPD) are aggressive clinical conditions with poor prognosis. We have previously reported use of a ganciclovir (GCV), zidovudine (AZT), rituximab and dexamethasone regimen (GARD) that induced complete and durable responses in patients with primary CNS post-transplant lymphoproliferative disease (PTLD). Responses were associated with detection of two GCV/AZT viral drug targets, the EBV protein kinases BGLF4 and BXLF1. These viral proteins are normally expressed in lytic phase EBV, and the molecular basis for expression in latently infected EBV+ CNS-LPD is unknown. Methods: RNA expression of EBV genes LMP1, BZLF1, BXLF1, and BGLF4 was measured in 12 CNS-LPD biopsies (n=8/12 DLBCL-type) via qRT-PCR. Mass spectrometry-based EpiTYPER assay was used to quantify DNA methylation in EBV gene promoters. Biopsies from 25 patients with systemic PTLD were used for comparison. The BGLF4 locus was investigated in detail using luciferase reporters and 5’RACE mapping of transcription start sites in 3 EBV-infected cell lines and 4 CNS-LPD biopsy samples. Overall EBV methylation was assessed in a screening assay covering 31 EBV loci. Mechanistic involvement of active demethylation via TET2 was studied in a HEK293 CRISPR-Cas9 gene knockout model under EBV (strain M81) infection. Results: Gene expression analysis of biopsy specimens showed expression of LMP1, BXLF1, and BGLF4, but not BZLF1. Expression of BXLF1 and BGLF4 was significantly higher in CNS-LPD when compared to systemic EBV+ PTLD (p=0.043 and p=0.00022, respectively). EpiTYPER data showed significantly decreased promoter methylation at BGLF4 (p=0.00013). Luciferase reporter analysis of the BGLF4 upstream sequence revealed 3 regions of promoter activity which were silenced by in vitro methylation of reporters. 5’RACE identified transcription start sites at all 3 BGLF4 promoters and detected their activity in EBV+ cell lines and CNS-LPD biopsies. By overlaying transcriptional activity, 5’RACE and DNA methylation data, we identified DNA methylation loss at single CpG dinucleotides correlated with BGLF4 expression (r2=0.73). These sites were specifically demethylated in CNS-LPD, while surrounding EBV methylation remained high. Knockout of TET2 followed by EBV infection revealed that active demethylation is required for demethylation at BGLF4 promoters. Conclusions: The molecular determinant for success of the GARD regimen in CNS-LPD has remained poorly understood, with immunohistochemical detection of BGLF4/BXLF1 as the only predictor of treatment response. Our results provide the molecular basis for expression of BGLF4 in CNS-LPD by showing decreased promotor DNA methylation. We identified specific BGLF4 methylation sites that hold promise as potential DNA biomarkers for EBV+ CNS-LPD. Citation Format: Christoph Weigel, Haley L. Klimaszewski, Selamawit Addissie, Sarah Schlotter, Fode Tounkara, James P. Dugan, Bradley M. Haverkos, Lynda Villagomez, Mark Lustberg, Pierluigi Porcu, Timothy Voorhees, Michael A. Caligiuri, Ginny Bumgardner, Christopher C. Oakes, Robert Baiocchi. Specific activation of the Epstein-Barr Virus BGLF4 locus via lowered DNA methylation in EBV-associated CNS lymphoproliferative disease [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 LB422.

Discovery of a small-molecule inhibitor that traps Polθ on DNA and synergizes with PARP inhibitors

The DNA damage response (DDR) protein DNA Polymerase θ (Polθ) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Polθ inhibitor (Polθi) class with 4–6 nM IC50 that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Polθi selectively inhibits Polθ polymerase (Polθ-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Polθi fails to inhibit Polθ-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in the open configuration. Remarkably, Polθi binding to the Polθ-pol:DNA/DNA closed complex traps the polymerase on DNA for more than forty minutes which elucidates the inhibitory mechanism of action. These data reveal a unique small-molecule DNA polymerase:DNA trapping mechanism that induces synthetic lethality in HR-deficient cells and potentiates the activity of PARPi.

Abstract LB280: Enhancing glioblastoma radiotherapy by COMT inhibition

Abstract There is an urgent unmet medical need to discover novel therapeutics to enhance the efficacy of glioma radiotherapy. Here we assessed the roles of catechol - o - methyltransferase COMT, a key enzyme in dopamine metabolism and a drug target for Parkinson's disease, in glioma treatment. Analysis of TCGA data showed significantly higher COMT expression levels in glioma versus normal brain tissues. Inhibition of COMT by genetic knockout or FDA - approved COMT inhibitors significantly sensitized glioma cells to radiotherapy in vitro and murine glioma in vivo. Mechanistically, COMT inhibition in glioblastoma cells led to mitochondria dysfunction and increased mitochondrial RNA release into the cytoplasm, activating the cellular antiviral dsRNA sensing pathway and type I IFN response. Elevated type I IFNs stimulated microglial cells′ phagocytic capacity, enhancing radiotherapy efficacy. Because of the long - established safety record of the COMT inhibitors, our findings provide a solid rationale to evaluate them in combination with radiotherapy in glioma patients. Citation Format: Meng Jiao, Christopher J. Pirozzi, Chen Yu, Xuhui Bao, Mengjie Hu, Dong Pan, Sejiro Littleton, Nathan Reynolds, Daniel R. Saban, Fang Li, Chuan-yuan Li. Enhancing glioblastoma radiotherapy by COMT inhibition [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 LB280.

Knockdown of PGBD5 inhibits the malignant progression of glioma through upregulation of the PPAR pathway.

Glioma is the most common type of primary intracranial malignant tumor, and because of its high invasiveness and recurrence, its prognosis remains poor. The present study investigated the biological function of piggyBac transportable element derived 5 (PGBD5) in glioma. Glioma and para-cancerous tissues were obtained from five patients. Reverse transcription-quantitative PCR and western blotting were used to detect the expression levels of PGBD5. Transwell assay and flow cytometry were used to evaluate cell migration, invasion, apoptosis and cell cycle distribution. In addition, a nude mouse tumor transplantation model was established to study the downstream pathways of PGBD5 and the molecular mechanism was analyzed using transcriptome sequencing. The mRNA and protein expression levels of PGBD5 were increased in glioma tissues and cells. Notably, knockdown of PGBD5 in vitro could inhibit the migration and invasion of glioma cells. In addition, the knockdown of PGBD5 expression promoted apoptosis and caused cell cycle arrest in the G2/M phase, thus inhibiting cell proliferation. Furthermore, in vivo experiments revealed that knockdown of PGBD5 expression could inhibit Ki67 expression and slow tumor growth. Changes in PGBD5 expression were also shown to be closely related to the peroxisome proliferator-activated receptor (PPAR) signaling pathway. In conclusion, interference with PGBD5 could inhibit the malignant progression of glioma through the PPAR pathway, suggesting that PGBD5 may be a potential molecular target of glioma.

Abstract LB215: Polymerase θ (POLθ) inhibitors (novobiocin, ART-558, RP6685) were tested with 22 approved and investigational agents in multi-cell type spheroids of genetically selected patient-derived human tumor cell lines

Abstract The potential of novobiocin to augment cancer chemotherapy was explored in the late 1980s and early 1990s in tumor cells and tumor-bearing mice and in Phase 1 clinical trials with cyclophosphamide or cisplatin. The molecular target of novobiocin was identified to be DNA POLθ which has a role in repair of DNA double strand breaks (DSB) during mitosis. Genetic alterations which may increase or decrease POLθ inhibitor effects have been elucidated. Thirty patient-derived tumor cell lines with known BRCA, ATM, ATR, POLQ, XRCC1, PALB2, PARP1, and LIG3 alterations were screened in a complex spheroid assay (tumor cells, endothelial cells, mesenchymal stem cells) with a POLθ inhibitor (novobiocin, ART-558, or RP6685), alone or in simultaneous combination with a FDA approved or investigational anticancer small molecule, using a 7-day exposure and a CellTiter-Glo 3D luminescence endpoint. As single agents, the POLθ inhibitors had little or no cytotoxicity. Potentiation of the topoisomerase II inhibitors doxorubicin (DOX) and etoposide by the POLθ inhibitors ART-558 or RP6685 was observed in spheroids grown from the serous endometrial adenocarcinoma 922993-354-T-J3, which has ATM and BRCA2 variants. The 379773-124-R-J2 endometrioid endometrial adenocarcinoma which harbors mutant POLθ demonstrated antagonism with ART-558 in combination with PARP inhibitors and less than additive to additive cytotoxicity in combination with DOX. Activity of the Chk1/2 inhibitor prexasertib was potentiated by either ART-558 or RP6685 in the 922993-354-T-J3 complex spheroids. The combination of POLθ inhibitor ART-558 and the Chk1/2 inhibitor prexasertib produced up to 1 log increase in cytotoxicity in the 922993-354-T-J3 complex spheroids. KRAS G12D inhibitor MRTX-133 cytotoxicity against 186277-243-T-J2 colon adenocarcinoma with the KRAS G12D variant was potentiated by either ART-558 or RP6685. Potential combinations to advance into in vivo studies will be presented. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Beverly A. Teicher, Thomas S. Dexheimer, Thomas Silvers, Nathan P. Coussens, J Paul Eder, James H. Doroshow. Polymerase θ (POLθ) inhibitors (novobiocin, ART-558, RP6685) were tested with 22 approved and investigational agents in multi-cell type spheroids of genetically selected patient-derived human tumor cell lines [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 LB215.

Resting State Brain Networks under Inverse Agonist versus Complete Knockout of the Cannabinoid Receptor 1.

The cannabinoid receptor 1 (CB1) is famous as the target of Δ9-tetrahydrocannabinol (THC), which is the active ingredient of marijuana. Suppression of CB1 is frequently suggested as a drug target or gene therapy for many conditions (e.g., obesity, Parkinson's disease). However, brain networks affected by CB1 remain elusive, and unanticipated psychological effects in a clinical trial had dire consequences. To better understand the whole brain effects of CB1 suppression we performed in vivo imaging on mice under complete knockout of the gene for CB1 (cnr1-/-) and also under the CB1 inverse agonist rimonabant. We examined white matter structural changes and brain function (network activity and directional uniformity) in cnr1-/- mice. In cnr1-/- mice, white matter (in both sexes) and functional directional uniformity (in male mice) were altered across the brain but network activity was largely unaltered. Conversely, under rimonabant, functional directional uniformity was not altered but network activity was altered in cortical regions, primarily in networks known to be altered by THC (e.g., neocortex, hippocampal formation). However, rimonabant did not alter many brain regions found in both our cnr1-/- results and previous behavioral studies of cnr1-/- mice (e.g., thalamus, infralimbic area). This suggests that chronic loss of cnr1 is substantially different from short-term suppression, subtly rewiring the brain but largely maintaining the network activity. Our results help explain why pathological mutations in CB1 (e.g., chronic pain) do not always provide insight into the side effects of CB1 suppression (e.g., clinical depression), and thus urge more preclinical studies for any drugs that suppress CB1.

Identification of prospective aging drug targets via Mendelian randomization analysis.

Aging represents a multifaceted process culminating in the deterioration of biological functions. Despite the introduction of numerous anti-aging strategies, their therapeutic outcomes have often been less than optimal. Consequently, discovering new targets to mitigate aging effects is of critical importance. We applied Mendelian randomization (MR) to identify potential pharmacological targets against aging, drawing upon summary statistics from both the Decode and FinnGen cohorts, with further validation in an additional cohort. To address potential reverse causality, bidirectional MR analysis with Steiger filtering was utilized. Additionally, Bayesian co-localization and phenotype scanning were implemented to investigate previous associations between genetic variants and traits. Summary-data-based Mendelian randomization (SMR) analysis was conducted to assess the impact of genetic variants on aging via their effects on protein expression. Additionally, mediation analysis was orchestrated to uncover potential intermediaries in these associations. Finally, we probed the systemic implications of drug-target protein expression across diverse indications by MR-PheWas analysis. Utilizing a Bonferroni-corrected threshold, our MR examination identified 10 protein-aging associations. Within this cohort of proteins, MST1, LCT, GMPR2, PSMB4, ECM1, EFEMP1, and ISLR2 appear to exacerbate aging risks, while MAX, B3GNT8, and USP8 may exert protective influences. None of these proteins displayed reverse causality except EFEMP1. Bayesian co-localization inferred shared variants between aging and proteins such as B3GNT8 (rs11670143), ECM1 (rs61819393), and others listed. Mediator analysis pinpointed 1,5-anhydroglucitol as a partial intermediary in the influence LCT exhibits on telomere length. Circulating proteins play a pivotal role in influencing the aging process, making them promising candidates for therapeutic intervention. The implications of these proteins in aging warrant further investigation in future clinical research.

Systematic proteome-wide Mendelian randomization using the human plasma proteome to identify therapeutic targets for lung adenocarcinoma

BackgroundLung adenocarcinoma (LUAD) is the predominant histological subtype of lung cancer and the leading cause of cancer-related mortality. Identifying effective drug targets is crucial for advancing LUAD treatment strategies.MethodsThis study employed proteome-wide Mendelian randomization (MR) and colocalization analyses. We collected data on 1394 plasma proteins from a protein quantitative trait loci (pQTL) study involving 4907 individuals. Genetic associations with LUAD were derived from the Transdisciplinary Research in Cancer of the Lung (TRICL) study, including 11,245 cases and 54,619 controls. We integrated pQTL and LUAD genome-wide association studies (GWASs) data to identify candidate proteins. MR utilizes single nucleotide polymorphisms (SNPs) as genetic instruments to estimate the causal effect of exposure on outcome, while Bayesian colocalization analysis determines the probability of shared causal genetic variants between traits. Our study applied these methods to assess causality between plasma proteins and LUAD. Furthermore, we employed a two-step MR to quantify the proportion of risk factors mediated by proteins on LUAD. Finally, protein–protein interaction (PPI) analysis elucidated potential links between proteins and current LUAD medications.ResultsWe identified nine plasma proteins significantly associated with LUAD. Increased levels of ALAD, FLT1, ICAM5, and VWC2 exhibited protective effects, with odds ratios of 0.79 (95% CI 0.72–0.87), 0.39 (95% CI 0.28–0.55), 0.91 (95% CI 0.72–0.87), and 0.85 (95% CI 0.79–0.92), respectively. Conversely, MDGA2 (OR, 1.13; 95% CI 1.08–1.19), NTM (OR, 1.12; 95% CI 1.09–1.16), PMM2 (OR, 1.35; 95% CI 1.18–1.53), RNASET2 (OR, 1.15; 95% CI 1.08–1.21), and TFPI (OR, 4.58; 95% CI 3.02–6.94) increased LUAD risk. Notably, none of the nine proteins showed evidence of reverse causality. Bayesian colocalization indicated that RNASET2, TFPI, and VWC2 shared the same variant with LUAD. Furthermore, NTM and FLT1 demonstrated interactions with targets of current LUAD medications. Additionally, FLT1 and TFPI are currently under evaluation as therapeutic targets, while NTM, RNASET2, and VWC2 are potentially druggable. These findings shed light on LUAD pathogenesis, highlighting the tumor-promoting effects of RNASET2, TFPI, and NTM, along with the protective effects of VWC2 and FLT1, providing a significant biological foundation for future LUAD therapeutic targets.ConclusionsOur proteome-wide MR analysis highlighted RNASET2, TFPI, VWC2, NTM, and FLT1 as potential drug targets for further clinical investigation in LUAD. However, the specific mechanisms by which these proteins influence LUAD remain elusive. Targeting these proteins in drug development holds the potential for successful clinical trials, providing a pathway to prioritize and reduce costs in LUAD therapeutics.